JOURNAL OF APPLIED HORTICULTURE Vol. 14, No. 2, July-December, 2012

ISSN 0972-1045 Vol. 14, No. 2, July-December, 2012 Appl Hort Journal of THE SOCIETY FOR ADVANCEMENT OF HORTICULTURE JOURNAL OF APPLIED HORTICULTURE Vol. 14, No. 2, July-December, 2012 CONTENTS Effect of root substrates and seed cover materials on the germination and growth of organic tomato transplants —Kurt O. Taylor, Muchha R. Reddy, Carl E. Niedziela Jr., Mary M. Peet and Godfrey Gayle (USA) 83 Ginger juice enhanced growth of aromatic chilli during in vitro culture and acclimatization —K. Bodhipadma, S. Noichinda, P. Luangsriumporn, C. Meenapa, K. Nathalang, and D.W.M. Leung (Thailand/New Zealand) 88 Water retention characteristics of soil bio-amendments used as growing media in pot culture —S.S. Kukal, Debasish Saha, Arnab Bhowmik and R.K. Dubey (India) 92 Isolation of biomolecules of pharmacological importance from Garcinia indica fruit and evaluation of total antioxidant activity —P. Gayathri and P. Govindaraju (India) 98 Postharvest microbial diversity on major cultivars of Indian mangoes —S.N. Jha, Pranita Jaiswal, K. Narsaiah, Rishi Bhardwaj, Poonam Preet Kaur, Ashish Kumar Singh, Rajiv Sharma and R. Kumar (India) 102 Effect of dehydration on keeping quality of white button mushroom, Agaricus bisporus Lange (Sing.) —M.P. Singh, H.S. Sodhi, A. Singh and P.K. Khanna (India) 110 Relationship of nutritional status of field grown Thompson Seedless grapevines with powdery mildew incidence —Jagdev Sharma, A.K. Upadhyay, Indu S. Sawant and S.D. Sawant (India) 114 Effect of growing media on seed germination and seedling growth of papaya (Carica papaya) cv. ‘Red lady’ —R.L. Bhardwaj (India) 118 Effect of plant bio-regulators on physico-chemical characteristics of three apple varieties during ambient storage —B.L. Attri, Hare Krishna, B. Das, N. Ahmed and Akhilesh Kumar (India) 124 Influence of biofertilizers on plant growth, fruit yield, nutrition and rhizosphere microbial activity of pomegranate (Punica granatum L.) cv. Kandhari Kabuli —Muzaffar Mir and Som Dev Sharma (India) 129 Resistance evaluation of the pistachio rootstocks to Meloidogyne species in Iran —Mehrdad Madani, Ahmad Akhiani, Mahmoud Damadzadeh and Ahmad Kheiri (Iran) 134 Biology and seasonal activity of semilooper, Dichromia orosia (Cramer) (Lepidoptera: Noctuidae) on anthmool, Tylophora asthmatica Wight and Arn. —L. Saravanan and Vipin Chaudhary (India) 139 Occurrence of false smut on date palm (Phoenix dactylifera L.) in the southern coastal plains of Yemen —M.H. Abdul Sattar, A. Rashid Yassin Ibrahim and Watheq A. Aulaqi (Yemen) 144 Resource use efficiency of orange and kinnow cultivation in Jammu region of J&K state —Jyoti Kachroo, Anil Bhat and Dileep Kachroo (India) 146 Enhancing water relations and vase life of cut tulip (Tulipa gesneriana L.) using floral preservatives —R. Kumar, N. Ahmed, D.B. Singh and O.C. Sharma (India) 152 Forthcoming Papers Growth control and flower promotion of salvia with benzyladenine foliar sprays —Dennis J. Carey, Barbara A. Fair, Wayne Buhler, Ingram McCall, and Brian E. Whipker (USA) Characterization and biostimulation of benzene biodegradation in the potting-mix of indoor plants —Torpy, F.R., P.J. Irga, D. Moldovan, J. Tarran and M.D. Burchett (Australia) Differential response of citrus rootstocks to copper concentration in sand culture —Joseph P. Albano, Kim D. Bowman, P. Chris Wilson and Michael G. Bausher (USA) Transformation of cabbage (Brassica oleracea var. capitata) expressing a synthetic cry1F gene resistant to diamondback moth (Plutella xylostella) Lepidoptera: Yponomeutidae —H.M. Mahadeva Swamy, S.N. Nagesha, Riaz Mahmood, T.K.S. Gowda and R. Asokan (India) Molecular biology of tomato spotted wilt virus: an update —Saurabh Kulshrestha, Anshul Sharma and Chandrika Attri Seth (India) Improvement of somatic embryogenesis and plant regeneration of seven date palm (Phoenix dactylifera L.) cultivars: Effect of cytokinins and activated charcoal —Mona M. Hassan, Ibrahim A. Ibrahim, Mohsen K.H. Ebrahim, Ewald Komor (Egypt) Growth and development response of Antirrhinum to plant growing media —Farhat Naz, Jalal-Ud-Din Baloch, M. Munir and A.A. Khakwani(UK & Pakistan) Grafting onto African eggplant enhances growth, yield and fruit quality of tomatoes in tropical forest ecozones —G.O. Nkansah, A.K. Ahwireng, C. Amoatey and A.W. Ayarna (Ghana) Ecophysiological performances of eight Jatropha curcas L. provenances cultivated in Tunisia —Z. Nasr, M.L. Khouja, R. Aini, A. Hammadi, H. Manai and B. Mimouni (Tunisia) Performance of different potato genotypes under aeroponics system —Margaret Chiipanthenga, Moses Maliro, Joyce Njoloma, Paul Demo and Navin Khumar (Malawi) Effect of irrigation levels on fruit quality of the Picual olive (Olea europaea L.) cultivar — M.M. Khattab, A.E. Shaban, I. Hussein and O.H. Elgamaal (Egypt) Overcoming the seasonality of production by growing two types of indeterminate tomato (Solanum lycopersicum) varieties under cooled plastic house conditions in Sudan —Randa B.M. Ali and Saifeldin Mohamed El-Amin (Sudan) Seasonal changes in endogenous hormone and sugar contents during bud dormancy in tree peony —Philip M.P. Mornya and Fangyun Cheng (China) Assessment of strength of self-incompatibility in the S-allele lines of cabbage (Brassica oleracea var. capitata L.) —Saurabh Singh and Vidyasagar (India) The role of avocado in coffee based farming system of south western Ethiopia: The case of Jimma Zone —Berhnau Megerssa (Ethiopia) Effect of different growth media on the growth and flowering of beef steak Begonia (Begonia erythrophylla) —Henry A. Akintoye, Olusola O. AdeOluwa, Olukemi Y. Akinkunmi (Nigeria) Growth, yield and nutrient uptake responses of snake tomato (Trichosanthes cucumerina L.) to organo-mineral fertilizer rates and leaf harvest methods —O.O. Olubode, R.I. Adedeji, and O.O. Oyegbola (Nigeria) QTL analysis associated with oleoresin content in intraspecific RIL population of chilli (Capsicum annuum L.) —Neeraj Dwivedi, Rajesh Kumar, Rakesh Kumar Singh and Major Singh (India) Journal Journal of Applied Horticulture, 14(2): 83-87, 2012 Appl Effe c t of root subst rat e s a nd se e d c ove r m at e ria ls on t he ge r m inat ion a nd grow t h of orga nic t om at o t ra nspla nt s Kur t O. Ta ylor a , M uchha R. Re ddy a * , Ca rl E. N ie dzie la J r. b , M a r y M . Pe e t c a nd Godfre y Ga yle a a Department of Natural Resources and Environmental Design, North Carolina Agricultural and Technical State University, 1601 East Market Street, Greensboro, NC 27411. bSchool of Natural Sciences and Mathematics, Ferrum College, PO Box 1000, Ferrum, VA 24088. cNational Institute of Food and Agriculture, USDA, 800 9th Street SW, Washington, DC 200242220. *E-mail: [email protected] Abstract In two experiments, seeds of tomato (Solanum esculentum L.) cultivar ‘Celebrity’ were planted in four root substrates (Grower’s Mix 20, Fafard 4P, Johnny’s 512 Select and Sunshine Planter’s) in 72-cell plastic plugs trays using different cover materials. In physical property evaluations, the four substrates had similar total porosity. However, Johnny’s 512 Select had the highest container capacity and bulk density while Fafard 4P and Sunshine Planter’s had the largest air space. There was some seasonal variation between the germination and growth results of the two studies. The use of root substrate, coir, or vermiculite resulted in better germination than leaving the seeds uncovered, with the exception of the seeds germinated in Johnny’s 512 Select in Experiment 1. Also, in Experiment 1, tomato seedlings were the tallest and heaviest when grown in Grower’s Mix 20. Using newspaper to cover seeds reduced germination in Experiment 2. Tomato seedlings grown in Grower’s Mix 20 and Johnny’s 512 Select were equal or greater in shoot height or weight as compared to those grown in the conventional substrate Fafard 4P. Introduction U.S. demand for organic foods and beverages has increased from $1 billion in 1990 to $24.8 in 2009 (Organic Trade Association, 2010). Organic food and beverage sales in 2009 increased 5.1 percent above 2008 sales. The largest sales increase in 2009 was in organic fruit and vegetable sales, which increased 11.4 percent above 2008. To be certified as organic, farms must use organically produced seeds and transplants (National Organic Program, 2012). However, these transplants are expensive and difficult to produce. Cantliffe (1998) concluded that optimizing seed germination is a critical step to ensure economic returns in a transplant operation, but relatively little work has been performed on optimizing germination in substrates approved for organic production. Russo (2005) compared shoot height and dry weight of bell pepper, onion, and watermelon transplants grown in approved organic substrates to conventional substrates. Arancon et al. (2003 and 2004) and Atiyeh et al. (2000) conducted studies on the effect of vermicompost as a rooting substrate component on emergence and growth of a wide range of seedlings, including tomato. Larrea (2006) suggested that covering the seed with vermiculite might have increased germination in organic substrates containing vermicompost; however, this hypothesis was not tested in a sideby-side comparison of germination rates. Compost and other organic materials are commonly used on organic and sustainable farms as a soil conditioner and source of mineral nutrients. One barrier to large-scale use of compost and similar soil amendments from waste materials is the need for consistent and predictable quality (Baarth, 1999). Arancon et al. (2004) stated that there are possible adverse environmental and economic impacts of agrichemicals on ornamental plant production and suggested a greater utilization of organic amendments such as composts or vermicompost for greenhouse production of bedding plants. With decreased use of synthetic nutrient sources, which are typically applied in a more soluble form than organic sources, the run-off of nitrogen (N), phosphorus (P) and potassium (K) could potentially be reduced. The use of a seed cover material provides the small-scale farmer with a technique that may improve germination; however, studies on the use of cover materials other than the substrate itself to increase germination are limited. Materials with unique properties, such as coconut coir and vermiculite, need to be evaluated for cover materials in organic transplant production. Coconut (Cocos nucifera L.) coir dust is an agricultural waste product that is produced from the mesocarp or husk of the coconut (Evans et al., 1998). There is limited research on the effectiveness of coconut coir as a seed cover material. Vermiculite is frequently used for this purpose. This research was conducted to provide research-based results on organic transplant production to meet the growing demand for organically and sustainably-grown produce. The main objectives of this study were: 1) to compare the effects of commercial and custom organically blended substrates on tomato seed germination and growth and 2) to evaluate coconut coir and vermiculite as seed cover materials on tomato seed germination in organic transplants production. Complementary Copy Key words: Solanum esculentum, vermicompost, feather meal, kelp meal, seedlings, root media. Effect of root substrates and seed cover materials on the germination and growth of organic tomato Materials and methods Two experiments were conducted in the Reid Greenhouse at North Carolina Agricultural and Technical State University in Greensboro, NC, USA (36° N latitude). Temperatures were set at day and night minimums of 25 and 21°C, respectively. Tomato (Solanum esculentum ‘Celebrity’) seeds were germinated and grown in 72-cell trays (Landmark Plastic Corp., Akron, OH) in both experiments. Plants were watered as needed based on the physical appearance of the substrate and seedlings. Four substrates were selected for both experiments: a conventional, commercial transplant substrate (Fafard 4P, Conrad Fafard, Inc., Agawam, MA), two commercial organic substrates (Sunshine Planter’s, Sun Gro Horticulture, Bellevue, WA and Johnny’s 512 Select, Johnny’s Selected Seeds, Winslow, ME), and a modified organic substrate mixed for this research (Grower’s Mix 20). Fafard 4P contained 45% Canadian sphagnum peat moss combined with aged pine bark, vermiculite, Dolomitic limestone, gypsum, wetting agent, starter nutrients, and slow release nitrogen (Fafard Product Guide, 2011). Johnny’s 512 Select was a blend of sphagnum and sedge peat mosses, compost, and perlite (Johnny’s Selected Seeds, 2012). The Grower’s Mix 20 components and mixing specifications were adopted from Larrea (2006). Grower’s Mix 20 was a 4 peat: 1 perlite (v/v) amended with feather meal (Boer Commodities.Inc., Fresno, CA) and kelp meal (Algit, P.B. Ohrstrom & Sons Inc., Arlington Heights IL) at 19.53 and 8.40 L.m-3, respectively. Tomato seeds were sown on 21 Aug. and 6 Dec. 2007 for Experiments 1 and 2, respectively. In Experiment 1, each 72cell tray was covered with one of the four seed cover materials: vermiculite (Peaceful Valley, Grass Valley, CA), coconut coir (Down to Earth Distributors, Eugene, OR), the same substrate in which the seed were planted, or left uncovered. In Experiment 2, a single layer of wet newspaper was added as a fifth seed cover material treatment over uncovered seeds. The wet newspaper remained over the flat until ten days after cotyledons were visible (20 Dec). Sample and data collection: Samples of the soilless substrates were collected for physical properties prior to planting. Germination data were collected on 31 Aug. and 20 Dec. in Experiments 1 and 2, respectively. Germination percentage was calculated as the total number of seedlings that emerged divided by the total number of seed sown multiplied by 100 (Bradford, 1986). Height was measured on 20 randomly selected transplants from each flat on 18 Sept 2007 (18 days after sowing) and 3 Jan 2008 (14 days after sowing) in Experiments 1 and 2, respectively. Shoot tissue was sampled by cutting the stem at the base of 20 transplants using a hand pruner. Plant samples were collected 28 days after sowing in Experiment 1 and 18, 32 and 39 days after sowing in Experiment 2. The composited fresh weight of the 20 sample transplants was recorded. Plant samples were rinsed in distilled water for 30 s. The samples were then placed in brown paper bags and dried at 70°C in a forced air oven until a constant weight was attained (approximately 48 h). The dry weight of each sample was then recorded. Dry plant samples were ground to pass a 1 mm screen (20-mesh) using the Thomas-Wiley Laboratory Stainless Steel Mill Model No. 4 (Thomas Scientific, Philadelphia). Substrate and tissue analyses: All substrates were analyzed for physical properties (total porosity, container capacity, air space and bulk density) in the Horticultural Substrates Laboratory at North Carolina State University using the procedures described by Fonteno (1996). Nitrogen was determined on 10 mg plant tissue samples with a Perkin Elmer 2400 CHN elemental analyzer (Norwalk, CT). Another 0.5 mg sample was placed in the Erlenmeyer flask for dry ashing. The sample was then placed in the Muffle Furnace for eight hours at 550 °C (Mills and Jones, 1996). Nitric acid (2.5mL) was added to the ash. The Erlenmeyer flask was then rinsed twice and filtered in a 50 mL volumetric flask. The ash solution was diluted to 50 mL with deionized water. The sample was then analyzed for potassium (K), phosphorous (P), calcium (Ca) and magnesium (Mg), using an Inductively Coupled Plasma Spectrometer (ICP-OES; Perkin-Elmer Optima 3300DV, Norwalk, CT). All tissue analyses were conducted at the Analytical Services Laboratory, School of Agriculture and Environmental Sciences, NC A&T State Univ. Experimental design and statistical analysis: Experiment 1 was a factorial design with four substrates and four cover materials arranged in a randomized complete block with four replications for a total of 64 experimental units (72-cell flats). Experiment 2 was a factorial design with four substrates and five cover materials arranged in a randomized complete block with four replications for a total of 80 experimental units (72-cell flats). Data were subjected to analysis of variance using PROC ANOVA (SAS 9.3, SAS Inst., Cary, NC). Means were separated using the Fisher’s Protected LSD test at the 0.05 level of significance. Results and discussion Substrate physical properties: Total porosity was similar for the four root substrates (Table 1). Container capacity was greatest in Johnny’s 512 Select followed by Grower’s Mix 20 and least in Fafard 4P and Sunshine Planter’s. Air space was greatest in Fafard 4P and Sunshine Planter’s, intermediate in Grower’s Mix 20, and least in Johnny’s 512 Select. Bulk density varied from greatest to least in the following order: Johnny’s 512 Select, Grower’s Mix 20, Fafard 4P, and Sunshine Planter’s. Table 1. Total porosity, container capacity, air space and bulk density of four root substrates used for growing ‘Celebrity’ tomato transplants Substratez Porosity Container Air Space Bulk capacity (% by density (% by volume) (% by volume) volume) (g.cm-3) Grower’s Mix 20 88.37 76.73 11.57 0.17 Fafard 4P 87.60 70.23 17.37 0.14 Sunshine Planter’s 87.37 70.40 16.97 0.09 Johnny’s 512 Select 86.93 80.53 2.16 6.43 2.14 0.26 0.03 LSD0.05 NS z Grower’s Mix 20, Sunshine Planter’s and Johnny’s 512 Select were substrates formulated to meet the National Organic Standards for transplant production. Fafard 4P was a substrate used commercially for conventional transplant production. Transplant production practices, such as frequency of irrigation, must be adjusted based on a root substrate’s physical properties and vice versa (Nelson, 2011). For example, root substrates with a high container capacity and low percent air space, such Complementary Copy 84 Effect of root substrates and seed cover materials on the germination and growth of organic tomato 85 as Johnny’s 512 Select, require less frequent watering during plant production and retain more water during plant shipping and marketing. However, greater care is required with these substrates that plants are not over-watered during production. Well-drained substrates such as Fafard 4P and Sunshine Planter’s, require more frequent irrigations, which also affects the leaching of nutrients. The greater bulk density of Johnny’s 512 Select may also increase the shipping cost for the bulk substrate and finished transplants. There was no root substrate x seed cover material interaction or cover material effect on the shoot height. The tallest plants were recorded in Grower’s Mix 20 followed by Fafard 4P and Johnny’s 512 Select (Table 2). The shortest plants were produced in Sunshine Planter’s. There was a root substrate x seed cover material interaction effect on shoot dry weight. With all four cover materials, shoots were heaviest in Grower’s Mix 20 and lightest in Sunshine Planter’s (Fig. 1B). Within plants grown in Fafard 4P and Sunshine Planter’s, plants had similar dry weights regardless of seed cover material. In Grower’s Mix 20, plants from seeds covered with vermiculite were heavier than those covered with coir or left uncovered. However, in Johnny’s 512 Select, plants from seeds covered with vermiculite were lighter than those covered with substrate or coir and those left uncovered weighed less than those covered with substrate. There was no root substrate x seed cover material interaction or cover material effect on the shoot nutrient concentrations. Nutrient concentration data is shown in Table 2. Shoot N Table 2. Effect of root substrate on the shoot height and nutrient concentrations of ‘Celebrity’ tomato transplants at 28 days after planting in Experiment 1 Height N P K Ca Mg Substratez (cm) (%) (%) (%) (%) (%) Grower’s Mix 20 31.9 3.50 0.97 3.17 1.78 0.55 Fafard 4P 19.4 2.05 0.50 2.94 1.15 0.68 Sunshine Planter’s 4.9 2.01 0.23 0.56 1.09 0.62 Johnny’s 512 Select 19.6 3.90 0.60 2.96 1.83 0.32 LSD0.05 1.3 1.01 0.08 0.39 0.24 0.08 z Grower’s Mix 20, Sunshine Planter’s, and Johnny’s 512 Select were substrates formulated to meet the National Organic Standards for transplant production. Fafard 4P was a substrate used commercially for conventional transplant production. Fig. 1. Germination and shoot dry weight of ‘Celebrity’ tomato transplants in four root substrates (Grower’s Mix 20, Fafard 4P, Sunshine Planter’s, and Johnny’s 512 Select) in combination with four seed cover materials in Experiment 1. Vertical bars indicate LSD0.05 for comparing root substrate within each seed cover material or for comparing seed cover materials within each root substrate. concentration was higher in plants grown in Grower’s Mix 20 and Johnny’s 512 Select than those grown in Fafard 4P and Sunshine Planter’s. Shoot P concentration went from highest to lowest in the following order: Grower’s Mix 20, Johnny’s 512 Select, Fafard 4P, and Sunshine Planter’s. Shoot K was lower in plants grown in Sunshine Planter’s than the other three root substrates. Shoot Ca concentration was higher in plants grown in Grower’s Mix 20 and Johnny’s 512 Select than those grown in Fafard 4P and Sunshine Planter’s. Plants grown in Johnny’s 512 Select had the lowest shoot Mg concentration and plants grown in Gower’s Mix 20 had a lower shoot Mg concentration than those grown in Fafard 4P. Experiment 2: There was no root substrate x seed cover material interaction effect on seed germination. However, seed germination rate was greater in Sunshine Planter’s and Johnny’s 512 Select than Grower’s Mix 20 (Table 3). Also, seed germination rate was greater using the root substrate, coir or vermiculite than with newspaper or uncovered seeds (Table 4). There was no root substrate x seed cover material interaction effect on shoot height. However, plants grown in Sunshine Planter’s were shorter than those in the other three substrates (Table 3). Also, plants were taller when seeds were covered with vermiculite than when seed were not covered and plants Complementary Copy Experiment 1: There was a root substrate x seed cover material interaction effect on seed germination. In Johnny’s 512 Select, seed germination was similar for coir, vermiculite, and no cover (Fig. 1A). However, using the Johnny’s 512 Select substrate as a cover resulted in a lower germination rate than with vermiculite. In Sunshine Planter’s, not covering seeds resulted in the lowest germination rate. In Fafard 4P, the uncovered seeds germinated at a similar rate to those covered with coir but a lower rate than those covered with the substrate or vermiculite. In Grower’s Mix 20, seeds covered with vermiculite or uncovered germinated at a lower rate than those covered with the substrate or coir. These results indicate that the best choice of cover material for germinating tomato seeds was dependent on the root substrate. Johnny’s 512 Select was a heavier substrate with greater container capacity and bulk density, the germinating seedlings benefited from being covered by the lighter vermiculite or leaving the seeds uncovered in Experiment 1. Whereas, the other substrates were lighter and more prone to drying out so leaving the seeds uncovered was detrimental. 86 Effect of root substrates and seed cover materials on the germination and growth of organic tomato Table 3. Effect of root substrate on the germination rate, shoot height, and shoot nutrient concentrations of ‘Celebrity’ tomato transplants at 32 days after planting in Experiment 2 Germination (%) Height (cm) N (%) P(%) K(%) Ca(%) Mg(%) Substratez Grower’s Mix 20 84.4 14.6 5.36 0.15 1.61 0.39 0.15 Fafard 4P 88.2 13.9 3.68 0.08 0.96 0.17 0.15 Sunshine Planter’s 90.6 4.2 3.19 0.08 0.62 0.39 0.25 Johnny’s 512 Select 89.8 13.4 4.55 0.13 1.61 0.71 0.16 LSD0.05 4.3 1.4 1.96 0.06 0.63 0.21 0.05 z Grower’s Mix 20, Sunshine Planter’s, and Johnny’s 512 Select were substrates formulated to meet the National Organic Standards for transplant production. Fafard 4P was a substrate used commercially for conventional transplant production. Table 4. Effect of seed cover material on the germination rate and shoot height of ‘Celebrity’ tomato transplants at 32 days after planting in Experiment 2 Cover Substratez Coir Vermiculite No cover Newspaper LSD0.05 Germination (%) 92.9 89.9 90.7 83.9 83.8 4.8 Height (cm) 12.0 12.3 12.6 10.9 9.8 1.5 z The substrate used for the seed cover material was the same as the respective root substrate material. There was a root substrate x seed cover material interaction effect on shoot dry weight (Fig. 2). Plants grown in Sunshine Planter’s were lighter than those grown in the other three substrates regardless of seed cover material. Plants grown in Grower’s Mix 20 and Sunshine Planter’s had similar dry weights regardless of seed cover material. In Fafard and Johnny’s 512 Select, plants grown from seed covered with coir or vermiculite were heavier than those with other three cover materials. There was no root substrate x seed cover material interaction or cover material effect on the shoot nutrient concentrations (Table 3). Shoot N concentration was higher in plants grown in Grower’s Mix 20 than those grown in Sunshine Planter’s. Shoot P concentration was higher in plants grown in Grower’s Mix 20 than those grown in Fafard 4P and Sunshine Planter’s. Shoot K concentration was higher in plants grown in Grower’s Mix 20 and Johnny’s 512 Select than those grown in Fafard 4P and Sunshine Planter’s. Shoot Ca concentration was highest in plants grown in Johnny’s 512 Select followed by Grower’s Mix 20 and Sunshine Planter’s and lowest in those grown in Fafard 4P. Shoot Mg concentration was higher in plants grown in Sunshine Planter’s than the other three substrates. Differences in the results between the two experiments can be attributed to seasonal variation. Experiment 1 was begun in August when daylengths were longer than 12 hours, light levels were high, and greenhouse temperatures were hot. Experiment 2 was conducted in December and January when daylengths were shortest, light levels were low, and greenhouse temperatures were cooler. Thus, tomato seedlings were taller and weighed more in Experiment 1 than Experiment 2. Although total porosity was similar between the four root substrates tested, container capacity and bulk density were greatest in Johnny’s 512 Select and air space was greatest in Fafard 4P and Sunshine Planter’s. The use of root substrate, coir, or vermiculite resulted in better germination than leaving the seeds uncovered, with the exception of the seeds germinated in Johnny’s 512 Select in Experiment 1. Using newspaper to cover seeds also reduced germination. Tomato seedlings grown in Grower’s Mix 20 and Johnny’s 512 Select were equal or greater in shoot height or weight as compared to those grown in a conventional substrate (Fafard 4P). Acknowledgements This paper is a portion of a thesis submitted by K.O. Taylor. This research was supported in part by the NCA&TSU Agricultural Research Program (Evans-Allen Funds). The use of trade names in this publication does not imply endorsement or criticism of the products named, or criticism of similar ones not mentioned. References Fig. 2. Shoot dry weight of ‘Celebrity’ tomato transplants in four root substrates (Grower’s Mix 20, Fafard 4P, Sunshine Planter’s, and Johnny’s 512 Select) in combination with five seed cover materials in Experiment 2. Vertical bars indicate LSD0.05 for comparing root substrate within each seed cover material or for comparing seed cover materials within each root substrate. Arancon, N.Q., C.A. Edwards, P. Bierman, J.D. Metzger, S. Lee and C. Welch, 2003. Effects of vermicomposts on growth and marketable fruits of field-grown tomatoes, peppers and strawberries. Pedobiologia, 47: 731-735. Arancon, N.Q., C.A. Edwards, R. Atiyeh and J.D. Metzger, 2004. Effects of vermicompost produced from food waste on the growth and yields of greenhouse peppers. Bioresource. Technol., 93: 139-144. Atiyeh, R.M., C.A. Edwards, S. Subler and J.D. Metzger, 2000. Earthworm-processed organic wastes as components of horticultural potting media for growing marigold and vegetables. Compost Sci. Utilization, 8: 215-223. Baarth, J. 1999. Compost quality, quality assurance and use: the basis for sustainable organic waste management in Europe. Proceedings of the International Composting Symposium. (P.R.Warman and B.R. Taylor, ed.), 1: 14-31. Complementary Copy were taller when seeds were covered with root substrate, coir or vermiculite than when covered with newspaper (Table 4). Effect of root substrates and seed cover materials on the germination and growth of organic tomato Larrea, E.S. 2006. Optimizing Substrates for Organic Tomato Transplant Production. M.S. Thesis., North Carolina State University, 2006. 87 pp. Mills, A.H. and J.B. Jones, 1996. Plant Analysis Handbook II: A Practical Sampling, Preparation, Analysis, and Interpretation Guide. MicroMacro Publishing, Inc, Athens, Georgia. Nelson, P.V. 2011. Greenhouse Operation and Management. 7th Ed. Prentice-Hall, Upper Saddle River, New Jersey. Organic Trade Association, 2010. 2010 Organic Industry Survey. Organic Trade Assn., Brattleboro, VT. Russo, V.M. 2005. Organic vegetable transplant production. HortScience 40: 623-628. Received: May, 2012; Revised: October, 2012; Accepted: October, 2012 Complementary Copy Bradford, K.J. 1986. Manipulation of seed water relations via osmotic priming to improve germination under stress conditions. HortScience 21: 1105-1112. Cantliffe, D. J. 1998. Seed germination for transplants. HortTechnology, 8: 499-503. Evans, M.R., J.N. Smith and R.A. Cloyd, 1998. Fungus gnat population development in coconut coir and sphagnum peat based substrates. HortTechnology, 8: 406-408. Fonteno, W.C., 1996. Growing media: Types and physical/chemical properties. In: A Grower’s Guide to Water, Media, and Nutrition for Greenhouse Crops. Ed. D.W. Reed.: Ball Publishing, Batavia, IL. Johnny’s Selected Seeds, 2012. <http://www.johnnyseeds.com/p-6693johnnys-512-mix-og-2-yard-bulk.aspx> 87 Journal Journal of Applied Horticulture, 14(2): 88-91, 2012 Appl Ginge r juic e e nha nc e d grow t h of a rom at ic chilli during in vit ro c ult ure a nd a c clim at izat ion K . Bodhipa dm a a , S. N oichinda a , P. Lua ngsrium por n a , C. M e e na pa a , K . N at ha la ng b a nd D.W.M . Le ung c * a Department of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangsue, Bangkok 10800 Thailand. bDepartment of Biology, Faculty of Science, Mahidol University, Rama VI Rd., Rajathevi District, Bangkok 10400, Thailand. cSchool of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand. *E-mail: [email protected] Abstract Stem explants excised from seedlings of aromatic chilli (Capsicum frutescens L) grown under aseptic conditions were cultured on basal medium alone (control), and basal medium supplemented with 5, 10 or 20 mL/L juice of ginger rhizome of 6 or 10 months old (herein referred to as YGE and OGE, respectively). At the end of 6 weeks of culture, the average number of roots formed per stem explant was higher when cultured on media supplemented with the three different levels of YGE or OGE (except 5 mL/L) compared to the control. Roots, formed in stem explants cultured on media containing the different levels of YGE (except 20 mL/L) and OGE, were longer than those cultured on basal medium. Particularly notable was that the average length of roots formed in stem explants cultured on medium supplemented with 5 mL/L OGE was more than double that of the control. Prior culture on media containing the different levels of YGE had no promotive effect on the number of leaves per exflasked plantlet compared to the control at the end of three weeks of acclimatization but the plantlets cultured previously on 5 or 10 mL/L YGE were taller than the control. The best performance of plantlets regarding leaf number and stem height after acclimatization was exhibited by those cultured previously on medium containing 10 mL/L OGE as they had at least 20% more leaves and were taller than the control. Introduction substances that can be sourced locally such as ginger rhizomes in Thailand for micropropagation of plants. Chemical analysis of ginger juice has shown that it has antimicrobial potential and it is a good source of antioxidants, carbohydrates and some minerals (Shirin and Prakash, 2010). Here, we evaluated the hypothesis that different concentrations of ginger juice had some beneficial effect not only on in vitro culture but also on acclimatization of exflasked aromatic chilli plantlets. In addition, the effect of juice from ginger rhizome of two different ages was compared as it might be possible that biological activity could vary with different ages of ginger rhizomes. Many complex food extracts have been added to plant tissue culture medium for different purposes. For example, there are reports showing use of banana and apple juice to promote growth of cymbidium protocorms (Kusumoto and Furukawa, 1977), pineapple juice for plantlet development of several orchid species (Kitsaki et al., 2004), coconut water and tomato juice for shoot multiplication from encapsulated buds of Pogostemon cablin (Swamy et al., 2009), banana extract for rapid plant regeneration of Dendrobium lituiflorum (Vyas et al., 2009), yeast extract, casein hydrolysate and potato extract for callus induction of Stevia rebaudiana (Das and Mandal, 2010) and sugarcane juice for shoot proliferation and growth of Pogostemon cablin and bananas (Swamy et al., 2010; Buah et al., 2011). Rooting of shoots derived from juvenile stem explants of avocado was promoted in medium supplemented with peptone and an auxin (NAA or 1-naphthlene acetic acid) but not with the auxin alone (Nhut et al., 2008). There is no report on use of complex natural substances to improve performance of exflasked plantlets during acclimatization, particularly, if these substances are commonly available at a relatively low cost. This is an important practical objective of micropropagation of plants. Materials and methods In our preliminary work, a protocol was developed for regenerating plantlets from stem explants of aromatic chilli, an important ingredient in many Thai cusine. It is of interest to the plant production industry to find low-cost natural organic complex Preparation of ginger juice: Ginger rhizomes (Zingiber officinale Rosc. Cv. Yai) of about 6- and 10-months old, herewith referred to as young and old ginger rhizomes, respectively, were purchased from a local market in the Nonthaburi province, Plant materials and surface-sterilization: Seeds of aromatic chilli (Capsicum frutescens L.) were purchased from Thai Seed and Agriculture Co., Ltd., Bangkok, Thailand. The following sequence of steps was carried out to surface-sterilize the seeds: (1) soaking in distilled water overnight, (2) washing briefly with tap water and liquid soap, (3) immersing in 15% (v/v) Clorox (a commercial bleach solution containing 5.25% (w/w) sodium hypochlorite as available chlorine) for 15 min, (4) 10% (v/v) Clorox containing 3-4 drops of Tween-20 for 5 min, and (5) rinsing 3 times (1 min each time) with sterile distilled water. Complementary Copy Key words: Acclimatization, aromatic chilli, ginger juice, root elongation, root induction Ginger juice enhanced growth of aromatic chilli during in vitro culture and acclimatization Germination of seeds, explant preparation and culture: Surface-sterilized aromatic chilli seeds were placed onto basal MS (Murashige and Skoog, 1962) medium gelled with 0.9% agar (w/v) and kept under 16 h of illumination with white fluorescent lamps (47.31 μmol/m2/s light intensity) and 8 h of darkness in a growth room at 25±2 °C. After 3 weeks of culture, stem explants (2 cm long comprising the shoot tip and 2-3 nodes) were excised from aromatic chilli seedlings which were approximately 3 to 4 cm tall. These explants (one per culture jar and there were six replications) were transferred to MS medium supplemented with different concentrations (5, 10 and 20 mL/L ) of juice from young and old ginger rhizomes. The pH of all the media was adjusted to 5.6 before they were autoclaved. The cultures were kept for 6 weeks under the same conditions as already described for aseptic germination of the surfaced-sterilized seeds. Plantlet acclimatization: Plantlets were taken out of culture jars and the agar adhered to the roots were removed by shaking gently in running tap water for a couple of minutes before they were placed in plastic pots (5.8 cm tall, 3.2x4 cm on top and 2x2.5 cm at the base of a pot) containing a soil mixture (soil:sand:chopped rice straw in 5:4:1 ratio). One plantlet was placed in each pot (there were six replications) filled to the top with the soil mixture which was kept watered to capacity throughout the experiment. Each pot was covered loosely with a clear plastic bag and lifted briefly daily throughout the experiment so that the leaves were sprayed with water using a hand-held water spray bottle. At the beginning of the second week of acclimatization, two holes (1.5 cm diameter each) were made on the side of the bag and six more holes of the same size were made at the beginning of the third week. All the plantlets were grown in a plant nursery under a plastic shade net with 12 hours of natural sunlight a day and average day temperature around 30-32oC and night temperature around 22-23oC. Number of leaves and stem height (measured from top of a pot to the shoot apex) per plantlet were determined after 4 weeks of acclimatization under ex vitro conditions. Data analysis: Means of root number, root length, leaf number and stem height ± SE per plantlet were analyzed. One-way ANOVA (STATISTIX for Windows 2.0 analytical software) was first performed at the significance level of 0.05. After this, when appropriate, Duncan comparison of means was carried out at P<0.05. endogenous auxin or hormonal balance within these explants was sufficient for adventitious root organogenesis (Bodhhipadma et al., 2010). However, compared to control more roots per explant were induced when the explants were cultured on basal MS medium supplemented with different concentrations of YGE or OGE with the exception of 5 mL/L OGE (Fig. 1). Interestingly, YGE or OGE at the concentrations used in this study had no effect on root formation in stem explants of Kaffir lime (Citrus hystrix DC.), another plant that rooted in medium without exogenous plant growth regulators (data not shown). With the exception of 20 mL/L of YGE, different concentrations of YGE or OGE added to basal MS medium promoted root elongation in aromatic chilli stem explants compared to control (Fig. 2). The promotive effect of 5 mL/L of YGE on elongation of roots formed in explants was more than that of the higher concentrations of YGE (Fig. 2). This was also observed among different concentrations of OGE. Overall, the most effective treatment was 5 mL/L of OGE as root length was more than double a b c a b c a b c d b c d Fig. 1. Number of roots formed per stem explant (n=6) of aromatic chilli cultured for 6 weeks on different media supplemented with different concentrations of juice of young or old ginger rhizome (YGE or OGE respectively). Values are mean number of roots±SE and those assigned with different letters are significantly different (P<0.05). Different media: 1 = basal MS; 2= MS + 5 mL/L YGE; 3= MS + 10 mL/L YGE; 4= MS + 20 mL/L YGE; 5= MS + 5 mL/L OGE; 6= MS + 10 mL/L OGE; 7= MS + 20 mL/L OGE. a b c d e b c d e c d Results and discussion Root formation and growth in vitro: Adventitious root formation is a crucial step in micropropagation from stem explants. Many studies have been carried out to investigate the factors influencing rooting in vitro. An auxin such as IAA, IBA, NAA or 2,4-D is generally added to plant tissue culture media for root initiation in vitro (Saxena et al., 2000; Ashrafuzzaman et al., 2009). In the present study, root formation occurred in stem explants of aromatic chilli cultured on basal MS medium without any exogenous plant growth regulators (control), suggesting that Fig. 2. Elongation of roots formed by stem explants (n=6) of aromatic chilli cultured for 6 weeks on different media supplemented with different concentrations of juice of young or old ginger rhizome (YGE or OGE respectively). Values are mean root length±SE and those assigned with different letters are significantly different (P<0.05). Different media: 1 = basal MS; 2= MS + 5 mL/L YGE; 3= MS + 10 mL/L YGE; 4= MS + 20 mL/L YGE; 5= MS + 5 mL/L OGE; 6= MS + 10 mL/L OGE; 7= MS + 20 mL/L OGE Complementary Copy Thailand. Juice was extracted from 300 g of ginger rhizome (of a particular age) which had been peeled and chopped into small pieces using a juicer blender (Severin Entsafter Juicy 300 ES 3557). Young and old ginger rhizomes yielded 167 and 151 mL of juice, respectively, which was added to plant tissue culture medium as described below. 89 Ginger juice enhanced growth of aromatic chilli during in vitro culture and acclimatization Table 1. Per cent survival (A), number of leaves (B) and stem height (C) of aromatic chilli plantlets from different media at the time of exflasking (day 0) and after acclimatization for 4 weeks (A) Survival of ex flasked aromatic chilli plantlets (%) MS medium Basal +5 mL/L YGE +10 mL/L YGE +20 mL/L YGE +5 mL/L OGE +10 mL/L OGE +20 mL/L OGE Day 0 100+0a 100+0a 100+0a 100+0a 100+0a 100+0a 100+0a 4 weeks 100+0a 100+0a 100+0a 100+0a 100+0a 100+0a 100+0a (B) Number of leaves per exflasked aromatic chilli plantlet MS medium Day 0 4 weeks Basal 18.5 ± 0.922a 24.66 ± 1.406c +5 mL/L YGE 15.83 ± 0.543b 25.0 ± 1.693c +10 mL/L YGE 15.83 ± 0.401b 25.0 ± 0.730c +20 mL/L YGE 14.66 ± 0.494bc 25.16 ± 0.401c +5 mL/L OGE 18.33 ± 0.882a 29.5 ± 1.258b +10 mL/L OGE 19.16 ± 0.307a 31.5 ± 0.719ab +20 mL/L OGE 20.0 ± 1.0a 33.5 ± 1.408a (C) Stem height (mm) per exflasked aromatic chilli plantlet MS medium Day 0 4 weeks Basal 40.0 ± 0.775c 50.0 ± 2.236cd +5 mL/L YGE 41.16 ± 0.477b 65.0 ± 3.416a +10 mL/L YGE 43.5 ± 1.204ab 60.0 ± 0ab +20 mL/L YGE 41.0 ± 0.931b 52.5 ± 1.708cd +5 mL/L OGE 45.16± 0.477a 63.33 ± 2.108a +10 mL/L OGE 46.16 ± 1.014a 59.16 ± 0.833ab +20 mL/L OGE 45.83 ±1.400a 55.0 ± 1.291bc Values are means of 6 replications±SE and those sharing the same letter are not significantly different. (YGE = young ginger juice, OGE = old ginger juice) that of control. Taken together, depending on concentrations and age of ginger rhizome, juice of ginger rhizome added to basal MS medium could generate more beneficial effects than basal MS medium alone as far as formation and growth of roots in stem explants of aromatic chilli was concerned. Besides ginger juice, several complex natural substances have also been shown to have beneficial effects on root formation and elongation in vitro. It was hypothesized that extracellular extracts of cyanbacterial culture might add some plant growth regulators or other factors that act directly in promoting rooting response (Manickavelu et al., 2006). Peptone together with NAA was found better than NAA alone for root initiation and elongation in stem explants of avocado (Nhut et al., 2008). It was thought that peptone had an indirect rooting effect as it prevented deterioration of the stem explants so that NAA could initiate root formation. In the present study, ginger juice might have substances that augment the action of endogenous auxin in the aromatic chilli stem explants. Effect of culture treatments on ex vitro acclimatization: There was virtually no significant difference in the survival rates of all the exflasked plantlets of aromatic chilli under the ex vitro acclimatization conditions in this study, regardless of the previous in vitro culture treatments (Table 1A). This suggests that the acclimatization conditions used in the present study were adequate to ensure high survival rates of aromatic chilli plantlets following exflasking. Leaf production: At the time of exflasking, the aromatic chilli plantlets developed on YGE-supplemented media had fewer leaves per plantlet than control or those developed on media supplemented with OGE (Table 1B). After 4 weeks of acclimatization, exflasked plantlets previously cultured on media supplemented with different concentrations of OGE formed 2030% more leaves per plantlet than control and those previously cultured on media supplemented with YGE (Table 1B). Therefore, it would seem more beneficial to add OGE rather than YGE to MS basal medium as far as boosting leaf production in exflasked plantlets during acclimatization was concerned. Stem height: At the end of in vitro culture, the aromatic chilli plantlets in control were shorter than those developed on media supplemented with YGE or OGE (Table 1C). After acclimatization, exflasked plantlets previously cultured on media supplemented with 5 or 10 mL/L but not 20 mL/L YGE or OGE were taller than control. Plantlets from the OGE treatments (5 or 10 mL/L) were about 15-30% taller than control. These results were consistent with the notion that in vitro treatments could have lasting or long-term effects on in vivo growth of exflasked plants (Economou and Read, 1987; Nowak and Shulaev, 2003; Iacona and Muleo, 2010). In conclusion, this is the first report on utility of ginger juice to influence growth and development of other plants. Aromatic chilli plantlets exflasked from medium supplemented with OGE (10 mL/L), generally grew better than those previously cultured with YGE in terms of both increase in leaf number and stem height during acclimatization. It would seem worthwhile in future to identify the chemical basis of this effect of OGE. It has been shown that ginger juice has antioxidants, carbohydrates and some minerals (Shirin and Prakash, 2010). These are likely to vary with different ages of the ginger rhizome from which the ginger juice is extracted. It would be of interest to investigate more closely if these substances present in ginger juice might promote root initiation, root elongation and improved performance of exflasked aromatic chilli plantlets after acclimatization. In addition, since it is well-known that auxin and cytokinin are generally involved in plant regeneration (Bodhipadma et al., 2011), it seems worthwhile to investigate the occurrence and levels of these plant hormones in ginger juice. Although it remains to be determined what factors in ginger juice might be responsible for its effects on plant growth and development, the practical benefit of ginger juice for improving growth of exflasked aromatic chilli plantlets during acclimatization has been clearly demonstrated which was the primary objective of this study. It would be of further interest to determine if ginger juice has any effect on other in vitro processes including callus induction and growth, somatic embryogenesis etc. References Ashrafuzzaman, M., M.M. Hossain, M.R. Ismail, M.S. Haque and S.M. Shahin-uz-zaman, 2009. Regeneration potential of seedling explants of chilli (Capsicum annuum). Afr. J. Biotechnol., 8: 591-596. Bodhipadma, K., S. Noichinda, I. Yadbuntung, W. Buaeiam and D.W.M. Leung, 2010. Comparison of in vitro and in vivo inflorescence of common cockscomb (Celosia argentea var. cristata). Science Asia, 36: 68-71. Bodhipadma, K., S. Noichinda, W. Padyencheun, T. Khunthacharoen, and U. Chikhunthod, 2011. Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose). Plant Cell Tiss. Organ Cult., 105: 465-469. Complementary Copy 90 Ginger juice enhanced growth of aromatic chilli during in vitro culture and acclimatization Nowak, J. and V. Shulaev, 2003. Priming for transplant stress resistance in in vitro propagation. In vitro Cell Devel. Biol. Plant., 39: 107124. Nhut, D.T., N.N. Thi, B.L.T. Khiet and V.Q. Luan, 2008. Peptone stimulates in vitro shoot and root regeneration of avocado (Persea americana Mill.). Sci. Hort., 115: 124-128. Saxena, C., S. Samantaray , G.R. Rout and P. Das, 2000. Effect of auxins on in vitro rooting of Plumbago Zeylanica: Peroxidase activity as a marker for root induction. Biol. Plant., 43: 121-124. Shirin, A.P.R. and J. Prakash, 2010. Chemical composition and antioxidant properties of ginger root (Zingiber officinale). J. Med. Plants, 4: 2674-2679. Swamy, M.K., S. Balasubramanya and M. Anuradha, 2009. Germplasm conservation of patchouli (Pogostemon cablin Benth.) by encapsulation of in vitro derived nodal segments. Int. J. Biodiversity Conservat., 1: 224-230. Swamy, M.K., K.M. Sudipta, S. Balasubramanya and M. Anuradha, 2010. Effect of different carbon sources on in vitro morphogenetic response of patchouli (Pogostemon cablin Benth.). J. Phytol., 2: 11-17. Vyas, S., S. Guha, M. Bhattacharya and I.U. Rao, 2009. Rapid regeneration of plants of Dendrobium lituiflorum Lindl. (Orchidaceae) by using banana extract. Sci. Hort., 121: 32-37. Received: March, 2012; Revised: July, 2012; Accepted: November, 2012 . Complementary Copy Buah, J.N., J.W. Tachie-Menson, G. Addae and P. Asare, 2011. Sugarcane juice as an alternative carbon source for in vitro culture of plantains and banans. Am. J. Food Technol., 6: 685-694. Das, A. and N. Mandal, 2010. Enhanced development of embryogenic callus in Stevia rebaudiana Bert. by additive and amino acids. Biotechnol., 9: 368-372. Economou, A.S. and P.E. Read, 1987. Light treatments to improve efficiency of in vitro propagation systems. Hort. Sci., 22: 751-754. Iacona, C. and R. Muleo, 2010. Light quality affects in vitro adventitious rooting and ex vitro performance of cherry rootstock Colt. Sci. Hort., 125: 630-636. Kitsaki, C.K., D. Zygouraki, M. Ziobora and S. Kintzios, 2004. In vitro germination, protocorm formation and plantlet development of mature versus immature seeds from several Ophrys species (Orchidaceae). Plant Cell Rep., 23: 284-290. Kusumoto, M. and J. Furukawa, 1977. Effect of organic matter on the growth of Cymbidium protocorms cultured in vitro. J. Japan Soc. Hort. Sci., 45: 421-426. Manickavelu, A., N. Nadarajan, S.K. Ganesh, R. Ramalingam, S. Raguraman and R.P. Gnanamalar, 2006. Organogenesis induction in rice callus by cyanobacterial extracellular product. Afr. J. Biotechnol., 5: 437-439. Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15: 473497. 91 Journal Journal of Applied Horticulture, 14(2): 92-97, 2012 Appl Wat e r re t e nt ion cha ra c t e rist ic s of soil bio-a m e ndm e nt s use d a s grow ing m e dia in pot c ult ure S.S. Kuk a l 1 * , De ba sish Sa ha 1 , Ar na b Bhow m ik 1 a nd R.K . Dube y 2 Department of Soil Science, 2Department of Floriculture and Landscaping, Punjab Agricultural University, Ludhiana, 141004, India. *E-mail: [email protected] 1 Abstract Key words: Bio-amendments, growing media, bulk density, water retention, air filled porosity, easily available water, water buffering capacity Introduction The commercial use of different organic growing media as bioamendments in soil used in nurseries and greenhouse crops is very common due to their healthy effects on the soil physical environment. The suitable use of growing media is of importance for production of quality greenhouse crops. It directly influences the growth, development, ramification and functioning of rooting system (Argo, 1998; Richards and Beardsell, 1986). It is important to maintain the optimum aeration and water status of the media to avoid the drought or excess water stress due to the shallow depth and limited volume of soil in the containers. The ability of the growing media to make a healthy balance between water content and gaseous exchange is critical for the keeping quality of the ornamental plants (Dressboll, 2010). Thus, the organic soil amendments are usually mixed with soil to provide the optimal physical and chemical environment (Yangyuoru et al., 2006) to the growing plants. This also buffers the temporary water stress and plant failure during the establishment stage (De Boodt, 1990; Johnson and Leah, 1990). Solely field soils are generally not recommended for pot culture purpose as they do not provide the required aeration, drainage and water holding capacity which results in suppression of root growth and susceptibility to soil borne diseases (Beattie and White, 1992). Thus, several bio-amendments have recently been used in nurseries and greenhouses. The coir, farmyard manure (FYM), sewage sludge, vermicompost are the most commonly used growing media in the greenhouses and pot culture. Recently the use of coir or coconut mesocarp as a substitute of sphagnum peat has been well accepted in horticultural nurseries (Yau and Murphy, 2000; Pickering, 1997) because of its better physical properties (Evans et al., 1996; Prasad, 1997) and excellent ability to make a balanced air and water supply to the roots. These non-soil materials can be manipulated or processed in different ratios and combinations to provide the superior physical and chemical environment for optimum plant growth. Thus the success of plants grown in pot culture can be summed up by the physical attributes of the growing media that influences its ability to provide sufficient water to the root systems without any oxygen shortage (Michel, 2010; Khayyat et al., 2007) which is the most determining factor in containerised cultivation. The good physical environment means the physical make up of the growing media viz., maximum water holding capacity (WHC), bulk density of the mixture, air-filled porosity (AFP), water retention characteristics at a particular matric potential and the mechanical support to the plant growth. The AFP is the volume of air in a media mix after it has been watered heavily and then allowed to drain. If the media mix is very dense and there is too little air in the media mix (less than 10%) then the roots will not be properly aerated and the plant will suffer, especially in a medium like pot having limited drainage capacity. Although plants can extract more than the available water, they will need to exert extra energy to do so and this restricts their growth. The balance between the available water and the air space depends on the size and shape of the particles in the media or more precisely the pores Complementary Copy The efficacy of the natural bio-amendments in improving physical condition as well as water retention characteristics of the growing media in pot culture was studied on ten different compositions of growing media. The treatments comprised of (i) soil as sole medium; (ii) soil + sewage sludge (SS) in the ratio of 1:1; (iii) soil + SS + coir (CP) in the ratio of 1:1:1; (iv) soil + vermicompost (VC) in the ratio of 1:1; (v) soil + VC + CP in the ratio of 1:1:1; (vi) soil + farmyard manure (FYM) in the ratio of 1:1; (vii) soil + FYM + CP in the ratio of 1:1:1; (viii) soil + SS + VC in the ratio of 1:1:1; (ix) soil + SS + FYM in the ratio of 1:1:1 and (x) soil + FYM + VC in the ratio of 1:1:1. The bulk density of media composition soil+SS+CP, soil+VC+CP and soil+FYM+CP was 24.2, 27.5 and 27.5% lower than the media containing only soils (1.32 mg m-3), respectively. The water holding capacity (WHC) was lowest (45.4%) in sole soil treatment and it was 6.3, 5.6 and 6.1 times higher in soil+SS+CP, soil+VC+CP and soil+FYM+CP, respectively. The volumetric water retention at various suctions was significantly improved by addition of the organic amendments with soil. The magnitude of the differences in water retention among the treatments became wider at the higher suctions. The combination of soil+VC+CP showed the highest amount of water retention among all the treatments at all the suctions. The air filled porosity was highest (190.7%) in soil+FYM+CP media and lowest (25.3%) in sole soil media. Significant increase in easily available water was observed with the incorporation of coir with sewage sludge, vermicompost and FYM. The water buffering capacity was lowest in media with only soil (7.56%) and the media containing soil+VC+CP recorded the highest (24.7%) water buffering capacity. Water retention characteristics of soil bio-amendments used as growing media in pot culture Materials and methods The experiment was conducted at Punjab Agricultural University, Ludhiana, India. Four different soil bio-amendments viz. FYM, vermicompost, coir and sewage sludge were used for evaluating their effect on soil water retaining characteristics on mixing with soils at different proportion and combinations. The treatments consisted of ten different combinations of soil and non-soil organic amendments in equal proportion. The treatments comprised of (i) soil as sole medium; (ii) soil + sewage sludge (SS) in the ratio of 1:1; (iii) soil + SS + coir (CP) in the ratio of 1:1:1; (iv) soil + vermicompost (VC) in the ratio of 1:1; (v) soil + VC + CP in the ratio of 1:1:1; (vi) soil + farmyard manure (FYM) in the ratio of 1:1; (vii) soil + FYM + CP in the ratio of 1:1:1; (viii) soil + SS + VC in the ratio of 1:1:1; (ix) soil + SS + FYM in the ratio of 1:1:1 and (x) Soil + FYM + VC in the ratio of 1:1:1. The organic amendments were dried under shade and then different treatment combinations were prepared by mixing them thoroughly by hand at equal proportions (w/w) to make it homogeneous. In case of coir, the raw sample was used without any grinding except removal of the knots. Pot filling: The growing media was packed in the pots with a preselected compaction level. The soil mixed with bioamendments in the desired proportions was filled in 30 cm high polyvinyl chloride (PVC) pots having internal diameter of 20 cm. The PVC pots had a hole of 1 cm diameter at the bottom end so as to drain the excess water. The soil along with bioamendments were filled in the pots in small lots with 30 strokes using a wooden rod with cone-shaped pointed end. This was done to attain a uniform packing of different mixtures of soil and bioamendments. The pots were irrigated till near saturation and were then allowed to attain the equilibrium for 2-3 days. The treatments of the mixtures of growing medium were replicated thrice. In-situ sampling was done for determination of different physical characteristics from these pots. Bulk density: The bulk density was measured by core method (Blake and Hartge, 1986). A metallic core of 5 cm inner diameter and 5 cm height was inserted in the packed pots to take out undisturbed sample of the growing media mixture. The sample was then dried in an oven at 105oC for 24 h or till the sample weight became constant. The ratio of oven-dry weight of the sample in the core and the internal volume of metallic core was expressed as bulk density. Water holding capacity: The water holding capacity (%) was determined as per Richard (1954). It was computed as follows: WHC (%) = 100 x Wsw - Wsd - Wf Wsd - Wk - Wg Where, Wsw is the weight of watch glass + Keen’s box + filter paper + saturated sample in g; Wsd is the weight of watch glass + Keen’s box + filter paper + oven dry sample in g; Wk is the weight of Keen’s box + filter paper in g; Wg is weight of watch glass in g; and Wf is weight of water absorbed by filter paper. Water retention characteristics: Water retention at various soil matric suctions was determined with the help of Richard’s pressure plate apparatus (Richard, 1949). For this purpose, the cores sampled for bulk density measurement were sliced so as to fit in retainer rubber rings on the porous, saturated ceramic plate. These were then saturated overnight along with the ceramic plates with an excess of water maintained in the tray. The ceramic plate along with the saturated samples was placed in the pressure plate chamber and connected to an outflow tube. The required pressure equivalent to desired soil matric potential was maintained by the air compressor fitted with suitable pressure regulator so as to equilibrate the sample at required suction levels. After the equilibrium was attained, the samples were immediately transferred to the moisture box and the moisture content at particular suction was determined gravimetrically by drying the samples in an oven at a constant temperature of 1050C for 24 hours. The relationships between volumetric water content versus the soil matric suction were expressed in the form of soil moisture characteristics curves. These water retention curves were used to estimate the following parameters as suggested by Michel (2010). The air-filled porosity, defined as the volumetric proportion of the water contained at saturation (water potential = 0 kPa) in the coarsest pores and therefore, readily released and replaced by air at water potentials between 0 and -1 kPa. The easily available water, which is the volumetric proportion of water retained in the growing medium by forces which are compatible with root extraction capacity (defined for the range of water potential from -1 kPa to -10 kPa). The water buffering capacity, defined as the volumetric proportion of water released by the growing medium between -5 and -10 kPa, enabling physiological adaptation of the plant to the changing water potential. Statistical analysis: The data was analyzed using analysis of variance (ANOVA) in a completely randomised design, so as to estimate the critical difference among the treatments. Regression analysis was carried to study the significance of relationship between water holding capacity (WHC) and bulk density. Least significant difference (LSD) at 95% probability level was employed to compare the treatment means. Results and discussion Bulk density: Analysis of variance showed significant (P<0.05) differences in bulk density among the growing media (Fig.1). The influence of bio-amendments incorporation with soil on media bulk density was more pronounced in case of coir treatments. The bulk density of media containing soil+SS+CP, Soil+VC+CP and soil+FYM+CP was significantly lower than the other treatments. It was 24.2, 27.5 and 27.5% lower than the media with sole soil (1.32 Mg m-3), respectively. Sudhagar and Sekar (2009) reported the decrease in bulk density and particle density with increase of coir could be due to light weight of coir and increased pore space (Saravanan and Nambisan, 1995). On the contrary the application Complementary Copy between the solid particles. Therefore, the ideal mix should have a balance between medium and coarse particles with a minimum of fine particles. Thus, the present study was conducted to evaluate the physical and water retention characteristics of different combinations of soil and bio-amendments so as to identify the best blend for easy availability of water and air to the plants. 93 Water retention characteristics of soil bio-amendments used as growing media in pot culture Fig.1. Bulk density of soil as affected by different non-soil bioamendments. (Vertical columns are means and the bars on each column are ± standard errors of mean; lower case letters indicate differences at the 0.05 probability level in bulk density among the treatments) of vermicompost and FYM slightly increased the media bulk density as compared to control (only soil). The application of sewage sludge (soil+SS) reduced the media bulk density by 6% than the control media. Subsequent addition of coir further reduced the bulk density of media composition soil+SS+CP by 19.3%. On the contrary, the application of FYM increased the bulk density by 23% in Soil+SS+FYM media combination than the Soil+SS treatment. Differences in results obtained here are most likely due to the variation in particle-size distribution of the material (Richards and Beardsell, 1986). Being fibrous, the coir fibers evenly spread in the soil and due to its lighter weight it reduces the bulk density with concomitant increase in total porosity (Awang et al., 2009). The lower bulk density with coir media facilitates the mixing and incorporation of the media, easy transportation and sufficient aeration to the plants. Water holding capacity: The water holding capacity (WHC) significantly differed among the different media combinations (Fig. 2). With the application of organic amendments there was a significant increase in WHC among the treatments as compared to Fig. 2. Water holding capacity of soil as affected by different non-soil bioamendments. (Vertical columns are means and the bars on each column are ± standard errors of mean; lower case letters indicate differences at the 0.05 probability level in WHC among the treatments) the control with no amendments. The prominent beneficial effect of coir on soil physical make up is manifested by Fig. 2. The three coir treatments, soil+SS+CP, soil+VC+CP and soil+FYM+CP exhibited significantly higher WHC as compared to the other treatments. The WHC was lowest (45.4%) in media with sole soil and 6.3, 5.6 and 6.1 times higher in soil+SS+CP (283%), soil+VC+CP (256%) and soil+FYM+CP (276%), respectively. There was non-significant difference in WHC with the application of sewage sludge, FYM and vermicompost addition with the soil as in soil+SS, soil+VC and soil+FYM treatments. The differences in the WHC among the media could be due to the diversity in total porosity and pore-size distribution. The light weight, fibrous coir adds higher porosity and reduced bulk density that helps in holding higher amount of water. The higher WHC of coir based media due to its higher total porosity has also been reported by Evans et al. (1996) and Prasad (1997). Several experiments have revealed that use of coir alone or as a component of soil medium is suitable for various potted plants (De Kreij and Leeuven, 2001; Treder, 2008) as well as vegetable crops. There was a linear correlation (R2=0.75) (Fig. 3) between bulk density and WHC of the growing media. With the increase in bulk density there was a linear decrease in WHC. Thus the significantly lower bulk density of the coir treatments renders the significantly higher WHC as compared to other treatments. Water retention characteristics and aeration: The water retention characteristics are basically the capability of the media to retain water at different moisture potentials. The water retention curves of different media compositions have been presented in Fig. 4. Significant differences were observed in water retention among the treatments under different soil moisture suctions except in Fig. 4d, where there was not much difference among the treatments. The difference in water retention among the treatments is narrow in the lower suction range and the magnitude of difference increases with the increase in suction value. Awang et al. (2009) reported that at the initial values of soil water suction, the volume of water held by different media was more or less similar. Among all the treatments the media with only soils as growing media retained lowest amount of water at all the suctions. The application of sewage sludge with soil (soil+SS) caused 2 times increase in water retention at 1bar of soil water suction whereas, with the incorporation of coir with soil and sewage Fig. 3. Relationship between bulk density and water holding capacity of the growing media Complementary Copy 94 Water retention characteristics of soil bio-amendments used as growing media in pot culture a b d Complementary Copy c 95 Fig. 4. Water retention characteristics of different growing media as affected by different non-soil bio-amendments in different proportions and combinations sludge (soil+SS+CP) caused 4.3 times higher water retention at the same soil water suction (Fig. 4a). With the increase in suction the volume of water retained decreased in all the media but the magnitude of differences among the treatments increased. This indicated that the application of organic amendments improves water retention even at higher suctions as compared to sole soils. This is one of the important attributes of using bio-amendments as growing media that could be exploited to avoid the drought stress in the greenhouses. This is mainly due to the restricted movement of water from subsurface to surface because of reduced evaporation (Ouchi et al., 1990). The combination of soil+VC+CP showed the highest amount of water retention among all the treatments at all the suctions. The effect of coir was less prominent when it is mixed with soil and FYM (soil+FM+CP) (Fig. 4c). The remarkable increase in water retention of media with non-soil organic amendments (especially coir) as compared to media with sole soil is due to the predominant role of the organic amendments on soil physical properties. The microbial decomposition of the organic amendment compounds releases by-products like polysaccharides and polyuronoides (Hillel, 1980) which helps Water retention characteristics of soil bio-amendments used as growing media in pot culture in stabilising aggregates by enmeshing the soil particles within the aggregates. The water retention, air-filled porosity and gas diffusion are mainly dependent on particle size of the growing media and the pores between the particles (Caron and Nkongolo, 1999; Caron et al., 2005). The pore continuity is the most critical physical factor influencing water dynamics and gas exchange by roots (Gruda and Schnitzler, 2004). Evans et al. (1996) and Verhagen (2004) have shown the increased water retention of peat media with the addition of coco peat or coir dust. The positive effect of coir on soil physical environment has also been reported by Managecraft (2001). The air filled porosity as calculated from the water retention curve was significantly differed among the treatments (Table 1). It was highest (190.7%) in soil+FYM+CP combination and lowest (25.3%) in sole soil treatment. The application of coir with sewage sludge (soil+SS+CP) and vermicompost (soil+VC+CP) caused 6.9 and 4 times increase in air filled porosity as compared to media with only soil, respectively. Ensuring proper aeration for proper root growth in pots in greenhouse and nurseries is a real challenge for the growers because of impeded drainage due to limited soil volume in the container. Aeration basically depends on the media pore size. Watering in the pots causes saturation of the total porosity. As the media dries up, water first depletes from the larger pores. Application of coir improves the macro porosity of the media and helps in draining excess water and maintains the proper aeration status. Increased proportion of larger pores enhances more aeration after drainage (Handreek and Black, 2007). Khayyat et al. (2007) observed significant differences on growth performances of Epipremnum aureum Lindl. and Andre (‘Golden Pothos’) grown on coir based rooting media. They explained the large differences on rooting quality and shoot characteristics by the direct effect of growth substrate on the basal portion of cutting rather than the indirect or earlier physiological changes. Improved root formation and growth in coir mixtures could be due to better aeration, drainage conditions and water maintenance capability of the substrate (Eleni et al., Table 1. Air-filled porosity and water availability in different compositions of bio-amendments in soil Water Water Media composition Air filled buffering availability porosity capacity (%) (%) (%) Soil 25.3a 10.2a 7.36a Soil+SS (1:1) 41.9b 13.3a 8.17a Soil+SS+CP (1:1:1) 175.4c 30.3b 19.3b Soil+VC (1:1) 66.0d 13.5a 10.0a Soil+VC+CP (1:1:1) 101.3e 64.7c 24.7c Soil+FYM (1:1) 53.21f 10.3a 6.90a Soil+FYM+CP (1:1:1) 190.7g 25.4d 17.7b Soil+SS+VC (1:1:1) 75.3d 20.0e 13.8a Soil+SS+FYM (1:1:1) 65.5d 14.1a 10.8a Soil+FYM+VC (1:1:1) 79.8h 19.6e 11.7a The dissimilar lower case letters indicate significant differences among the treatments at P<0.05 SS= sewage sludge; CP = cocopeat; VC = vermicompost; FYM = farmyard manure 2001; Noguera et al., 2000) which are critical for the first phase of the root initiation. The effect of sewage sludge, vermicompost and FYM on increasing water availability only happened when they were associated with coir (Table 1). Mere application of sewage sludge, vermicompost and FYM did not significantly affect the water availability in soil+SS, soil+VC and soil+FYM treatments as compared to sole soils. Significant increase in water availability was observed with the incorporation of coir with sewage sludge, vermicompost and FYM. The effect of coir application on water availability is very clear in soil+SS+CP, soil+VC+CP and soil+FYM+CP treatment combinations where there were 2.9, 6.3 and 2.5 times higher water availability as compared to the control treatment, respectively (Table 1). Similar trend was observed in case of water buffering capacity of the media (Table 1). The media with only soil had the lowest (7.36%) water buffering capacity and the media containing vermicompost and coir associated with soil recorded the highest (24.7%) water buffering capacity. The higher water availability of the coir containing media indicates more available water to the plants. At the same time high water buffering capacity of the coir makes it more flexible in providing irrigation and less restrictive in terms of water management (Michel, 2010). The study of organic amendments on water retention properties showed that the non soil organic amendments have a positive effect on physical environment of the media and hence improve the water retention and aeration properties of the media. The impact of coir, which is environmentally safe and cheap, was much more pronounced among the organic amendments. The application of coir in association with vermicompost, FYM or sewage sludge rather than their individual use is found to be a viable option for providing better aeration, water availability to the plants as well as water buffering capacity of the media which has long been a challenge for pot culture in greenhouses and nurseries. References Argo, W.R. 1998. Root medium physical properties. Hort. Technol., 8: 481-485. Awang, Y., A.S. Shaharom, R.B. Mohamad and A. Selamat, 2009. Chemical and physical characteristics of cocopeat-based media mixtures and their effects on the growth and development of Celosia cristata. Am. J. Agric. Biol. Sci., 4: 63-71. Beattie, D.J. and J.W. White, 1992. Lilium–hybrids and species. In: The Physiology of Flower Bulbs. A. De Hertogh and M. Le Nard (eds.). Elsevier, Amsterdam. Blake, G.R. and K.H. Hartge, 1986. Bulk density. In: Methods of Soil Analysis, Part I, A. Klute (ed). ASA Monograph No 9. Madison, WI. p.363-376. Caron, J., L.M. Riviere and G. Guillemain, 2005. Gas diffusion and airfilled porosity: effect of some oversize fragments in growing media. Canadian J. Soil Sci., 85: 57-65. Caron, J. and N.V. Nkongolo, 1999. Aeration in growing media: recent developments. Acta Hort., 481: 545-551. De Boodt, M. 1990. Application of polymeric substances as physical soil conditioners. In: Soil Colloids and their Association in Soil Aggregates. (M. De Boodt et al., ed.), Planum Publishing Corporation, London, New York. p. 580-592. De Kreij, C. and G.J.L. Leeuven, 2001. Growth of pot plants in treated coir dust as compared to peat. Commun. Soil Sci. Plant Anal., 32: 2255-2265. Complementary Copy 96 Water retention characteristics of soil bio-amendments used as growing media in pot culture Ouchi, S., A. Nishikawa and E. Kameda, 1990. Soil-improving effect of a super-water-absorbent polymer. II. Evaporation, leaching of salts and growth of vegetables. Jap. J. Soil Sci. Pl. Nutr., 61: 606-613. Pickering, J.S. 1997. An alternative to peat. The Garden, 122: 428429. Prasad, M. 1997. Physical, chemical and biological properties of coir dust. Acta Hort., 450: 21-29. Richard, L.A. 1949. Pressure membrane apparatus construction and use. Agri. Engg., 28: 451-454. Richard, L.A. 1954. Diagnosis and Improvement of Saline and Alkali Soils: Agriculture Handbook No. 60. USDA. New York. Richards, D.M.L. and D.V. Beardsell, 1986. The influence of particlesize distribution in pinebark:sand:brown coal potting mixes on water supply, aeration and plant growth. Scientia Hort., 29: 1-14. Saravanan, A. and K.M.P. Nambisan, 1995. Utilization of coco peat as pot culture medium for Begonia semperflorense. Madras Agric. J., 82: 587-589. Sudhagar, R. and K. Sekar, 2009. Effect of coco peat medium on growth and quality of poinsettia (Euphorbia pulcherrima. Wild.). The Asian J. Hort., 4: 52-56. Treder, J. 2008. The effect of cocopeat and fertilization on the growth and flowering of oriental Lily ‘Star Gazer’. J. Fruit Ornamental Plant Res., 16: 361-370. Verhagen, J.B.G.M. 2004. Effectiveness of clay in peat based growing media. Acta Hort., 644: 115–122. Yau, P.Y. and R.J. Murphy, 2000. Biodegraded cocopeat as a horticultural substrate. Acta Hort., 517: 275-278. Yangyuoru, M., E. Boateng, S.G.K. Adiku, D. Acquah, T.A. Adjadeh and F. Mawunya, 2006. Effects of natural and synthetic soil conditioners on soil moisture retention and maize yield. West Africa J. Applied Ecol., 9: 1-8. Received: February, 2012; Revised: October, 2012; Accepted: December, 2012 Complementary Copy Dressboll, D.B. 2010. Effect of growing media composition, compaction and periods of anoxia on the quality and keeping quality of potted roses (Rosa sp.). Scientia Hort., 126: 56-63. Eleni, M., K. Sabri and Z. Dimitra, 2001. Effect of growing media on the production and quality of two rose varieties. Acta Hortic., 548: 79-83. Evans, M.R., S. Konduru and R.H. Stamps, 1996. Source variation in physical and chemical properties of coconut coir dust. Hort. Sci., 31: 965-967. Gruda, N. and W.H. Schnitzler, 2004. Suitability of wood fiber substrate for production of vegetable transplants. I. Physical properties of wood fiber substrates. Scientia Horti., 100: 309-322. Handreck, K.A. and N.D. Black, 2007. Growing Media for Ornamental Plants and Turf. 3rd Edn., UNSW Press, Sydney, ISBN:13:9780868407968. Hillel, D. 1980. Fundamentals of Soil Physics. Academic Press, New York. p.113-117. Johnson, M.S. and R.T. Leah, 1990. Effects of super absorbent polyacrylamide on efficacy of water use by crop seedlings. J. Sci. Fd Agric., 52: 431-434. Khayyat, M., F. Nazari and H. Salehi, 2007. Effects of different pot mixtures on pothos (Epipremnum aureum Lindl. and Andre ‘Golden Pothos’) growth and development. American-Eurasian J. Agric. Environ. Sci., 2: 341-348. Managecraft, 2001. Results of Analysis of Coconut Husk Fibre or CocoPeat. Managecraft (GH) Ltd., Box 207. Takoradi, Ghana. Michel, J. 2010. The physical properties of peat: a key factor for modern growing media. Mires Peat., 6: 1-6. Noguera, P., M. Abad, V. Noguera, R. Puchades and A. Maquieira, 2000. Coconut coir waste, a new and viable ecologically friendly peat substitute. Acta Hort., 517: 279-286. 97 Journal Journal of Applied Horticulture, 14(2): 98-101, 2012 Appl I solat ion of biom ole c ule s of pha r m a c ologic a l im por t a nc e from Ga rc inia indic a fruit a nd eva luat ion of t ot a l a nt iox ida nt a c t ivit y P. Ga yat hri a nd P. Govinda ra ju* Department of Biochemistry, Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore – 641 003, Tamil Nadu, India. *E-mail: [email protected] Abstract An investigation was undertaken to study the antioxidant activity of various solvent extracts of the fruit of Garcinia indica using FRAP assay and to separate the compounds in the potential extract through TLC, HPLC and analyse using GC-MS. The study revealed that methanol and ethyl acetate extracts showed a higher antioxidant value than the other extracts. The compounds present in the methanol extracts were separated by TLC, HPLC and analysed using GC-MS. The results of TLC revealed the separation of two different spots in case of phenols and a single spot in case of alkaloids. The eluted compounds, subjected to HPLC, separated into 8 peaks in case of phenolics and 8 peaks in case of alkaloids with varying retention time. The HPLC fractions were subjected to GC-MS to identify the compounds in comparison with the Wilcon-NIST library. The study is useful in identifying the bioactive compound for anticancer activity using cell lines. Introduction Garcinia indica Linn belongs to family Clusiaceae commonly called as ‘Kokum’ in Maharashtra and cultivated in Konkan, Goa and the western region of India. Fruits of G. indica have been suggested in the Indian system of medicine for a number of diseases. These include its usefulness as an infusion, in skin rashes caused by allergies, to relieve sunstroke, remedy for dysentery, an appetizer, liver tonic, to allay thirst and as a cardiotonic (Khare, 2007). The fruit rind contains polyisoprenylated benzophenones, garcinol, its isomer isogarcinol, xanthochymol, and isoxanthochymol (Kirtikar and Basu, 1991). Garcinol has antioxidative, chelating, free radical scavenging, antiglycation, anticancer, anti-inflammatory and antiulcer activities (Sheth et al., 2006; Chattopadhyay et al., 2006). Kokum contains other compounds with potential antioxidant properties which include citric acid, malic acid, polyphenols, carbohydrates, anthocyanin flavonoids and ascorbic acid (Ho et al., 2002; Yamaguchi et al., 2000). Free radicals include hydroxyl, superoxide, nitric oxide, nitrogen dioxide, peroxyl, lipid peroxyl and hydrogen peroxide, which are generated by products of normal cellular metabolism (Nordberg and Arner, 2001). The mitochondrial leakage of these reactive oxygen species leads to oxidative damage of cell components such as proteins, lipids and nucleic acids (Block et al., 1992). Increased oxidative stress has been proposed to be one of the major causes of the pathogenesis of cancer, cardiovascular disease, diabetes mellitus, neurodegenerative diseases (Alzheimer’s disease and Parkinson’s disease), autoimmune disorders, rheumatoid arthritis and ageing (Leifert and Abeywardena, 2008; Leja et al., 2003; Li et al., 2007). Natural antioxidants such as vitamins and polyphenols, in vegetables and fruits, are considered to be responsible for health benefits (Li et al., 2008; Stangeland et al., 2009). As an important category of phytochemicals, phenolic compounds universally exist in plants, and have been considered to have high antioxidant ability and free radical scavenging capacity, with the mechanism of inhibiting the enzymes responsible for ROS production and reducing highly oxidised ROS. In recent years, considerable interest has been shown in determining total phenolic contents and antioxidant activity of vegetables, fruits, spices, medicinal plants and algae (Gan et al., 2010; Ghasemzadeh et al., 2010; Cai et al., 2004; Devasagayam et al., 2006; Rezaeizadeh et al., 2011). The aim of this study was to evaluate the total antioxidant activity of the various extracts of G. indica fruits and to separate the components of the extract with high activity through TLC, HPLC and analysed by GC-MS. Materials and methods Chemicals and reagents: 2,4,6-tripyridyl-S-triazine (TPTZ) from M/s. Sigma-Aldrich Chemicals, Bangalore; HPLC grade methanol, ascorbic acid, bismuth nitrate, sodium acetate, glacial acetic acid, ferric chloride, potassium iodide, sodium carbonate, folin ciocalteau reagent, hexane, chloroform, methanol and ethyl acetate from M/s Merck, Mumbai were used in the study. Deionized water was used for HPLC analysis. Ready made plates coated with alumina from M/s Merck, Mumbai was used for the separation of active components. Plant material: G. indica fruits were collected from the local market in Coimbatore. Preparation of various solvent extracts of G. indica fruit: Fruits were cut open and the pulp was separated from the rind. The fruit rinds and pulp were allowed to dry in the shade. The dried fruit (rind and pulp) was subjected to size reduction to a Complementary Copy Key words: Garcinia indica, methanol, ethylacetate, chloroform, hexane, FRAP, antioxidant Isolation of biomolecules of pharmacological importance from Garcinia indica fruit Total antioxidant activity: The total antioxidant activity of the extract of G. indica was assayed by FRAP method (Ferric Reduction Antioxidant Power) (Benzie and Strain, 1996). One hundred microlitre of the diluted sample was added to 3 mL of the FRAP reagent. The absorbance of the mixture was measured at 593 nm in 1cm light path at 37º C using ELICO SL159 UV–Vis spectrophotometer after 4 min. Briefly, the FRAP reagent was prepared from sodium acetate buffer (300 mM, pH 3.6), 10 mM TPTZ solution (40 mM HCl as solvent) and 20 mM iron (III) chloride solution in a volume ratio of 10:1:1, respectively. The FRAP reagent was prepared fresh every time and warmed to 37° C in a water bath before use. A standard solution of 1 mM ascorbic acid was tested in parallel and the results were expressed as mg ascorbic acid equivalents/g dry weight of fruit. Thin layer chromatography: TLC of various extracts of G. indica whole fruit was performed using readymade plates coated with alumina. In brief, the plates were cut to the required dimension (8 x 4 cm) and the extract was spotted using a capillary tube leaving a distance of 1 cm from the bottom, for 10-15 times to get a concentrated spot. The spotted plates were placed in a solvent system of Chloroform: acetic acid (9:1) in a closed chamber and allowed for the run till the solvent mixture reached three-fourth of the plate. The plate was air dried and sprayed using the spray reagent Folin’s Ciocalteau (1:1 dilution) reagent followed by 20% Sodium carbonate, air dried followed by subsequent drying using drier to identify the presence of phenols. The solvent system chloroform : methanol (9:1) was used for the run and the Dragendorff’s reagent was used for spray to identify the presence of alkaloids. Dragendorff’s reagent was prepared as under (Sarkozi et al., 2006): Dragendorffs reagent: Solution A - 1.7 g bismuth nitrate in 100 mL distilled water: acetic acid (8:2); Solution B - 40 g KI in 100 mL distilled water; Solution C- 20 mL glacial acetic acid + 70 mL distilled water; Solution D - Mix 5 mL of solution A, 5 mL of olution B and 90 ml of solution C just before use. High performance liquid chromatography (HPLC) analysis conditions: Methanolic G. indica extract fractions separated by TLC were filtered using a 0.45 μm nylon filter. 15 μL of the extract was separated on a Waters dual-pump high-performance liquid chromatograph (HPLC) using a Waters C-18 Symmetry column (Milford, MA) and Diode Array Detector. The method used HPLC grade methanol (mobile phase A) and HPLC grade water (mobile phase B) with the following linear gradient programme: 85% A, 0 min.; 75% A, 15 min.; 70% A, 20 min.; 45% A, 24 min.; 10% A, 28 min.; 0% A, 30 min.; and 85% A, 35-40 min. Analytes were detected at 280 nm and the mobile phase flow rate was 1 mL/min. GC-MS conditions: The HPLC fractions of phenolics and alkaloids of G. indica fruit extract were subjected to GC-MS on a Perkin-Elmer Clarus 500 of mass range between 50-450 m/z with a flow rate of 1 mL/min. The injection volume was 1 μL and the Library used was Willey & NIST. Results and discussion The results of the total antioxidant activity determined by the FRAP assay revealed that the methanol extracts of G. indica fruit had the highest antioxidant activity in terms of mg ascorbic acid equivalents compared to the other solvent extracts. This may be due to the efficient extraction of various active components of antioxidant property especially the phenolics in the methanol extract. Table 1. Total antioxidant activity of G. indica fruit S.No Extract (1g dried sample) Total antioxidant activity 1. Aqueous 1.32 ± 0.13 2. Chloroform 0.63 ± 0.08 3. Ethyl acetate 6.53 ± 0.62 4. Methanol 7.60 ± 0.51 5. Petroleum ether 0.79 ± 0.25 6. Hexane 2.87 ± 0.39 Values are mean ± SD of triplicates and expressed as equivalents of mg ascorbic acid Phenolic compounds have specific health effects even though they are nonnutritive compounds. Antioxidant properties of phenolic compounds play a vital role in the antioxidative defence mechanisms of biological systems (Wright et al., 2001). The antioxidative effect of phenolics is due to a direct free radical scavenging activity (Halliwell et al., 2000; Halliwell, 1996), reducing activity and an indirect effect arising from chelation of prooxidant metal ions. The chelation of metal ions generally requires ortho-dihydroxylation on the phenyl ring in phenolic acids and flavonoids or the presence of a 3- or 5-hydroxyl group in flavonoids (Shahidi, 2000; Shahidi, 2007). Natural antioxidants can protect the human body against free radicals, and have also been shown to retard the progress of a variety of chronic diseases (cancer, heart disease, diabetes, etc.), as well as ameliorating or retarding lipid rancidity in foods (Wettasinghe and Shahidi, 1999; Kinsella et al., 1993). Thin Layer Chromatography of methanol extract of G. indica whole fruit was performed using various solvent systems to identify the presence of secondary metabolites viz., phenols and alkaloids. The results revealed the presence of phenolic compounds and alkaloids which was confirmed by the blue and orange colour spots obtained after spraying with the spray reagent. HPLC analysis of TLC phenolics fraction of G. indica whole fruit revealed the separation of the TLC phenol fraction into 4 major peaks and 5 minor peaks (Fig. 1) with retention time as mentioned in the Table 2. HPLC analysis of TLC alkaloid fraction of G. indica revealed the separation of the TLC alkaloid fraction into 3 major peaks and 5 minor peaks (Fig. 2) with retention time as mentioned in the Table 3. The phenolic HPLC fraction of G. indica extract was subjected to GC-MS and the compounds identified in the fraction upon comparison with the library are shown in Table 4. The alkaloid HPLC fraction of G. indica extract was subjected to GC-MS and Complementary Copy coarse powder. The coarsely powdered form of shade dried whole fruit was extracted using various solvents viz., hexane, chloroform, methanol, ethyl acetate using mechanical grinder. Then the extracts were centrifuged and the supernatant was collected and the solvent was evaporated at room temperature. The semisolid extract obtained was stored in an airtight container in refrigerator for further use. The solution of aqueous extract was prepared by using normal saline as solvent for experiment. The suspension of hexane, chloroform, methanol and ethyl acetate extract of G. indica fruit was prepared in distilled water. 99 100 Isolation of biomolecules of pharmacological importance from Garcinia indica fruit Table 2. HPLC peaks of TLC phenolics fraction with retention time Peak No 1 2 3 4 5 6 7 8 9 Retention time 2.635 3.147 5.005 5.621 6.208 7.125 9.248 18.400 20.970 Table 3. HPLC peaks of TLC alkaloid fraction with retention time Fig. 1. HPLC of TLC phenolics fraction- G. indica Peak No 1 2 3 4 5 6 7 8 Retention time 3.135 4.133 5.312 6.144 9.621 18.325 20.853 34.630 Fig. 2. HPLC of TLC alkaloid fraction- G. indica the compounds identified in the fraction upon comparison with the library are shown in Table 5. Compound Trans, Trans-3,12-Dimethoxy-1,6,10dodecatriene Tridecane,2,2,4,10,12,12-Hexamethyl7(3,5,5-trimethylhexyl Tetradecanoic Acid, 10,13-Dimethyl,Methyl Ester Octadecane M.W Formula 224 C14H24O2 1,7-Dimethoxy-1,6-Heptadiene 394 C28H58 270 C17H34O2 254 C18H38 156 C9H16O2 Based on the study, it is concluded that G. indica fruit is a potent source of antioxidants. The compounds identified through GCMS can be tested for antioxidant and anticancer activity using different cell-lines. Further identification of active principles present in the extract and structural elucidation can be done using LC-MS and NMR. Table 5. GC-MS of G. indica extract-Alkaloid-HPLC fraction Compound M.W Formula 6-Methyl-7-(Trimethylsilyl)-5-Hepten-1-ol 200 C11H24OSi 2R,3R,4R,5S,6R,8S,10S)-5-Acetoxy-10Benzyloxy-8-Benzyloxymethyl 498 C29H38O7 References Tetradecanoic Acid, 10,13-Dimethyl, Methyl ester 270 C17H34O2 Benzie, I.F.F. and J.J. Strain, 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239: 70–76. Block, G., B. Patterson and A. Subar, 1992. Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutrition and Cancer, 18: 1-29. Cai, Y.Z., Q. Luo, M. Sun and H. Corke, 2004. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Science, 74: 2157-2184. Chattopadhyay, S.K. and S. Kumar, 2006. 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Reactive oxygen specie, antioxidants and the mammalian thioredoxin system. Free Radical Biology and Medicine, 31: 287-1312. Rezaeizadeh, A., B.Z. Zuki, M. Abdollahi, Y.M. Goh, M.M. Noordin, M. Hamid and T.I. Azmi, 2011. Determination of antioxidant activity in methanolic and chloroformic extracts of Momordica charantia, Afr. J. Biotechnol., 10 (24): 4932-4940. 101 Journal Journal of Applied Horticulture, 14(2): 102-109, 2012 Appl Post ha r ve st m ic robia l dive rsit y on m a jor c ult iva rs of I ndia n m a ngoe s S.N . J ha * , Pra nit a Ja isw a l, K . N a rsa ia h, Rishi Bha rdw a j, Poona m Pre e t K a ur, Ashish Kum a r Singh, Ra jiv Sha r m a a nd R. Kum a r Agricultural Structures and Environmental Control Division, Central Institute of Postharvest Engineering & Technology, Ludhiana 141004, India. *E-mail: [email protected] Abstract Microbial diversity on fruit surface of nine mango cultivars (Alphonso, Banganapalli, Chausa, Dashehri, Kesar, Langra, Mallika, Maldah and Neelam) harvested from orchards of nine Indian states (Andhra Pradesh, Bihar, Gujarat, Karnataka, Maharashtra, Orissa, Punjab, Tamil Nadu and Uttar Pradesh) were studied using standard methods. A total of 47 fungal and 123 bacterial isolates were purified from 761 mango samples, which included 63 Gram positive and 60 Gram negative bacterial isolates. The relative abundance of Gram positive, Gram negative bacteria and different filamentous fungi varied among cultivars. Gram positive bacteria dominated on Langra of Uttar Pradesh, while Dashehri from Punjab showed dominance of Gram negative bacteria. Among total fungal isolates, the common genera were Aspergillus and Fusarium, while among bacterial isolates, the most common genera were Bacillus, Aeromonas, Pseudomonas, Lactobacillus, Citrobacter, Mycobacterium and Serratia. Alphonso and Kesar variety from Maharashtra showed maximum and minimum fungal diversity, respectively. Genera and species identified include members known for spoilage of fruits; having all types of pectinase and cellulase activities and those used in biocontrol of plant pathogens. Introduction Mango (Mangifera indica) is an important tropical fruit and India is the largest mango producer contributing 37% of 30.5 million tons of total global mango production. Annually, India supplies 50,000 tons of mangoes to different parts of the world including Japan, Middle East, Europe and United States and the demand is increasing year by year (Pandit et al., 2009). However, tropical and subtropical fruits such as mango present greater problems in transportation and storage due to its perishable nature and presence of numerous microflora on its surface (Mitra and Baldwin, 1997) which may cause spoilage of fruits. The postharvest loss of such perishable commodities is estimated to be as high as 50% (Mitra and Baldwin, 1997). This can partly be reduced by knowing the spectrum of microbial community and devising the measures to reduce their effect. Microorganisms associated with postharvest spoilage of fruits have drawn attention of scientists for years (Verma et al., 1991; Okigbo, 2001). Mango fruit is also susceptible to many postharvest diseases such as anthracnose (Colletotrichum gloeosporoides) and stem end rot (Lasiodiplodia theobromae) during storage under ambient conditions or even at low temperature (Arauz, 2000). Spoilage microbes are capable of colonizing and creating lesions on healthy, undamaged plant tissue (Tournas, 2005). Many spoilage microorganisms use their extra cellular lytic enzymes to degrade plant polymers into simpler fractions which can be used as nutrients for their growth. Fungi and many bacterial strains produce an abundance of extracellular pectinases and hemicellulases, which play a major role in spoilage (Miedes and Lorences, 2004). Besides, many microbes isolated from the fruit surface have been identified to be useful agents in postharvest treatment (Wisniewski and Wilson, 1992; Wilson et al., 1993). Extensive studies have been conducted on the diversity of epiphytic microbes on annuals bearing deciduous leaves (De Jager et al., 2001; Joshi, 2008). Recently microbial population on long living leaves of evergreen trees like mangoes have also been studied (Pruvost and Luisetti, 1991; De Jager et al., 2001; Ngarmsak et al., 2006). Information on microbial diversity on Indian varieties of mangoes is missing, thererefore the objective of this investigation was to study the microbial diversity on the surface of mango fruit for deciding strategies to reduce postharvest spoilage of commercially important varieties. Materials and methods Sampling procedure: Major mango cultivars (Alphonso, Banganapalli, Chausa, Dashehri, Kesar, Langra, Maldah, Mallika and Neelam) were collected from orchards of nine Indian states (Andhra Pradesh, Bihar, Gujarat, Karnataka, Maharashtra, Orissa, Punjab, Tamil Nadu and Uttar Pradesh) using complete randomized block design (Jha et al., 2010). Fruits with 5-10 cm stalk were manually plucked directly in pre-sterilized zip locked plastic bags in the forenoon, from each side and also from centre of tree canopy in random manner. Sampling schedule along with abbreviations used for each cultivar is presented in Table 1. Zip locked plastic bags were transported to the laboratory in the well ventilated corrugated fiber board boxes along with partially refrigerated gel packs placed in between the two layers of mangoes to minimize the quality loss (Jha et al., 2010). Isolation and growth of bacterial and fungal isolates: Microbial communities from the mango fruit were isolated by washing and dilution plating method (Jha et al., 2010). In order to isolate total microbial diversity from mango surface, three Complementary Copy Key words: Bacteria, biochemical, diversity, filamentous fungi, mango, relative abundance Postharvest microbial diversity on major cultivars of Indian mangoes mangoes from each cultivars were used in experimentation. The mangoes were washed and suitable dilutions of wash water were prepared. Inoculums from different dilutions containing surface micro flora from mango were plated on Nutrient Agar (NA) and Potatao Dextrose Agar (PDA, Himedia, India) plates in triplicate (Atlas and Snyder, 2006). NA plates were incubated at 37oC and PDA plates at 28oC. After incubation, the individual colonies were picked up and purified with streak plate method. The isolates were grown on their respective media in petriplates or slants to study their morphological features. Bacterial isolates were characterized based on shape, color, surface and edge of the bacterial colonies while filamentous fungi were characterized based on their hyphal and spore characteristics according to Hawksworth et al. (1995). Identification of filamentous fungi was further confirmed at NTCC (National Type Culture Collection Center), Indian Agricultural Research Institute, New Delhi based on morphological features. Biochemical characteristics: Each bacterial isolate was propagated in nutrient broth before use and an overnight culture was employed in the tests. Standard staining procedures (such as Gram’s staining, Negative staining, Acid fast staining, Spore staining) and biochemical tests namely citrate, urease, hydrogen sulfide production, carbohydrate utilization test, lactose fermentation test, glucose oxidation and fermentation test, indole production test, methyl red test, oxidase test, catalase test, flouroscein production test, cellulose degradation test and pectin hydrolysis test were used for characterization of microbial isolates using commercially available media (Himedia, India). The isolated organisms were purified and assayed as recommended in Bergey’s manual (Holt et al., 1994). Statistical analysis: Evenness/Relative abundance (RA): Evenness is defined as a measure of the relative abundance of different species which was calculated as below: RA (%) = (N x 100) /T Where, N = Total number of isolates belonging to one group T = Total number of isolates belonging to all groups Richness was calculated as number of species per sample. Simpson’s Index (D) was calculated as below: D = ∑ (n/N)2 Where, n = Total no. of organisms of a particular species N = Total no. of organisms of all species Simpson’s Index of Diversity Simpson’s index of diversity = 1 – D Where, D = Simpson’s index Results and discussion Bacterial diversity: Microbial diversity describes complexity and variability at different levels in an ecosystem where, microbes play a crucial role in biological organization. Special interest in the fruit surface microorganisms exist in view of identifying microbes responsible for spoilage of mango fruit and further development of biosensor by generating antibodies against them for quick identification. Further, microbes present on plant surface have been reported to fix atmospheric nitrogen, compete with plant parasites, produce plant growth regulator such as gibberellic acid and produce lipases which degrade surface waxes (Chun and Mc Donald, 1987). If such organism also resides on fruit, they may predispose fruit to moisture loss and decay during long term storage. In current scenario, the adverse effects of synthetic chemical residues on human health and the environment have lead scientists from all over the world to develop alternative control strategies such as studies on natural microbial diversity on fruit surface and their role in plant protection. A total of 123 bacterial strains were isolated from the mango fruit samples of nine major cultivars collected from nine Indian states. All the isolates were characterized initially based on the biochemical nature of cell wall using standard Gram staining procedure. Results indicated that among total 123 isolates, Gram positive and Gram negative bacterial isolates were found to be 51.22 and 48.78%, respectively. The RA of Gram positive bacteria varied from 11.11% in CU to 75.00 % in LU (Table 2), while that of Gram negative bacteria varied from 25.00 % in LU to a maximum of 100.00 % in DP. In variety BA and NT, the RA of Gram positive and Gram negative was found to be almost same. Results showed great variation in RA of Gram positive and Gram negative bacteria among different mango cultivars (except BA, MO and NT) which might be attributed to variation in environmental condition (humidity) at the time of sampling at different sampling site. Silva et al. (2000) also reported that the percent composition of Gram positive and Gram negative bacteria is mainly governed by moisture content of the atmosphere, with Gram positive bacteria dominated in wet and Gram negative bacteria in dry atmosphere. The percentage of rods was found to be twice that of cocci in both Gram positive and Gram negative bacteria in all the mango cultivars indicating the dominance of spoilage causing microflora, as most of the bacterial strains responsible for spoilage of fruits and vegetables are reported to be Gram positive or Gram negative rods (Frazier and Westhoff, 2008). Among total bacterial isolates, the colonies of 55 isolates were found to be pigmented (Table 3). Such chromogenic isolates potentially have selective advantage over other inhabitants because their pigmentation protects them from ultra violet radiation and are frequently isolated and had been reported to colonize plant surface in large numbers (Crosse, 1971; Hirano and Upper, 1991; Mansvelt and Hattingh, 1987). Biochemical characterization of all the 123 bacterial isolates showed presence of 63 Gram positive and 60 Gram negative isolates (Table 3). Among Gram positive isolates, 40 were found to be rod shaped and the rest 23 were cocci form. Further, among Complementary Copy Table 1. Abbreviations used for mango cultivars harvested from different locations Name of cultivar Place of procurement Abbreviations used Alphonso Karnataka AK Alphonso Maharashtra AM Banganapalli Andhra Pradesh BA Banganapalli Orissa BO Chausa Punjab CP Chausa Uttar Pradesh CU Dashehri Punjab DP Dashehri Uttar Pradesh DU Kesar Gujarat KG Kesar Maharashtra KM Langra Uttar Pradesh LU Maldha Bihar MB Mallika Orissa MO Neelam Tamil Nadu NT 103 104 Postharvest microbial diversity on major cultivars of Indian mangoes Table 2. Number and Relative abundance of Gram positive and Gram negative bacteria from different mango cultivars AK AM BA BO CP CU DP DU KG KM LU MB MO NT Gram positive Gram positive Gram negative Gram negative Gram positive Gram positive Gram negative Gram negative (number) (RA) (number) (RA) rods (RA) cocci (RA) rods (RA) cocci (RA) 9 13 5 4 4 1 0 1 6 5 3 1 4 7 52.94 72.22 50.00 57.14 66.67 11.11 0.00 25.00 60.00 62.50 75.00 16.67 44.44 50.00 8 5 5 3 2 8 1 3 4 3 1 5 5 7 47.06 27.78 50.00 42.86 33.33 88.89 100.00 75.00 40.00 37.50 25.00 83.33 55.56 50.00 Gram positive rods, 14 isolates were found to form spores and 26 were non-spore formers. These spore forming strains were purified under purely aerobic conditions, therefore, the isolates identified belonged to genera Bacillus. Among the 26 non-spore formers, 10 showed positive acid fast reaction, which may be from genus Mycobacterium and remaining 16 showed the negative acid fast reaction. Further, results of catalase test showed that all the 16 bacterial isolates with negative acid fast reaction belonged to Lactobacillus genus, out of which 13 bacterial isolates showed acid formation on glucose fermentation test, which gave indication that these isolates may be L. casei or L. delbrueckii and remaining 3 bacterial isolates gave no reaction, indicating that they belong to other species of Lactobacillus genera. Bacillus spp. as a group is one of the important components of soil microbial community (Prescott et al., 2006). Many Bacillus spp. had been reported to secrete wide range of degradative enzymes such as cellulases, amylases, pectinases and proteases (Silva et al., 2000). Both pectinase and pectate lyase had been reported from Bacillus spp. (Soriano et al., 2000). In current investigation many identified isolates as Bacillus (according to Bergey’s manual) have shown presence of hydrolytic enzymes cellulases and pectinases, which might endow them with the advantage of colonizing the fruit surface. De Jager et al. (2001) also reported the predominance of Bacillus pumilis on mango leaf surface besides other bacterial genera (Cornyform, Pseudomonas, Xanthomonas and Erwinia) found to be present on mango phylloplane. All 23 isolated Gram positive cocci showed negative catalase activity indicating that they may either belong to Streptococcus or Enterococcus genus (Table 3). Among 60 Gram negative bacterial isolates, 37 were rod shape and rest were cocci. Among Gram negative rods 26 isolates showed positive oxidase test, and other 11 showed negative oxidase test. The isolates showing negative oxidase test belonged to family Enterobacteriaceae. Out of 26 isolates with positive oxidase test, 24 exhibited acid production on glucose fermentation, further, they didn’t require presence of sodium in the medium for growth. Therefore, isolates represent characteristics of genus Aeromonas as given in Bergey’s manual and rest of bacterial strains (2), although were able to ferment glucose, didn’t produce acid in the medium. Thereby, indicating that these isolates can belong to genus Pseudomonas. Among the Gram negative bacterial isolates, Aeromonas was found to 35.29 38.89 40.00 42.86 50.00 0.00 0.00 25.00 30.00 62.50 50.00 0.00 11.11 35.71 17.65 33.33 10.00 14.29 16.67 11.11 0.00 0.00 30.00 0.00 25.00 16.67 33.33 14.29 29.41 22.22 30.00 28.57 16.67 44.44 100.00 50.00 20.00 37.50 25.00 50.00 33.33 28.57 17.65 5.56 20.00 14.29 16.67 44.44 0.00 25.00 20.00 0.00 0.00 33.33 22.22 21.43 be the most dominant genera as it has been isolated from 12 mango cultivars (AK10, AK11, AK17, AM04, AM06, AM07, AM12, BA05, BA09, BA10, BO03, CP06, CU01, CU02, CU07, DU03, KG09, KM03, KM08, MB02, MB06, LU03, NT07 and NT10), while Pseudomonas was found on BO02, MO07 and NT05 cultivars. Aeromonas is widely found in nature including decomposing vegetable matter, while Pseudomonas is well known for their ability to metabolize a variety of diverse nutrients (Frazier and Westhoff, 2008). Many strains have been reported to play an important role in environmental biotechnological applications (Walsh et al., 2001; Mark et al., 2006; Ali Khan and Ahmad, 2006). However, on the contrary, many other have been reported to be phytopathogen and are widely dispersed on plants mainly on leaves and rhizosphere (Silva et al., 2000). Bacterial isolates belonging to Enterobacteriaceae family were further sub grouped based on the lactose fermentation test and results indicated that 7 (MB03, DU02, MO08, CU08, KG01, MO09 and NT01) were able to ferment lactose (Table 3). Further MB03 showed indole production by oxidizing an essential amino acid tryptophan, which utilized citrate as sole carbon source and showed negative VP test which is characteristic of genus Citrobacter. DU02 and MO08 were able to ferment lactose, showed indole production but did not show citrate reaction, which is characteristic of genus Escherichia. CU08, KG01, MO09 and NT01 did not oxidize tryptophan but KG01 showed positive reaction for both MRVP tests indicating that KG01 may be Enterobacter intermedius. CU08 & MO09 showed positive test for methyl red only and NT01 showed positive VP test only indicating that CU08 and MO09 may be Serratia fonticola / Klebsiella pneumoniae (subsp. Ozaenae) / Citrobacter freundii and NT01 may be Klebsiella pneumoniae (subsp. pneumoniae)/ Enterobacter spp. / Erwinia caotovora / Serratia rubidaea. The other four isolates of Enterobacteriaceae family showing negative reaction for lactose fermentation were able to oxidize tryptophan to indole and showed negative result for hydrogen sulfide production test, may belong to genus Morgenella /Providencia. Enterobacter is a common inhabitant of soil and sewage. Erwinia spp. is another common Gram negative spoilage microbe associated with fresh-cut vegetables. Erwinia, a genus within the family Enterobacteriaceae are small rods and facultative anaerobes. Erwinia is reported to cause rapid necrosis, progressive tissue maceration called “soft-rot”, occlusion of vessel elements called “vascular wilt,” and hypertrophy leading to gall or tumor formation in plant tissues (Margaret et al., 2009). Brocklehurst et al. (1987) and Manvell and Ackland (1986) identified E. carotovora as Complementary Copy Cultivar Table 3. Morphological and biochemical characteristics of bacterial isolates from nine different mango cultivars Morphological characteristic Biochemical characteristics Isolate Group G N S A P T S 1 2 3 4 5 6 7 8 9 10 11 AK1 A + R + + + + + + AK2 A + R + + + + + + + + AK3 A + R + + + + + AK4 A + R + + + + + + + + + + AK5 A + R + + + + + + AK6 B + C + + + + + + + + + + AK7 B + C + + + + + + + + AK8 B + C + + + + + + + + AK9 A + R + + + + + + + + + AK10 C R + + + + + + + AK11 C R + + + + + + + + + + AK12 C R + + + + + + + + + AK13 D C + + + + + + AK14 D C + + + + + + + + AK15 D C + + + + + + + AK16 C R + + + + + + + + + AK17 C R + + + + + + + + + AM1 B + C + + + + + + AM2 A + R + + + + + AM3 B + C + + + + + + + + AM4 C R + + + + + + + AM5 B + C + + + + + + AM6 C R + + + + + + + + AM7 C R + + + + + + + + AM8 B + C + + + + + + AM9 A + R + + + + + + AM10 A + R + + + + AM11 A + R + + + + + + AM12 C R + + + + + + AM13 A + R + + + + + + AM14 A + C + + + + + + AM15 A + C + + + + + AM16 D C + + + + + + AM17 A + R + + + + + + AM18 A + R + + + + + + + BA1 A + R + + + + + + + + + BA2 B + C + + + + + + BA3 D C + + + + + + BA4 A + R + + + + + BA5 C R + + + + + + + BA6 D C + + + + + + + BA7 A + R + + + + + + BA8 A + R + + + + + + + + BA9 C R + + + + + + + + + + BA10 C R + + + + + + + BO1 D C + + + + + + BO2 C R + + + + + + BO3 D C + + + + + + + BO4 A + R + + + + + + + + BO5 B + C + + + + + + + BO6 A + R + + + + + + BO7 A + R + + + + + + + + + + CP1 B + C + + + + + + CP2 A + R + + + + + CP3 A + R + + + + + + + CP4 D C + + + + + + + + + + + CP5 A + R + + + + + + + CP6 C R + + + + + + + + + CU1 C R + + + + + + + + CU2 C R + + + + + + CU3 D C + + + + + CU4 B + C + + + + + - 105 12 - 13 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 14 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - 15 + + + + + + + + + - 16 + + + + + + + + + - 17 + + + + + + + + - Complementary Copy Postharvest microbial diversity on major cultivars of Indian mangoes CU5 CU6 CU7 CU8 CU9 DP1 DU1 DU2 DU3 DU4 KG1 KG2 KG3 KG4 KG5 KG6 KG7 KG8 KG9 KG10 KM1 KM2 KM3 KM4 KM5 KM6 KM7 KM8 MB1 MB2 MB3 MB4 MB5 MB6 LU1 LU2 LU3 LU4 MO1 MO2 MO3 MO4 MO5 MO6 MO7 MO8 MO9 NT1 NT2 NT3 NT4 NT5 NT6 NT7 NT8 NT9 NT10 NT11 NT12 NT13 NT14 Postharvest microbial diversity on major cultivars of Indian mangoes D D C C D C D C C A C B D A D B A B C A A A C A C A A C B C C D D C A A C B B A D B D B C C C C A D D C A C A A C A B D B + + + + + + + + + + + + + + + + + + + + + + + + + + + C C R R C R C R R R R C C R C C R C R R R R R R R R R R C R R C C R R R R C C R C C C C R R R R R C C R R R R R R R C C C + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + - G=Gram reaction, N=Negative staining, S=Spore staining, A=Acid fast staining, P=Pigmented colony, T=Transparent colony, S=Smooth edge on colony. Biochemical characteristics: 1=Lactose fermentation 2=Casein Hydrolysis 3=Starch hydrolysis 4=Flouroscein production 5=Peroxidase reaction 6=Gelatin hydrolyis 7=Methyl red test 8=Voges-Proseuker test 9=Urease test 10= Indole production test 11=Citrate utilization test 12= Hydrogen sulphide production test 13=Glucose fermentation test 14= Oxidase test 15 = Cellulose degradation test 16=Pectate lyase production test 17= Pectinase production test Complementary Copy 106 Postharvest microbial diversity on major cultivars of Indian mangoes 107 Mango cultivar Alphonso, Maharashtra Isolate AM01 AM02, 07, 10 AM08 AM03, 04, 06 AM05, 13 AM09 Alphonso, AK02,0 4 Karnataka AK03 Baganpalli, BA02 Andhra Pradesh BA08 Baganpalli, Orissa BO03 Chausa, Punjab CP01, 03 CP02 Chausa, CU01 Uttar Pradesh CU04 CU03 CU06 Dashehri, Punjab DP01 DP02 DP03 DP04 Kesar, Gujarat KG01 KG02 KG03 Kesar, Maharashtra KM01, 02 Langra, LU01 Uttar Pradesh LU02 LU03 Maldah, Bihar MB01 MB03 Mallika, Orissa MO05 MO01 MO06 MO02 MO03 Neelam, NT01 Tamil Nadu NT02 NT03, 4 Fungus Evenness / Relative Abundance, RA (%) Species 9.09 A. fumigatus 27.27 A. flavus 9.09 A. niger 27.27 Fusarium F. solani 18.18 Alternaria A. alternata 9.09 Cladosporium C .cladosporioides 66.66 Aspergillus A. niger 33.33 A. flavus 50 Aspergillus A.terreus 50 Pencillium P. citrinum 100 Aspergillus A. niger 66.67 Aspergillus A. fumigatus 33.33 Pencillium P. chrysogenum 25 Fusarium F. moniliforme 25 F. oxysporum 25 Aspergillus A. flavus 25 A. fumigatus 25 Fusarium F. pallidoroseum 25 Aspergillus A. niger 25 A. nidulans 25 A. fumigatus 33.33 Fusarium F. moniliforme Aspergillus A. fumigatus 33.33 33.33 A. flavus 100 Aspergillus A. niger 33.33 Alternaria A. alternata 33.33 Aspergillus A. niger 33.33 Heminthosporium H. spiciferum 50 Aspergillus A. fumigatus 50 Trichoderma T. longibrachiatum 20 Fusarium F. pallidoroseum 20 Pencillium P. chrysogenum 20 P. oxalicum 20 Aspergillus A. terreus 20 A. niger 25 Aspergillus A. flavus 25 A. terreus 50 A. niger Genera Aspergillus a principal spoilage microbe of both fresh-cut and fresh vegetables. Buick and Damoglou (1987) found that E. carotovora was the dominant spoilage microorganism on sliced carrots packed in air, consisting of more than 80% of total detectable microflora. Robbs et al. (1996) identified Erwinia in 5 of 16 soft-rot samples of fresh-cut celery. Erwinia had been reported to produce pectinolytic enzymes, which cause degradation of cell wall leading to soft rot disease (Lund, 1983). All the bacterial isolates were tested for presence of hydrolytic enzymes. Out of 123 isolates, 50 showed cellulase activity and 32 showed pectin hydrolysis out of which 17 showed production of pectatae lyase and 15 showed production of polygalacturonase. Such microbes having capability of producing hydrolytic enzymes may use it to overcome plant defence mechanisms and gain access to plant nutrients. The pectolytic enzymes, including pectin methyl esterase (PME), polygalacturonase (PG), pectin lyase (PNL), and pectate lyase (PL), can degrade pectins in the middle lamella of the cell, thereby resulting in liquefaction of the plant tissue leading to conditions such as soft rot. Besides, many strains of Bacillus and Erwinia had been reported to show antagonistic effect against pathogens causing black spot Species Richness Simpson’s index (D) Simpson’s index of Diversity (1-D) 6 0.17 0.83 2 0.56 0.44 2 0.50 0.50 1 1.00 0.00 2 0.56 0.44 4 0.25 0.75 4 0.25 0.75 3 0.33 0.66 1 1.00 0.00 3 0.33 0.66 2 0.50 0.50 5 0.20 0.80 3 0.37 0.63 of mango (Pruvost and Luisetti, 1991). Okigbo and Osuinde (2003) reported bio-control of fungal leaf spot disease of mango with Bacillus subtilis. In South Africa, concerted efforts had been made to develop biocontrol agent against anthracnose disease of mango (Burger and Korsten, 1988). Korsten et al. (1991) reported effective reduction in the incidence of the disease when Bacillus licheniformis was used as either pre or postharvest application. They further reported that the efficacy of the biocontrol agent could be further improved when it was applied with recommended fungicide used at lower concentration (Korsten et al., 1992, Silimela and Korsten, 2001). Bacillus licheniformis is now available in commercial formulation (Mango green), and is reported to effectively reduce the fungal population from mango surface (Govender et al., 2005). Fungal diversity: Altogether 47 filamentous fungal isolates were purified from mango fruit surface of nine mango cultivars and among them the most abundant genera was Aspergillus followed by Fusarium and Pencillium with a relative abundance (RA) of 60.00, 17.00 and 8.00 %, respectively. Other fungi identified in minority belonged to genus Alternaria, Cladosporium, Helminthosporium and Trichoderma. Complementary Copy Table 4. Mold diversity on different mango cultivars from different parts of India Postharvest microbial diversity on major cultivars of Indian mangoes Among the genus Aspergillus, the dominant species were A.niger, A. flavus and A. fumigates., while the genus Fusarium was dominated by F. monoliforme. Aspergillus species are highly aerobic and widespread in nature, being found on fruits, vegetables and other substrates which may provide nutriment, where they commonly grow as molds on the surface of a substrate, as a result of the high oxygen tension. Some species of this genus are involved in food spoilage (Pelczar et al., 2008). Acknowledgement Aspergillus niger and Aspergillus flavus were found to be most common on mango cultivars representing 23.00 and 15.00 % of the total fungal isolates, respectively. Aspergillus niger is reported to cause black mould disease on certain fruits and vegetables such as grapes, onions and peanuts and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments (Frazier and Westhoff, 2008). Fungal composition did not vary much among the cultivars however, maximum species richness was found on AM followed by MO, CU and DP while lowest on BO and KM. Alphonso mango harvested from Maharastra (AM) showed presence of 11 fungi belonging to genus Aspergillus, Fusarium, Alternaria and Cladosporium with RA 45.45, 27.23, 18.18 and 9.09 %, respectively, thereby exhibiting highest Simpson’s index of diversity (measure of the probability that two individuals randomly selected from a sample will belong to the same species.) amongst various mango cultivars under study (Table 4). While cv. BO and KM showed the presence of Aspergillus niger only and hence, depicting lowest species richness and Simpson’s diversity index (Table 4). Tournas and Katsoudas (2005) also reported the dominance of Alternaria, Cladosporium, Penicilium, Fusarium, Trichoderma, Geotrichum, Rhizopus and A. niger in their study with citrus fruits. Ali Khan, M.W. and M. Ahmad, 2006. Detoxification and bioremediation potential of a Pseudomonas fluorescens isolate against the major Indian water pollutants. J. Environ. Sci. Health Part A Toxic/ Hazardous Subsances Environ. Eng., 41(4): 659-674. Atlas, R.M. and J.W. Snyder, 2006. Handbook of Media for Clinical microbiology. CRC Press, London. Arauz, L.F. 2000. Mango anthracnose: economic impact and current options for exported mango. Plant Disease, 84: 600-611. Barnby, F.M., F.F. Morpeth and D.L. Pyle, 1990. Endopolygalacturonase production from Kluyveromyces marxianus. I. Resolution, purification and partial characterization of the enzyme. Enzym. Microb. Tech., 12: 891-897. Brocklehurst, T.F., C.M. Zaman-Wong and B.M. Lund, 1987. 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Frazier, C.W. and C.D. Westhoff, 2008. Food Microbiology. Fourth Edition. McGraw Hill, New Delhi. Govender, V., L. Korsten and D. Sivakumar, 2005. Semi-commercial evaluation of Bacillus licheniformis to control mango postharvest disease in South Africa. Postharvest Biol. Technol., 38: 57-65. Hawksworth, D.L., P.M. Kirk, B.C. Sutton and D.N. Pegler, 1995. Ainsworth and Bisby’s Dictionary of the Fungi. CAB International, Wallinford, UK. Hirano, S.S. and C.D. Upper, 1991. Bacterial community dynamics. In: Microbiol. Ecology of leaves. J.H. Andrew and S.S. Hirano (eds.).Springer-Verlag, New York. p. 271-294. Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Stanley and S.T. Williams, 1994. In: Bergey’s Manual of Determinative Bacteriology. M.D. Baltimore, (eds.). Ninth edition. Williams & Wilkins. p. 787. Hoondal, G.S, R.P. Tiwari, R. Tewari, N. Dahiya and G.K. Beg, 2002. Microbial alkaline pectinases and their industrial applications - A review. Appl. Microbiol. Biotechnol., 59: 409-418. Jha, S.N., P. Jaiswal, K. Narsaiah, R. Bhardwaj, R. Sharma, R. Kumar and A. Basedia, 2010. Postharvest micro-flora on major cultivars of Indian mangoes. Scientia Hort., 125(4): 617-621. Joshi, S.R. 2008. Influence of roadside pollution on the phylloplane microbial community of Alnus nepalensis (Betulaceae). Rev. de boil. Trop., 56(3): 1521-1529. Korsten, L., J.H. Lonsdale, E. De Villiers and E.S.De Jager, 1992. Preharvest Biological Control of Mango Diseases. South African Mango Growers Association Yearbook, 12: 72-74. The fungal isolates were tested for presence of hydrolytic enzymes (cellulases and pectinases) and results indicated that 6 out of 47 isolates showed presence of one or more hydrolytic enzymes (data not shown). Majority of these isolates belonged to genus Aspergillus except one, which belonged to genus Alternaria. Aspergillus is well known fungus for its hydrolytic activity. Almost all the commercial preparartions of pectinases are produced from fungal sources (Singh et al., 1999). Aspergillus niger is the most commonly used fungal species for industrial production of pectinolytic enzymes (Kotzekidov, 1991; Barnby et al., 1990; Naidu and Panda, 1998). Current study showed that mango surface harbored a huge microbial diversity, which comprised varietal as well as regional variation. In general, Gram positive rods showed predominance in bacterial community and Aspergillus spp. in fungal community. The highest fungal diversity was observed in variety AM, followed by MO, CU, DP, KG, LU, NT, BA, MB, AK, CP, BO, KM. This study is an important step in identification of spectrum of microbial community on mango surface which will be helpful in devising measures to reduce the effect of spoilage and pathogenic microbes. Further, the available bio-resources on mango surface can be used to screen the potential micro-flora with a role in biocontrol of plant diseases, for production of metabolite/enzyme of commercial importance or for the development of instrumental methods such as biosensors, spectroscopic instruments, etc. for rapid detection of microbial as well as physico-chemical characteristics. This research was supported by the National Agricultural Innovation Project (NAIP), Indian Council of Agricultural Research (ICAR) through its subproject entitled “Development of non-destructive systems for evaluation of microbial and physico-chemical quality parameters of mango” Code number “C1030”. References Complementary Copy 108 Korsten, L., M.W.S. Van harmelen, A. Heitmann, E. DeVilliers and E.S. De Jager, 1991. Biological Control of Postharvest Mango Fruit Diseases. South African Mango Growers Association Yearbook, 11: 65-67. Kotzekidov, P. 1991. 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K ha nna Department of Microbiology, Punjab Agricultural University, Ludhiana, *Department of Processing and Food Engineering, Punjab Agricultural University, Ludhiana-141004, India. E-mail: [email protected], [email protected] Abstract Key words: Agaricus bisporus, cabinet drying, microwave-oven drying, color index, texture index, carbohydrates, proteins, lipids Introduction drying had been combined with conventional hot air drying to reduce the drying time, optimize energy efficiency and improve product quality (Zhang et al., 2006). Pretreatments of mushrooms before drying by, washing in water, KMS, sugar, salt either alone or in combination help in checking enzymatic browning, stabilizing color, enhancing flavor retention and maintaining textural properties (Singh et al., 2001). The optimum conditions of drying were established by Kar et al. (2004) on the basis of rehydration ratio and sensory evaluation. Drying at microwave intensity of 400 W with pretreatment of blanching in boiling water for 3 min., followed by steeping in solution of 0.1% KMS + 0.2% CA + 6% sugar + 3% NaCl at room temperature for 15 min. yielded an acceptable dehydrated product in about 45 min. The rehydration ability of the dried product is a critical parameter indicating the degree of damage caused by physiochemical treatments (Krokida and Marinos, 2003). Present investigation was undertaken to study the effect of drying by different methods on color, texture, biochemical and microbiological quality parameters of the dehydrated white button mushrooms. White button mushroom (Agaricus bisporus) is devoured by mankind for its characteristic aroma, texture and nutritional value (Arumuganathan et al., 2003). Dehydration appears to be a promising and cost effective method of mushroom preservation for the Indian conditions as it is easy to transport the dried product compared to canned, pickled and frozen products. The dehydrated products, apart from the increased shelf life, offer an advantage of decreased weight and volume with a potential for saving the cost of packaging, handling, storage and transport (Amuthan et al., 1999). Dried mushrooms, packed in airtight containers can have a shelf life of above one year (Bano et al., 1992). Mushrooms could be dried to a moisture level down below 10% at drying temperature of 55˚C to give the end product with the desired qualities of texture, color and rehydration. At a drying temperature of 55-60˚C, the insects and microbes on the mushrooms would be killed in a few hours, which give the dehydrated final product of lower moisture content with longer shelf life. A cabinet drier with proper air circulation has been reported to be superior by Mudahar and Bains (1982). Materials and methods Microwave oven drying is an alternative way which generates very rapid heat and mass transfer resulting in quick drying of mushrooms. It offers the product with good organoleptic and nutritional values (Sahni et al., 1997). Heating of foods by microwave energy sources are instantaneous. Microwave-vacuum Two strains S11 and U3 of A. bisporus, were harvested from the Mushroom Research Farm, PAU, Ludhiana. Half the sample of mushrooms (500g) from each strain was treated with 1% KMS for 10 minutes in order to inactivate the enzymes and improve its color characteristics while other half was left untreated as Complementary Copy White button mushrooms, Agaricus bisporus (strains U3 and S11) were dried in cabinet at two temperatures (45 and 55°C) and microwave oven at 380W for 30 minutes. Dried mushrooms were subjected to physical (color, texture, rehydration ratio, dehydration ratio), biochemical (carbohydrates, proteins and lipids) and microbiological (total bacterial count) parameters after three months of storage period. In strain U3, carbohydrate content was highest in 0.1% KMS treated mushrooms dried at 45˚C, protein ranged between 3.43 to 3.89 g/100 g of fresh mushrooms, lipid content ranged between 0.06 to 0.30 g/100 g of mushrooms and the total bacterial count ranged between 1.48 to 2.07 log cfu/g which was within the permissible limits of dried fruit products while in microwave oven dried mushrooms there was no significant difference in two strains in terms of carbohydrate, protein and lipid contents. Bacterial count was found to be within the permissible limit of dried fruit products (1.85-2.17 log cfu/g). The weight of dried mushrooms remained almost constant throughout the storage period of 3 months. However, cabinet drying was preferred for most of the color and texture index parameters. Springiness was maximum for microwave oven dried mushrooms of S11 strain treated with 0.1% KMS, followed by the unwashed mushrooms. Resilience ranged between 0.23 to 0.33 in all the treatments. Cohesiveness was maximum in unwashed mushrooms of U3 dried at 55˚C, followed by cabinet dried mushrooms of S11 strain (55˚C) both unwashed and 0.1% KMS treated. Chewiness and gumminess were also maximum for cabinet dried unwashed mushrooms of U3, followed by microwave oven dried 0.1% KMS treated mushrooms. A. bisporus was most acceptable in cabinet drying for 0.1% KMS treated U3 strain at both 45˚C and 55˚C while in case of microwave oven drying, total color difference (2.88 for U3 and 2.58 in S11) was minimum and rehydration ratio (1.91 to 3.06) was found to be maximum for U3 strain. Effect of dehydration on keeping quality of white button mushrooms Cabinet drying: Mushrooms were dried in cabinet tray drier (Kilbron Oven, Macneil and Magor Ltd.) where mushrooms were kept in series of trays and warm air temperature of 45oC and 55oC was passed with air velocity of approximately 1m / sec. Weight of tray was noted regularly. Readings were taken at an interval of 2 hours till 7-8% constant moisture was achieved. Microwave drying: Mushrooms were dried in microwave [Electrolux (250-700)] at 380W for 30 minutes. The dried mushrooms were packed in airtight polyethylene bags of 150 gauze. The observations were made on wt. loss (%), appearance, color, optical density, dehydration ratio, rehydration ratio, biochemical and microbiological properties in comparison to the unwashed mushrooms as control. Rehydration was done by dipping weighed sample (5g) of dried mushroom in distilled water. Dehydration and rehydration ratio was calculated using the formula of Ranganna (1986). Color and texture analysis: The color of freshly harvested and stored samples was measured by using Miniscan XE plus Hunter Lab Colorimeter (Burton et al., 1987; Gormley, 1974). L, a, b values for samples were obtained in triplicate. From these values of ‘L’, ‘a’ and ‘b’ total color difference was obtained using the formula: ΔE =√ΔL2 + Δa2 +Δb2 Where, ΔL, Δa and Δb are deviations from L, a and b values of the fresh sample. ΔL = L sample - L standard; + ΔL means sample was lighter than standard, -ΔL means sample was darker than standard. Δa = a sample - a standard; +Δ a means sample was redder than standard, -Δa means sample was greener than standard. Δb = b sample - b standard, + Δb means sample was yellower than standard, -Δb means sample was bluer than standard. The hue (H), chroma (C) and browning index (BI), which represented the purity of brown color (Polou et al., 1999), were also calculated according to the given equation: Hue= tan-1 (b/a), Chroma = (a2 + b2)1/2 BI= 100(x - 0.31) /0.172, Where, x= (a + 1.75L) / (5.645 + a-3.012b) The textural behavior of the whole mushroom was estimated in terms of the texture profile analysis (TPA) curve. The parameters of brittleness, hardness, cohesiveness, adhesiveness, chewiness, springiness and gumminess were calculated from the plot of two cyclic compression text performed on mushroom sample of about 5 mm thickness using an aluminium cylinder known as P/75 cylindrical probe having 75 mm diameter. The conditions set for TPA were: Load cell: 50 kg, Test mode: measure force in compression, Pretest speed: 10 mm/s, Test speed: 5 mm/s, Post test speed: 10 mm/s, Time lag between 2 compressions: 2s, Test strain: 75% of sample height, Trigger force: 0.05 N, Data acquisition rate: 200 pps, Probe: SMS P/75 cylindrical probe, 75 mm diameter. Total bacterial count: Total bacterial count (cfu/ g fresh mushrooms) was determined by standard method of serial dilution and plating. Biochemical analysis: Estimation of total sugars was done by Dubois et al. (1956) method. Extraction was done by taking carbohydrates from dried samples of A. bisporus. Estimation of proteins was done by method given by Lowry et al. (1951) and extraction and estimation of total lipids was done by Folsch et al. (1957) method. Results and discussion Biochemical and microbiological analysis: Fruit bodies of A. bisporus strains S11 and U3 were subjected to cabinet drying (45˚C and 55˚C) and microwave oven drying (380W, 30 minutes) followed by their biochemical and microbiological analysis. In strain U3, carbohydrate content was highest in 0.1% KMS washed and mushrooms dried at 45˚C followed by mushrooms received same pretreatment and dried at 55˚C. The protein content ranged between 3.43 to 3.89 g/100 g of fresh mushrooms whereas lipid content was 0.06 to 0.30 g/100 g of mushrooms (Table 1). The total bacterial count ranged between 1.48 to 2.07 log cfu/g which was within the permissible limits of dried fruit products (less than 2.7 log cfu/g, non pathogenic). In microwave oven dried mushrooms, there was no significant difference in two strains in terms of carbohydrates, proteins and lipids contents and bacterial count was found to be within the permissible limits of dried fruit products (1.85-2.17 log cfu/g) (Table 1). The weight of dried mushrooms remained almost constant throughout the storage period of 3 months. Dehydration ratio for all the treatments ranged between 9.25 to 13.78 with lowest for 0.1% KMS treated mushrooms (S11 and U3) dried in microwave oven. The rehydration ratio for all the treatments ranged between 1.91 to 3.06 with highest in 0.1% KMS treated U3 mushrooms dried in microwave oven (Table 1). Color index: The mushrooms from two different strains received different pretreatments and dried by two different methods along with control were compared for color index for L, a, b, ΔE, hue, chroma, browning index. In each treatment, L value for 0.1% KMS treated mushrooms was better with highest value of 41.45 L for U3 strain. There were no significant differences in a value for different treatments. ΔE value was lowest for microwave oven dried mushrooms in comparison to cabinet dried mushrooms. The hue index showed increased value from 0.1% KMS pretreated to unwashed mushrooms of S11 strain in cabinet drying whereas contrast was true for mushrooms of U3 in cabinet drying and both S11 and U3 for microwave oven dried mushrooms. The browning index for cabinet dried unwashed mushrooms of S11 was higher whereas it was low for unwashed U3 mushrooms. However, there was no significant difference in browning index in microwave oven dried mushrooms (Table 2). Texture profile: The texture profile of dried mushrooms indicated adhesiveness ranging from -1.57 to -6.98 for all treatments. In cabinet dried mushrooms, maximum hardness (texture) was in 0.1% KMS treated mushrooms from U3 strain. In microwave oven dried mushrooms, maximum hardness for both 0.1%KMS treated and unwashed mushrooms was in U3 strain. Springiness was maximum for microwave oven dried mushrooms of S11 strain treated with 0.1% KMS, followed by the unwashed mushrooms. Complementary Copy control. The mushrooms were then cut longitudinally into pieces, blanched and dried in cabinet drier and in microwave. Five replicates for each set of experiment were observed and analyzed statistically for ANOVA at P= 0.05. 111 112 Effect of dehydration on keeping quality of white button mushrooms Table 1. Physico-chemical and microbiological properties of dried A. bisporus using different techniques Drying Strain Treatment Temperature Carbohydrate Proteins Lipids Bacterial Change in Dehydration Rehydration type (g/100g of (g/100g of (g/100g of count ratio (x) ratio (x) wt. during mushrooms) mushrooms) mushrooms) (log cfu/g) storage (%) Cabinet S11 0.1%KMS 45˚C 2.04 3.79 0.06 1.70 7.24 13.47 2.39 Unwashed 2.89 3.58 0.12 2.07 6.66 9.93 2.22 drying 0.1%KMS 55˚C 2.47 3.61 0.21 1.61 15.75 13.78 2.76 Unwashed 2.50 3.52 0.30 2.0 5.5 12.63 2.56 U3 0.1%KMS Unwashed 0.1%KMS Unwashed 45˚C 55˚C CD (P=0.05) Microwave S11 Unwashed 380W(30min) oven drying 0.1%KMS U3 Unwashed 380W(30min) 0.1%KMS CD (P=0.05) 4.39 2.17 3.02 2.01 0.73 2.48 3.64 3.89 3.43 3.77 NS 3.80 0.06 0.09 0.08 0.07 0.35 0.10 1.48 2.07 1.31 2.0 0.17 2.17 4.32 6.20 4.51 12.61 0.34 -13.0 11.32 11.42 11.80 12.13 0.34 10.22 2.48 3.03 2.25 1.91 0.35 2.41 2.50 2.13 2.14 NS 3.63 3.50 3.43 0.38 0.13 0.10 0.08 NS 1.91 2.07 1.85 0.12 -.0015 12.01 -28.70 1.63 9.25 11.12 9.25 0.37 2.61 2.70 3.06 0.18 NS= Non Significant in all the three table Resilience ranged between 0.23 to 0.33 in all the treatments. Cohesiveness was maximum in unwashed mushrooms of U3 dried at 55˚C, followed by cabinet dried mushrooms of S11 strain (55˚C) both unwashed and 0.1% KMS treated. Chewiness was also maximum for cabinet dried unwashed mushrooms of U3, followed by microwave oven dried 0.1% KMS treated mushrooms. This lot also indicated maximum gumminess (Table 3). Singh et al. (2007) also showed that button mushroom samples dehydrated at 50˚C gave better quality. They reported diffusivity of 1.05x10-08 to 7.48x10-09 m2/s and it increased with drying air temperature. Drying using microwave oven has been reported as non satisfactory mainly because of lack of temperature control and high time of exposure resulting in charring of mushrooms particularly at the edges (Walde et al., 1997). However, the time taken for drying from 7.5% (db) moisture to 2% (db) has been reported more for cabinet than for microwave oven drying (Walde et al., 2006). A comparative new technique ‘Microwave-vacuum drying’ had resulted in 70-90% reduction in the drying time with better rehydration characteristics as compared to convective air drying (Giri and Prasad, 2007). Observations after three months of storage indicated that cabinet drying at temperatures of 45 and 55˚C gave best results particularly in 0.1% KMS treated mushrooms of U3 strain. The drying temperature of 55˚C in the plenum chamber gave the end product with the desired qualities of texture, color and rehydration (NRCM, 2008). Pre-drying treatments had a significant effect on whiteness and color change of the dried mushroom slices. Whiteness is reported to be high in blanched mushrooms compared to other treatments but it gave very low rehydration ratio (Nour et al., 2011). In present study, cabinet drying gave superior results with respect to color and texture index, while microwave drying required lesser time for drying to achieve same Table 2. Color index of dried A. bisporus using different techniques Drying type Strain Treatment Temperature L Cabinet drying S11 0.1%KMS 45˚C Unwashed 0.1%KMS Unwashed U3 0.1%KMS Unwashed 0.1%KMS Unwashed C.D (P=0.05) Microwave oven drying 55˚C 45˚C 55˚C S11 Unwashed 380W (30 min) 0.1%KMS U3 Unwashed 380W (30 min) 0.1%KMS CD (P=0.05) Use of cabinet and microwave-oven drying for the dehydration of A.bisporus (strains U3 and S11) suggested that, in cabinet drying, out of two strains, 0.1% KMS treated mushrooms of U3 strain gave good results for most of the parameters at both drying temperature of 45˚C and 55˚C while in case of microwave oven Color index ΔE a b 28.66±0.83 5.27±0.22 -1.10±0.96 22.04±0.68 4.27±0.16 6.18±0.64 34.99±0.61 6.41±0.13 5.20±0.25 30.02±0.62 39.47±0.47 32.02±0.15 41.45±1.16 34.46±0.39 5.41±0.12 7.27±0.18 5.81±0.07 8.00±0.34 6.59±0.01 6.20±0.21 8.42±1.06 2.51±0.16 6.39±0.59 3.49±0.21 3.13 28.80±1.67 30.49±1.67 NS 6.5±0.12 8.18±0.14 0.94 4.53±0.15 3.53±0.15 2.58 2.88 9.890 5.16 9.62 7.69 HUE CHROMA BI -11.79 5.38 8.91 55.35 7.51 46.05 39.09 8.25 28.64 48.89 49.19 23.37 38.62 27.91 8.22 11.12 6.33 10.24 7.46 35.45 36.58 20.64 29.99 23.81 0.47 34.87 23.34 0.57 7.92 8.91 0.19 32.60 30.64 30.15±0.04 6.7±0.02 5.68±0.09 40.28 8.78 36.12 32.15±0.02 8.53±0.05 4.68±0.09 28.75 9.73 33.90 NS NS NS 0.20 NS 0.77 Complementary Copy level of moisture content in the end product (Table 2 & 3). Effect of dehydration on keeping quality of white button mushrooms Cabinet drying S11 0.1%KMS U3 C.D (P=0.05) Microwave oven drying Unwashed 0.1%KMS Unwashed 0.1%KMS Unwashed 0.1%KMS Unwashed 45˚C 55˚C 45˚C 55˚C S11 Unwashed 380W (30 min) 0.1%KMS U3 Unwashed 380W (30 min) 0.1%KMS CD (P=0.05) Springiness (mm) Resilience Cohesiveness Chewiness Gumminess (g x mm) (g) -3.89 10562 0.68 0.27 0.03 281 411 -5.00 -5.33 -5.30 -6.98 -2.08 -6.79 -3.42 0.36 -1.57 -1.99 -4.93 -4.02 0.19 12274 14487 14400 197140 35485 170870 88010 628626.0 44550 44965 109876 103921 47.64 0.78 0.94 0.90 0.42 0.61 0.001 0.40 0.16 1.00 1.10 0.14 0.15 0.22 0.29 0.31 0.25 0.33 0.24 0.32 0.28 0.37 0.23 0.24 0.29 0.30 NS 0.02 1.96 1.80 0.07 0.75 0.80 2.18 0.17 0.57 0.75 0.07 0.09 0.19 195 26851 23509 6044 16450 226 77033 13.84 25616 37657 1239 1573 0.60 250 28396 25920 14391 26862 137550 191863 3531.0 25393 34039 8438 9997 0.60 drying, L Value and rehydration ratio were highest for mushrooms of U3 strain (0.1% KMS treated and untreated) as compared to S11 (0.1% KMS treated and untreated). Among the two drying methods, cabinet drying should be preferred for texture and whiteness index (L value) while to save time, microwave drying should be preferred for lower ΔE value and better rehydration ratio of the product. References Amuthan, G., R. Visvanathan, R. Kailappan and V.V. Sreenarayanan, 1999. Studies on osmo-air drying of milky mushroom, Calocybe indica. Mush. Res., 8: 49-52. Arumuganathan, T., R.D. Rai, C. Induranic and A.K. Hemkar, 2003. Rehydration characteristics of the button mushroom (Agaricus bisporus) dried by different drying methods. Mush. Res., 12: 121-23. Bano, Z., S. Rajarathnam, Shashi and M.N. Rekha, 1992. Mushroom as the unconventional single cell protein for a conventional consumption. Indian Fd. Packer, 46: 20-31. Burton, K.S., C.E. Frost, P.T. Atkey, 1987. Effect of vacuum cooling on mushrooms browning. Intl. J. Fd. Sci. Technol., 22: 599-606. Dubois, M., K.A. Gill, J.K. Hamilton, P.A. Roberts and F. Smith, 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Folsch, J., M. Less and G.H. Stanley, 1957. A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Giri, S.K. and S.K. Prasad, 2007. Drying kinetics and rehydration characteristics of microwave-vacuum and convective hot air dried mushroom. J. Fd. Eng., 78: 512-521. Gormley, R. 1974. Chill storage of mushrooms. J. Sci. Fd. Agr., 26: 401-411. Kar, A., P. Chandra, R. Prasad and S.K. Dash, 2004. Microwave drying characteristics of a button mushroom (Agaricus bisporus). J. Fd. Sci. Technol., 41: 636-641. Krokida, M.K. and K.D. Marinos, 2003. Rehydration kinetics of dehydrated products. J. Fd. Eng., 57: 1-7. Lowry, O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with folin phenol reagent. J. Biol. Chem., 193: 265-275. Mudahar, G.S. and G.S. Bains, 1982. Pre-treatment effect on quality of dehydrated Agaricus bisporus mushrooms. Indian Fd. Packer, 28: 19-27. Nour V., I. Trandafir and M.E., Ionica, 2011. Effects of pretreatments and drying temperatures on the quality of dried button mushrooms. South-West. J. Hortic. Biol. Environ., 2(1): 15-24. NRCM, 2008. Annual Report of All India Coordinated Mushroom Research Project. Solan, p. 16-19. Palou, E., M.A. Lopez, C.G.V. Barbosa, C.J. Welti and G.B. Swanson, 1999. Polyphenoloxidase activity and colour of blanched and high hydrostatic pressure treated banana puree. J. Fd. Sci., 64: 42-45. Ranganna, S. 1986. Handbook of Analysis and Quality Control for Fruits and Vegetable Products. Tata McGraw Hill publishing Co. Ltd., New Delhi. Sahni, C.K., D.S. Khwidiya, M.A. Dalal and S.B. Maini, 1997. Microwave processing of foods-potentialities and prospects. Indian Fd. Packer, 51: 32-42. Singh, S.K., M. Narain and B.K. Kumbhar, 2001. Effect of drying air temperatures and standard pretreatments on the quality of fluidized bed dried button mushroom (Agaricus bisporus). Indian Fd. Packer, 55: 82-86. Singh, U., S.K. Jain, R.C. Verma, A. Doshi and M.K. Jaipal, 2007. Dehydration characteristics and quality analysis of button mushroom slices (Agaricus bisporus). Agr. Eng. Today, 31: 43-46. Walde, S.G., K. Balaswamy, R. Shivaswamy, A. Chakkaravarthi and D.G. Rao, 1997. Microwave drying and grinding characteristics of gum karaya (Sterculia urens). J. Fd. Eng., 31: 305-13. Walde, S.G., V. Velu, T. Jyothirmayi and R.G. Math, 2006. Effect of pretreatment and drying methods on dehydration of mushroom. J. Fd. Eng., 74: 108-115. Zhang, M., J. Tang, A.S. Mujamdar and S. Wang, 2006. Trends in microwave related drying of fruits and vegetables. Trends Fd. Sci Technol., 17: 524-534. Received: April, 2012; Revised: October, 2012; Accepted: October, 2012 Complementary Copy Table 3. Texture profile of dried A. bisporus by different techniques Drying type Strain Treatment Temperature Adhesiveness Hardness (g x mm) (g) 113 Journal Journal of Applied Horticulture, 14(2):114-117, 2012 Appl Re lat ionship of nut rit iona l st at us of fi e ld grow n T hom pson Se e dle ss gra pevine s w it h pow de r y m ilde w inc ide nc e Ja gdev Sha r m a * , A.K . U pa dhya y, I ndu S. Sa w a nt a nd S.D. Sa w a nt National Research Centre for Grapes, Pune (MS)-412307, India. *E- mail: [email protected] Abstract Relationship between nutritional status of open field grown Thompson Seedless grapevines and powdery mildew incidence was studied for two years at two growth stages. Amongst different nutrients, potassium showed highest degree of significant and negative correlation with the powdery mildew disease rating (r= -0.817 and -0.875) at two growth stages. Regression analysis also revealed the importance of potassium nutrition in powdery mildew incidence. During the first year of the study, N, P, K, Ca, Mg and Na when regressed together accounted for 82.7 % (R2 = 0.826) variation in disease incidence and potassium alone accounted for 66.8 % variation in disease incidence (R2 = 0.667). During the second year N, P, K, Ca, Mg and Na when regressed together accounted for 85.7 % (R2 = 0.857) variation in disease incidence and potassium alone accounted for 76.6 % variation in disease incidence (R2 = 0.765). Introduction Materials and methods Powdery mildew is one of the major diseases of grapevines in India. The disease is mainly managed by fungicide application. However, fungicidal efficacy varies from vineyard to vineyard. It is known that the nutritional status of plants has a substantial impact on their predisposition to pests and diseases. All essential mineral elements are reported to influence disease incidence or severity (Huber, 1978 and 1980; Graham and Webb, 1989) and potassium is of particular importance. Perrenoud (1990) reviewed almost 2450 literature references on this subject and concluded that the use of potassium (K) decreased the incidence of fungal diseases in 70% of the cases. Potassium alters the compatibility relationship of the host-parasite environment within the plant (Marschner, 1995). Sweeny et al. (2000) have found reduced incidence of diseases as a result of K fertilization in different crops. Increased incidence of powdery mildew in vines exhibiting inward leaf curling (symptoms of potassium deficiency) has been observed (Sharma et al., 2009). Leaf curling interferes in fungicide coverage thereby promoting the disease development. Crop loss in field grown vines due to downy mildew was also higher in potassium deficient grapevines (Sawant et al., 2010). Petiole samples exhibiting varying degree of inward leaf curling (potassium deficiency symptom) and powdery mildew incidence were sampled on 45th and 90th day after foundation pruning during 2009 and 2010, respectively from field grown vines in experimental plots of NRC for Grapes, Pune. In order to get wide range of nutrient contents, vines grown on different stock scion combinations were sampled and rated for disease severity. Nutrient composition of petioles was studied as per standard methods of analysis. All the vines were grown under uniform package of practices, including the fungicides applied for the control of powdery mildew. The 5th leaf position was selected for nutrient analysis on 45th day after pruning. Each sample was collected from a set of three vines. Severe powdery mildew incidence was observed on the basal leaves at 90th day after pruning. The leaves at 3rd to 7th nodes (middle representative) were selected for analysis from vines having severe disease incidence as well as those having fewer incidences. The basal 10 leaves on each shoot were rated for disease incidence (visually active fungal growth) on a scale of 0-4 (Fig.1). The data were analyzed by correlation and stepwise regression using SAS software. Data on micronutrients is not presented as they were not related to the disease incidence and the vines were sprayed with micronutrients. The effect of mineral nutrition on diseases has been determined either through observing the effects of fertilization on disease severity, comparing the mineral concentration in resistant and susceptible cultivars or correlating conditions influencing mineral availability with disease incidence or severity or combination of these factors. Since very little information is available on the relation of the nutritional status of the grapevines with powdery mildew incidence, relationship between nutritional status of the open field grown Thompson Seedless vines raised in different stock scion combinations and powdery mildew incidence at two different growth stages of grapevines was worked out. Results and discussion The data on nutrient contents and powdery mildew ratings are presented in Table 1 and 2 and correlation coefficients in Table 3. The nutrient contents particularly that of N, P and K varied greatly in different stock scion combinations. Nitrogen has been implicated in many studies to increase the disease incidence. Nitrogen exhibited positive but non-significant correlation with disease rating (r=0.3536) and (r=0.3056). Although there was strong and positive correlation between N:K ratio and disease rating during both the years, nitrogen content might not have played a direct role in increasing the disease rating in the present Complementary Copy Key words: Powdery mildew, nutritional status, grapevines, potassium, disease incidence Relationship of nutritional status of field grown Thompson Seedless grapevines with powdery mildew incidence Table 2. Nutrient status of vines and disease incidence 90th day after foundation pruning during 2010 Plot number N (%) P (%) K (%) Ca (%) Mg (%) Na (%) 1. 1.06 0.52 2.04 2.04 0.96 0.44 Cl Powdery (%) mildew rating 0.35 1.5 2. 1.13 0.33 2.55 2.17 0.86 0.73 0.35 1.2 3. 1.21 0.3 1.85 2.05 0.83 0.63 0.42 1.3 4. 0.85 0.48 2.35 2.15 0.92 1.15 0.7 1.3 5. 1.13 0.45 1.78 2.21 1.12 0.5 0.4 1.5 6. 0.96 0.35 2.65 2.13 0.95 0.8 0.35 1.3 7. 1.1 0.28 1.8 1.85 0.84 0.55 0.35 1.2 8. 1.03 0.52 2.16 2.06 1.02 1.03 0.42 1.2 9. 1.24 0.5 1.85 2.29 0.93 0.46 0.46 1.3 10. 1.13 0.3 1.95 1.95 0.91 0.72 0.32 1.2 Table 1. Nutrient content of vines and powdery mildew ratings on 45th day after foundation pruning during 2009 Plot N number (%) P (%) K (%) Ca (%) Mg (%) Na (%) 1. 0.78 0.27 0.71 0.63 0.97 0.18 Cl Powdery (%) mildew rating 0.11 1.5 2. 0.76 0.22 1.02 0.71 0.75 0.23 0.18 1.2 3. 0.63 0.25 0.73 0.77 0.88 0.23 0.25 1.5 4. 0.62 0.26 1.00 0.69 0.77 0.28 0.21 1.2 5. 0.90 0.26 0.66 0.65 1.00 0.18 0.11 3.0 6. 0.99 0.25 0.66 0.61 1.00 0.15 0.18 7. 0.89 0.25 0.64 0.68 0.96 0.23 8. 0.95 0.35 1.65 0.52 0.91 9. 0.78 0.33 1.53 0.56 0.83 10. 0.50 0.42 1.85 0.57 11. 0.62 0.40 1.63 12. 0.76 0.32 13. 0.74 14. 11. 1.15 0.35 1.69 1.89 0.95 0.56 0.4 1.2 12. 0.95 0.56 2.4 2.32 1.18 1.09 0.75 1.5 13. 1.18 0.23 1.28 1.85 0.80 0.65 0.4 3.2 14. 1.12 0.48 1.06 2.14 0.86 0.42 0.35 3.8 15. 1.12 0.2 1.17 2.05 0.63 0.75 0.35 3.6 16. 1.17 0.6 1.33 2.06 0.92 0.37 0.42 3.2 17. 1.21 0.28 1.02 1.85 1.02 0.65 0.46 3.2 18. 1.03 0.52 1.15 2.21 1.12 0.39 0.32 3.7 19. 1.13 0.22 1.1 2.15 1.13 0.7 0.42 3.2 20. 1.16 0.32 1.02 2.16 0.73 0.68 0.46 3.3 21. 1.24 0.26 1.16 2.06 0.94 0.68 0.32 3.2 Table 3. Correlation coefficient (r) between powdery mildew rating and petiole nutrient content Nutrient (%) 45th days after pruning 90th day after pruning N 0.3536 0.3056 P -0.4631* -0.1909 3.0 K -0.8170* -0.8750* 0.11 3.2 Ca 0.4125* -0.03878 0.33 0.50 1.0 Mg 0.4902* -0.1678 0.33 0.46 1.1 Na -0.5999* -0.3390 0.96 0.38 0.64 1.0 0.65 0.85 0.35 0.64 1.1 Cl -0.5837* -0.2211 0.78 0.64 1.17 0.16 0.18 2.1 0.30 0.57 0.70 0.95 0.19 0.21 2.1 0.62 0.32 0.60 0.72 0.89 0.20 0.21 2.0 15. 0.62 0.29 0.53 0.67 1.15 0.15 0.14 2.4 16. 0.74 0.34 0.75 0.68 0.95 0.19 0.11 2.3 17. 0.71 0.40 1.55 0.53 0.83 0.24 0.21 1.0 18. 0.66 0.40 1.52 0.55 0.85 0.25 0.21 1.0 19. 0.75 0.32 1.30 0.56 0.90 0.25 0.25 1.2 20. 0.72 0.35 1.60 0.53 0.78 0.20 0.21 1.0 21. 0.66 0.32 0.80 0.64 1.10 0.19 0.21 2.0 22. 0.71 0.34 0.75 0.64 0.96 0.24 0.14 2.2 23. 0.64 0.36 0.62 0.56 1.20 0.18 0.21 2.4 24. 0.64 0.32 0.58 0.62 1.02 0.21 0.18 2.3 25. 0.64 0.31 0.68 0.66 1.09 0.15 0.18 2.1 * Significant at P<0.05 vigorously growing vines in a vineyard and more vigorous shoots in a vine vineyards where the K applications are either inadequate or not made (Sharma et al., 2009a). Further leaves of vines having severe powdery mildew incidence also exhibited ‘shiny spots’ on leaves associated with potassium deficiency (Sharma et al., 2009b). Leaf curling interferes in fungicide coverage thereby promoting the disease development. Further shaded conditions are always favourable for spore germination and mycelial growth than sunny conditions (Willocquet et al., 1996). Increased sensitivity to powdery mildew incidence exhibiting leaf curling (potassium deficiency symptoms) has also been reported by Mundankar et al. (2008) and Sharma et al. (2009a). Nutrition alters the compatibility relationship of the host-parasite environment within the plant. However, K is especially critical in the production and transport of fungus inhibiting phenolic and Complementary Copy situation since its content was not high at 45th day (Table 1) as per the petiole nutrient standards (Sharma and Shikhamany, 2008). Further, at 90th day during 2010, nitrogen content in some of the healthy vines was higher than that in affected vines. Amongst different nutrients, potassium showed highest degree of significant and negative correlation with the disease rating (r=-0.8170*) followed by P (r=-0.4631*) at 45th day after pruning. At 90th day K again exhibited significant and negative correlation (r=-0.8750*) with disease rating (Table 3). Other nutrients did not exhibit significant correlation. Since more than one nutrient exhibited significant correlation at 45th day of sampling with disease rating stepwise regression analysis was carried out. Regression analysis also revealed the importance of potassium nutrition in powdery mildew incidence. N, P, K, Ca, Mg and Na when regressed together accounted for 82.7 % variation in disease incidence. Potassium alone accounted for 66.8 % variation in disease incidence (R2 = 0.6678) on 45th day of sampling. During the second year, N, P, K, Ca, Mg and Na when regressed together accounted for 85.7 % (R2 = 0.8573) variation in disease incidence. Potassium alone accounted for 76.6 % variation in disease incidence on 45th day of sampling. Potassium is one of the essential and major nutrients in viticulture involved in a variety of functions in grapevines. Its deficiency in grapes causes inward leaf curling. Inward leaf curling symptoms are commonly observed during the vegetative growth period in 115 116 Relationship of nutritional status of field grown Thompson Seedless grapevines with powdery mildew incidence Fig. 2. Severe leaf curling and higher powdery mildew incidence in unfertilized vines (RHS) compared to vines fertilized with potassium (LHS). flavonoid compounds and a shortage of K reduces the amount of the plants’ natural antifungal compounds at the site of infection (Marschner, 1995). Bowen et al. (1992) found no difference in Uncinula necator infection in water sprayed and K2HPO4 sprayed leaves of potted grapevines. However, their studies did not mention K and P content of the leaves. In their studies, the amount of K2HPO4 used in the spray was not enough to change the P and K content of the vines. However, in studies carried out by Saleh et al. (2007), vines fertilized with low NPK concentration were more susceptible to disease than those fertilized with high levels. Reuveni et al. (1993) observed that number of grape clusters infected by U. necator and the severity of infection were greater on vines fertilized with a low level of NPK (50% infection) than in plants fertilized with a high level (9.5% infection). During 2010, one set of Thompson Seedless vines (20 vines) was fertilized with recommended doses of K and also sprayed with K at fortnightly interval (Fig. 2) during the foundation pruning and the other set of vines was neither fertilized nor sprayed with potassium. Disease incidence was recorded on 90th day when the environmental conditions for powdery mildew were highly favourable. Unfertilized vines exhibited severe inward leaf curling symptoms and more incidence of powdery mildew as compared to fertilized vines (Fig. 2). The average petiole K content in vines having more disease incidence was lower (1.21%) than those having less incidence (1.95%). These results suggest that powdery mildew incidence is related to nutritional status of the vines. Amongst different nutrients, potassium was the most important nutrient whose deficiency increased the sensitivity of grapevines to powdery mildew disease. Thus maintaining the optimum level of potassium in grapevines should become a part of the integrated disease management strategy for Indian vineyards. References Bowen, P., J. Menzies, D. Ehret, L. Samuels and A.D.M. Glass, 1992. Soluble silicon sprays inhibit powdery mildew development on grape leaves. J. Amer. Soc. Hort. Sci., 117(6): 906-912. Graham, R.D. and M.J. Webb, 1991. Micronutrienis and disease resistance and tolerance in plants. In: Micronutrienis in Agriculture. R.M. Welch (ed). 2nd Edition. Soil Sci. Soc. America, Madison, WI. pp 329-370. Huber, D.M. 1978. Disturbed mineral nutrition. In: Plant pathology – An Advanced Treatise, Vol. 3: Academic Press, NY. p.163-181. Huber, D.M. 1980. The role of mineral nutrition in defence. In: Plant Pathology – An advanced Treatise, Vol. 5: Academic Press, NY. p. 381-406. Huber, D.M. and D.C. Arny, 1985. Interactions of potassium with plant disease. In: Potassium in Agriculture, R.D. Munson (ed). American Soc. Agronomy, Madison, WI. pp 467-488. Mundankar, K.Y., S.D. Sawant, Indu S. Sawant and J. Sharma, 2008. An expert system for the management of powdery mildew disease of grapes in India. Acta Hort., 785: 297-300. Marschner, H. 1995. Mineral Nutrition of Higher Plants. 2nd edition. Academic Press. Complementary Copy Fig. 1. Disease rating 1/4 (LHS), disease rating 2/4 (middle) and 4/4 (RHS) on 90th day Relationship of nutritional status of field grown Thompson Seedless grapevines with powdery mildew incidence Sharma, J., A.K. Upadhyay, S.D. Sawant and Indu S. Sawant, 2009b. Studies on shiny spot symptom development on leaves and its effect on fruitfulness, disease incidence and vine yield. Indian J. Hort., 66: 48-52 Sharma, J. and S.D. Shikhamany, 2008. Petiole nutrient standards for Thompson Seedless vines on Dog Ridge rootstock. Acta Hort.,785: 379-382. Sweeney, D.W., G.V. Granade, M.G. Eversmeyer and D.A. Whitney, 2000. Phosphorus, potassium, chloride, and fungicide effects on wheat yield and leaf rust severity. J. Plant Nutr., 23(9): 12671281. Willocquet, L., D. Colombet, M. Rougier, J. Fargues and M. Clerjeau, 1996. Effects of radiation, especially ultraviolet B, on conidial germination and mycelial growth of grape powdery mildew. European J. Plant Pathol., 102(5): 441-449. Received: February, 2012; Revised: July, 2012; Accepted: November, 2012 Complementary Copy Perrenoud, S. 1990. Potassium and plant health. In: IPI Research Topics No. 3, 2nd revised edition. Basel/Switzerland. Reuveni, M., A. Naor, R. Reuveni, M. Shimoni and B. Bravdo, 1993. The Influence of NPK fertilization rates on susceptibility to powdery mildew of field-grown winegrapes. Journal of Small Fruit and Viticulture, 2(1): 31-41. Saleh, M.M.S., N.E. Ashour, M.H. El-Sheikh and M.A.A. El- Nagaar, 2007. Foliar sprays of potassium dihydrogen phosphate and their impact on yield, quality and controlling powdery mildew disease of Thompson Seedless Grapevines. American Eurasian J. Agric. Environ. Sci., 2(2): 133-140. Sawant, Indu S., S.D. Sawant, Anuradha Upadhyay, J. Sharma, A.K. Upadhyay, D. Shetty and R. Bhirangi, 2010. Crop loss in grapes due to downy mildew infection on clusters at pre- and post-bloom stages under non-epiphytotic conditions. Indian J. Hort., 67(4): 425-432. Sharma, J., A.K. Upadhyay, Indu S. Sawant and S.D. Sawant, 2009a. Association of mineral nutrients with vein reddening and necrosis in Thompson Seedless grape. Indian J. Hort., 66: 154-162. 117 Journal Journal of Applied Horticulture, 14(2): 118-123, 2012 Appl Effe c t of grow ing m e dia on se e d ge r m inat ion a nd se e dling grow t h of pa pa ya (Ca ric a pa pa ya ) c v. ‘Re d La dy’ R.L. Bha rdw a j Krishi Vigyan Kendra-Sirohi (Rajasthan ), India. 307001. E-mail: [email protected], Abstract The effect of three types of media and three levels of cocopeat were studied under agronet house conditions on germination and development of papaya seedling in a Complete Randomized Design with nine treatment combinations. The results showed that the medium of vermicompost + sand + pond soil (1 : 1 :1) with 2 cm cocopeat on the top (T9) gave maximum speed of emergence (277.6 and 709.09), highest germination per cent (95.27 and 90.15), highest seed vigour (91.97 and 86.69), maximum germination index (7.15 and 7.22), germination value (17.33 and 33.83) and least time required for imbibtion (9.45 and 9.30 days) with minimum germination period (3.70 and 2.75 days), respectively in both years of experimentation. This medium was also found to be the best medium for the growth of papaya seedlings as it gave the highest parameters in terms of seedling height (23.43 cm and 22.67 cm), leaf area (349.33 cm2 and 329.20 cm2), number of leaves (10.02 and 9.67), stem girth (3.16 mm and 3.48 mm), number of roots (17.20 and 16.17), root length (10.20 cm and 9.67 cm), production of total biomass (5.02 g/plant and 4.77 g/plant) and least root/shoot ratio (0.22 and 0.20). This treatment also significantly reduced the seedling mortality and produced maximum healthy seedlings (92.23 % and 93.15%) in minimum days (35.33 and 35.15) with highest net profit (Rs. 3493.30/1000 seedling and Rs. 3448.00/ 1000 seedling) and B:C ratio (1.85 and 1.84) of seedlings, in both years (2008-09 and 2009-10), respectively. Introduction Use of suitable growing media or substrates is essential for production of quality horticultural crops. It directly affects the development and later maintenance of the extensive functional rooting system. A good growing medium would provide sufficient anchorage or support to the plant, serve as reservoir for nutrients and water, allow oxygen diffusion to the roots and permit gaseous exchange between the roots and atmosphere outside the root substrate (Abad et al., 2002; Argo, 1998a; Argo, 1998b; Bunt, 1988; Richards and Beardsell, 1986). Nursery potting media influence quality of seedlings produced (Agbo and Omaliko, 2006; Baiyeri, 2005; Sahin et al., 2005). The quality of seedling obtained from a nursery influences re-establishment in the field (Baiyeri, 2006) and the eventual productivity of an orchard (Baiyeri and Ndubizu, 1994). Papaya is an important fruit crop which is propagated by seeds only. Seed germination is affected by many factors, which include type of substrate used, environmental factors such as oxygen, water, temperature and for some plant species, light (Hartmann et al., 2001). The germination of seed of papaya (C. papaya) is frequently reported to be slow, erratic and incomplete (Chacko and Singh, 1966). Freshly harvested seed gave only 6 per cent germination (Koyamu, 1951). Red Lady is choicest variety of papaya grown due to hermaphrodite nature and prolonged shelf life of fruits but the seed cost of this variety is very high (Rs. 2.0 lakh/kg). The germination of papaya (C. papaya) cv. Red Lady seeds faces certain problems in germination and has high seedling mortality due to damping off disease in nursery stage. Initial mortality and incomplete germination is also one of the causes of reduced survival per cent of papaya plants. In heavy soil, without enough drainage, the development of root system is suppressed and plants are more susceptible to soil borne diseases (Beattie and White, 1992). The papaya seed is enclosed within a gelatinous sarcotesta (aril or outer seed coat which is formed from the outer integument). While this sarcotesta can prevent germination, dormancy is also observed in seeds from which the sarcotesta has been removed (Lange, 1961; Yahira, 1979). Growing media plays important role for seed germination. It not only acts as a growing place but also as a source of nutrient for plant growth. Media composition influences the quality of seedling (Wilson et al., 2001). Generally, media for fruit crop seedling are composed of soil, organic matter, pond soil and sand. The pond soil is usually used as a basic medium because it is cheapest and easy to procure. Supplementing the sand is aimed to make media more porous while the organic matter (FYM and vermicompost) is added so as to enrich adequate nutrients for the seedling. There is better rooting in manuare rather than conventional soil mix and less susceptibility of the seedling to soil borne pests and diseases (Akanbi et al., 2002). Cocopeat is an agricultural by-product obtained after the extraction of fiber from the coconut husk. It is considered as a good growing media component with acceptable pH, electrical conductivity and other chemical attributes (Abad et al., 2002). Cocopeat has good physical properties, high total pore space, high water content, low shrinkage, low bulk density and slow biodegradation (Evans et al., 1996; Prasad, 1997). Due to usually high initial level of potassium and sodium in cocopeat, the fertilization program should be adjusted carefully to plant requirements. Keeping in view the influence of media on germination and seedling growth of papaya, the present investigation was carried out to study the effect of different media viz., sand, pond soil, FYM, vermicompost and cocopeat on seed germination, seedling growth and vigour of papaya seedlings. Complementary Copy Key word: B: C ratio, cocopeat, plant growth, pond soil, seedling, vermicompost Effect of growing media on seed germination and seedling growth of papaya Seed material and treatment: Seed germination and seedling growth experiments of papaya were carried out at NHM Model nursery (average temperature, 32 + 10 0C and RH, 60-80%) of Krishi Vigyan Kendra-Sirohi (Rajasthan), India during two successive season from July to August 2009 and 2010. Experimental treatments comprised of nine treatment combinations consisting of different combination of growth media and cocopeat filling at the top of seedling polybags (10 x 12 cm) namely, T1 – sand + pond soil (1:1) without cocopeat, T2- sand + pond soil (1:1) with 1 cm cocopeat, T3- sand + pond soil (1:1) with 2 cm cocopeat, T4- FYM + sand + pond soil (1:1:1) without cocopeat, T5- FYM + sand + pond soil (1:1:1) with 1 cm cocopeat, T6- FYM + sand + pond soil (1:1:1) with 2 cm cocopeat, T7- vermicompost + sand + pond soil (1:1:1) without cocopeat, T8- vermicompost + sand + pond soil (1:1:1) with 1 cm cocopeat, T9- vermicompost + sand + pond soil (1:1:1) with 2 cm cocopeat. The seed sowing was done in month of July about 1 cm deep in different media as per treatments. The polybags were irrigated immediately after seed sowing and repeated every day till the final emergence. After the completion of germination the bags were irrigated once in 2 days. Experimental design and measured parameters: Experiment was conducted in Complete Randomized Design with three replications. Each treatment was composed of 100 polybags. All the observation on germination parameters were recorded at the time of germination and growth parameter at the time of transplanting (45 days after seed sowing) from 100 seeds for germination parameter and randomly selected 10 seedling for growth parameters. Data on germination was recorded from the first germination until no further germination at two days interval. The imbibtion period, number of days from sowing to commencement of germination, was recorded for all studied treatments. The speed of emergence (SE) was calculated according to Islam et al. (2009) using the following formula: Speed of emergence = No. of seedlings emerged 5 days after sowing x 100 No. of seedlings emerged 15 days after sowing The germination percentage was calculated as the percent of germinating seeds starting from the first germination to no further germination. Germination percentage was calculated by number of germinated seeds divided by the total number of seeds sown in polybags and multiplied by 100. The germination period was calculated as the difference between initial and final emergence (number of days) recorded. Seed vigour was calculated by total number of healthy seedling divided by the number of total seedlings and multiplied by 100. The germination index was calculated as described in the Association of Official Seed Analysis (1983) by the following formula: Germination index= Σ (GT/Tt) or Germination index = No. of germinating seeds Days of first count + . . . .+ No. of germinating seeds Days of final or last count The germination value (GV) was calculated according to Hossain et al. (2005) by the following formula: Germination value = (Σ DGs/N) X GP/10. Where, GP is the germination percentage at the end of experiment, DG is the daily germination speed obtained by dividing the cumulative germination percentage by the number of days since sowing, (Σ DGs) is the total germination obtained by adding every DGs value obtained from the daily counts, (N) is the total number of daily counts starting from the first germination and (10) is constant. Counting of number of leaves was done at the end of experiment when the true leaves have emerged. Stem girth was measured 1 cm from the base of the stem using vernier calipers. Plant height was measured from polybag top soil surface upto the highest leaf tip by straightening all leaves. Leaf area was calculated by the leaves traced on a graph paper. Number of roots, root length was measured by destructive method of uprooting the plants and taking measurement by standard method. Stem and root were weighed to record stem, root fresh weight, root/shoot ratio, and total weight per plant (g) was recorded at time of transplanting. Survival per cent (after transplanting in main field) was recorded by following formula: Survival percent = Total survival transplanted plants Total transplanted plants x 100 The net return was calculated by subtracting cost of each treatment from the gross return and benefit: cost ratio = Gross income/Cost of seedling production. All data was subjected to analysis of variance (ANOVA) to determine significant differences followed by Tukey’s test for the comparison of means at significant level of 5 per cent. Result and discussion The results showed that growing media and cocopeat had beneficial effect on seed germination and growth of papaya seedling. Seed germination parameters: Seed germination parameters of papaya (Carrica papaya) as affected by growing media and use of cocopeat are presented in Table 1. The treatment T9 was found to be best followed by T8 with regard to germination behaviour as these media have suitable physical properties and good water holding capacity that supports the germination of papaya seeds (Table 1 and Fig. 1). Coir dust when mixed with organic manure acts as the best media because coir dust has good physical characteristics (Garcia and Daverede, 1994) and also successfully tested as a growing medium in ornamentals (Van Holm, 1993). Germination started at the 9.45 days and 9.30 days after sowing in vermicompost medium with 2 cm cocopeat (T9) for both year of experimentation, respectively. Germination continued until the 25.72 days and 22.03 days from sowing thereafter further germination was not noticed in both year of experimentation, respectively. For both years of experimentation, the maximum speed of emergence (277.6 and 709.0), highest germination per cent (95.27 and 90.15), highest seed vigour (91.97 and 86.69), maximum germination index (7.15 and 7.22), germination value (17.33 and 33.83) and least time required for imbibtion (9.45 and 9.30 days) with minimum germination period (3.70 and 2.75 days) were obtained in vermicompost + sand + pond soil (1:1:1) with 2 cm filling with cocopeat of seedling polybags (T9) in both year of experimentation, respectively. The sand + pond soil (1:1) Complementary Copy Materials and methods 119 Effect of growing media on seed germination and seedling growth of papaya without cocopeat showed the poor results in most cases. The vermicompost medium with 2 cm cocopeat allowed increased values of germination parameters from the beginning to the end of experiment compared to other media combination in both years of experimentation. The reason for the best performance of pond soil and vermicompost was high organic matter content which increased the water and nutrient holding capacity of the medium, which improved the water utilization capacity of plant. Joiner and Nell (1982) found similar results in peat + perlite mixture for Aglaonema and Dieffenbachia. Vermicompost is reported to have bioactive principles which are considered to be beneficial for root growth and this has been hypothesized to result in greater root initiation, higher germination, increased biomass, enhanced growth and development (Bachman and Metzger, 2008) and also balanced composition of nutrients (Zaller, 2007). The well decomposed organic matter (vermicompost) may preserve soil humidity, increase nutrient content and improve soil structure which increase water absorption and maintains the cell turgidity, cell elongation and increase respiration at optimum level, leading to conditions favourable for seed sprouting. Vermicompost mixed with pond soil affects properties of soil, since organic matter acts as glue for soil aggregate and source of soil nutrient (Soepardi,1983). Vermicompost may improve soil aggregation leading to improved permeability and airflow in the polybags. Vermicompost and pond soil (due to high organic matter) may decrease fluctuation of soil temperature. Further, seed germination and root growth becomes easier to the particular depth so that plant grows well and may absorb more water and nutrient (Jo, 1990). Organic matter may also improve nutrient availability and improve phosphorus absorption (Karama and Manwan, 1990). All these factors were favourable for seed germination and ultimately increased seed germination per cent, speed of emergence, seed vigour, germination index, germination value and reduce imbibtion period. Combined application of vermicompost and cocopeat in the treatment T9 showed significant effect on germination, seedling growth and plant biomass probably due to the synergistic effect of both the factors in improving physical condition of the media and nutritional factors (Sahni et al., 2008). Seedling growth and development parameters: Data presented in Table (2 and 3) and Fig. 1 show that growth and development of papaya seedling was significantly affected by growing media and cocopeat. Significant differences were observed among the different treatments with regard to seedling growth characters and maximum number of leaves was observed in T9 treatment (10.02 and 9.67) which was at par with T6 treatment (9.20 and 9.00) respectively, in both years (Table 2). Maximum seedling girth (3.16 and 3.48 mm), highest seedling height (23.43 and 22.67 cm), largest leaf area (349.33 and 329.20 cm2), longest root length (10.20 and 9.67 cm) and highest fresh weight of plants (5.02 and 4.77 g) were recorded in T9 treatment in both years of experimentation, respectively. Similarly maximum number of roots per plant was also higher in T9 treatment (17.20 and 16.17) which was at par with T8 treatment (16.83 and 15.20). Highest fresh weight of shoot (4.12 and 3.98 g), fresh weight of roots (0.89 and 0.78 g) and least root/shoot ratio (0.22 and 0.20) was also reported in T9 treatment in both years of experimentation (200809 and 2009-10), respectively. Manure (vermicompost) provides adequate nutrients and enhances both the physical properties and the water holding capacity (Soegiman, 1982). The similar result was also reported by Supriyanto et al. (1990) working on orange seedling that media containing manure produced the growth Table 1. Effect of seedling growing media and cocopeat on the germination parameters of papaya Treatments T1 T2 T3 T4 T5 T6 T7 T8 T9 SEm+ CD at 5% Imbition period 2008-09 2009-10 16.67 14.67 14.60 12.60 12.35 11.75 16.45 14.25 13.55 13.62 11.75 11.42 14.30 13.38 11.13 12.12 9.45 9.30 0.377 0.618 1.117 1.829 Speed of Germination Germination Seed Germination Germination emergence (%) period vigour index value 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 129.51 125.22 61.70 57.67 9.05 7.35 52.80 57.67 2.00 2.73 1.66 2.21 133.12 205.99 69.14 66.50 7.35 6.60 62.72 65.60 2.81 3.46 3.20 3.79 166.74 285.68 79.36 81.30 6.50 5.25 73.78 77.69 3.70 4.97 5.64 8.02 152.02 156.21 69.45 72.13 7.90 6.65 62.08 67.30 2.94 3.53 2.53 4.20 188.06 366.19 79.70 83.40 5.65 4.10 73.18 80.33 4.23 4.78 5.59 9.84 206.08 445.04 84.65 86.65 4.72 3.17 80.22 85.39 5.30 5.60 9.01 15.92 231.47 273.54 80.33 74.90 6.95 5.35 73.92 73.60 3.86 3.51 4.44 5.59 257.91 603.16 90.13 85.00 4.55 3.60 84.90 81.62 5.69 6.29 10.35 18.64 277.65 709.09 95.27 90.15 3.70 2.75 91.97 86.69 7.15 7.22 17.33 33.83 5.874 12.833 1.830 2.173 0.264 0.265 1.804 1.853 0.224 0.136 0.278 0.278 17.385 37.979 5.416 6.432 0.783 0.786 5.338 5.484 0.665 0.404 0.822 0.834 Table 2. Effect of seedling growing media and cocopeat on the growth parameters of papaya Treatments Number of Stem girth Seedling height leaves (mm) (cm) 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 T1 4.00 3.07 0.96 1.28 7.20 9.33 T2 6.05 5.13 1.28 1.60 8.45 10.33 T3 7.11 8.25 1.92 2.24 10.45 12.33 T4 7.23 6.00 1.28 1.80 9.05 9.17 T5 8.25 8.15 2.24 2.44 12.20 12.17 T6 9.20 9.00 2.87 2.76 17.45 17.17 T7 8.13 6.63 1.89 2.21 16.43 11.67 T8 9.02 7.60 2.53 2.84 20.18 19.67 T9 10.02 9.67 3.16 3.48 23.43 22.67 SEm+ 0.318 0.386 0.083 0.119 0.422 0.448 CD at 5% 0.941 1.142 0.245 0.353 1.251 1.326 Leaf area Number of Root length Fresh weight of (cm2) roots (cm) plants (g) 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 18.30 15.14 5.00 6.60 3.01 3.90 0.60 0.63 33.35 30.30 9.45 10.35 3.55 4.21 0.80 0.79 51.23 50.37 12.03 12.30 6.02 5.45 1.00 1.02 41.25 59.45 7.10 7.83 5.25 4.90 0.72 0.65 84.31 99.00 11.13 9.69 7.37 6.47 1.20 0.80 134.00 134.30 12.17 12.35 7.60 6.94 1.65 1.23 126.17 149.12 13.80 12.00 8.15 7.19 2.67 2.17 216.25 249.25 16.83 15.20 9.45 8.58 3.82 3.30 349.17 329.20 17.20 16.17 10.20 9.67 5.02 4.77 3.457 3.457 0.581 0.536 0.218 0.196 0.046 0.051 10.232 10.232 1.721 1.587 0.647 0.580 0.137 0.152 Complementary Copy 120 Table 3. Effect of seedling growing media and cocopeat on the growth parameters, Treatments Fresh weight Fresh weight of Survival Root /Shoot of shoot root per cent ratio (g) (g) 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 2008-09 2009-10 0.39 0.39 0.21 0.23 77.30 78.12 0.55 0.59 T1 T2 0.53 0.51 0.27 0.27 80.37 82.14 0.50 0.54 T3 0.69 0.68 0.31 0.34 84.35 86.00 0.45 0.50 T4 0.54 0.47 0.18 0.18 81.80 82.60 0.33 0.37 T5 0.95 0.61 0.27 0.19 84.88 86.65 0.28 0.31 T6 1.27 0.96 0.35 0.27 86.86 90.61 0.27 0.28 T7 2.12 1.74 0.50 0.43 87.20 84.30 0.24 0.25 T8 3.11 2.77 0.71 0.60 90.25 89.35 0.23 0.22 T9 4.12 3.98 0.89 0.78 92.23 93.15 0.22 0.20 SEm+ 0.057 0.057 0.016 0.016 1.633 1.570 0.009 0.009 CD at 5% 0.170 1.170 0.048 0.048 4.835 4.649 0.029 0.028 and roots better than those containing sawdust and rice hulls. Purbiati et al. (1994) proved that soil + manure (1:1) was the best medium for the growth components of salacca cv. Pondoh and Bali. Erwin (1998) also reported that in case of L. longiflorum forcing media such as cocopeat and rice hulls have produced equal or superior crops compared to many existing commercial media. Combined application of vermicompost and cocopeat in the treatment T9 showed significant effect on seedling growth parameters and plant biomass probably due to the synergistic effect of both the factors in improving the physical conditions of the media and nutritional factors (Sahni et al., 2008). Thus based on the results of this study, it can be concluded that the treatment T9 (Vermicompost: pond soil: Sand 1:1:1 with 2 cm cocopeat) showed better water holding capacity, favourable pH, increased accumulation of nutrients which helped in better nutrient availability to the growing plants and hence supporting enhanced seed germination and seedling growth compared to the other treatments. This result is akin to the findings of Campos Mota et al. (2009) and Abirami et al. (2010) who suggested that since coir dust is low in nutrients when mixed with vermicompost provides a better growth medium for plant establishment. Air filled porosity (AFP), easily available water (EAW) and aeration of vermicompost and FYM were not at the recommended level which in turn limit the root growth and lowered the water holding capacity. Therefore, the medium with vermicompost and cocopeat is more suitable than vermicompost alone because of the better physical properties and enhanced nutrient level. Treatment,T9 was also helpful in reducing damping off disease in seedling due to proper aeration in root zone of the seedling and produced highest survival per cent of seedling (92.23 and 93.15 %, respectively) which was at par with T8 treatment (90.25 and 93.15 %, respectively). Because of the better physical properties and enhanced nutrient level in T9 treatment growth of seedling was rapid and minimum days required for gaining transplanting size (35.33 and 35.15 days) were at par with T6 (37.62 days) in second year (2009-10) of experimentation (Table 3 and Fig. 1). Vermicompost with cocopeat may improve soil porosity, water content, drainage pores, soil permeability and water availability, whereas weight of soil may decrease. This may develop soil aggregation, and improves permeability and air flow in the soil. This type of condition sharply reduced damping off disease in nursery stage and provided support to fast growth of the seedling due to availability of better nutrition with water and air in root zone of the seedling. Ultimately the seedling gained transplanting survival per cent, Days required for transplanting size of seedling 2008-09 2009-10 47.35 45.30 45.30 42.35 42.50 41.20 43.60 40.60 40.50 39.50 38.58 37.62 40.25 40.30 38.30 38.35 35.33 35.15 1.006 1.006 2.979 2.979 net return and B: Net return (Rs./1000 seedlings) 2008-09 2009-10 805.00 755.0 1005.0 955.0 1905.0 1855.0 1093.0 1043.0 1893.0 1843.0 2393.0 2343.0 2193.3 2148.0 2993.3 2948.0 3493.3 3448.0 60.324 45.057 178.53 133.34 121 C ratio of papaya B:C ratio 2008-09 2009-10 1.17 1.15 1.19 1.18 1.35 1.34 1.20 1.19 1.34 1.33 1.41 1.40 1.65 1.64 1.78 1.77 1.85 1.84 0.066 0.066 0.198 0.198 size very soon in this treatment combination then other treatments. It seems that good physical and biological conditions in cocopeat and vermicompost had positive effect on root development, which is helpful in increased survival per cent of seedling in main field after transplanting. Beneficial effects of cocopeat on root system was observed on nutmeg seedling (Abirami et al., 2010), Osteospermum cuttings (Nowak, 2004), salvia, viola (Pickering, 1997) and Impatiens (Smith, 1995). Application of vermicompost: pond soil: sand (1:1:1) with 2 cm cocopeat media (T9) for preparation of papaya seedling proved profitable and showed maximum net return (Rs. 3493.30/1000 seedlings and Rs. 3448.00/1000 seedlings) and benefit: cost ratio (1.85 and 1.84) for the first and second year of experimentation, respectively due to higher germination percent and survival percent obtained (Table 1 and 3). This treatment was significantly superior to rest of the treatments during both year but benefit: cost ratio was at par with T8 treatment. At the end, it can be concluded that vermicompost and cocopeat could be used successfully in production of papaya seedlings due to suitable physical, chemical and biological properties. Vermicompost+ pond soil+ sand (1:1:1) with 2 cm cocopeat was considered the best media as the germination, seedling growth and development parameters were higher as compared to other media. Therefore, it can be suggested that vermicompost, pond soil and sand with cocopeat should be used as growing media for higher germination per cent, quick seedling growth of papaya and more profit by sale of seedlings. References Abad, M., P. Noguera, R. Puchades, A. Maquieira and V. Noguera, 2002. Physico-chemical and chemical properties of some coconut dusts for use as a peat substitute for containerized ornamental plants. Bioresearch Technology, 82: 241-245. Abirami, K., J. Rema, P.A. Mathew, V. Srinivasan, and S. Hamza, 2010. Effect of different propagation media on seed germination, seedling growth and vigour of nutmeg (Myristica fragrans Houtt.). Journal of Medicinal Plants Research, 4: 2054-2058. Agbo, C.U. and C.M. Omaliko, 2006. Initiation and growth of shoots of Gongronema latifolia Benth stem cuttings in different rooting media. African Journal of Biotechnology, 5: 425-428. Akanbi, B.W., A.O. Togun, and R.A. Baiyewn, 2002. Suitability of plant residue compost as nursery medium for some tropical fruit tree seedlings. Moor Journal of Agriculture Research, 3: 24-29. A.O.S.A. 1983. Seed vigour testing handbook. Contribution No. 32 to handbook on seed testing. Association of Official Seed Analysis. Complementary Copy Effect of growing media on seed germination and seedling growth of papaya Effect of growing media on seed germination and seedling growth of papaya Complementary Copy 122 Fig. 1. Effect of growing media and cocopeat on germination (1a), survival (1b), plant height (1c), root growth & leaf area (1d) of papaya. In Fig. 1a, 1b, 1c results only under T3, T6, T9 are depicted. Argo, W.R. 1998a. Root medium physical properties. HortTechnology, 8: 481-485. Argo, W.R. 1998b. Root medium chemical properties. 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Media and Mixes for Container- Grown Plants. 1st Edn., Springer, London, ISBN:10: 0046350160. Campos Mota, L., U. Van Meeteren and C. Blok, 2009. Comparison of physical properties of vermicompost from paper mill sludge and green compost as substitutes for peat based potting media. Acta Horticulture, 819: 227-234. Chako, E.K. and R.N. Singh, 1966. Studies on the longevity of Papaya, Phalsa, Guava and Mango seeds. Pro. International Seed Testing Association, 36: 147-158. Erwin, J.E. 1998. Easter lily production. Minn. Commune Flower Grower Bulletin, 47: 1-10. Evans, M.R., S. Konduru and R.H. Stamps, 1996. Source variation in physical and chemical properties of coconut coir dust. HortiSciences, 31: 965-967. Garcia, M. and C. Daverede, 1994. Dust from coir fibres: New substrate for soilless culture. PHM Revue Horticole, 348: 7-12. Hartmann, H.T., D.E. Kester, F.T. Davies and R.L. Geneve, 2001. 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Penggunaan pupuk organic pada tanaman pangan. Makalah pada Lokakarya Nasional Efisiensi Penggunaan Pupuk. Cisarua Bogor, 12-13 November 1990, p. 44. Koyamu, K. 1951. A preliminary note on the germination of Papaya seed. Madras Agriculture Journal, 38: 348-349. Lange, A.H. 1961. Effect of Sarcotesta on the germination of Papaya (C. papaya). Bot. Gazette, 122: 305-311. Nowak, J. 2004. The effect of rooting media and CO2 enrichment, P – nutrition and micorrhizal inoculation on rooting and growth of Osteospermum. Acta Horticulture, 644: 589-593. Pickering, J.S. 1997. An alternative to peat. The Garden, 122: 428429. Prasad, M. 1997. Physical, chemical and biological properties of coir dust. Acta Horticulture, 450: 21-29. 123 Purbiati, T., Q.D. Ernawanto and S.R. Soemarsono, 1994. Pengaruh Komposisi Media Tumbuh and Ukuran Pot terhadap Keberhasilan and Pertumbuhan Tunas Anakan Salak yang Diperbanyak secara Vegetatif Penel. Horticulture, 6: 1-12. Richards, D.M.L. and D.V. Beardsell, 1986. The influence of particlesize distribution in pinebark: sand: Brown coal potting mixes on water supply, aeration and plant growth. Sciences of Horticulture, 29: 1-14. Sahin, U., S. Ors, S. Ercisli, O. Anapali and A. Esitken, 2005. Effect of pumice amendment on physical soil properties and strawberry plant growth. Journal of Central European Agriculture, 6: 361-366. Sahni, S., B.K. Sarma, D.P. Singh, H.B. Singh and K.P. Singh, 2008. Vermicompost enhances performance of plant growth promoting rhizobacteria in Cicer arietinum rhizosphere against Sclerotium rolfsii. Crop Protection, 27: 369-376. Smith, C. 1995. Coir: a viable alternative to peat for potting. Horticulturist, 4: 25-28. Soegiman, M. 1982. Ilmu tanah. Terjemahan dari. The Nature and Properties of Soils. Buckman and Brady. Bhatara Karya Aksara. Jakarta. 788 hal. Soepardi, G. 1983. Sifat dan cirri tanah. Department Ilmu-ilmu Tanah, IPB. Bogor. Supriyanto, A., Q.D. Ernawanto and L. Setiono, 1990. Media Tumbuh Pembibitan Jeruk. Penelitian Hortikultura, 5: 1-8. Van Holm, L. 1993. Coir as a growing medium. 7th floricultural symposium, Oct. 11, Institute of fundamental studies: Hantana, Kandy, Srilanka, p. 1-23. Wilson, S.B., P.J. Stoffella and D.A. Graetz, 2001. Use of compost as a media amendment for containerized production of two subtropical perennials. Journal of Environment and Horticulture, 19: 37-42. Yahira, M. 1979. Effects of seed pretreatments on the promotion of germination in Papaya (C. Papaya L.). Memoirs of the Faculty of Agriculture, Kago Shima University, 15: 49-54. Zaller, J.G. 2007. Vermicompost as a substitute for peat in potting media: Effects on germination, Biomass allocation, Yields and fruit quality of three tomato varieties. Scientia Hort., 112: 191-199. Received: November, 2011; Revised: March, 2012; Accepted: July, 2012 Complementary Copy Effect of growing media on seed germination and seedling growth of papaya Journal Appl Journal of Applied Horticulture, 14(2): 124-128, 2012 Effe c t of pla nt bio-re gulat ors on physic o-che m ic a l cha ra c t e rist ic s of t hre e a pple va rie t ie s during a m bie nt st ora ge B.L. At t ri* , H a re K rishna , B. Da s, N . Ahm e d a nd Ak hile sh Kum a r Central Institute of Temperate Horticulture, Regional Station, Mukteshwar – 263 138, Uttarakhand, India. *E-mail: [email protected] Abstract For extending the shelf life, a study was carried out on the effect of bio-regulators viz., salicylic acid and Ca-EDTA on three apple varieties viz., Fanny, Golden Delicious and Vance Delicious. The selected fruits were dipped for 30 minutes in aqueous solution of salicylic acid @ 200 ppm, Ca-EDTA @ 0.4% ppm and control (distilled water dip). The treated fruits were stored in CFB boxes at ambient temperature (18-20oC) for 60 days. During storage, the effect of bio-regulators on various physico-chemical characteristics such as TSS, acidity, ascorbic acid, sugars and antioxidants of apple fruits were studied at 10 days interval. The results revealed that the fruits treated with bio-regulators had significantly better retention of firmness and low PLW (12.10, 12.80 and 13.69%) as compared to control (20.26, 18.75 and 19.35%) during storage for 60 days. The TSS, acidity, ascorbic acid, sugars and antioxidant contents in the treated fruits were stable, whereas in untreated ones the conversion rate was faster. During storage, salicylic acid and Ca-EDTA slowed down respiration rate resulting better shelf life of apple. The treated fruits of Golden Delicious had a shelf life of 60 days as compared to 40 days in control. The study revealed that the shelf life of the apple fruits could be increased with better physico-chemical characteristics using bio-regulators like salicylic acid and Ca-EDTA. Introduction In India, the North-West Himalayan Region (NWHR) comprising Jammu & Kashmir, Himachal Pradesh and Uttarakhand, have mountainous, low and mid hills with valleys, which are suitable for a number of temperate fruit crops. Uttarakhand is very rich in the plant diversity because of its congenial climatic and geographic conditions suitable for cultivating a number of temperate fruits like apple, pear, peach, plum, apricot, walnut etc. Apple (Malus domestica Bork.) is the largest cultivated fruit in the region covering an area of 32,050 ha with a production of 1.30 lakh tonnes (Anon., 2009). Besides local consumption, the apples are also preferred in the distant markets of the country due to the crisp texture and pleasant flavour. However, due to poor postharvest facilities available in the hilly regions and lack of proper transport, the fruit do not reach distant market in good condition, thus resulting in high postharvest losses (Sharma and Singh, 2010). The costly and improved techniques such as cold storage, controlled atmospheric storage and modified atmospheric packaging are used in developed countries to check these losses. Under Indian conditions, simple and inexpensive techniques such as postharvest chemical treatments offer better prospects for reducing the post harvest losses of fruits (Randhawa et al., 1980). In Uttarakhand, a number of apple varieties including Delicious group as well as spur types are being grown. However, only a few of them had a better shelf life because of hard fruit skin whereas others lack in postharvest shelf life restricting their entry to distant markets and ensuring availability for a longer duration. Further, harvesting of fruits in a limited period create a glut in the market because of which the growers don’t get remunerative returns for their produce. The apple in Uttarakhand matures early as compared to Himachal Pradesh and Jammu & Kashmir, when both temperature and humidity in the air are quite high. The onset of monsoon and poor roads in the state also restrict the fast transport of the harvested fruits to the distant markets thereby causing losses to the growers. Bio-regulators like salicylic acid and calcium play a significant role in enhancing the postharvest shelf life of a number of fruits (Issar et al., 2011). Salicylic acid (C6H4 (OH) COOH), which is also known as 2-hydroxybenzenecarboxcylic acid, is an organic acid and functions as plant hormone. Several studies show that it has beneficial effects on the shelf and storage life of several temperate fruits. Calcium is thought to be the most important mineral element determining the fruit quality. It helps in maintaining fruit firmness and decreases the incidence of physiological disorders (Conway et al., 1998). Although, it is one of the most significant fruit tree nutrients but it is very difficult to accumulate in enough concentrations in the fruit because of its less mobility in the plant. Its accumulation is more during the fruit development but tends to decrease in the latter phase (Cline and Hanson, 1991). In several studies, it has been demonstrated that Ca improves fruit quality and shelf life of apples. Hence, the present investigations were carried out to study the effect of salicylic acid and Ca-EDTA on the physico-chemical characters of three apple cultivars during storage at ambient conditions. Materials and methods The present investigation was undertaken during 2009 at Central Institute of Temperate Horticulture, Regional Station, Mukteshwar, situated at 2,200 m above mean sea level in Nainital district of Uttarakhand, India. Complementary Copy Key words: Apple varieties, salicylic acid, Ca-EDTA, physico-chemical characters, ambient storage, anti-oxidants Plant bio-regulators and physico-chemical characteristics of apple varieties Treatments: The total treatments were 9 as T1= Fanny 200 ppm Salicylic acid, T2= Fanny 0.4% Ca-EDTA, T3= Fanny control, T4= Golden Delicious 200 ppm Salicylic acid, T5=Golden Delicious 0.4% Ca-EDTA, T 6= Golden Delicious control, T7= Vance Delicious 200 ppm Salicylic acid, T8= Vance Delicious 0.4% Ca-EDTA, T9= Vance Delicious control. The concentration of the bio-regulators was selected on the basis of earlier work carried out in other temperate fruits. 30 fruits of each variety were dipped for 30 minutes in aqueous solutions of bio-regulators. The dipping duration was based on the earlier work. The treated fruits were subjected to air drying in shade at room temperature followed by storage in the corrugated fibre board boxes (CFB) at ambient temperature (18-20oC) having relative humidity of 85-90%. The storage study was conducted for 60 days. Physico-chemical characters: The physiological loss in weight (%PLW) of the marked 5 fruits in each treatment was recorded at regular interval (10 days) on initial weight basis. The firmness of the fruits was observed with fruit pressure tester model FT 327 and the results were expressed as lb/in2. The fruits as well as extracted juice was subjected to physical characters analysis viz., weight (g), length (mm), breadth (mm), juice (%), and pomace (%) by following standard methods (Ranganna, 1997). Similarly, the total soluble solids (TSS oBrix) of the juice were recorded with hand refractometer (Erma, Japan) corrected at 20oC, acidity (%) by titrating a known volume of aliquot against N/10 NaOH using phenolphthalein as indicator as described by Ranganna (1997). The reducing and total sugars (%) of the juice were estimated as per the methods of AOAC (1990). The ascorbic acid contents (mg/100g) were recorded by titrating a known volume of juice with metaphosphoric acid against 2,6 dicholophenol indophenol dye (Ranganna, 1997) and were expressed as mg/100g juice. Antioxidant activity: Antioxidant activity was measured by following the method of Apak et al. (2004), which measures the copper (II) or cupric ion reducing ability at 450 nm after 30 min. of sample reagent mixing. To a test tube were added 1 mL each of copper (II) chloride solution (10-2M, 1.705g in 1 L distilled water), Neocuproine solution of 7.5 x 10-3M (1.562 g in 1 L ethanol), and ammonium acetate buffer, pH 7 (19.27 g in 250mL distilled water) solutions. Antioxidant sample (or standard) solution (x mL) and H2O (1.1- x mL) were added to the initial mixture so as to make the final volume 4.1 mL. The tubes were stoppered and after one hour, the absorbance at 450 nm was recorded against a reagent blank. The antioxidant activity was expressed as m mol Trolox® per litre, or mM TE. Statistical analysis: The experiment was undertaken in completely randomized design, replicated four times. The data on physico-chemical characteristics of different varieties of apple recorded during storage at ambient temperature were analysed as per method described by Panse and Sukhatme (2000). Results and discussion All the physico-chemical characteristics of the three varieties of apple varied significantly (Table 1). Fruit firmness was maximum (15.0 lb/in2) in Golden Delicious followed by Vance Delicious. Fruit weight was highest in Vance Delicious (177.76 g) and lowest in Fanny (78.00g). The recovery of extracted juice was maximum (62.82%) in Fanny followed by Vance Delicious (55.64%). The highest TSS of the juice (15.0oB) was observed in Fanny and lowest (13.0oB) in Golden Delicious. The ascorbic acid content in the juice was maximum (13.6 mg/100g) in Fanny and minimum (5.0 mg/100g) in Vance Delicious. The total antioxidants in the extracted juice of different varieties were recorded and found highest in Fanny (10.51 mMTE/L) and lowest in Vance Delicious (6.89 mMTE/L). The variation in the physico-chemical characters in different varieties of apple may be attributed to their genetic characterstics (Farooqui et al., 2004). Table 1. Physico-chemical characters of different apple varieties (mean±S.E.) Characters Pressure (lb/in2) Weight (g) Length (mm) Breadth (mm) Juice (%) Pomace (%) TSS (oB) Acidity (%) Ascorbic acid (mg/100g) Reducing sugars (%) Total sugars (%) Total antioxidants mMTE/L TSS acid ratio Fanny 9.20±0.057 78.00±1.154 47.60±0.346 57.67±0.387 62.82±0.242 37.18±0.242 15.0±0.115 0.67±0.0057 13.6±0.196 6.25±0.057 7.69±0.103 10.510.008 Golden Delicious 15.00±0.115 125.00±2.886 58.50±0.288 65.56±0.323 54.59±0.225 45.41±0.225 13.0±0.057 0.50±0.0011 7.0±0.016 5.88±0.046 7.14±0.034 8.61±0.008 Vance Delicious 12.60±0.230 177.76±1.154 66.45±0.144 76.25±0.144 55.64±0.138 44.36±0.138 14.0±0.230 0.40±0.0011 5.0±0.057 7.14±0.023 8.00±0.057 6.89±0.008 22.38±0.375 25.89±0.178 34.82±0.386 The physiological loss in weight (% PLW) of different varieties of apple was recorded at different intervals and a significant difference was found among different treatments (Table 2). Irrespective of the variety, the lowest PLW (%) was recorded in the fruits treated with Ca-EDTA (Ca) followed by salicylic acid (SA) and control. Among the varieties, the maximum PLW was recorded in Fanny (13.69,15.46 and 20.26%) and minimum in Golden Delicious (12.10, 13.32 and 18.75%) in the fruits treated with Ca, SA and control, respectively after storage for 60 days at ambient conditions. The minimum PLW in the fruits treated with Ca and SA may be attributed to the restrictions in the respiration process thereby decreasing the loss in weight during storage. Han et al. (2003) had reported similar findings in peaches after exogenous application of salicylic acid. Similarly, Kumar et al. (2005) reported significant effect of Ca(NO3)2 in maintaining the physico-chemical characters of aonla during storage. The firmness value of the fruits, irrespective of the variety and treatments declined significantly during storage period of 60 days. The maximum loss in firmness was recorded in the fruits under control in each variety whereas minimum in the fruits treated with Ca (Table 3). Among the varieties, the highest firmness loss was recorded in Golden Delicious (43.83%) and lowest in Fanny (37.50%) after 60 days of storage (Fig. 1). The maximum retention of the firmness in the fruits treated with Ca may be due to creating a barrier for the loss of water and checking the breakdown of insoluble protopectin thereby maintaining the firmness better as compared to other treatments. The exogenous application of salicylic acid inhibited the respiration rate and delayed the ethylene production peak of ripening peaches at Complementary Copy Raw material: The matured and uniform fruits of Fanny, Golden Delicious and Vance Delicious varieties were procured from the orchard of the Regional Station. After sorting out, the uniform matured fruits were selected on the basis of water dip method. 125 Plant bio-regulators and physico-chemical characteristics of apple varieties Table 2. Physiological loss in weight (%PLW) of different varieties of apple during storage Treatments Storage period (Days) 0 10 20 30 40 50 60 T1 0.0 1.92 5.35 8.74 11.10 13.79 15.46 T2 0.0 1.27 4.74 7.19 9.10 11.78 13.69 T3 0.0 3.84 7.47 11.30 14.66 17.51 20.26 T4 0.0 1.45 4.70 7.87 10.35 11.85 13.32 T5 0.0 1.02 3.92 6.40 8.30 10.58 12.10 T6 0.0 2.75 6.72 9.65 12.68 15.67 18.75 T7 0.0 1.56 5.10 8.26 10.86 12.46 14.38 T8 0.0 1.15 4.24 6.90 8.90 11.10 12.80 T9 0.0 3.08 7.18 10.95 13.20 16.75 19.35 CD 0.05 0.03 0.03 0.06 0.05 0.04 0.05 Table 3. Effect of salicylic acid and calcium on firmness (lb/in2) of different varieties of apple during storage Treatments T1 T2 T3 T4 T5 T6 T7 T8 T9 CD 0.05 0 9.20 9.40 9.60 14.80 15.00 14.60 12.40 12.60 12.80 0.10 10 9.00 9.20 9.00 14.20 14.60 13.40 12.00 12.20 12.00 0.09 Storage period (Days) 20 30 40 8.80 8.20 7.80 9.00 8.40 8.00 8.70 7.80 7.00 13.60 12.80 12.00 14.00 13.40 12.60 12.00 11.00 10.20 11.40 10.80 10.00 11.80 11.20 10.60 11.20 10.00 9.40 0.16 0.17 0.14 50 7.20 7.40 6.60 11.20 12.00 9.40 9.40 10.00 8.80 0.16 60 6.80 7.00 6.00 10.40 11.20 8.20 8.60 8.20 7.90 0.19 ambient storage conditions (Han et al., 2003). The plum fruits treated with Ca(NO3)2 (0.5, 1.0 and 2.0%) and stored at low temperature revealed that the fruits treated with 2% Ca(NO3)2 recorded the highest firmness throughout the storage as compared to control signifying the utility of calcium nitrate during transit and marketing (Mahajan et al., 2008). Similarly, the PLW (%) was also less in fruits treated with calcium nitrate revealing that calcium is helpful in maintaining fruit firmness and tissue rigidity thereby checking the moisture loss from the fruit surface. The retention of higher firmness with postharvest application of calcium nitrate may probably be due to the role of calcium in maintaining the cellular organization and regulating the enzyme activities (Jones and Lent, 1967). A significant increase in the total soluble solids of the fruits in different treatments of apple was recorded upto 30 days followed by decrease during storage. The highest increase followed by decrease was found in the fruits under control and lowest in Ca (Table 4). The minimum increase in the TSS in the fruits treated with Ca may be due to the restriction in the physiological process and respiration during storage thereby maintaining the total soluble solids better as compared to the fruits under control. The results are in conformity with those reported by Mahajan et al. (2004) in Asian pear. During storage, the acidity (%) of the fruits in different treatments of apple was found to reduce significantly. It was observed that the acidity reduced faster in the control as compared to SA and Table 4. Effect of salicylic acid and calcium on total soluble solids (TSS o B) of different varieties of apple during storage Treatments T1 T2 T3 T4 T5 T6 T7 T8 T9 CD 0.05 0 15.0 15.2 15.0 13.0 13.2 13.0 14.0 14.0 14.2 0.1 10 15.2 15.2 15.4 13.2 13.4 13.4 14.2 14.2 14.4 0.1 Storage period (Days) 20 30 40 15.4 15.8 15.2 15.6 16.0 15.6 15.8 16.2 15.0 13.4 13.8 13.4 13.6 14.0 13.8 13.8 14.4 13.0 14.4 14.8 14.2 14.6 15.0 14.6 15.0 15.4 13.8 0.2 0.2 0.2 50 14.6 14.8 14.0 13.0 13.4 12.6 13.6 14.0 13.0 0.2 60 14.0 14.2 13.2 12.6 13.0 12.0 13.2 13.6 11.8 0.2 Table 5. Effect of bio-regulators on acidity (%) of different varieties of apple during storage Treatments T1 T2 T3 T4 T5 T6 T7 T8 T9 CD 0.05 0 0.67 0.67 0.67 0.50 0.50 0.50 0.40 0.40 0.40 0.01 10 0.60 0.59 0.57 0.48 0.47 0.44 0.37 0.34 0.32 0.01 Storage period (Days) 20 30 40 0.59 0.54 0.44 0.57 0.50 0.40 0.54 0.44 0.37 0.47 0.40 0.37 0.44 0.37 0.34 0.40 0.34 0.32 0.34 0.32 0.30 0.32 0.30 0.28 0.30 0.28 0.23 0.01 0.01 0.01 50 0.37 0.34 0.27 0.35 0.32 0.28 0.28 0.27 0.22 0.01 60 0.34 0.27 0.24 0.34 0.30 0.27 0.27 0.23 0.20 0.01 Table 6. Ascorbic acid (mg/100g) of different varieties of apple as affected by salicylic acid and calcium during storage Treatments Fig.1. Per cent loss in firmness of apple in different treatments during storage T1 T2 T3 T4 T5 T6 T7 T8 T9 CD 0.05 0 13.6 13.6 13.7 7.0 7.0 7.0 5.0 5.0 5.0 0.1 10 12.5 12.0 11.5 6.5 6.3 6.0 4.8 4.6 4.4 0.1 Storage period (Days) 20 30 40 12.0 11.4 11.0 11.5 11.1 10.5 11.0 10.7 10.0 6.3 6.1 6.0 6.1 6.0 5.8 5.8 5.4 5.2 4.7 4.4 4.2 4.5 4.2 4.1 4.2 4.1 4.0 0.1 0.1 0.1 50 10.6 10.1 9.5 5.7 5.1 5.1 4.1 4.0 3.9 0.1 60 10.0 9.6 9.0 5.2 5.0 4.9 4.1 4.0 3.8 0.1 Complementary Copy 126 Plant bio-regulators and physico-chemical characteristics of apple varieties The ascorbic acid content of the different varieties of apple treated with SA and Ca was recorded during storage and it was found that the fruits treated with the bio-regulators retained significantly higher ascorbic acid content than fruits dipped in water (control), irrespective of the variety (Table 6). As SA and Ca had restricted the process of ripening the retention of the ascorbic acid was attributed due to slow rate of ripening and oxidation. The postharvest dip in calcium chloride, calcium lactate and calcium propionate helped to increase significantly the shelf life with reduced ethylene production in peach stored in cold storage for 4 weeks (Manganarisa et al., 2007). Ascorbic acid breakdown is accelerated by amino acids and in the presence of amine as it is oxidized to dehydroascorbic acid that is the reactive intermediate in the pathway to furfural and brown pigment production (Sharma et al., 2006). The reducing and total sugars (%) in the fruits of different treatments of apple under study showed significantly increasing trend irrespective of the variety upto 30 days followed by reduction. The increase was recorded faster in the fruits of control as compared to SA and Ca (Table 7 & 8). The increase in the reducing and total sugars was probably due to the hydrolysis of starch as well as other complex carbohydrates to simpler sugars. Further, the reduction in the sugars during storage may be due to the utilization of the same in the respiration process. The results are in conformity with those reported by Knee and Smith (1989) in apple fruits. On complete hydrolysis of starch, no further Table 7. Effect of salicylic acid and calcium on reducing sugars (%) of different varieties of apple during storage Treatments Storage period (Days) 0 10 20 30 40 50 60 T1 6.25 6.32 6.36 6.49 6.28 6.17 6.02 T2 6.25 6.36 6.41 6.57 6.25 6.09 5.88 T3 6.25 6.41 6.57 6.84 6.17 6.02 5.74 T4 5.88 5.95 6.09 6.25 6.25 5.95 5.74 T5 5.88 6.09 6.25 6.41 6.17 5.88 5.61 T6 5.88 6.25 6.57 6.84 6.09 5.74 5.49 T7 7.14 7.24 7.40 7.57 7.29 7.19 6.94 T8 7.14 7.35 7.51 7.69 7.24 7.14 6.84 T9 7.14 7.46 7.69 7.93 7.19 7.04 5.37 CD 0.05 0.04 0.05 0.03 0.08 0.05 0.04 0.03 Table 8. Effect of bio-regulators on total sugars (%) of different varieties of apple during storage Treatments Storage period (Days) T1 T2 T3 T4 T5 T6 T7 T8 T9 CD 0.05 0 7.69 7.70 7.68 7.14 7.15 7.14 8.00 8.05 8.00 0.04 10 7.78 7.84 8.00 7.24 7.29 7.40 8.06 8.13 8.26 0.05 20 8.00 8.16 8.34 7.35 7.40 7.54 8.19 8.34 8.47 0.05 30 8.26 8.51 8.69 7.54 7.69 8.00 8.34 8.51 8.69 0.04 40 8.13 8.00 7.90 7.40 7.35 7.24 8.16 8.09 8.00 0.05 50 8.00 7.93 7.78 7.29 7.24 7.19 8.00 7.90 7.78 0.06 60 7.90 7.78 7.63 7.19 7.14 7.09 7.93 7.84 7.69 0.04 Table 9. Total antioxidants (mMTE/L) of different varieties of apple affected by salicylic acid and calcium during storage Treatments T1 T2 T3 T4 T5 T6 T7 T8 T9 CD 0.05 0 10.52 10.52 10.52 8.61 8.62 8.61 6.89 6.89 6.90 0.01 10 10.49 10.45 10.39 8.59 8.54 8.49 6.85 6.81 6.72 0.01 Storage period (Days) 20 30 40 10.37 10.22 10.01 10.31 10.15 9.94 10.27 10.11 9.82 8.47 8.34 8.16 8.40 8.27 8.07 8.38 8.22 7.97 6.71 6.56 6.38 6.66 6.50 6.32 6.60 6.45 6.26 0.01 0.01 0.01 50 9.79 9.69 9.56 7.93 7.85 7.79 6.19 6.07 5.95 0.01 60 9.51 9.39 9.26 7.73 7.61 7.49 6.01 5.89 5.77 0.01 increase in TSS and sugars occurs and consequently a decline in these parameters is predictable as they are the primary substrates in respiration (Wills et al., 1980). The total antioxidants in fruits of all the varieties of apple under the study exhibited a significant decrease during storage. The reduction was found highest in control in each variety and lowest in fruits treated with SA. Among the varieties, the maximum (9.51, 9.39 and 9.26 mMTE/L) antioxidants were recorded in Fanny whereas minimum (6.01, 5.89 and 5.77 mMTE/L) in Vance Delicious after storage for 60 days (Table 9). The reduction in the antioxidant contents may be attributed to the over ripening of the fruit thereby breakdown of these contents as well as oxidation during respiration. The shelf life of different cultivars of peach was found to increase significantly by post-harvest application of SA and Ca as compared to control (Attri et al., 2010). From the present study, it can be concluded that the apple varieties like Fanny, Golden Delicious and Vance Delicious can be stored for more than 60 days at ambient temperature after treating with either salicylic acid or Ca. There was minimum loss in physicochemical parameters and PLW with better firmness in Golden Delicious as compared to other two varieties. References Anonymous, 2009. Statistical Database (2008-09) on area and production of fruits and vegetables. Directorate of Horticulture and Food Processing, Chaubattia, Ranikhet (Uttarakhand). A.O.A.C. 1990. Official Methods of Analysis. Association of Official Analytical Chemists. 13th Edn, Washington, DC. Apak, R., K. Guclu, M. Ozyurek and S.E. Karademir, 2004. Novel total antioxidant capacity index for dietary polyphenols and vitamin C and E, using their cupric ion reducing capability in presence of neocuproine: CUPRAC method. J. Agr. Fd. Chem., 52: 79707981. Attri, B.L., H. Krishna, B. Das, J.K. Ranjan, Pragya and N. Ahmed, 2010. Effect of bio-regulators on the shelf life of different cultivars of peach (Prunus persica L. Batsch.). Natl. Symp. on Conservation Hort. (21-23 March, 2010, Dehradun, India), Book of Abstracts, pp. 262. Banik, D., R.S. Dutta, S.K. Ghosh and S.K. Sen, 1988. Studies on extension of storage life of sapota (Achras sapota L.). Indian J. Hort., 45: 241-48. Cline, J.A. and E.J. Hanson, 1991. Calcium accumulation in Delicious apple fruit. J. Plant Nutr., 14(11): 1213-1222. Conway, W.S., C.E. Sams and K.D. Hickey, 1998. Pre and post harvest calcium treatment on apple fruit and its effect on quality. J. Agr. Fd. Chem., 46(7): 2452-2457. Complementary Copy Ca treated fruits (Table 5). As the respiration was checked due to exogenous application of bio-regulators the acidity was found to remain under control. The results are in line with those reported by Banik et al. (1988) where it has been mentioned that retention of high acidity means slow ripening. Further, the decline in acidity during storage might account for use of organic acids in respiration (Ulrich, 1974). 127 Plant bio-regulators and physico-chemical characteristics of apple varieties Farooqui, K.D., K.M. Bhat and A. Mehmood, 2004. Effect of different levels of fertilization on quality and production of apple cultivars. Progressive Hort., 36(2): 216-220. Han, T., W.L. Li and X. Ge, 2003. Effect of exogenous salicylic acid on post harvest physiology of peaches. Acta Hort., 628: 583-589. Issar, K., M.C. Nautiyal, S.K. Sharma and T.S. Bisht, 2011. Effect of chemicals, GA treatment and packaging on shelf life and quality of apple. J. Hill Agr., 2(1): 63-73. Jones, R.C.W. and O.R .Lent, 1967. The role of calcium in plants. Botl. Rev., 33: 407-26. Knee, M. and S.M. Smith, 1989. Variation in quality of apple fruits stored after harvest on different dates. J. Hort. Sci., 64: 413-419. Kumar, S., A. Kumar, M.J. Baig and B.K. Chaubey, 2005. Effect of calcium on physico-chemical changes in aonla (Emblica officinalis Gaertn), Indian J. Hort., 62(4): 324-326. Mahajan, B.V.C., A.S. Dhatt and W.S. Dhillon, 2004. Effect of prestorage treatments on the quality and storage of Asian pear. Indian J. Hort., 61(3): 342-344. Mahajan, B.V.C., J.S. Randhawa, H. Kaur and A.S. Dhatt, 2008. Effect of post harvest application of calcium nitrate and gibberellic acid on the storage of plum. Indian J. Hort., 65(1): 94-96. Manganarisa, G.A., M.Vasilakakisa, G. Diamantidisa and I. Mignani, 2007. The effect of post harvest calcium application on tissue calcium concentration, quality attributes, incidence of flesh browning and cell wall physicochemical aspects of peach fruits. Fd. Chem., 100 (4): 1385-1392. Panse,V.G. and P.V. Sukhatme, 2000. Statistical Methods for Agricultural Workers. ICAR Pub., New Delhi. Randhawa, J.S., B.S. Dhillon, S.S. Sandhu and J.S. Bhullar, 1980. Effect of post harvest application of GA3, cycocel and CaCl2 on storage behaviour of Le Conte pear. J. Res. Punjab Agr. Uni., 17: 363-365. Ranganna, S. 1997. Handbook of Analysis and Quality Control for Fruit and vegetable Products. 2nd Edn. Tata McGraw Hill Pub. Co. Ltd., New Delhi. Sharma, S.K., B.B.L. Kaushal and P.C. Sharma, 2006. Reduction of non-enzymatic browning of lemon juice concentrates by removal of reaction substrates. J. Fd. Sci. Technol., 43(1): 83-88. Sharma, R.R. and D. Singh, 2010. Effect of different packaging materials on shelf-life and quality of apple during storage. Indian J. Hort., 67(1): 94-101. Ulrich, R. 1974. Organic acids. In: Biochemistry of Fruits and Their Products. A.C. Hulme (ed.). Academic Press, New York, pp. 89118. Wills, R.B.H., P.A. Cambridge and K.J. Scott, 1980. Use of flesh firmness and other objective tests to determine consumer acceptability of delicious apples. Austral. J. Exp. Agri. Ani. Husb., 20: 252-56. Received: November, 2011; Revised: March, 2012; Accepted: July, 2012 Complementary Copy 128 Journal Journal of Applied Horticulture, 14(2): 129-133, 2012 Appl I nfl ue nc e of biofe r t ilize rs on pla nt grow t h, fruit yie ld, nut rit ion a nd rhizosphe re m ic robia l a c t ivit y of pom e gra nat e (Punic a gra na t um L.) c v. K a ndha ri K a buli M uza ffa r M ir* a nd Som Dev Sha r m a Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (H.P), India-173230. *E-mail: [email protected] Abstract The present study represents the positive response of biofertilizers in pomegranate cuttings followed by their transplantation in field conditions. Nursery and field experiments were carried out to assess the effectiveness of selected N2-fixing bacteria, phosphate solubilizing bacteria and AM fungi alone or in combination, on the growth and biomass production of Punica granatum. In both experiments, the combined treatment of Azotobacter chroococcum + Glomus mosseae was found to be the most effective. Besides enhancing the rhizosphere microbial activity and concentration of various metabolites and nutrients, these bioinoculants helped in better establishment of pomegranate plants under field conditions. A significant improvement in the plant height, plant canopy, pruned material and fruit yield was evident in 6-year-old pomegranate plants in field conditions. In view of the above results, use of biofertilizer technology may be adopted for the establishment and development of other horticultural plant species in rainfed agroecosystem. Introduction Pomegranate (Punica granatum L.) is an economically important commercial fruit plant species belonging to family Punicaceae. The plant is drought tolerant, winter hardy and can thrive well under rainfed conditions. Pomegranate is a good source of protein, carbohydrate, minerals, antioxidants, vitamins A, B and C, also useful in controlling diarrhea, hyperacidity, tuberculosis, leprosy, abdominal pain and fever. Pomegranate juice contains antioxidants such as soluble polyphenols, tannins, anthocyanins and antiatherosclerotic properties (Michel et al., 2005), and can be used in the treatment of cancer and chronic inflammation (Ephraim and Robert, 2007). Commercial pomegranate orchards are found in Indian rainfed areas, which is characterized by nutrient-deficient soils, low organic matter, irregular distribution of rainfall and generally experience water deficit during plant growth period (Panwar and Tarafdar, 2006). Vegetation of rainfed areas is often hindered by the lack of resident microflora, which acts as both source and sinks for essential plant nutrients and is fundamental to the transformation of various nutrients. Production of horticultural crops has undergone significant changes in recent years due to development of innovative technologies including integrated nutrient management practices involving biofertilizers, which include phosphate-solubilizing bacteria (PSBs), symbiotic and non-symbiotic N2-fixing bacteria and arbuscular mycorrhizal (AM) fungi. The use of biofertilizers in enhancing plant growth and yield has gained momentum in recent years because of higher cost and hazardous effect of chemical fertilizers. Nitrogen-fixing bacteria and arbuscular mycorrhizal fungi were found to enhance the growth and production of various fruit plants significantly (Khanezadeh et al., 1995), besides improving the microbiological activity in the rhizosphere (Kohler et al., 2007). Though there are many reports on the effect of different biofertilizers on various fruit plants, no information is available on usefulness of these biofertilizers with respect to pomegranate. Hence, an attempt has been made to examine the effect of biofertilizers namely Azotobacter chroococcum (AC), phosphate-solubilizing bacteria (PSB), Glomus fasciculatum (GF), and Glomus mosseae (GM) on growth and nutrient uptake as well as rhizosphere microbial activity of pomegranate under field conditions. Materials and methods The field experiment was conducted on pomegranate cv. ‘Kandhari Kabuli’ during 2008-11. The experimental farm is located at 30° 50’ 45” latitude and 77° 88’ 33” longitude at an elevation of 1320 m above mean sea level, representing mid hill zone of state. The climate of the area is typically sub-temperate. The annual rainfall ranges between 800-1300 mm. The orchard soil was sandy in texture with pH 6.55, 0.59 dS/m electrical conductivity and 0.61 % organic carbon content. Water holding capacity, bulk density and porosity of surface soil at 15 cm depth were 31.65 %, 1.30 % and 48.50 %, respectively. The initial available N, P and K contents of the soil were 309.85, 11.75 and 342.82 kg/ha, respectively. DTPA extractable micro-nutrients viz., zinc (Zn), manganese (Mn), iron (Fe), copper (Cu) and boron (B) were 1.97, 45.72, 57.24, 2.47 and 0.70 ppm, respectively. Strains of Azotobacter (a free living non-symbiotic N2-fixing bacterium) were isolated using N-free semisolid malate medium (Day and Dobereiner, 1976) and Jensen’s N-free agar medium (Jensen, 1954), respectively, from the roots and rhizosphere soil of field-grown pomegranate. The identification of strains was carried out by Indian Agriculture Research Institute (IARI), New Delhi (India) as PSB and A. chroococcum. The pure cultures of these strains were maintained in Microbiology Department, University of Horticulture and Forestry, Solan, (H.P.), using above-mentioned media. After 3 days of growth, the cells were Complementary Copy Key words: Pomegranate (Punica granatum L.), azotobacter, mycorrhizal fungi, PSB, soil enzymes. Influence of biofertilizers on growth, yield, nutrition and rhizosphere microbial activity of pomegranate centrifuged, washed twice in sterile distilled water and suspended in 0.15 M phosphate buffer at pH 7.0. Ten milliliters of cell suspension having 108 cell mL-1 was used as inoculums for both bacterial types at the time of planting pomegranate cuttings. Spores of AM fungi were extracted from the rhizosphere soils of field grown pomegranate plants by wet sieving and decanting technique (Gerdeman and Nicolson, 1964). Total number of spores was estimated by the method of Gaur and Adholeya (1994) and spore densities were expressed as the number of spores per 50 g of soil. Taxonomic identification of AM spores up to species level was based on spore size, spore colour, wall layers and hyphal attachment and was made using the identification manual of Schenck and Perez (1990) and the description provided by the International Collection of Vesicular and Arbuscular Mycorrhizal Fungi (http://invam.wvu.edu). The AM fungi were identified as G. fasciculatum, G. margarita and G. mosseae. The pure cultures were maintained on pearl millet/wheat plant roots under sterile condition. Ten grams of soil including root bits containing about 10-12 viable AM fungal propagules per gram soil were used as inoculum and spread as a thin layer 2 cm below soil surface in polythene bag (30 × 10 cm) containing 900 g substrate (soil:farm yard manure:pond silt, in the proportion of 3:1:1 by volume). In case of control treatment, similar amount of inoculum was sterilized in autoclave and added to polythene bag in same manner. Semi-hard wood cuttings of pomegranate (5-month-old cuttings) were kept in 250 ppm indole butyric acid solution for 12 h and then planted in polybags one each. The experiment was laid out in a complete randomized design consisting of eight treatments with 20 replications during the years 2008-2011. Dual inoculation of A. chroococcum + G. mosseae, A. chroococcum + G. fasciculatum and A. chroococcum + PSB, were selected based on the data obtained on various combinations of AM fungi, nitrogen-fixing bacteria and phosphate solubilizing bacteria under sterile condition during our preliminary study. The plants were watered on alternate days to field capacity. After sampling rhizosphere soil along with root fragments of 3-month-old seedlings grown in polythene bags, 10 plants from each treatment were harvested and leaf area (leaf area meter, C203, USA) and total chlorophyll (Arnon, 1949) were measured. Total phenols, reducing sugars and amino nitrogen in the leaves were analyzed from the alcohol extracts (Mahadevan et al., 1965). Shoot along with leaves were dried at 65 °C till constant weight and dry weight were recorded. The percent root colonization was determined by root slide technique (Read et al., 1976) after clearing 1 cm root segment with KOH and staining with trypan blue. Dehydrogenase activity, a measure of microbial activity, was assayed by the method of Tabatabi (1982). The soil samples were incubated with 2,3,5-triphenyl tetrazolium chloride, and production of triphenyl formazone was determined spectrophotomitrically. Hydrolysis of fluorescein diacetate (FDA) was determined by the standard procedure of Schnurer and Rosswell (1982) and the fluorescein released was quantified spectrophotomitrically. For this, soil sample (0.1 g) was placed in plastic tubes and 10 mL sterile potassium buffer (pH 7.6, 60 mM) was added to it. The reaction started after adding fluorescein diacetate (1 mg mL-1 in acetone). Tubes were sealed and kept in an incubator at 37 °C four 4 h. After incubation, 10 mL acetone was added to stop the reaction and after centrifugation (3200 rpm), supernatant’s optical density was determined at 490 nm. Total nitrogen-fixing potential (nitrogenase activity) of the soil was determined by incubating 50 mg of soil in 7 mL test tube containing 3 mL nitrogen-free semisolid malate medium for 48 h at 30 °C. The cotton plugs were replaced with suba seals and 10% of air was replaced with C2H2 and later ethylene (C2H4) produced was estimated by an AIMIL-Nucon Gas chromatograph fitted with Porapak-N column (2 × 0.003 m) using N2 as carrier gas at a flow rate of 25 mL min-1. Nitrogenase (N2-ase) activity was expressed as n mol C2H4 produced per hour (Rao and Venkateswarlu, 1982). For activity of alkaline phosphatase in the rhizosphere soil, the p-nitrophenyl phosphate was used as a substrate in a borex-NaOH (pH 9.4) buffer (Tabatabi and Bremner, 1969). Dry powder of shoot was used for analyzing total nitrogen (by microkjeldhal method), phosphorus (by vanado-molybdo phosphoric yellow colour method), potassium (by flame photometer), calcium and magnesium (by titrimetry employing disodium salt of EDTA) after di/tri acid digestion (Jakson, 1967). Estimates of copper, zinc, manganese and iron were made by using atomic absorption spectrophotometer (AAS 4141). Out of the remaining seedlings, 30 healthy plants of each treatment were transplanted at research field of Department of Fruit Science, UHF, Solan during January 2009 in rows with 6.0 m inter and intra row spacing. For this purpose, pits with depth and diameter of 0.6 m were dug and filled with 30 kg of the same soil mixture used for the plantlets production added with 100 g endosulfan dust (4%). The plants were irrigated at fortnightly intervals. Immature fruits along with few leaves and young branches were removed during the first season (November-February, 2009). After every winter season pruning was done for which weak and dead limbs and basal suckers were removed to give proper shape to plants. Pruning is encouraged for the growth of new spurs in each season and it helps the plant to sustain more fruit weight (Panwar et al., 1994). Pruned material (weak and dead shoot portion along with leaves) weights were recorded after drying in a hot-air oven to a constant weight and ground to a fine powder. Plant canopy was calculated using the formula πr2 in which radius mean was measured from all the directions of crown. Plant height, canopy, pruned material and fruit yield were recorded yearly for next 3 years (2009, 20010 and 2011). Results Sprouting of pomegranate occurred in 6-9 days in the biofertilizers inoculated cuttings while it took 8-10 days longer (data not given) in non-inoculated controls, resulting in a higher number of branches (Table 1). However, significant differences were not observed in number of branches between various biofertilizers used. A significant enhancement (27.4-57.6%) was observed in leaf area. The increase was maximum in dual inoculated seedlings with A. chroococcum + G. mosseae followed by A. chroococcum + G. fasciculatum. Biofertilizer inoculation enhanced shoot dry weight by 16-36%. The inoculation effect varied among treatments with the maximum in dual inoculation treatment and minimum with control. Inoculation resulted in a significantly higher total chlorophyll content as well as accumulation of reducing sugars, total phenols and amino nitrogen in 4 months old inoculated plants (Table 2). Total chlorophyll was observed Complementary Copy 130 Influence of biofertilizers on growth, yield, nutrition and rhizosphere microbial activity of pomegranate AC Branches (number plant-1) 4 ± 0.54 Leaf area (cm2 plant-1) 289.9 ± 0.57 Shoot dry wt. (g plant-1) 1.63 ± 0.012 GF 9 ± 0.36 241.2 ± 0.67 1.60 ± 0.022 GM 9 ± 0.56 264.4 ± 0.43 1.61 ± 0.097 PSB 9 ± 0.57 261.8 ± 0.22 1.52 ± 0.023 AC+GF 12 ± 0.43 291.3 ± 0.57 1.90 ± 0.066 AC+GM 12 ± 0.58 293.7 ± 0.57 1.92 ± 0.076 AC+PSB 12 ± 0.21 290.1 ± 0.57 1.72 ± 0.034 Control 4 ± 0.34 180.4 ± 0.44 1.39 ± 0.006 Treatments Table 2. Effect on inoculation with different biofertilizers on chlorophyll content and various metabolites (mg g-1 fresh weight) in pomegranate after 4 months Treatments AC Total Total Amino Reducing chlorophyll phenols nitrogen sugars 5.60 ± 0.017 1.95 ± 0.009 3.06 ± 0.029 3.19 ± 0.45 GF 5.12 ± 0.007 1.90 ± 0.076 3.02 ± 0.045 3.15 ± 0.78 GM 5.45 ± 0.023 1.91 ± 0.064 3.05 ± 0.027 3.17 ± 0.35 PSB 5.10 ± 0.058 1.89 ± 0.029 3.00 ± 0.023 3.14 ± 0.93 AC+GF 6.12 ± 0.078 2.11 ± 0.073 3.54 ± 0.08 3.74 ± 0.018 AC+GM 6.25 ± 0.099 2.14 ± 0.059 3.57 ± 0.45 3.75 ± 0.065 AC+PSB 6.0 ± 0.008 2.08 ± 0.065 3.51 ± 0.78 3.71 ± 0.010 Control 5.05 ± 0.016 1.78 ± 0.064 2.68 ± 0.085 2.85 ± 0.019 AC=A. chroococcum, GF=G. fasciculatum, GM=G. mosseae, PSB= Phosphate solubilizing bacteria. ± represents standard error of means. highest in dual inoculated seedlings with A. chroococcum + G. mosseae followed by A. chroococcum + G. fasciculatum. A similar trend was found in total phenols, amino nitrogen and reducing sugars contents. Four months after inoculation, AM fungi spore population and percent root colonization by AM fungi was enhanced by 103.57-183.92% and 267.77-444.45%, respectively over the control (Table 3). Uninoculated control also showed infection by mycorrhiza which could be attributed to the presence of native AM fungi in the soil. Dual inoculation resulted in maximum mycorrhizal root infection. Soil inoculation with biofertilizers significantly enhanced the activities of dehydrogenase, alkaline phosphatase and nitrogenase (N2-ase) and hydrolysis of fluorescein diacetate in rhizosphere soils of pomegranate compared to that of uninoculated plants (Table 3). Uptake of various nutrients was significantly higher in plants upon inoculation with various beneficial microorganisms (Table 4). In general, dual inoculation led to maximum uptake of N, P, K and micronutrients in pomegranate seedlings. In case of N, dual inoculation with A. chroococcum + G. mosseae followed by A. chroococcum + G. fasciculatum treatment and in case of P, dual inoculation with A. chroococcum + PSB treatments, respectively recorded higher uptake of nutrients. K, Cu, Zn, Mn and Fe were found in significantly higher concentration in dual inoculation treatment with A. chroococcum + G. mosseae followed by A. chroococcum + PSB treatments, however, significant variations were noticed in efficiency of different biofertilizers. Among nitrogen fixers and AM fungi, A. chroococcum and G. mosseae, respectively, Table 3. Effect of inoculation with biofertilizers on AM fungal spore density, percent mycorrhizal root colonization, rhizosphere enzyme activity and hydrolysis of FDA Percentage root Dehydrogenase Nitrogenase (n Alkaline phosphatase FDA-hydrolysis Treatments AM spores colonization (%) (p kat g-1 soil) mol C2H4 h-1) (n kat100 g-1 soil) (p kat g-1 soil) (50 g-1 soil) AC 236 ± 2.72 72 ± 1.59 8.26 ± 0.023 392 ± 1.19 13.2 ± 0.86 2231 ± 3.18 GF 232 ± 2.57 70 ± 1.35 8.06 ± 0.053 389 ± 1.23 13.6 ± 0.26 2223 ± 3.14 GM 233 ± 2.52 71± 1.29 8.21 ± 0.022 390 ± 1.13 13.5 ± 0.29 2291 ± 3.19 PSB 228 ± 2.72 68 ± 1.45 8.01 ± 0.065 387 ± 1.16 14.9 ± 0.98 2211 ± 3.18 AC+GF 315 ± 6.64 96 ± 2.55 9.15 ± 0.021 410.5 ± 2.37 16.9 ± 0.147 2710 ± 2.39 AC+GM 318 ± 6.56 98 ± 2.79 9.68 ± 0.086 412.5 ± 2.49 16.5 ± 0.145 2772 ± 2.53 AC+PSB 313± 6.69 96 ± 2.32 9.25 ± 0.028 411.5 ± 2.35 16.8 ± 0.167 2715 ± 2.53 Control 112 ± 1.63 18 ± 0.49 6.25 ± 0.078 217 ± 1.78 9.6 ± 0.132 1695 ± 2.68 ± represents standard error of means Table 4. Nutrient uptake (mg plant-1) by pomegranate plants as influenced by inoculation with various biofertilizers after 4 months Treatments AC N (%) 23.7 ± 0.12(1.39) P (%) 3.4 ± 0.12(0.21) K Cu Zn Mn (%) (ppm) (ppm) (ppm) 18.1 ± 0.06(1.10) 0.026 ± 0.001(16.5) 0.15 ± 0.02(91.1) 0.26 ± 0.01(157) Fe (ppm) 0.22 ± 0.01(135) GF 22.0 ± 0.06(1.37) 4.83 ± 0.09(0.30) 18.6 ± 0.09(1.16) 0.030 ± 0.001(19.1) 0.14 ± 0.02(91.3) 0.25 ± 0.01(158.4) 0.24 ± 0.01(150) GM 22.5 ± 0.11(1.38) 5.57 ± 0.12(0.34) 20.3 ± 0.09(1.24) 0.027 ± 0.001(17.4) 0.15 ± 0.02(95.1) 0.26 ± 0.01(160.6) 0.26 ± 0.01(157) PSB 22.8 ± 0.13(1.38) 3.07 ± 0.12(0.20) 18.6 ± 0.09(1.16) 0.026 ± 0.001(16.5) 0.14 ± 0.02(91.3) 0.26 ± 0.01(157) 0.23 ± 0.01(139) AC+GF 26.5 ± 0.29(1.42) 6.1 ± 0.19(0.32) 22.4 ± 0.02(1.20) 0.036 ± 0.001(18.6) 0.16 ± 0.01(89.2) 0.28 ± 0.04(156.4) 0.29 ± 0.02(160) AC+GM 27.0 ± 0.29(1.43) 6.4 ± 0.15(0.34) 22.8 ± 0.06(1.21) 0.037 ± 0.001(19.6) 0.17 ± 0.01(90.3) 0.29 ± 0.02(158.6) 0.30 ± 0.02(161) AC+PSB 26.5 ± 0.29(1.42) 7.4 ± 0.15(0.36) 22.6± 0.54(1.21) 0.036 ± 0.001(18.6) 0.16 ± 0.01(89.2) 0.29 ± 0.02(156.2) 0.31 ± 0.01(164) Control 18.9 ± 0.23(1.36) 2.5 ± 0.09(0.18) 13.2 ± 0.18(0.95) 0.022 ± 0.001(16.3) 0.12 ± 0.01(86.9) 0.21 ± 0.02(152.4) 0.15 ± 0.02(106) ± represents standard error of means. Complementary Copy Table 1. Effect of inoculation with different biofertilizers on growth of pomegranate after 4 months 131 Influence of biofertilizers on growth, yield, nutrition and rhizosphere microbial activity of pomegranate were found to enhance maximum uptake of aforesaid nutrients as compared to control. The results of field study revealed that inoculated plants resulted in a significant enhancement in plant height and fruit yield (Fig. 1) with a maximum increase in dual inoculation with A. chroococcum + G. mosseae treatment. Enhanced plant height, canopy, pruned plant material and fruit yield were recorded in first year (2008-2009) and almost similar trend was observed afterwards (2009-2011). Further appraisal of field study data indicates that A. chroococcum + G. mosseae enhanced all field study parameters among N2-fixing bacteria and AM fungi, respectively. Plant height Fruit yield Fig.1. Effect of inoculation with various biofertilizers on plant height and fruit yield of pomegranate grown in field conditions. AC=A. chroococcum, GF=G. fasciculatum, GM=G. mosseae, PSB= Phosphate solubilizing bacteria, Cont=Control Discussion During the present study, sprouting of pomegranate occurred in 7-10 days in the biofertilizers inoculated cuttings while it took 8-10 days longer (data not given) in non-inoculated controls. It may be due to the action of plant growth regulators mainly indole butyric acid secreted by both nitrogen fixers and AM fungi (Luis et al., 2003). Occurrence of maximum branching and enhanced rooting in 2-year-old trifoliate orange (Poncirus trifoliate L.) cutting with AM fungi inoculation (Yao et al., 2009) has also been reported. Increased leaf area and shoot dry weight upon inoculation were observed in this study. Similar observations of enhanced leaf area and shoot dry weight have been made in different fruit plants upon inoculation with AM fungi (Khanezadeh et al., 1995), PSB (Karlidag et al., 2007) and Azotobacter + G. fasciculatum (Sharma et al., 2008). During the present study it was observed that dual inoculation with A. chroococcum + G. mosseae had resulted in a higher biomass production compared to that of uninoculated or single inoculation treatment. Sharma et al. (2009) reported that root inoculation of citrus seedlings with AM fungi and A. chroococcum could produce large and better plants. The higher vegetative growth (seedling’s height, diameter and leaf area) of AMF treated plants might be due to the growth promoting effect of fungus and Azotobacter that improved P and N availability and thereby causing higher protein synthesis resulting in improved morphological growth. A significantly higher total chlorophyll content as well as higher accumulation of various metabolites (reducing sugar, total phenol and amino nitrogen) might have resulted from enhanced plant growth and biomass production (Kohler et al., 2007). These observations are in conformity with those from Aseri et al. (2008) who reported higher chlorophyll content and photosynthesis in pomegranate upon inoculation with N2-fixing bacteria. Mathur and Vyas (2000) reported enhanced synthesis of reducing sugars, free amino acids, soluble protein and chlorophyll in Ziziphus mauritiana seedlings upon inoculation with AM fungi. The increase observed in dehydrogenase, alkaline phosphatase, nitrogenase and hydrolysis of fluorescein diacetate may be mainly due to increase in the rhizosphere microbial population as a consequence of the inoculation treatments (Skujins, 1973) and (Aseri et al., 2008]). The enhanced phosphatase activity may help the plant to mobilize P and thereby increase the biomass production (Tarafdar and Gharu, 2006). A significant enhancement in total nitrogen-fixing potential as reflected by increased nitrogenase activity in rhizosphere soils was observed. The measurement of these enzymatic activities can provide an early indication of changes in soil fertility, since they are related to mineralization of important nutrient elements as N, P and C (Ceccanti et al., 1994). The degree of root colonization and sporulation in rhizosphere soils varied significantly among different treatments. The degree of mycorrhizal colonization and sporulation was higher in dual inoculation with A. chroococcum + G. mosseae as compared to single inoculation treatment. This suggests that plant growth promoting rhizobacteria act as a helping tool for AM fungi for better establishment and functioning of symbiosis (Kohler et al., 2007). Inoculation of soil with Azotobacter increased microflora population whereas; addition of AM fungi inoculum had synergistic effect on Azotobacter. The synergistic host response could mainly be due to the production of phytohormones or growth regulators produced by these microbes. It has been reported that spores of AM fungi seems to give Azotobacter an operational base in the vicinity of roots and supply of carbon that increases the efficiency of both AM fungi and Azotobacter (Saxena and Tilak, 1994). There was a good agreement between AM fungal spore density and percent root colonization. Higher uptake of nutrients was due to the synergistic effect of improved biomass and higher nutrient concentration in the inoculated plants (Table 4). Maximum N and P uptake was observed in dual inoculation treatment which may be due to the improved symbiotic N2 fixation as well as due to improved phosphatase activity and thereby P mobilization and subsequent P uptake by mycorrhizal hyphae. These results confirmed the earlier findings of Singh and Sharma (1993) who reported enhanced N and P concentration in sweet orange when inoculated with biofertilizers. Inoculated seedlings were found to contain significantly higher amount of other nutrients viz., K, Ca, Mg, Cu, Fe, Zn and Mn mainly in dual inoculation treatment. This could be attributed to the N-fixation ability of Azotobacter, better P mobilization and enhanced uptake of micro-nutrients due to AM fungi application. Because of enhanced accumulation of various metabolites, increased rhizosphere activity and higher nutrient uptake, transplanted pomegranate grew significantly better in field conditions and growth trend were almost the same between all the Complementary Copy 132 treatments as found during nursery period. Similar observations of increased plant growth and fruit production under field conditions were made in banana and tomato by Jeeva et al. (1988) and Kohler et al. (2007), respectively. The present study represents the positive response of biofertilizers in nursery seedlings followed by their transplanting in rainfed conditions. In conclusion our results showed that biofertilizers technology play a vital role in helping pomegranate to establish rainfed soils. The pre-inoculation of nursery seedlings with selected N2-fixing bacteria, phosphate solubilizing bacteria or AM fungi or combination of synergistically interacting species may be helpful in producing vigorous plants to survive and thrive under rainfed conditions. References Arnon, D.I. 1949. Copper enzymes in isolated chloroplast: polyphenol oxidase in Beta vulgaris. Plant Physiol., 24: 1-15. Aseri, G.K., Neelam Jain, Jitendra Panwar, A.V. Rao and P.R. Meghwal, 2008. Biofertilizers improve plant growth, fruit yield, nutrition, metabolism and rhizosphere enzyme activities of pomegranate (Punica granatum L.) in Indian Thar Desert. Scientia Horticulturae,117: 130-135. Ceccanti, B., B. Pezzarossa, F.J. Gallardo-Lancho and G. Masciandaro, 1994. Bio-tests as markers of soil utilization and fertility, Geomicrobiol. J., 11: 309-316. Day, J.M. and J. Dobereiner, 1976. Physiological aspects of N2-fixation by a Spirillum from Digitaria roots. Soil Biol. Biochem., 8: 45-50. Ephraim, P.L. and A.N. Robert, 2007. Punica granatum (Pomegranate) and its potential for prevention and treatment of inflammation and cancer. J. Ethanopharm., 109: 177-206. Gaur, A. and A. Adholeya, 1994. Estimation of VAMF spores in soil: a modified method. Mycorrhiza News, 6: 10-11. Gerdemann, J.W. and T.H. Nicolson, 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc., 46: 235-244. Jakson, M.L. 1967. Soil Chemical Analysis. Prentice-Hall of India Private Limited, New Delhi. Jeeva, S., M., Kulasekaran, K.G. Shanmugavelu and G. Oblisami, 1988. Effect of Azospirillum on growth and development of banana cv. poovan (AAB). South Indian Hort., 36: 1-4. Jensen, H.L. 1954. The Azotobacteriace. Bact. Rev., 18: 195-214. Karlidaga, H., Ahmet Esitkenb, Metin Turanc and Fikrettin Sahind, 2007. Effects of root inoculation of plant growth promoting rhizobacteria (PGPR) on yield, growth and nutrient element contents of leaves of apple. Scientia Horticulturae, 114(1): 16-20. Khanizadeh, S., C. Hamel, H. Kianmehr, D. Buszard and D.L. Smith, 1995. Effect of three arbuscular mycorrhizal fungus species and phosphorus on productivity and vegetative growth of three strawberry cultivars. J. Plant Nutr., 18: 1073-1079. Kohler, J., F. Caravaca, L. Carrasco and A. Rolden, 2007. Interactions between a plant growth-promoting rhizobacterium, an AM fungus and phosphate-solublizing fungus in the rhizosphere of Lactuca sativa. Appl. Soil Ecol., 35: 480-487. Luis, J., M. Martinez, S.U. Lucia, E.B. Beatriz, A. Jose and A. Sanchez, 2003. Indole 3-butyric acid (IBA) production in culture medium by wild strain Azospirillum brasilense, FEMS Microbiol. 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Arid Zone., 33: 45-47. Rao, A.V. and B. Venkateswarlu, 1982. Nitrogen fixation by Azospirillum isolated from tropical grasses native to Indian Desert. Indian J. Exp. Biol., 20: 316-318. Read, D.J., N.K. Kouckeki and J. Hodgson, 1976. Vesicular arbuscular mycorrhizae in natural vegetative system. The occurrence of infection. New Phytol., 77: 641-653. Saxena, A.K. and K.V.B.R. Tilak, 1994. Interaction among beneficial soil microorganisms. Indian Journal of Microbiology, 34(2): 91-103. Schenck, N.C. and Y. Perez, 1990. Manual for the Identification of VA Mycorrhizal Fungi. 3rd ed., Synergistic Publications, Gainesville, Florida, USA. Schnurer, J. and T. Rosswall, 1982. Fluorescein di-acetate hydrolysis as a measure of total microbial activity in soil and litter. Appl. Environ. Microbiol., 43: 1256-1261. Sharma, S.D., P. Kumar, S.K. Singh and V.B. Patel, 2009. Indigenous AM fungi and Azotobacter, and their screening from citrus seedlings at different levels of of inorganic fertilizers application. Indian Journal of Horticulture, 66(2): 183-89. Sharma, S.D., N. Sharma, C.L. Sharma, R. Sood and R.P. Singh, 2008. Studies on correlation between endomycorrhizal and Azotobacter population with growth, yield and soil nutrient status of apple (Malus domestica Borkh) orchards in Himachal Pradesh. Acta Horticulturae, 696: 283-87. Singh, C. and B.B. Sharma, 1993. Leaf nutrient composition of sweet orange as affected by combined use of bio and chemical fertilizers. South Indian Hort., 41: 131-134. Skujins, J. 1973. Dehydrogenase: an indicator of biological activities in arid soils. Bull. Ecol. Res. Commun., 17: 235-241. Tabatabai, M.A. and J.M. Bremner, 1969. Use of P-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem., 1: 301307. Tabatabai, M.A. 1982. Soil enzymes. In: Methods of Soil Analysis. Part 2. 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Received: February, 2012; Revised: July, 2012; Accepted: October, 2012 Complementary Copy Influence of biofertilizers on growth, yield, nutrition and rhizosphere microbial activity of pomegranate Journal Journal of Applied Horticulture, 14(2): 134-138, 2012 Appl Re sist a nc e eva luat ion of t he pist a chio root st ock s t o M e loidogyne spe c ie s in I ra n M e hrda d M a da ni 1 * , Ahm a d Ak hia ni 2 , M a hm oud Da m a dza de h 2 a nd Ahm a d K he iri 3 1 University of Tarbiat Modares, College of Agriculture, Plant Pathology Department, Tehran, Iran. Present address: University of Manitoba, Soil Science Department, Winnipeg, MB, Canada. 2Plant Pest and Disease Research Institute. Ministry of Agriculture and Jahad, Esfahan, Iran, 3University of Tehran, College of Agriculture, Plant pathology department, Karaj, Iran. *E-mail: [email protected]. Abstract Pistachio (Pistacia vera) is a edible nut native to Iran, the country that ranks first in worldwide pistachio production. Root-knot nematodes (RKN), Meloidogyne species, are among the most important pathogens that restrict the cultivation of pistachio in Iran. The objective of this study was to evaluate resistance of native pistachio rootstocks for resistance to isolates of M. incognita. Greenhouse experiment was conducted to determine the reaction of eleven cultivars of P. vera and six accessions of wild pistachio viz P. mutica, P. khinjuk, P. terebintus, P. atlantica, P. atlantica sub sp mutica and P. atlantica sub sp cabilica, against five selected populations of RKN. Meloidogyne incognita and M. javanica were identified based on the morphological characters, and esterase isozyme phenotype. Resistance was characterized based on root gall and egg mass indices and nematode reproduction. Resistance to M. incognita was detected among the cultivars and wild accessions of pistachio. There was a significant interaction among nematode populations and host genotypes, suggesting the presence of virulent pathotypes among the M. incognita isolates. These data suggest that it will be possible to development cultivars with resistance as a means of suppressing damage to pistachio that is caused by RKN. Introduction Pistachio originated from central part of the Middle East located in North Eastern part of the Iran. The pistachio tree (Pistacia vera L.) is heterozygous, deciduous, dioucious with separate male and female plants and is cross-pollinated. Iran is the leading producer of pistachio nuts with more than 252,790 hectares under cultivation with an annual production of 190,000 metric tons. The main areas of pistachio cultivation are located in central Iran in the provinces of Kerman and Esfahan; however, the provinces of Yazd, Semnan and Ghazvin also produce pistachio. The wild species P. khinjuk and P. mutica are found in mountainous regions of the country especially in the west and south. Root-knot nematodes (RKN) Meloidogyne (Nematoda: Heteroderidae), including M. incognita and M. javanica are the most destructive plant-parasitic nematodes found throughout subtropical, tropical and temperate regions of the world. Pistachio vera is the principal rootstock used and is susceptible to both of these species. Estimated production losses in pistachio worldwide caused by Meloidogyne species is US $118 million (Koenning et al., 1994). In Iran, M. incognita and M. javanicva has been reported attacking pistachio roots (Abivardi et al., 1979; Akhiani et al., 1986; Banihashemi and Kheiri, 1995; Kargar, 1989, Mojtahedi and Barooti, 1976; Madani et al., 1988). These species were also reported from pistachio roots in California (McKenry and Kretsch, 1984). Although several reports indicate wide presence of RKN in pistachio orchards in Iran and the occurrence of M. incognita and M. javanica, there is no systematic study on damage caused by RKN and/or resistance/susceptibility of pistachio cultivars and species in Iran. Therefore, the objective of this study was to screen diffierent pistachio cultivars including P. vera and wild relative accessions against Meloidogyne spp. Materials and methods The experiments were carried out at the Plant Pest and Disease Institute of Ministry of Agriculture, Esfahan, Iran. Test for identification of nematodes were performed at the Plant Research International, Wageningen, The Netherlands. A survey was conducted in the main cultivation areas of pistachio in Iran to collect pistachio seeds and nematode isolates. Infested trees were easily identified based on stunted growth habit with moderate to severely necrotic and chlorotic leaves. Thirteen seed samples from different cultivars of P. vera and 16 samples of wild species were collected from trees or provided by local growers. Additionally, three samples of wild pistachio seeds previously identified as P. atlantica, P. terebintus and P. sp. were received from Dr. Sh. Dehghani, University of Adelaide, Australia). A sample of wild species collected from Hormozgan province were kindly provided by Dr. Z. Banihashemi, Shiraz University, Shiraz, Iran. Sampling location and information on host plants are presented in Table 1. Collected seeds were kept in paper bags, placed in a cool dry container and transferred to the laboratory where they were maintained at 4°C until use. Nematode isolates were isolated from 30 soil and root samples collected from naturally infested pistachio orchards from different locations in Iran (Table 2). Two or three sub-samples were collected from different parts of each orchard, and mixed together to make a composite sample of about 1 kg containing feeder roots with rhizosphere and bulk soil from a depth of 50-70 cm. One composite sample was taken from each Complementary Copy Key words: Pistachio vera, root knot nematodes, gall index, eggmass index, cultivar Resistance evaluation of the Pistachio rootstocks to Meloidogyne species in Iran No. Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Pv-Mes Pv-Jan Pv-Fa-Kh Pv-Bo1 Pv-Bo2 Pv-Bad-Ra Pv-Fa-Ra Pv-Kh-Ra Pv-Sar Pv-Ard Pv-DM Pv-Kh-Dam Pv-Gh Pw-Kh-sem Pw-Mu-sem Pw-Kh-Bam Pw-Mu-Bam Pw-Kh-Jir Pw-At-Jir Seed source (Province- location) Esfahan-Mesr Esfahan-Jandagh Esfahan-Khor Va Biabanak Esfahan-Borkhar Esfahan-Borkhar Kerman-Rafsanjan Kerman-Rafsanjan Kerman-Rafsanjan Khorasan-Sarakhs Yazd-Ardekan Semnan-Damghan Semnan-Damghan Ghazvin Esfahan-Semirom Esfahan-Semirom Kerman-Bam Kerman-Bam Kerman-Jiroft Kerman-Jiroft 20 21 22 23 24 25 26 27 28 Pw-Kh-Sar Pw-Mu-Sar Pw-Mu-Mar Pw-Mu-Mok Pw-Mu-Ghir Pw-Mu-Siv Pw-Mu-Khan Pw-Mu-Koh Pw-At-Cab Khorasan-Sarakhs Khorasan-Sarakhs Fars-Marvdasht Fars-Mok va kavar Fars-Ghir Fars-Sivand Fars-Khanemein Fars-Kohanjan Hormozgan-Geno 29 30 31 32 33 Pw-Mu-Ker Pw-Mu-Koh Pw-Ter-Aus2 Pw-At-Aus3 Pw-sp Kermanshah Kohkiloieh Australia Australia Australia Species / cultivar P. vera-Mesri P. vera-Jandaghi P. vera-Fandoghi-Khor P. vera-Borkhar P. vera-Borkhar P. vera-Badami P. vera-Fandoghi P. vera-Khanjari P. vera-Sarakhs P. vera-Ardekani P. vera-Mamoli P. vera-Khanjari P. vera-Ghazvini P. khinjuk P. mutica P. khinjuk P. mutica P. khinjuk P. atlantica sub sp mutica P. khinjuk P. mutica P. mutica P. mutica P. mutica P. mutica P. mutica P. mutica P. atlantica sub sp cabulica P. mutica P. mutica P. terebinhtus P. atlantica Pistachio sp orchard and placed in a plastic bag and transported to laboratory in cooler box at 7-12°C. For locations where no distinct gall symptoms were observed on pistachio roots, an extra soil samples were collected from weeds infected with RKN close to trees. No soil sample was collected from wild Pistachio spp. Collected seeds were surface sterilized with 1% NaOCl solution for 5 minutes followed by soaking overnight in sterilized water. Seeds of P. vera cultivars were then placed between two layers of wet cotton tissue in a plastic tray and kept at 23-28°C until germination. To prevent contamination with common saprophytic fungi, seeds were sprayed with solution of 0.002% pentachloronitrobenzene (PCNB) fungicide every two days during the incubation period. After one week, germinated seeds were transferred to plastic containers (20×25×10 cm), filled with a sterilized mixture of sand, Perlite and peat moss (50:25:50 v/v) and then covered with 1 cm layer of sand. Containers were then kept in a greenhouse at 25-27°C until seedlings emerged. Seedlings with two leaves were then transplanted to a steam pasteurized clay pot (15×25 cm) containing approximately 500 cm 3 of sterilized river sand, vermiculate and peat moss (50:25:25 v/v). Pots were kept in a greenhouse with temperature ranging from 22 to 28 °C and 65-85% relative humidity, for a week, and then inoculated with nematodes. Seeds of wild species were dormant, therefore, they were soaked overnight in water and green coat was removed. Seeds were then transferred to plastic container filled with sterile river sand, wrapped container with aluminum sheets and maintained at 4°C for 30 to 40 days until germination. Each germinated seed with two leaves were then transplanted to a clay pots and kept at greenhouse under conditions described above. As an alternative, a method based on H2SO4 treatment was also tested to overcome the seed dormancy. For this, seeds were subjected to a solution of 1% acid for 1-2 minutes, rinsed with water and then planted. To obtain pure populations of nematodes, a single egg mass from each sample was collected and used to inoculate a susceptible tomato (Lycopersicum esculantum Mill. cv. Rutgers) seedling. Several egg masses from pistachio roots were used separately for inoculation of tomato seedlings at 2-4 leaf stage. For pistachio root samples, where it was difficult to find an egg mass, tomato seedlings were planted in collected soil and/or an egg mass was picked from the roots of weeds. Inoculated tomatoes were maintained in a greenhouse, separated from each other by plastic sheets to prevent any cross contamination of the nematode species. After 55 days tomato plants were harvested, soil washed from the roots and several egg masses and female nematodes were collected for species identification. Perineal pattern from at least 20 mature females were prepared using lactic acid, mounted in glycerin (Brito et al., 2004) and examined for identification of species of each RKN isolate. Based on preliminary species identification, four isolates identified as M. incognita from the main geographical region of pistachio production including (Kerman, Davaran (RD), Esfahan, Khor Va Biabanak (KH) and Kashan, (K and P) and one M. javanica isolate (MJ) from the province of the Yazd (Ardekan). Only M. incognita isolates were selected for subsequent experiments. Further confirmation of species was performed on these isolates using esterase and malate dehydrogenase protein isoelectrophoresis (IEF) of white female (Karssen et al., 1995), electron microscopy examination of perineal pattern and J2 morphology and morphometric characters (Courtney et al., 1995). Populations of the selected isolates of M. incognita population collected from Kashan, race 2, (Mi-K), Kashan race 4 (Mi-P), Khor Va Biabanak, Esfahan, (Mi-KH), and Rafsanja, Davaran (Mi-RD) and M. pistachio from Ardekan, Yazd (MJ) were raised from single egg masses and propagated on tomato seedlings for two generations. Inocula were prepared by macerating infected roots using a blender. Eggs and J2 were collected on a 25-μm pore sieve and gently rinsed with sterilized water into a 250-mL glass beaker. Three 1-mL samples were taken with a pipette to estimate numbers of eggs and J2 under a stereomicroscope. Eleven cultivars of P. vera and six accessions of wild Pistachio spp. were selected for evaluation of resistance to these isolates. Seedling pots were arranged in a randomized complete block design with four replications of each plant genotype and nematode isolate combination. P. vera seedlings were at the 4-6 leaf stage of development and wild accessions were 3-month-old seedlings when inoculation was done. Each seedling was inoculated with 1x104 eggs and J2 of nematode isolate. Control plants were inoculated with water and maintained in a greenhouse. Pots were watered every three days and fertilized weekly with a 0.5% Hoagland solution for 135 days. Complementary Copy Table 1. Origin, species and location of pistachio seeds used 135 136 Resistance evaluation of the Pistachio rootstocks to Meloidogyne species in Iran population, soil from each pot was transferred onto a tray, mixed and Species Species identification a sub sample of 250 cm3 taken for Perineqal pattern, J2 M. incognita nematodes extraction by elutriation and Peineal pattern, J2 M. incognita centrifuge (Lo´pez-Pe´rez et al., 2005). M. incognita Peineal pattern, J2-IEF* The host reaction was determined as resistant (GI ≤ 2, RF <1), tolerant (GI Peineal pattern, J2 M. incognita ≤ 2, RF >1), hyper-susceptible (GI ≥ M. sp (un identified) Peineal pattern, J2 2, RF <1), and susceptible (GI > 2, RF Peineal pattern, J2 M. incognita >1) (Canto-Saenz, 1987). Rootstock reaction was interpreted either based Peineal pattern, J2 M. incognita on GI and EI (Taylor and Sasser, 1978), Peineal pattern, J2 M. incognita or using index of R (Trudgill, 1991). Table 2. Nematode sampling location, host and test applied to identify Meloidogyne population used Host 1 Esfahan-Ardestan P. vera 2 Esfahan-Natanz P. vera 3 Esfahan-Khor va Biabanak P. vera 4 Esfahan-Khoram dasht-1 P. vera 5 Esfahan- Khoram dasht-2 P. vera 6 Esfahan-Chopanan P. vera 7 Esfahan-Mazrae Nemoneh P. vera 8 Esfahan-Alah Abad P. vera 9 Esfahan-Amir Abad P. vera M. incognita Peineal pattern, J2 10 Esfahan-Mesr P. vera, Cabbage M. incognita Peineal pattern, J2 11 Esfahan-Frah zad P. vera, Cabbage M. incognita Peineal pattern, J2 12 Esfahan-Jandagh P. vera, weeds M. incognita Peineal pattern, J2 13 Esfahan-Arosan P. vera M. incognita Peineal pattern, J2 14 Esfahan-Golestan P. vera M. incognita Peineal pattern, J2 15 Esfahan-Neishabor P. vera M. incognita Peineal pattern, J2 16 Esfahan-Kashan-Sabahi P. vera M. incognita Peineal pattern, J2-IEF 17 Esfahan-Kashan-Kaghazi P. vera M. incognita Peineal pattern, J2-IEF 18 Esfahan-Kashan-Ghale gosheh P. vera M. incognita Peineal pattern, J2 19 Yazd-Ardekan P. vera M. javanica Peineal pattern, J2-IEF Peineal pattern, J2 20 Kerman-Zarand P. vera M. incognita 21 Kerman-Zarand P. vera M. sp (un identified) Peineal pattern, J2 22 Kerman-Anar P. vera M. incognita Data analysis: Data on gall and egg mass numbers (GN and EN) per root system were subjected to analysis of variance using SAS, version 7.1. Mean comparison of data were performed and significance differences in means of nematode reproduction were separated using Duncan’s test with significant differences at 5% probability and differences among treatments were determined for each nematode population by cultivar. Results A total of 36 indigenous P. vera and 16 wild accessions were collected during the survey, from which 11 Peineal pattern, J2 P. vera M. incognita 23 Kerman-Rafsanjan cultivars and six wild accessions were Peineal pattern, J2 P. vera M. incognita 24 Kerman-Heidar abad selected for resistance assessment Peineal pattern, J2 P. vera M. incognita 25 Kerman-Heidar abad (Table 1). Preliminary identification of RKN from soil samples revealed P. vera M. incognita Peineal pattern-IEF 26 Kerman-Davaran the presence of two species, M. Peineal pattern, J2 P. vera M. javanica 27 Kerman-Davaran incognita and M. pistachio in 27 Peineal pattern, J2 P. vera Meloidogyne (un 28 Kerman-Davaran of sampling locations (Table 2). Peineal pattern, J2 , J2 P. vera M. incognita 29 Kerman-Naserieh Meloidogyne incognita was identified in 25 sampling areas from either Kerman-Naserieh Peineal pattern, J2 P. vera M. incognita 30 pistachio roots or weeds, whereas Species in bold were used for the final experiment on resistance test. *IEF: Isoelctrofocusing M. javanica was identified from the pistachio roots in two locations from Yazd province (Ardekan) After harvest, the roots were gently washed with tap water, and and Kerman province (Davaran). Three populations collected placed in beakers containing approximately 400 mL of 0.05 % from pistachio roots, one from province of Esfahan (Khoramdasht Phloxin B solution for 10 to 15 min to stain the egg masses a 2), and two from province of Kerman (Zarand and Davaran), bright red color. Each root system was scored for nematode galls could not be identified to species level due their unusual perineal (gall index=GI) based on 0 to 5 scale where 0= no gall, 1=1 to pattern morphology. Esterase and malate dehydrogenase IEF 2, 2=3 to 10, 3=11 to 30, 4=31 to 100 and 5= >100 galls (Safdar protein profile, electron microscopy of perineal pattern, J2 and McKenry, 2007). The length and weight of each root and morphology and morphometric obtained for the selected isolates stem were measured. of M. incognita (Mi-K, Mi-P, Mi-KH, Mi-RD) and M. javanica The reproduction factor (RF) was quantified based on R=Pf/Pi, (MJ), were in agreement with published data (Karssen et al., where Pf and Pi were final and initial population densities, 1995) and confirmed the identity of these isolates. respectively (Zhou and Starr, 2003). For calculation of Pf, the In the greenhouse screening test, egg mass number per seedling J2 per root system was estimated by macerating the roots using ranged from 1 to 167 for P. vera cultivars inoculated with M. a blender with 1% NaOCl solution. Because eggs viability was incognita (Table 3). The mean number of eggs masses across not a concern the higher concentration of 1% NaOCl was used to increase the extraction efficiency (Zhou et al., 2000). Released all pistachio cultivars for the four isolates of M. incognita ranged eggs and J2 were collected on a 25-μm pore sieve and then from 41.4 to 61.4, whereas the means across all four nematode counted under a stereomicroscope. To quantify the soil nematode isolates for the cultivars ranged from 11 to 114.7. For the egg Peineal pattern, J2 Complementary Copy No. Province-Location Resistance evaluation of the Pistachio rootstocks to Meloidogyne species in Iran Mi-Kh Mi-RD Mi-K Mi-P Table 4. Root gall production by four isolates of M. incognita on eleven cultivars of pistachio in a greenhouse test. Abbreviation for pistachio cultivar are given in table 1. Cultivars with the same letter not differ significantly (P <0.05) Mean Mi - Kh Mi-RD Mi-K Mi-P Mean Kh-Ra 28.0 4.0 1.0 3.3 11a Kh-Ra 147.5 30.8 39.0 21.3 59.7c Bad-Ra 139.7 65.5 24.3 12.5 60.5d Bad-Ra 281.5 170.7 69.0 22.8 136.0ab Ard 29.3 18.5 37.7 21.3 26.7b Ard 51.7 67.5 75.7 48.7 60.9c Gh 7.3 7.0 4.0 52.5 17.7a Gh 15.8 29.5 10.0 58.3 28.4d Bo1 54.0 57.7 21.7 25.0 39.6bc Bo1 82.6 89.8 72.7 56.5 75.4bc Dam-Mam 15.3 16.7 15.0 72.0 29.8b Dam-Mam 47.0 47.0 21.0 93.5 52.1c Fa-Ra 10.5 68.5 73.5 20.7 43.3c Fa-Ra 190.0 147.3 88.0 55.0 120.1b Bo2 30.3 50.5 72.3 28.0 45.3c Bo2 71.7 143.0 85.5 58.0 89.6c Sar 28.5 9.5 15.3 39.3 23.2b Sar 69.3 34.7 18.5 42.5 41.3d Fa-Kh 54.0 58.7 68.0 33.8 53.6c Fa-Kh 130.0 98.5 167.0 63.3 114.7e 61.4a 42.3c Kh-Dam Mean 47.9a 41.4a 45.4a mass production in P. vera cultivars, there was no significant differences among isolates across the cultivars; however, a significant difference in mean value of egg mass number was observed by cultivar among the four nematode isolates (Table 3). Two-way analysis of variance for the gall number also revealed significant differences in gall production among cultivars across all nematode isolates. Differences were observed in gall production among nematode. Nematodes isolate of Mi-K and Mi-P both collected from Kashan province were significantly different in gall production and separated from the rest of populations (Table 4). Analysis of data from the wild accessions showed there were no significant differences among nematodes isolates across the accessions for either gall and egg mass production (Table 5). Mean value of gall number in Pw-Te-Aus3 and Pw-AtCab accessions showed significant differences with the rest of accession. Mean value of egg mass number in Pw-At-Mu and Pw-At-Aus2 accession showed significant difference compared to other accessions for production of egg mass. Discussion The main aim of the present study was to identifying possible source of resistance through screening of cultivated pistachio root stocks and wild accession. During the survey and based on our observation and results of the experiments, RKNs obviously are among the most important pistachio root disease in Iran which 51.5 113.7 80.0 117.0 Kh-Dam 187.5 148.7 237.5 89.3 Mean 108.7a 93.0b 72.4abc 60.3c 90.6bc 165.8a 83.6ab can restrict planting of pistachio, especially in loamy sandy soil. Although sites with potential infestation of RKN were targeted in this study, wide distribution of RKN in pistachio orchards, especially in nurseries, were observed causing damage and reduction of yield in pistachio trees. M. incognita was identified in most of the sampling areas and detected in more than 80% of soil samples. This species was the most prevalent RKNs found in pistachio orchards. Although M. javanica was identified only in two locations, it appears to be the second important member of RKNs in terms of spreading and causing damage in pistachio orchards. Possible presence of other Meloidogyne species on pistachio is likely, where an unusual perineal patterns morphology in three populations collected from pistachio roots in Esfahan province (Khoramdasht 2), and Kerman province (Zarand and Davaran) was observed. More study needed to confirm the identity of these populations. Usefulness of wild germplasm as source of resistance to plant parasitic nematodes has been emphasized by other researches (Yaghoobi, et al., 1995). From this preliminary study it appears that a wide range of reaction from resistance to highly susceptible is present in pistachio root stocks against M. incognita. In addition, identifying the pistachio cultivar used as rootstocks is crucial in resistance experiments, especially when study of different flora from Iran and neighbor countries showed the differences in nomenclature or speciation of pistachio cultivars. In resistance experiments, study of cultivars and nematodes originated from the same geographic Table 5. Mean comparison of egg mass (EN) and gall numbers (GN), of six accessions of wild Pistachio species against Meloidogyne incognita in green house experiment. Data are from four replicates. Abbreviation for pistachio cultivar are given in Table 1. Cultivars with the same letter not differ significantly (P <0.05) Cultivar/code Pw-At-Aus2 Pw-Te-Aus3 Pw-At-Mu Pw-At-Cab Pw-Kh1 Pw-Kh2 Mean Mi-Kh GN 22.3 7 6.5 7.5 8.3 9.3 10.2a EN 30.3 1.5 22 2.3 29.8 31.8 19.6a Mi-RD GN EN 27 23.5 7 1.8 5.8 6.3 5.5 8 9.3 28.5 7.3 30.5 10.3a 16.4a Mi-K GN 23.5 6.3 6.3 4.5 9 9.8 9.9a Mi-P EN 20 1.3 1.5 0.5 45.3 46.5 19.2a GN 26.8 3 36 7.5 9 9.5 15.3a Mean EN 27 3.5 24.5 12 28.5 17.8 20.6a GN 25.2c 2.0a 13.6b 5.7a 33.0c 34.2c EN 24.9c 5.8a 13.7b 6.3a 8.9a 9.00a Complementary Copy Table 3. Egg mass production by four isolates of M. incognita on eleven cultivars of pistachio in a greenhouse test. Abbreviation for pistachio cultivar are given in table 1. Cultivars with the same letter not differ significantly (P <0.05) 137 Resistance evaluation of the Pistachio rootstocks to Meloidogyne species in Iran 0region is more validated due to co-evolution of the two organisms. In this study pistachio seeds and nematode samples were collected from the same location. It is assumed that the root galling is not more accurate indicator for the stability of RKN resistance (Safdar and McKenry, 2007; Zhou et al., 2000), therefore, in this study the virtual number of egg mass were also used for analysis of root stocks reaction. Nematode population from soil and roots were extracted and used for study of virulence and pathogenicity of the different population (data not shown). This provide valuable data for further study on resistance of pistachio root stocks against RKNs. In some sampling area severe disease symptoms were observed on above ground parts of pistachio trees while infestation of RKN was low and in some areas trees showing mild symptoms had severely infested roots to nematodes. This was in accordance with greenhouse experiments where the same phenomenon was observed in some of the cultivar nematode interaction. This indicates the presence of varying level of resistance/susceptibility among cultivars and existence of variation in nematode population in terms of pathogenicity and virulence. Range of variation was more limited in wild accession compare to the domestic pistachio cultivars. For most of the nematode cultivar interaction there was a positive correlation between gall and eggmass numbers, however it was observed that some cultivar with high number of galls did not support nematode reproduction. In addition, less variability in mean value of eggmass production was observed between nematode isolates than between cultivars. This shows that cultivars represent more variability in terms of supporting nematode reproduction. The same was observed for wild accession with nematodes isolates. Almost all of the nematode isolates caused galling and high rate of reproduction on pistachio cultivars. This is the first report on evaluation of pistachio cultivars used as root stocks in Iran. This provides preliminary information on identifying source of resistance. More studies are needed to understand the host pathogen interaction in orchard and trial plot. Study of differential interaction of nematode population will reveal more details on this phenomenon. Acknowledgements Authors are thankful to James, L. Starr and C. Abivardi for reviewing this paper carefully, Z. Banihashemi and Sh. Dehghani for providing some of the pistachio seeds. This work is dedicated to Ahmad Akhiani for his major participation in this work, who passed away while this research was being conducted. References Abivardi, C., M. Shahcheraghi and M. Sharafeh, 1979. Recent studies on distribution and control of the root-knot nematodes Meloidogyne spp in southern Iran. Proceedings of the 2nd research planning conference on root-knot nematodes, Meloidogyne spp., Region VII, Athens, Greece, (International Meloidogyne Project, Contract No. AID/ta-c-1234) Akhiani, A., H. Mojtahedi and A. Naderi, 1986. Hosts of the root knot nematode in Iran. Proc. Plant Pathol., p.134 . Banihashemi, Z. and A. Kheiri, 1995. The occurrence of root knot nematode (Meloidogyne javanica) on Pistachio in Damghan. Iranian J. Plant Pathol., 31: 37-38. Brito, J., T.O. Powers, P.G. Mullin, R.N. Inserra and D.W. Dickson, 2004. Morphological and molecular characterization of Meloidogyne mayaguensis isolates from Florida. J. Nematol., 36: 232-240. Canto-Saenz, M. and B. Brodie, 1987. Comparison of compatible and incompatible response of potato to Meloidogyne incognita. J. Nematol., 19: 218-221. Courtney, W.D., D. Polley and V.L. Miller, 1995. TAF, an improved fixative in nematode technique. Plant Disease Reporter, 39: 570-571. Hirchmann, H. 1985. The genus Meloidogyne and morphological characters differentiating its species. In: An Advanced Treatise on Meloidogyne, vol. 1. Biology and Control. J.N. Sasser and C.C. Carter (eds.). Raleigh: North Carolina State University. p. 79-93. Karssen, G., T. Vanhoenselaar, B. Verkerk-Bakker and R. Janssen, 1995. Species identification of cyst and root-knot nematodes from potato by electrophoresis of individual females. Electrophoresis, 16: 105-109. Kargar, A. 1989. Study on nematode fauna in pomegranate, pistachio and almond orchards in Yazd province. M.Sc. Thesis. Tarbiat Modaress University. 240 pp. Koenning, S.R., C.O. Verstreet, J.W. Noling, P.A. Donald, J.O. Becker and B.A. Fortnum, 1994. Survey of crop losses in response to phytoparasitic nematodes in the United States for 1999. J. Nematol., 31: 587-618. Lo´pez-Pe´rez, J.A., M.L. Strangeb, I. Kaloshian and A.T. Ploeg, 2005. Differential response of Mi gene-resistant tomato rootstocks to root-knot nematodes (Meloidogyne incognita). Crop Protection, 25: 382-388 Madani, M., A. Kheiri and A. Akhiani, 1988. Races and species of the Meloidogyne in Pistachio orchards in Iran. Proc.Plant Protection, 1989, Tehran, Iran, p. 87. McKenry, M.V. and J.O. Kretsch, 1984. Nematodes in pistachio orchards. California Agriculture, 38: 21. Mojtahedi, H. and S. Barooto, 1976. The chemical control of root-knot nematode in Esfahan. Iranian J. Plant Pathol., 12: 45-46. Safdar, A.A. and M.V. McKenry, 2007. Variability in reproduction of four population of Meloidogyne incognita on six cultivars of cotton, J. Nematol., 39(2): 105-110. Taylor, A.L. and J.N. Sasser, 1978. Biology, identification and control of root-knot nematodes (Meloidogyne spp.). Coop. Publ. Department of plant pathology, Library of Congress catalog card No. 77-94505, North Carolina State University, Raleigh, N.C. Trudgill, D.L. 1991. Resistance to and tolerance of plant parasitic nematodes in plants. Ann. Rev. Phytopatho., 29: 167-192. Yaghoobi, J., I. Kaloshian., Y. Wen and V.M. Williamson, 1995. Mapping a new nematode resistance locus in Lycopersicon peruvianum. Theorotical Applied Genetics, 91: 457-464. Zhou, E. and J.L. Starr, 2003. A comparison of the damage functions, root galling, and reproduction of Meloidogyne incognita on resistance and susceptible cotton cultivars. J. Cotton Sci., 7: 224-230. Zhou, T., A. Wheeler and J.L. Starr, 2000. Root galling and reproduction on Meloidogyne incognita isolates from Texas on resistance cotton genotypes. J. Nematol., 32: 513-518. Received: March, 2012; Revised: July, 2012; Accepted: October, 2012 Complementary Copy 138 Journal Appl Journal of Applied Horticulture, 14(2):139-143, 2012 Biology a nd se a sona l a c t ivit y of se m iloope r, Dic hrom ia orosia (Cra m e r) (Le pidopt e ra : N oc t uida e ) on a nt hm ool, Tylophora a st hm a t ic a Wight a nd Ar n. L. Sa rava na n 1 * a nd V ipin Cha udha r y 2 Directorate of Oil Palm Research, Pedavegi-534 450, Andhra Pradesh, India. 2Directorate of Medicinal and Aromatic Plants Research, Boriavi-387 310, Gujarat, India. *E-mail: [email protected] 1 Abstract Dichromia orosia (Cramer), a near monophagus pest was observed to cause severe defoliation to its host plant, anthmool (Tylophora asthmatica Wight and Arn.), an important medicinal plant used in Ayurvedic formulations to treat asthma world over. Biology and seasonal activity of the pest was studied during 2009-10 at Anand, Gujarat. Though incidence was observed throughout the year, however, the pest activity was more during July, August, December, January and February months. The pest completed its life cycle in 24.53±0.40 days (Eggs 3-4, larvae 10-14 and pupae 6-7 days). The longevity of the male and female was 15.70±0.68 and 19.70±0.42 days, respectively. Each female laid an average of 178.5±17.66 eggs, mostly on the under surface of the leaves in 12.20±0.49 days of oviposition period. The larvae developed through five instars in 12.9±0.35 days and pupal period lasted for about 6.8±0.11 days. Correlation of peak pest population periods with corresponding and previous Standard Meteorological Weeks (SMW) revealed that prevalence of maximum temperature (27.5-30.20C) mean temperature 29.31 0C, high RH and low rainfall recorded in increase of larval population. Introduction Material and methods Tylophora asthmatica Wight and Arn. (Synonym T. indica (Burm. f) Merr.) (Asclepiadaceae) is a climbing perennial plant indigenous to India. It grows wild in the southern and eastern regions and has a long-standing reputation as a remedy for asthma. Its leaves and roots are used as laxative, expectorant, diaphoretic, and purgative. The plant is also used for the treatment of bronchitis, colds, diarroahea, dysentery and joint pain (Prajapati et al., 2003). Over exploitation and lack of organized cultivation led to a rapid decline in the wild population of this species. Besides, the plants were reported to be severely defoliated by a semi looper, Dichromia orosia (Cramer) (=Hypena sagitta (Fabricus)) (Devaiah et al., 1983). The pest was observed to cause severe defoliation in the host plant throughout the year in the Herbal gardens of Directorate of Medicinal and Aromatic Plants Research, Anand, Gujarat, India. Since there was no previous report of this pest from Western India, its identity was confirmed by the Insect Identification Service, Indian Agricultural Research Institute, New Delhi (Registered under RRS No.67-69/10) as Dichromia orosia (Carmer). Studies on biology: The larvae of D. orosia (Noctuidae: Lepidoptera) were collected from field and reared on the host plant in laboratory in 20x15 cm plastic jars covered with muslin cloth. The food to larvae was replenished every day till the pupation. The pupae were collected from the jars and kept separately in a jar for adult emergence. The freshly emerged adults were sexed based on abdomen size and black markings at the tip of the abdomen of male insects. Ten pairs of male and female adults were kept in mating jars (15 x10 cm) provided with pieces of cotton soaked in 50 per cent honey fortified with few drops of multivitamin syrup and 2 to 3 twigs of T. athmatica dipped in water filled vials for egg laying. The twigs were carefully replaced with fresh twigs daily. The observations on fecundity, duration of oviposition and longevity of adults were recorded daily until the death of both the adults. Except for preliminary information on biology, feeding preference and predation of larvae by a pentatomid bug (Canthecona parva) (Sridhar and Jhansi Rani, 2003 and 2010), no other information is available. In view of this, systematic investigation on life traits, nature of damage, and seasonal abundance in relation to climatic factors was carried out. The present investigation certainly contributes to plan the strategy for effective management of the pest on T. asthmatica. The fresh batches of eggs were monitored regularly to find out incubation period. The neonate larvae (15 nos.) were carefully picked up with the help of a fine brush and transferred individually on the excised fresh leaves of T. asthmatica kept in 90 mm transparent plastic Petri dishes lined with moistened blotting paper to prevent the desiccation of leaves. Fresh leaves were replenished daily until the larvae reached pupal stage. The larvae were monitored regularly and moulting was confirmed by the presence of exuviae and head capsules. The duration and number of larval instars were determined. The newly formed pupae were moved to 200 mL plastic jar covered with muslin cloth and checked daily to record the adult emergence. The diameter of eggs, body length and width of I and II instar larvae and head capsule width of all the instars were determined with Complementary Copy Key words: Dichromia orosia, Tylophora asthmatica, biology, seasonal activity, longevity Biology and seasonal activity of semilooper, Dichromia orosia (Cramer) on anthmool, Tylophora asthmatica the help of ocular micrometer (calibrated with stage micrometer) fitted to a microscope. The body length and width of 3rd, 4th and 5th instar larvae, pre pupa, pupa and adults stage were measured using standard measuring scale. The width was taken from the thickest part of the body which coincided at the central part of it. The experiment was conducted in BOD incubator at 30±1 oC and 70±5 % RH. Studies on seasonal activity: The observation on the seasonal abundance of D. orosia was carried out on T. asthmatica plants grown in herbal garden of Directorate of Medicinal and Aromatic Plants Research, Anand, Gujarat during 2009-10. The experimental plot area was kept free from any plant protection measures throughout the study period to encourage natural insect incidence. The plot was divided into 6 quadrats (1m2 each) and total number of larvae feeding in each quadrat (includes larva present on plant as well as on the ground) was counted at weekly interval. The observation was taken at early hours of the day as the larvae tend to move to the soil surface and hide under plant debris, soil clods and crevices during the later part of the day. The relation between the weekly distribution of immature stages and prevailing weather parameters viz., rainfall, maximum and minimum temperature and relative humidity was assessed by simple correlation analysis using MS excel. Results and discussion Studies on biology Adult stage: Both male and female adults were nearly identical except in size, where males (32±0.17 mm at wing expanse) were slightly bigger than female (30±0.23 mm at wing expanse). The thorax was with grayish scales and abdomen was yellow coloured. Fore wings were grey with a large sub-triangular black patch with pale edges occupying the medial area, but not reaching the inner margin. Hind wings were yellow in colour with apical area dark brown. The female moths lived longer (19.70±0.42 days) than male moths (15.70±0.68 days) and the sex ratio was in favour of females (1: 1.1) (Table 1 and 2). Sridhar and Jhansi Rani (2010) reported adult longevity of 21.5±1.69 days. Eggs stage: Gravid female laid eggs mostly in groups and some time singly on the under surface of the leaves (often at the point where the leaf blade joins the petiole or either along side a major vein) and tender twigs. On an average, each female laid 178.5±17.66 eggs during the oviposition period of 12.20±0.49 days. The eggs were pale yellowish in colour, spherical and sculptured with a diameter of 0.75±0.01mm. The eggs hatched in 3-4 days. Soon after hatching the neonate larvae fed on epidermal tissue of leaves. Sridhar and Jhansi Rani (2010) reported duration of egg stage as 4-5 (av. 4.4±0.49) days and fecundity per female was 172.4 eggs. Larval stage: The Ist instar lasted for 2-3 (av. 2.3±0.13) days. Larvae grown to a length of 2.48±0.23 mm and width of 0.34±0.01 mm before moult. Its colour was pale yellow with pale black tubercles all over the body. Head was very minute with a head capsule width of about 0.34±0.01 mm. Instar II also lasted for 2-3 (av. 2.5±0.13) days and attained a length of 4.6±0.71 mm and width of 1.18 ± 0.22 mm. The head capsule width was 0.53±0.07 mm. The IIIrd instar was yellow in colour with prominent black coloured warts, this instar lasted for an average of 2.2±0.17 days. The length and width of the body was 11.8±0.87 and 1.7±0.49 Table 1. Duration of different life stages and reproductive phases of D. orosia on anthmool at 30±10C and RH 75±5% Stages Egg period (days) Larval period (days) 1st Instar 2nd Instar 3rd Instar 4th Instar 5th Instar Total larval period (days) Pre-pupal period (days) Pupal period (days) Total life cycle (days) Longevity of Adults a. Male b. Female Pre- oviposition period (days) Oviposition period (days) Post oviposition period (days) Fecundity (Nos.) Range (days) 3-4 Mean ± SE 3.4±0.13 2-3 2-3 1-3 1-3 2-4 10-14 1-2 6-7 21-26 2.3±0.13 2.5±0.13 2.2±0.17 2.2±0.14 3.6±0.16 12.9±0.35 1.4±0.13 6.8±0.11 24.53±0.40 12-18 18-22 4-6 10-15 2-5 88-292 15.70±0.68 19.70±0.42 4.6±0.22 12.20±0.49 2.9±0.0.28 178.5±17.66 Table 2. Morphometrics of different stages of D. orosia on anthmool (Mean ± SE) Stage Width of Length head capsule of body (mm) (mm) Width of body (mm) Wing expanse (mm) Diameter (mm) Egg 0.75±0.01 Larva 1st Instar 0.34±0.01 2.48±0.23 0.34±0.01 2nd Instar 0.53±0.07 4.6±0.71 1.18±0.22 3rd Instar 1.0±0.13 11.8±0.87 1.7±0.49 4th Instar 1.5±0.11 16.5±1.0 2.5±0.34 5th Instar 1.8±0.22 21.7±1.73 3.7±0.32 Pre-pupa 18.0±1.87 4.1±0.22 Pupa 12.2±0.65 3.9±0.2 Adult a.Male 32±0.17 b.Female 30±0.23 mm, respectively and the width of the head capsule was about 1.0±0.13 mm. The IVth instar lasted for 1-3 (av. 2.2±0.14) days, its length and width were 16.5±1.0 and 2.5±0.34 mm, respectively and the head capsule width measured about 1.5±0.11 mm. The Vth instar lasted for 2-4 (av. 3.6±0.16) days, grown to a length of 21.7±1.73 mm and a width of 3.7±0.32 mm. The colour of the larvae were dark yellow having dark black warts all over body with setae. The head was now quite distinct and measured about 1.8±0.22 mm (Table 1 and 2). The larvae is a semilooper having three pairs of true jointed legs just behind the head and four additional pairs of fleshy legs called ‘prolegs’, three pairs on the abdominal segments 4th,5th and 6th and fourth pair on the final abdominal segment. They move with a characteristic “looping” motion. Larvae were pale to dark yellow. The head and somites were having series of small black tubercles from where spines arisen. The larvae passed through five instars in 12.9±0.35 days and reached pupal stage. Sridhar and Jhansi Rani (2010) reported a total larval period of 25.5±0.65 days. The different instars lasted as, Instar-I, 3-4 days; Instar-II, 4-5 days; Instar-III, 4-6 days; Instar-IV, 5-7 days and Instar-V, 6-9 days. Nature of damage: All the instars inflicted severe damage to the host plants. On hatching, the neonate larvae immediately moved Complementary Copy 140 Biology and seasonal activity of semilooper, Dichromia orosia (Cramer) on anthmool, Tylophora asthmatica to the under surface of leaf and started feeding on tender leaves. The larvae of Ist to IIIrd instars scrapped the under surface of the leaves leaving the upper epidermal layer intact. It was observed that 5 to 6 early instar larvae were feeding on a single leaf. These larvae fed mainly on the chlorophyll content of the leaves from under surface of the leaves and leaving the upper epidermis intact. In a badly infested situation, the climbers presented a look, as if the plant had been scorched. Such leaves quickly dried up and fallen to the ground, resulting in complete denudation of the climber. The late instar larvae (IVth and Vth instars) fed voraciously on the 141 entire leaf tissues leaving major leaf veins. When the population of late instar larvae were high, the climber defoliated completely and giving the appearance as it was grazed by the cattle. Pupal stage: During the pre-pupal period of 1-2 days the full grown larvae stopped feeding. The body contracted and larvae settled in a place and spun cocoon with the help of silken threads, plant/leaf debris and excreta. The pre-pupae measured 18.0±1.87 mm in length and 4.1±0.22 mm in width. It transformed into pupae within 1-2 days. The pupae were brown in color and pupal Complementary Copy A B Fig.1. A. Average weekly incidence of Dichromia orosia on Tylophora asthamatica B. Environmental variables during the pest incidence period. Biology and seasonal activity of semilooper, Dichromia orosia (Cramer) on anthmool, Tylophora asthmatica Table 3. Correlation between mean weekly larval population (19-52nd & 1-18th SMW) and corresponding weekly weather parameters Weather Maximum Minimum Mean Relative Rain fall Parameters TemperatureTemperatureTemperature humidity Correlation -0.238 -0.210 -0.247 +0.016 -0.080 coefficient Table 4. Correlation between peak pest population periods (1st- 7th SMW) with corresponding week (1st- 7th SMW) weather parameters Weather Maximum Minimum Mean Relative Rain fall Parameters TemperatureTemperatureTemperature humidity Correlation +0.644** +0.933** +0.887** +0.057 0.0000 coefficient Table 5. Correlation between peak larval population and previous week weather parameters (46-52nd SMW) Maximum Minimum Mean Relative Rain fall Weather Parameters TemperatureTemperatureTemperature humidity Correlation -0.048 +0.348* +0.281* +0.469** -0.26 coefficient stage lasted for about 6-7 (av. 6.8±0.11) days (Table 1 and 2). It measured 12.2±0.65 mm in length and 3.9±0.2 mm in width at the broadest end. Sridhar and Jhansi Rani (2010) reported pupal period of 7-9 days (av. 7.7±0.64 days). The characters of full grown larvae observed in the present study are in conformity with those observed by Sridhar and Jhansi Rani (2010). The total development period from egg to adult emergence was determined as 24.53±0.40 days at about 30oC, thus permitting a maximum of 8 to 9 overlapping broods per year. This behavior is in line with the expectation of tropical butterflies to have a short life cycle, and multiple broods over the year. The developmental period of some stages determined in present study are not in conformity with the study made by Sridhar and Jhansi Rani (2010) on similar species under Bangalore, India conditions. Since climatic factors influence the instar duration and the overall development time (Mathavan and Pandian, 1975; Palanichamy et al., 1982; Pathak and Pizvi, 2003; Braby, 2003), the present findings may vary in other parts, depending upon prevailing climatic conditions. However, the development period of 24.53±0.40 is in good agreement with development period of related species H. laceratalis on lantana (Visalakshy and Jayanth, 1990). Seasonal activity: The mean incidence of D. orosia on anthmool is presented in Fig.1. The infestation of semilooper was first observed in 20th standard meteorological week (SMW), when the larval population was 1.6 larvae/m2. The maximum and minimum temperature during the week were 38.6 and 27.3oC, respectively and relative humidity (RH) was 63.0 per cent. The population increased slightly in 21st SMW to 3.2 larvae/m2 and reached to 22.67 larvae/m2 in 27th SMW, during this period slight rainfall (1.4 mm) in 24th SMW reduced the summer temperature to some extent and increased the RH from 51.6 to 69.3 per cent. Prevailing weather condition along with availability of foliage favoured the build up of larval population. This increased density of larval population at this particular point of time, resulted in complete defoliation of the climbers. Non availability of food and shelter coupled with monsoon rains (233.7 mm total rainfall during 27, 28, 29 and 30th SMW) resulted in mortality and forced pupation of larvae and hence nil population was observed from 28th to 32nd SMW. However, rainfall during these weeks increased the humidity up to 83.7 per cent (30th SMW), which resulted in rejuvenation of fresh flush of leaves. The congenial condition, like high RH (av. 74.80 per cent), nil rainfall and presence of fresh foliage triggered the emergence of adult from hibernating pupae, which resulted into increased egg laying, as egg mass and early instar larvae were abundant during 33th SMW (30.7 larvae/m2). In the subsequent 34th SMW population of late instar larvae was maximum (36.5 larvae/m2). Abundance of larvae per unit leaf area, left no space for adult moth to lay eggs. In general, some lepidopterans especially discriminate the egg load, larval competition on their host plants or relative abundance of host plants (Charnov, 1982; Wellings, 1991; Mugrabi and Moreira, 1996) either directly by vision (Wiklund and Ahrberg, 1978; Shapiro, 1981) or chemically through the presence of deterrent pheromones (Schoonhovan et al., 1990; Theiry et al., 1992). It was found that due to the scarcity of leaf tissues, the late instar larvae forcibly entered into pupal stage, as large number of pupae were observed among the dried leaves and on the soil surface during the period (35th SMW). Moreover, heavy down pour (131 mm) during the end of 35th SMW caused mortality in remaining larvae. The rain has been shown to cause the death of wide variety of insects in the pre imaginal as well as adult stages (Sappington and Showers, 1983). Owing to severe defoliation and denudation, the climbers did not recoup until 44th SMW. During 46th SMW few early instar larvae were seen (1.5 larvae/ 1m2quadrat) on the newly emerged flushes, the maximum and minimum temperature during the week were 28.5 and 18.9 oC, respectively and relative humidity was 73.4 per cent. The precipitation (4.8 mm) received during the later part of the 46th week resulted in mortality of those early stage larvae. Thereafter, the pest did not appear until 50th SMW. The intermittent precipitation and absence of pest activity for about 14 weeks favoured the climbers to recoup fully, which resulted in abundance of fresh foliage. The pest again appeared in 51st SMW, initially with the population of 8.2 larvae/m2, which increased steadily and a maximum of 163.2 larvae/m2 was observed during 6th SMW. At the end of that week the pest population was mixture of early (1-3rd instars) and late instars (4th5th instars). During the week maximum and minimum temperature were 31.0 and 16.2 0C, respectively and RH was 58.8 per cent. The abundance of pest completely defoliated or denuded the climbers and in the want of food and shelter the larval population declined to 16.7 larvae/m2 in 7th SMW due to mortality and forced pupation. As a result, during the period (7th SMW) large number of pupae was observed on the soil surface and among the dried leaves. Due to lack of green tissue, no pest population was observed upto 12th SMW. The moderate activity of pest was again observed from 13th SMW (1.33 larvae/m2), when fresh flush of leaves again rejuvenated and it remained continued upto 18th SMW. The temperature and humidity during the period ranged between 29.0 to 33.2 0C and 39.9 to 51.9 per cent, respectively. The correlation analysis between pest population and maximum, minimum and mean temperature (r= -0.24, -0.21 and -0.25, respectively) showed negative correlation. Relative humidity showed non-significant positive correlation (r=0.0164) (Table 3). Though rainfall could influence the insect population negatively (r= -0.081), but the correlation was not significant. Correlation of peak pest population periods with corresponding and previous SMW, revealed that out of 5 weather parameters tested 3 parameters viz. maximum temperature, RH and rain fall during 46-52nd and 1st -7th SMW were found to be effective in determining the level of infestation. The analysis inferred that prevalence of maximum temperature (27.5-30.20C), mean temperature 29.31 0C, high RH Complementary Copy 142 and low rainfall recorded in maximum increase of number of larval population (Table 4 and 5). The year round presence of immature stages on the host plant, T. asthmatica, showed that D. orosia breeds continuously. Weather conditions, especially RH and temperature though favoured the population build up of pest, yet their impact was non-significant, which contradicts the finding of several authors (Kaul and Kesar, 2003; Atluri et al., 2004 and 2010; Selvaraj et al., 2010), who found a significant effect of RH and temperature on population buildup of different lepidopteran pests. However, host abundance (particularly new flushes) and host quality favoured the population buildup of pest, as new flush facilitate the performance of larvae due to the likely higher levels of nitrogen and water content (Slansky and Feeny, 1977; Scriber, 1977; Mattson, 1980; Price, 2000; Awmack, 2002). Correlation of peak pest population periods with corresponding and previous SMW, revealed that prevalence of maximum temperature (27.5-30.20C), mean temperature 29.31oC, high RH and low rainfall recorded in maximum increase of number of larval population. However, the overall effect of weather on population trends is complex and difficult to predict, as also expressed by Pollard (1988). The semilooper, D. orosia has emerged as serious pest of anthmool, T.asthmatica and active almost round the year. The pest could generate another generation in 21-27 days. The weather parameters did not have significant effect on the built up of the pest. However, presence of new flushes favoured population built up. This knowledge on the pest will be helpful in formulating effective pest management strategies. Acknowledgements The authors are thankful to Director, Directorate of Medicinal and Aromatic Plants Research, Boriavi, Anand (Gujarat), India for providing facilities for the study and also to Dr. V.V. Ramamurthy, Division of Entomology, I.A.R.I., New Delhi for identifying this insect. References Atluri, J.B., B. Samantha, B.R. Matala, S.D. Devara and S.R. Chilakala, 2010. Ecobiology of the common castor butterfly Ariadne merione merione (Cramer) (Lepidoptera: Rhopalocera: Nymphalidae). J. Res. Lepidop., 42: 13-20. Atluri, J.B., S.P. Venkata Ramana and S.R. Chilakala, 2004. Ecobiology of Catopsilia pyranthe, a tropical pierid butterfly. Curr. Sci., 86(3): 457-461. Awmack, S.C. and S.R. Leather, 2002. Host plant quality and fecundity in herbivorous insects. Ann. Rev. Entomol., 47: 817-844. Braby, M.F. 2003. Larval and adult food plants for some tropical satyrine butterflies in northern Queensland. Austral. Entomol., 22(1): 5-13. Charnov, E.L. 1982. The Theory of Sex Allocation. Princeton University Press, Princeton. 355 pp. Devaiah, M.C., R.R. Gouda, Y.K. Kotikal and Y.S. Yelshett, 1983. A noctuid defoliator pest, Dichromia orosia Cramer (Noctuidae: Lepidoptera) of antamul, a medicinal plant. J. Bombay Natural History Soc., 80(3): 659. Kaul, V. and Y. Kesar, 2003. Incidence and management of lepidopteran fruit borers of Guava (Psidium Guajava L.) in Jammu, India. J. Asia-Pacific Entomol., 6(2): 201-205. Mathavan, S. and T.J. Pandian, 1975. Effect of temperature on food utilization in the monarch butterfly Danaus chrysippus. Oikos, 26: 60-64. 143 Mattson, W.J. 1980. Herbivory in relation to plant nitrogen content. Ann. Rev. Ecol. Systema., 11: 119-161. Mugrabi, O.E. and R.P. Moreira, 1996. Conspecific mimics and low host plant availability reduce egg laying by Heliconius eratophllis (Fab.) (Lepidoptera: Nymphalidae). Revista Brasialia Zool., 13(4): 929-937. Palanichamy, S., R. Ponnuchamy and T. Thangaraj, 1982. Effect of temperature on food intake, growth and conversion efficiency of Eupterote mollifera (Insect: Lepidoptera). Proceedings of Indian Academy of Sciences (Animal Sciences), 91: 417-422. Pathak, M. and P.Q. Pizvi, 2003. Age specific survival and fertility table of Papilio demoleus at different set of temperatures and host plants. Indian J. Entomol., 65(1): 123-126. Pollard, E. 1988. Temperature, rainfall and butterfly numbers. J. Appl. Ecol., 25: 819-828. Prajapati, N.D., S.S. Purohit, A.K. Sharma and T. Kumar, 2003. A Handbook of Medicnal Plants’ A Complete Source Book. First Edition. Agrobios (India). Price, W.P. 2000. Host plant resource quality, insect herbivores and biocontrol. In: Proceedings of the 10th International symposium on Biological control of weeds (Neal. R. Spencer ed.), Montana State University, Montana, USA, 1999, p.583-590. Sappington, T.W. and W.B. Showers, 1983. Effects of precipitation and wind on population of adult European corn borer, Ostrinia nublinalis (Lepidoptera: Pyralidae). Environ. Entomol., 12: 1193-1196. Schoonhoven, L.M., E.A.M. Beerling, J.W. Klijnstra and Y. Van Vuggt, 1990. Two related butterfly species avoid oviposition near eather’s eggs. Experimentia, 46: 526-528. Scriber, J.M. 1977. Limiting effects of low leaf water content on nitrogen utilization, energy budget, and larval growth of Hylaphora cecropia (Lepidoptera: Saturniidae). Oecologia, 28: 269-287. Selvaraj, S., D. Adiruobane, V. Ramesh and A.L. Narayanan, 2010. Impact of ecological factors on incidence and development of tobacco cut worm, Spodoptera litura Fabricus on cotton. J. Biopesti., 3: 043-046. Shapiro, A.M. 1981. Egg mimics of Streptanthus (Cruciferae) deter oviposition by Pieris sisyonbrii (Lepidoptera : Pieridae). Oecologia, 48: 142-143. Slansky, F. and P. Feeny, 1977. Stabilization of the rate of nitrogen accumulation by larvae of the cabbage butterfly on wild and cultivated food plants. Ecolol. Monographs, 47: 209-228. Sridhar, V. and B. Jhansi Rani, 2003. New record of Canthecona parva (Distant) (Heteroptera: Pentatomidae) as a predator of Hypena quadralis (Walker) a pest of Tylophora indica (Burm.f) Merr. J. Biol. Contr., 17(2): 179-180. Sridhar, V. and B. Jhansi Rani. 2010. Occurrence and biology of semilooper, Hypena sagitta (Fabricus)(=Dichromia orosia Cramer) (Lepidoptera: Noctuidae) on Indian Ipecae, Tylophora indica (Burm. F.) Merr. Pest. Mgt. Horti. Ecosys., 16(1): 78-79. Thiery, D., D. Gagel, P. Farkas and V. Pronier, 1992, Identification of an oviposition regulating pheromone in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae). Experimentia, 48: 697-699. Visalakshy, P.N.G. and K.P. Jayanth, 1990. Studies on biology and seasonal abundance of Hypena laceratalis Walker (Lepidoptera: Noctuidae) on Lantana camara L. in India. Entomon, 15 (3-4): 231-234. Wellings, P.W. 1991. Host location and oviposition on animals. In: Reproductive Behaviour of Insects: Individuals And Population, W.J. Bailey and J. Ridsdill- Smith (eds.). Chapman and Hall. p. 75-107. Wiklund, C. and C. Ahrberg, 1978. Host plants, nectar source plants and habitat selection of males and females of Anthocharis cardamines (Lepidoptera). Oikos, 31: 169-183. Received: June, 2012; Revised: October, 2012; Accepted: November, 2012 Complementary Copy Biology and seasonal activity of semilooper, Dichromia orosia (Cramer) on anthmool, Tylophora asthmatica Journal Journal of Applied Horticulture, 14(2): 144-145, 2012 Appl Oc c urre nc e of fa lse sm ut on dat e pa lm (Phoe nix da c t ylife ra L.) in t he sout he r n c oa st a l pla ins of Ye m e n M .H . Abdul Sat t a r 1 , A. Ra shid Ya ssin I bra him 2 a nd Wat he q A Aula qi 2 Plant Protection Section, 2Horticulture and Food Technology Section, El Kod Agricultural Research Station(AREA), Abyan Governorate, Republic of Yemen. E-mail: [email protected] 1 Abstract Twelve date palm cultivars were evaluated for field resistance to Graphiola leaf spot caused by Graphiola phaenicis (Moug) Poit. The disease incidence and number of sori were compared on both surface of leaf, pinnae position on leaves and plant age. Cultivars, Gizaz, Tha’al and Khodari showed negligable infection and fewer number of sori on the leaf surface and rachis. Symptom of disease was absent on leaves and rachis in cultivar Sagaee. These cultivars differed significantly from susceptible cultivars viz., Shahree, Soqotree and Khalas (P= 0.01). Abundant distribution of sori caused a drastic reduction of the leaf area covered by the fungus. Adaxial leaf surface trapped more number of sporidia and significant differences were detected among test cultivars (P=0.05). The temperature ranging between 32-38◦ C in summer and humidity accompanied by heavy dew in the night and early morning favored the development of infection. Correlation of age of cultivar “Shahree” and disease incidence revealed that older trees are more susceptible to disease. Key words: Date palm, Graphiola phaenicis, Southern coastal plain, Yemen. Graphiola phaenicis (Moug) Poit. causing leaf spot, infects older leaflets and rachis of the date palm (Phoenix dactylifera) and causes serious loss in yield (Guar, 2000). G. phaenicis has been extensively studied and reported from United States (Simone, 2004), Qatar (Abbas et al., 2004), Libya (Edongali, 1996), Canary Island (Cabrera et al., 1990) and India (Guar, 2000). Attempts have been made to control false smut by application of fungicides (Mehta et al., 1989) which is not desirable due to its negative impact on public health and environment. Cultivation of resistant and high yielding cultivars is the safe alternative to minimize pesticide use and consequently reduce the cost of production. Several reports indicated that date palm cultivars differ in their susceptibility to false smut disease (Mehta et al., 1989; Lodha, 2003). This study aimed on susceptibility of existing date palm cultivars in southern coastal plains to false smut disease in relation to age of tree and disease incidence. Materials and methods Present study involved several visits to date palm orchards in Lahej and Abyan Governorates which constitute the southern coastal plain of the Republic of Yemen. The date palm cultivars in each orchard were identified. Eight trees for each cultivar were randomly selected (Table 1). Leaflets from the apical, middle and base of the rachis were examined for infection by G. phaenicis. The number of sori were counted and recorded. Parts of these samples were transported to the laboratory, El Kod Agricultural Research Station for microscopic examination of basidiocarp and sporidia. Readings for incidence of disease for each cultivar were also taken by counting the number of infected trees for a given cultivar divided by total number of trees of a given cultivar in the orchard and expressed as percentage. A total of 360 samples representing the leaves and rachis were collected from three positions of pinnae and examined. The effect of age on the susceptibility of cultivars to false smut was studied utilizing the cultivar “Shahree” which is highly susceptible and widely grown in these areas. Trees 2, 12, 20 and 70 years old were selected for this study. Statistical analysis: Data were analyzed following standard procedures for analysis of variance and differences between means were compared for significance at P= 0.05 and P= 0.01. Correlation and regression analysis were conducted following standard procedures (Steel and Torrie, 1980). In all cases, the data were analyzed utilizing Genstat program (Genestat 5, No 3.1, 1996). Results and discussion Variation in the incidence of Graphiola leaf spot was apparent in 12 cultivars. Pathogen and symptom development appeared to be affected by the cultivar, age and position of leaf. Cultivars Gizaz, Tha’al and Khodari showed negligable infection and consequently less number of sori on the leaf surface (Table 1). Symptoms of the disease were almost absent on leaves and rachis in cultivar Sagaee, despite the presence of source of inoculum in the proximity of the cultivar. Although no significant difference (P= 0.05) was detected among these highly resistant cultivars, they differed significantly with the susceptible cultivars (P= 0.01). The symptoms (sori) exhibited a drastic reduction of the leaf area due to fungus covering. Singh et al. (1970) reported reduction in leaf area and decline in chlorophyll level in the leaves due to infection by G. phaenicis. Comparison of leaf surface revealed that the adaxial leaf surface trapped more number of sporidia than the abaxial surface (Table 1). Number of sori was higher and showed significant variation among test cultivars (P= 0.05). The distribution of sori in terms of leaf position was random (Table 2) and contradicts the trend reported by Lodha Complementary Copy Introduction Occurrence of false smut on date palm (Phoenix dactylifera L.) in the southern coastal plains of Yemen 145 Table 1. Disease incidence and number of sori at both surface of date palm leaves Table 4. Relationship between plant age and disease parameters on date palm cv. “Shahree” Cultivar Average number Average number Age of plant of sori on lower of sori on upper (years) leaf surface leaf surface 70 85.4 75.8 20 22.1 18.3 12 44.1 31.1 2 7.8 4.5 CV 16.596 28.120 20.669 17.172 LSD (P=0.05) 30.072 24.983 LSD (P= 0.01) Disease incidence (%) Shahree 95.0 Medini 9.0 Soqotree 65.0 Barhee 6.0 Gizaz 1.0 Zabidi 10.7 Mashtaf 4.0 Tha’al 2.0 Khodari 1.7 Ghahree 16.7 Khalas 40.0 Sagaee 0.0 12.3 LSD (P= 0.05) 16.6 LSD (P=0.01) CV 25.4 Number of sori on leaves Abaxial Adaxial 75.8 83.7 19.5 39.1 24.9 30.4 5.8 12.9 1.2 1.9 4.3 7.1 7.8 12.3 5.4 8.9 4.0 6.2 7.7 11.8 32.0 44.3 0.0 0.0 10.7 20.4 14.5 27.7 19.9 25.6 Disease reaction to false smut Highly susceptible Resistant Highly susceptible Resistant Highly resistant Resistant Resistant Highly resistant Highly resistant Resistant Highly susceptible Highly resistant Disease incidence (%) 95.0 30.0 30.0 3.0 17.5 14.2 20.73 (Table 3), where the disease incidence was highest in 70 years old trees (Table 4). Although the coefficient of determination are high and the slopes of the regressions are quite similar, the regressions differ principally in intercept values. This explains that older trees are more susceptible to Graphiola leaf spot than younger trees. Table 2. Position of leaves on the pinnae and number of sori on the leaves of date palm Cultivar Abaxial (number of sori) Adaxial (number of sori) Apical Mid Base Apical Mid Base Shahree 75.0 108.1 44.3 84.4 119.6 52.1 Medini 15.1 19.7 23.6 16.5 20.8 29.5 Soqotree 18.3 31.0 25.4 19.8 36.7 34.4 Barhee 4.8 5.7 7.0 12.3 13.7 12.7 Gizaz 0.9 1.4 1.2 2.0 1.9 1.7 Zabidi 4.0 3.9 5.0 6.7 7.0 7.5 Mashtaf 3.9 8.2 11.2 7.2 11.6 18.2 Tha’al 3.7 4.8 7.7 6.3 8.2 12.3 Khodari 5.9 3.1 3.1 8.2 5.2 5.0 Ghahree 5.5 8.9 8.8 8.7 15.8 10.8 Khalas 23.1 54.7 18.3 29.2 77.1 26.8 Sagaee 0.0 0.0 0.0 0.0 0.0 0.0 12.2 24.2 18.2 11.1 32.1 23.8 LSD (P=0.05) 16.5 32.8 24.7 15.1 43.6 32.3 LSD (P=0.01) CV 23.0 27.8 32.6 21.5 28.7 34.8 Table 3. Relationship between different variables (X) and age of date palm trees cultivar “ Shahree” (Y) Ŷ= a + b (x) Ŷ= 6.26+1.2784 x Ŷ= 7.00+0.978 x Ŷ = 13.09+1.028 x Ŷ = 11.12+0.939 x Ŷ = - 0.40+1.510 x Ŷ = 10.41+0.483 x Ŷ= 16.36+0.999 x Ŷ= 6.8+1.574 x Ŷ= 16.09+0.510 x F(prob) < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.038 < 0.001 < 0.001 0.083 R2 94.0 83.6 74.9 69.9 80.6 30.1 65.8 69.7 19.8 Variables Disease incidence (%) No of sori on upper surface of leaf No of sori on lower surface of leaf No of sori on apical upper surface of leaf No of sori on mid-upper surface of leaf No of sori on basal upper surface of leaf No of sori on apical lower surface of leaf No of sori on mid-lower surface of leaf No of sori on basal lower surface of leaf Abbas, E.H. and A.S. Abdulla, 2004. First report of false smut disease caused by Graphiola phaenicis on date palm trees in Qatar. Plant Pathology, 53(6): 815. Cabrera, R., F. Hodgson, M.De. Armas, C.T. Santiago, C.D. Lorenzo, C. Prendes and P. Plata, 1990. A fungus disease of canary palm (Phaenix canariensis chab.) produced by Graphiola phaenicis (Mong) Poit. Fisher in the Canary Islands. Phytoma Espana, 18: 21-25. Edongali, E.A. 1996. Disease of date palms (Phaenix dactilifera L.) of Libya. Arab Journal of Plant Protection, 14: 41-43. Elliot, Monica L. 2006. Graphiola leaf spot (False smut of palm). Florida Cooperative Extension Service. http: // edis.ifas.ufl. edu. Guar, V.K. 2000. Studies on Graphiola leaf spot disease of date palm. In: Proceedings of International Conference on “ Integrated Plant Disease Management for Sustainable Agriculture”, Vol I, New Delhi, India, 471-472. pp. Lodha, S. 2003. Influence of pinnae position, leaf age and some fungicides on the development of graphiola leaf spot on date palm. Arab Journal of Plant Protection, 21: 162-165. Mehta. N., P.C. Gupta, R.K. Tharija and J.K. Dang, 1989. Varietal behavior and efficacy of different fungicides for the control of date palm leaf spot caused by Graphiola phaenicis. Tropical Pest management, 35: 117-119. Simone, G.W. 2004. Graphiola leaf spot (false smut). In: Compendium of Ornamental Palm Diseases and Disorders. M.L. Elliot, T.K Broschat, J.Y. Uchida and G. W. Simone eds. American Phytopathological Society. St. Paul. MN. p. 26-27 Singh, M.K., R. Singh and R. Jeyarjan, 1970. Graphiola leaf spot on date palm (Phaenix dactilifera) , susceptibility of date varieties and effect on chlorophyll control. Plant Disease Reporter, 54: 617-619. Steel, R.G.D. and J.H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Approach . 2nd Edition. Mc. Graw Hill Book Company. 633 pp. Received: December, 2011; Revised: June, 2012; Accepted: July, 2012 Complementary Copy Reference (2003) who stated higher number of sori at the base on both the abaxial and adaxial surface of leaflets. Temperature ranging from 32-38° C in summer and 18-27° C in winter accompanied by heavy dew in the night and early morning hours regardless of duration are favorable for subsequent development of infection. No false smut is reported in Seiyun, Hadhramout governorate, which constitute the eastern plateau and is a major date palm cultivation area in Yemen is characterized by dry climate with low humidity (Personal communication, Ashoor AlZubeiri, 2009). This observation is in confirmity with findings of Elliot (2006). Regression of age of tree “Shahree” on disease incidence and the number of sori gave the highest coefficient of determination (r2) Journal Journal of Applied Horticulture, 14(2): 146-151, 2012 Appl Re sourc e use e ffi c ie ncy of ora nge a nd k innow c ult ivat ion in Ja m m u re gion of J & K st at e Jyot i K a chroo* , Anil Bhat a nd Dile e p K a chroo Sher-e-Kashmir University of Agricultural Sciences and Technology – Jammu, Chatha – 180009, Jammu and Kashmir, India. *E-mail: [email protected] Orange and kinnow occupy an important place in the horticultural industry of the country as well as in J&K state. In the present study, resource use efficiency of orange and kinnow was analysed. The regression coefficient values of selected inputs under orange orchards, mainly human labour, manures + fertilizers, irrigation, plant protection and training/ pruning varied significantly at the five age groups of five years from 5th to 28th years, corresponded to overall values as 0.955, 0.012, -0.012, 0.013 and -0.050, respectively. Out of which human labour, manures + fertilizers and plant protection with positive sign indicated that with one per cent increase in the use of these inputs, the output could be increased by 0.96 per cent in case of human labour and 0.01 per cent each in other two inputs. The regression coefficient of training/ pruning was statistically significant but negative indicating that one per cent increase in expenditures on training/ pruning could decrease the output to the extent of 0.05 per cent. The marginal value productivities of human labour, manures + fertilizers and plant protection were positive with their values at 0.185, 110.452, 0.076, respectively, whereas that of training/ pruning (-0.638) and irrigation (-0.054) were negative thereby indicated that there still existed scope of investing on human labour, manures + fertilizers and plant protection. The overall regression coefficient values obtained from kinnow cultivation were 0.029, -0.024, 0.016, 0.015 and 0.138 for human labour, manures + fertilizers, irrigation, plant protection and training/ pruning, respectively, out of which human labour and training/ pruning were statistically significant, indicating that one per cent increase in expenditures on these two inputs could increase the output to the extent of 0.03 per cent and 0.14 per cent, respectively. The regression coefficients of irrigation, plant protection and manures + fertilizers were non significant. The marginal value productivities of human labour, irrigation, plant protection and training/ pruning were positive with their values at 0.031, 0.025, 0.014 and 0.175, respectively, whereas that of manures + fertilizers (-0.027) was negative thereby indicating that there still existed scope in the investment on human labour, irrigation, plant protection and training/ pruning. Key words: Orange, kinnow, resource use efficiency, regression coefficient Introduction In a predominantly agricultural economy, the rate of growth in agriculture depends on the way farmers react to various programmes (Prasher et al., 2006). Diversification to horticultural crops has been found to be best option as they make more profit, generate additional employment for rural masses and conserve natural resources. Under present agricultural scenario, fruits in the food consumption play the key role in enhancing trade and business worldwide. The country’s population has almost tripled in the last five decades and its food grain production has more than quadrupled, significantly enhancing the per capita food grain availability. On the other hand, the share of agriculture in GDP has declined substantially from 55 per cent in early 1950s to about 42 per cent in the 1980s and further to 15.7 per cent in 2008-09 (Economic Survey, 2009-10). However, there is only a marginal decline in the number of resident in rural area. So, the government has identified horticultural crops as a means of diversification which are more profitable on the basis of efficient land use, optimum utilization of natural resources and creating employment opportunities for rural masses especially for women folk. India is the world’s second largest producer of fruits (57.73 million tonnes in 2008) which may increase to 98 million tonnes by the year 2020-2021 (Banerjee, 2009) and contributes 10.0 per cent in the world fruit production (Economic Survey, 200809). Productivity of horticultural crops has increased from 1.2 tonnes per hectare in 1953-54 to around 4.95 tonnes per hectare in 2007-08. Out of total production of fruits and vegetables about 65-70 per cent has been consumed domestically, two per cent for processing, and only one per cent exported while post harvest losses accounted to 20-30 per cent of the stored fruits (Wani, 2008). India’s export of fresh fruits and vegetables has increased from US$ 383.43 million in 2005-06 to US$ 605.33 million in 2007-08 i.e., an increase of 57.87 per cent (APEDA, 2008). Among the fruit crops, citrus is one of the major commercial fruit crop widely consumed both as fresh fruit and juice. Its global demand is attributed to its high vitamin C content and its antioxidant potential (Gorinstein et al., 2001). It is rich in folic acid, a good source of fiber, fat free, sodium free and cholesterol free with additional quality of containing potassium, calcium, folate, thiamin, niacin, vitamin B6, phosphorus, magnesium and copper. It may also help in reducing the risk of heart diseases and some types of cancer and has been found helpful in reducing the risk of pregnant women to have children with birth diseases (Economos and Clay, 1999). Essential and volatile oils are obtained from the citrus fruits’ peel sacks. So much so, it is used by the food industry to give flavor to drinks, foods and also acts as Complementary Copy Abstract Resource use efficiency of orange and kinnow cultivation in Jammu region of J&K state The state of Jammu and Kashmir, with its favourable climatic conditions for horticultural crops, is suitable for growing many fruits of commercial importance. The fruit industry is the second most important industry after tourism in Jammu and Kashmir state with 41.82 per cent area under horticultural crops out of the total net sown area of 7.34 lakh hectares and an income of Rs 2000.00 crore has been generated from fruits production during 2008-09 (Economic Survey, 2008-09). Yt = β0 ut (i = 1,2,3, …., n) Where, Y and Xi (i =1,2,3, …., n) are the output and levels of inputs. The constant β0 and βi’s (i = 1,2,3, ...., n) represent the efficiency parameters and the production elasticities of the respective input variables for the given population of fruit at a particular period, t. The fitted Cobb-Douglas production in present case with five input variables was written as: Y = a0 x1b1 x2b2 x3b3 x4b4 x5b5 Among the various fruit crops, growing of citrus has vast potential in Jammu region of Jammu and Kashmir state as it comprises highest area under its cultivation (11762 hectares) which is 99.62 per cent of total area under citrus in J&K, whereas its production has been realized to (19202 metric tonnes) which is 99.96 per cent of the total citrus production of J&K (Economic Survey, 2008-09). In Jammu region, the districts mainly Rajouri, Kathua, Jammu, Udhampur, Samba and Reasi are the prominent areas where it is grown. Keeping in view the importance of the orange and kinnow for our orchards and the facts described above, a study was undertaken with the following objectives: to estimate resource use efficiency of orange and kinnow in respect of important factor inputs. On log transformation, the above function then was transformed to a linear form as: Materials and methods x5 = Expenditure on training and pruning (Rs/acre) A multi stage sampling was adopted for the selection of samples, with districts, blocks, villages and orchardists as the first, second, third and fourth stage sampling units, respectively for orange and kinnow. Rajouri, Kathua, Jammu and Samba districts were selected because these four districts covered the maximum area under citrus cultivation (Rajouri covered 22.93 per cent, Kathua 20.56 per cent, Jammu 16.37 per cent and Samba 12.67 per cent of the total area under all citrus fruits cultivation in Jammu region). Three blocks from each district were selected except in case of Kathua where only two blocks were selected on the basis of area under orange and kinnow cultivation and from each block two villages were selected. The ultimate units, that is, orchardists were selected randomly from each village so as to constitute a total sample of 176 orchardists for the present study. a0 = Constant The primary data from growers of citrus was collected by survey method, using well-designed schedule. Collection of data was done by the personal interview method. The schedule was pretested before using for actual data collection. The reference period for this study was the year 2009. Production function analysis: In order to study the relationship between output and various inputs used, Cobb- Douglas production function was used. Here, gross product had been taken as dependent variable with manures + fertilizers, irrigation, plant protection, training/ pruning and human labour as the independent variables. The age of the orchards (orange and kinnow) were grouped into six groups viz. 5th to 9th year, 10th to 14th year, 15th to 19th year, 20th to 24th year, 25th to 28th year and overall group which included the whole age of the orchard. First four years were not taken in to account as these were the non fruit bearing years. The functional form applied is given as under: Log y = log a0 + b1 log x1 + b2 log x2 + b3 log x3 + b4 log x4 + b5 log x5 Or log y = log a0 + bi Where, Y i = Output (quintals/ acre) x1 = Human labour (man days per acre) x2 = Manure + fertilizers (kg/acre) x3 = Expenditure on plant protection (Rs/acre) x4 = Expenditure on irrigation (Rs/acre) b’s = Elasticities of production of respective resource categories To examine the productivity of different inputs used in production of studied fruits, marginal value productivities of inputs were estimated at geometric mean levels of inputs. To calculate Marginal Value Productivity (MVP) of resource xi, the following formula was used. × Py MVP = Where, MVP (xi)= marginal value productivity of ith resource = regression coefficient (estimated) GM (Y) = geometric mean of output GM (xi) = geometric mean of inputs Py = price of output Various combinations of variables were tried and the choice of the best equation was made on the basis of R2 explained and the relevance of the expected sign of coefficient. Some variables like irrigation and plant protection (in some age groups) were omitted during the analysis as the sample orchardists had not used these resources in those age groups. The marginal value productivity (MVP) of these resources used was worked out with the help of regression coefficients obtained. MVP of a particular resource represents the expected addition to the gross return caused by the additional one unit of the resource input while the other inputs were held constant. Complementary Copy a component for the pharmaceutical industry for the preparation of medicines, soaps, perfumes and other cosmetics, as well as for home cleaning products (Braddock, 1999). 147 Resource use efficiency of orange and kinnow cultivation in Jammu region of J&K state Results and discussion Table 1. Estimated regression coefficients of various factors, standard errors and MVP of orange and kinnow production (5th -9th year) Resource use efficiency of orange and kinnow: The results of regression function and marginal value productivity of orange and kinnow from 5th-9th year are depicted in Table 1. The table indicated that the output of orange orchards was regressed against human labour, manures + fertilizers, irrigation, plant protection and training/ pruning. For this group, production function was statistically significant having R2 value (0.622) meaning that 62.2 per cent of the total variations in the production of orange were explained by the explanatory variables under consideration. The regression coefficients of orange for human labour (0.771) and manures + fertilizers (0.010) with positive sign indicated that with one per cent increase in the use of these two inputs keeping all the other inputs constant, could increase the output by 0.77 per cent and 0.01 per cent, respectively. The contribution of irrigation (-0.014) and plant protection (-0.010) were found negative and non-significant. The regression coefficient of training/ pruning (-0.017) was also found negative and significant. All the regression coefficients were observed less than unity thereby indicating diminishing returns. It could be seen from the Table 1 that marginal value productivity of human labour (0.153) and manures + fertilizers (0.034) were noted positive while that of irrigation (-603.966), plant protection (-0.223) and training/ pruning (-260.371) were noted negative hence indicating their excess use and should be avoided to check the fall of returns. These results are in close conformity with Chinappa and Ramanna (1997). Variables The Table also depicted that the crop production function used from 5th-9th year of establishment was found statistically significant having R2 value (0.787) meaning that 78.7 per cent of the total variations in the production function for kinnow was explained by the explanatory variables under consideration. The table further revealed that the regression coefficients for human labour (0.316) and manures + fertilizers (0.320) with positively significant coefficient indicated that with one per cent increase in the use of these two inputs keeping all the other inputs constant, could increase the output by 0.32 per cent in both cases and at same time, both these inputs had significant contribution in the kinnow production. The regression coefficient of plant protection (-0.006) was however found negative and non significant. It could also be seen from the table that marginal value productivity of human labour (0.097), manures + fertilizers (0.469), irrigation (2.047) and training/ pruning (0.064) were positive and showed that additional one rupee spent on these inputs, could add to gross returns by Rs 0.10, 0.47, 2.05 and 0.06, respectively and hence there still existed a scope to invest more on these inputs. Similar findings were also reported by Iqbal (2009). The Table 2 in the age group of 10-14 years depicted that the R2 (0.884) value was statistically significant meaning that 88.4 per cent of the variation in the production of orange was due to the above mentioned explanatory variables. The regression coefficients of orange for human labour (0.867), plant protection (0.045) and training/ pruning (0.022) with positive sign indicated that with one per cent increase in Orange Kinnow Reg. Stand MVP Reg. Std. MVP Coeff. error Coeff. error Constant -0.057 0.254 0.796* 0.262 Manures + Fertilizers 0.010 0.013 0.034 0.320* 0.073 0.469 Irrigation -0.014 0.007 -603.966 0.004 0.024 2.047 Plant Protection -0.010 0.010 -0.223 -0.006 0.038 -0.104 Training/ Pruning -0.017** 0.008 -260.371 0.030 0.030 0.064 Human Labour 0.771* 0.080 0.153 0.316* 0.094 0.097 F Value 20.75 19.37 R2 0.622* 0.787* * and ** Significant at P=0.01 and P=0.05, respectively Table 2. Estimated regression coefficients of various factors, standard errors and MVP of orange and kinnow production (10th -14th year) Variables Orange Kinnow Reg. Stand MVP Reg. Std. MVP Coeff. error Coeff. error Constant -0.358** 0.145 0.884* 0.382 Manures + Fertilizers -0.056* 0.014 -1.845 0.573* 0.065 0.996 Irrigation 0.045** 0.018 1.354 -0.005 0.063 -0.0002 Training/ Pruning 0.022 0.013 512.188 0.124** 0.059 0.009 Human Labour 0.867* 0.040 0.085 0.245* 0.062 0.003 F Value 122.51 81.33 R2 0.884** 0.760* * and ** Significant at P=0.01 and P=0.05, respectively Table 3. Estimated regression coefficients of various factors, standard errors and MVP of orange and kinnow production (15th - 19th year) Variables Orange Kinnow Reg. Stand MVP Reg. Std. Coeff. error Coeff. error Constant -0.532* 0.158 1.837* 0.288 Manures + Fertilizers 0.001 0.014 0.040 0.263** 0.108 Plant Protection -0.002 0.012 -0.995 0.137** 0.064 Training/ Pruning -0.015 0.013 -622.236 0.026 0.069 Human Labour 0.874* 0.040 0.106 0.226** 0.097 F Value 129.03 24.65 R2 0.890* 0.889** * and ** Significant at P=0.01 and P=0.05, respectively MVP 9.071 0.021 0.007 0.012 the use of these inputs, keeping all the other inputs constant, could increase the output of the crop by 0.87 per cent in case of human labour, 0.05 per cent in case of plant protection and 0.02 per cent in case of training/ pruning. The contribution of manures + fertilizers was however found negative but significant (-0.056) thereby indicating that one per cent increase of this input, keeping all other inputs constant, could decrease the output by 0.06 per cent. It could be seen from the Table 2 that marginal value productivity of plant protection (1.354), training/ pruning (512.188) and human labour (0.085) were positive thereby indicated that these inputs were utilized sub-optimally hence additional use of these inputs could increase the output while that of manures + fertilizers was negative (-1.845) hence indicating its excess use and should be avoided to check the decrease in returns. These results are supported by the findings of Ahmad and Mustafa (2006). In case of kinnow in the age group of 10-14 years the R2 value worked out to be 0.760, thereby indicated that 76.0 per cent of the total variation in the production of kinnow, explained by the explanatory variables under consideration. The regression coefficients for human labour (0.245), manures + fertilizers (0.573) and training/ pruning (0.124) with significantly positive sign indicated that with one per cent increase in the use of these inputs, keeping all the other inputs constant, could Complementary Copy 148 Resource use efficiency of orange and kinnow cultivation in Jammu region of J&K state The result of regression function and marginal value productivity of orange and kinnow falling in the category of 15th-19th years have been given in Table 3. For this group, production function was statistically significant having R2 value of 0.890 meaning that 89.0 per cent of the total variations in the production of orange were explained by the explanatory variables under consideration. The regression coefficients of orange for human labour (0,874) and manures + fertilizers (0.001) respectively with positive sign indicated that with one per cent increase in the use of these two inputs, keeping all the other inputs constant, could increase the output of the orange crop to 0.87 and 0.01 per cent, respectively, though non significant coefficients for manures + fertilizers revealed that this input had negligible role in the production of orange during this period. These results have been found supported by the findings of Utomakili and Molua (1998) in banana. The contribution of plant protection (0.002) and training/ pruning (-0.015) were negative and non-significant. These findings have been in close conformity with Koujalagi et al. (1999). All the regression coefficients taken together were less than unity, thereby indicating operation of diminishing returns. It could be seen from the Table 3 that marginal value productivity of human labour (0.106) and manures + fertilizers (0.040) were positive while that of plant protection (-0.995) and training/ pruning (-622.236) were negative hence indicating their excess use should be avoided to have a check on fall of returns. The regression coefficients of kinnow (Table 3) for human labour (0.226), manures + fertilizers (0.263) and plant protection (0.137) with positive sign indicated their under utilization and with one per cent increase in the use of these inputs keeping all the other inputs constant, could increase the output of the kinnow crop to 0.23, 0.26 and 0.14 per cent, respectively. The production function used was statistically significant having R2 value as 0.889. The contribution of training/ pruning (0.026) was however positive but nonsignificant. All the regression coefficients were less than unity thereby indicating operation of diminishing returns. Here, the marginal value productivity of all the explanatory variables were positive with their value at Rs 0.01, 9.07, 0.02 and 0.07 in case of human labour, manures + fertilizers, plant protection and training/ pruning, respectively which meant that there is still a scope in investing in these inputs. These finding are in close conformity with the results of Iqbal (2009). The estimates of regression function and marginal value productivity of orange and kinnow falling in the category of 20th -24th year are presented in Table 4. The crop production Table 4. Estimated regression coefficients of various factors, standard errors and MVP of Orange and Kinnow production (20th-24th year) Variables Orange Kinnow Reg. Stand MVP Reg. Stand MVP Coeff. error Coeff. error Constant -1.178** 0.567 1.619** 0.643 Manures + Fertilizers 0.054 0.057 59.545 0.710* 0.105 0.004 Plant Protection -0.052 0.103 -0.003 0.156 0.135 0.006 Training/ Pruning 0.889* 0.074 0.098 0.005 0.090 0.020 Human Labour 43.86 46.29 F Value 0.733* 0.872* R2 0.890* 0.889** * and ** Significant at P=0.01 and P=0.05, respectively Table 5. Estimated regression coefficients of various factors, standard errors and MVP of Orange and Kinnow production (25th -28th year) Variables Orange Kinnow Reg. Stand MVP Reg. Stand MVP Coeff. error Coeff. error Constant -0.582 0.635 -0.575 0.785 Manures + Fertilizers 0.173* 0.064 203.462 0.025 0.084 0.042 Plant Protection -0.158 0.115 -0.010 -0.143 0.162 -1.079 Human Labour 0.723* 0.064 0.365 0.751* 0.086 0.498 F Value 33.76 58.35 R2 0.678* 0.727* * and ** Significant at P=0.01 and P=0.05, respectively Table 6. Estimated regression coefficients of various factors, standard errors and MVP of Orange and Kinnow production (overall) Variables Orange Kinnow Reg. Stand MVP Reg. Stand MVP Coeff. error Coeff. error Constant -0.399 0.387 2.991* 0.084 Manures + Fertilizers 0.012 0.013 110.452 -0.024 0.021 -0.027 Irrigation - 0.012 0.009 -0.054 0.016 0.011 0.025 Plant Protection 0.013 0.011 0.076 0.015 0.019 0.014 Training/ Pruning -0.050* -0.017 -0.638 0.138* 0.011 0.175 Human Labour 0.955* 0.094 0.185 0.029** 0.014 0.031 F Value 27.61 35.61 R2 0.687* 0.736* * and ** Significant at P=0.01 and P=0.05, respectively function used was found statistically significant having R2 value as 0.733 which means that 73.3 per cent of the total variation in the production of orange was explained by the explanatory variables under consideration. The results further showed that the regression coefficients of human labour and manures + fertilizers with positive sign were 0.889 and 0.054, respectively which indicate that with one per cent increase in the use of these two inputs, the output could be increased by their respective coefficients. The regression coefficient of plant protection (-0.052) was negative and non significant indicated its negligible role in production and over utilization. The marginal value productivity of manures + fertilizers (59.545) and human labour (0.098) indicated that an additional one rupee spent on them could increase the gross return by Rs 59.54 per acre and Rs 0.01 per acre, respectively. Hence, there was scope of investing more on manures + fertilizers and human labour. Results of regression coefficients of kinnow in the age group of 2024 years (Table 4) showed that the production function used was statistically significant having R2 value as 0.872 meaning that 87.2 per cent of the total variation in the production for kinnow was explained by the explanatory variables under consideration. The table further revealed that the regression coefficients of human labour (0.005), manures + fertilizers (0.710) and plant protection (0.156) with positive sign indicated that with one per cent increase in the use of these inputs, Complementary Copy increase the output of the crop by 0.25, 0.57 and 0.12 per cent, respectively. The contribution of plant protection (-0.005) was negative and non significant. Similar findings were reported by Koujalagi et al. (1999). It could be further seen from the Table 2 that marginal value productivity of human labour (0.003), manures + fertilizers (0.996), and training/ pruning (0.009) with positive sign revealed that one rupee spent on these inputs could increase the returns by Rs 0.003, 0.99 and 0.01, respectively while as plant protection (-0.0002) with negative sign indicated its excess use. 149 Resource use efficiency of orange and kinnow cultivation in Jammu region of J&K state the output could be increased by 0.01 per cent, 0.71 per cent and 0.16 per cent, respectively. The marginal value productivity of all the explanatory variables was observed positive. This showed that additional one rupee spent on these inputs would add to the gross returns by Rs 0.004, 0.01 and 0.02 in case of manures + fertilizers, plant protection and human labour, respectively. Ahmad and Mustafa (2006) also recorded the similar observations. The calculated values of regression analysis of orange falling in the age group of 25-28 years (Table 5) showed that the production function used was statistically significant having R2 value 0.678. It means that 67.8 per cent of the total variation in the production of orange was explained by the explanatory variables under consideration. The estimates further revealed that regression coefficients of human labour (0.723) and manures + fertilizers (0.173) with positive significant sign indicated that with one per cent increase in the use of these two inputs, could have increased output by 0.72 per cent and 0.17 per cent, respectively. The regression coefficient of plant protection (-0.158) was negative and non significant. It could be observed that MVP of manures + fertilizers (203.46) and human labour (0.365) indicated that an additional one rupee spent on manures + fertilizers could add to the gross return by Rs 203.46, while that of human labour indicated that an additional one rupee spent on human labour could increase the returns by Rs 0.37 per acre and hence there was scope of investing more on manures + fertilizers as well as on human labour. Regression coefficients of kinnow in the age group of 25-28 years depicted in Table 5 showed that the production function used was statistically significant having R2 value (0.727) meaning that 72.7 per cent of the total variations in the production for kinnow was explained by the explanatory variables under consideration. The regression coefficients of human labour (0.751) and manures + fertilizers (0.025) with positive sign indicated that with one per cent increase in the use of these two inputs, the return of output could be increased by 0.75 per cent and 0.03 per cent, respectively. The human labour contribution was significant in the kinnow production where as manures + fertilizers contribution was not significant in its production. The regression coefficient of plant protection (-0.143) was negative and non significant. The MVP of human labour (0.50) and manures + fertilizers (0.04) indicated that an additional one rupee spent on human labour could add to the gross return by Rs 0.50, while that of manures + fertilizers indicated that an additional one rupee spent on it could increase the returns by Rs 0.04 per acre and hence there was scope of investing more on human labour as well as on manures + fertilizers, but spending on plant protection could decrease the returns by Rs 1.08 per acre. The coefficients of regression analysis and MVP for overall groups of orange and kinnow are furnished in Table 6. It showed that the production function used was statistically significant having R2 value (0.687), indicating that 68.7 per cent of the total variations in the production function for orange was explained by the above mentioned explanatory variables. The regression coefficients for human labour, manures + fertilizers and plant protection with positive sign were 0.955, 0.012 and 0.013, respectively. It indicates that with one per cent increase in the use of these inputs, the output could be increased by 0.96 per cent in case of human labour and 0.01 per cent each in other two inputs. The regression coefficient of training/ pruning (-0.050) were found negative but significant. It indicates that this input was significant in the production of orange but it was used at more than optimum level, whereas that of irrigation (-0.012) was negative and non significant. The MVP as shown in Table 6 indicated that an additional one rupee spent on manures + fertilizers, human labour and plant protection could add to gross returns by Rs 110.45, 0.19 and 0.08, respectively, hence there was scope of investing more on these inputs. The MVP of irrigation (-0.054) and training/ pruning (-0.638) indicated that with an additional one rupee invested on these inputs would reduce the gross returns and hence should be checked. These findings are in close conformity with Chinappa and Ramanna (1997). The results of regression coefficients of overall group of kinnow depicted in Table 6 indicated that the production function used was statistically significant having R2 value as 0.736 meaning that 73.6 per cent of the total variations in the production function for kinnow was explained by the explanatory variables under consideration. Regression coefficients of human labour (0.029), irrigation (0.016), plant protection (0.015) and training/ pruning (0.138) with positive sign indicated that with one per cent increase in the use of these inputs, the output could be increased by 0.03 per cent, 0.02 per cent, 0.02 per cent and 0.14 per cent, respectively though only human labour and training/ pruning contributed significantly in kinnow production for the whole life of this orchard. The manures + fertilizers (-0.024) with negative and non significant regression coefficient implied its negligible contribution in the production of kinnow. These findings are in close conformity with Koujalagi et al. (1999). The MVP as shown in Table 6 indicated that an additional one rupee spent on human labour, plant protection, training/ pruning and irrigation may add Rs 0.03, Rs 0.01, Rs 0.18 and Rs 0.03 per acre, respectively, to gross returns, hence there was scope of investing more on these inputs. Conclusion and policy implications: The present study estimated the importance of various inputs in orange and kinnow production in Jammu region of J&K state. Estimates of Cobb-Douglas production function indicated that human labour was significant and underutilized in orange and kinnow in all age groups except in the age group of 20th to 24th of kinnow where human labour was found to be non significant and in the age group of 25th -28th, manures and fertilizers were found to be non significant. The farm resources such as human labour and manures + fertilizers were underutilized in case of orange whereas as in case of kinnow human labour, plant protection and irrigation were underutilized. There was overutilization of other farm resources also, so rational allocation of these resources is necessary. Also, growers must be convinced about the need and utility of application of manures + fertilizers in order to have more gains. The substitutability among different input factors has to be seen in depth and technologists should give enough thought in devising such strategies. References Ahmad, B. and K. Mustafa, 2006. Forecasting kinnow production in Pakistan: An econometric analysis. Intl. J. Agr. Biol., 8(4): 455458. APEDA, 2008. State-wise area, production and productivity of fruits. APEDA. www.apeda.com. Complementary Copy 150 Resource use efficiency of orange and kinnow cultivation in Jammu region of J&K state Iqbal, Mudasir, 2009. Investment Appraisal Of Mango And Ber Fruit Production in Jammu District of J&K state. M.Sc. Thesis. Shere-Kashmir University of Agricultural Sciences and Technology of Jammu, Jammu, India, 2009. 125pp. Koujalgi, C.B., R.S. Poddar and V.R. Kiresur, 1999. Profitability of production of marketing of pomegranate orchards in Bijapur district, Karnataka. Indian J. Agril. Economics, 58(4): 811. Prasher, R.S., M. Thomas and Y.S. Negi, 2006. Estimation of supply functions for Himachal apples. Indian J. Economics, pp. 433-451. Utomakili, J.B. and E. Molua, 1998. Analysis of resource use efficiency in banana Musa Sp. (AAA group) production in the south-east province of Cameroon: A case study. Intl. J. Trop. Agr., 16(1-4): 113-118. Wani, G.M. 2008. Past, Present and Future of Horticultural Development in J&K, India. www.buzzle.com. Received: October, 2011; Revised: June, 2012; Accepted: July, 2012 Complementary Copy Economic Survey, 2009. Directorate of Economics and Statistics, Ministry of Agriculture, Govt. of India. Economic Survey, 2010. Directorate of Economics and Statistics, Ministry of Agriculture, Govt. of India. Banerjee, G.D. 2009. Poised for a golden revolution. Times Agr. J., 20-21. Braddock, R.J. 1999. Handbook. of Citrus By-products and Processing Technology. John Wiley & Sons, Inc. p.247. Chinnapa, B. and R. Rammana, 1997. An economic analysis of guava production. Agril. Banker, 21(3): 29-33. Choubey, Manish and B.R. Atteri, 2000. Economic evaluation of litchi production in Bihar. The Bihar J. Agril. Mrktg., 8(2): 123-131. Economos, C. and W.D. Clay, 1999. Nutritional and health benefits of citrus fruits. FAO, Food, Nutrition Agr., 24: 11-16. Gorinstein, S., O. Martin-Belloso, Y. Park, R. Haruenkit, A. Lojek, I. Milan, A. Caspi, I. Libman and S. Trakhtenberg, 2001. Comparison of some biochemical characteristics of different citrus fruits. Food Chemistry, 74(3): 309-315. 151 Journal Journal of Applied Horticulture, 14(2): 152-156, 2012 Appl Enha nc ing w at e r re lat ions a nd va se life of c ut t ulip (Tulipa ge sne ria na L.) using fl ora l pre se r vat ive s R. Kum a r* , N . Ahm e d, D.B. Singh a nd O.C. Sha r m a Laboratory of Post Harvest Technology, Central Institute of Temperate Horticulture, Srinagar-190 007, Jammu and Kashmir, India. *E-mail: [email protected] Abstract The influence of different floral preservatives were assessed to determine their effect on the water relations and vase life of cut tulip cv. Yellow Purissima. Uniform size scapes of tulip at bud colour break stage were kept in ten different treatments of floral preservatives comprised of sucrose-(2, 4 and 6%), aluminium sulphate (100, 200 and 300 ppm) and 8-HQS (100, 200 and 300 ppm) along with control (distilled water). All the preservatives improved water relations and vase life of cut tulip significantly in comparison to control. The greatest cumulative water balance and maximum vase life were recorded in 8-HQS 300 ppm (10.5 g/scape and 10.1 days) followed by aluminium sulphate 300 ppm (9.67 g/scape and 8.9 days) over control (2.53 g/scape and 5.4 days), respectively. Maximum fresh weight change (10th day) was recorded in 8-HQS 300 ppm (105.13%) followed by aluminium sulphate 300 ppm (103.75%) in comparison to control (89.91%). The floral preservatives delayed the senescence of cut tulip by improving water uptake and post harvest physiology, thereby maintained better water balance leading to improved fresh weight and vase life. Introduction Tulip (Tulipa gesneriana L.) is an important bulbous flowering crop owing to its wide range of cultivars having attractive colours and exquisite shapes. It occupies 4th position among the top ten cut flowers in global floriculture trade (Jhon and Neelofar, 2006). In India, tulips are grown successfully in temperate regions of Jammu and Kashmir, Himachal Pradesh and Uttrakhand. Like other spring flowering bulbs, tulips are characterized by a short vase life by tepal senescence i.e. change in colour followed by dehydraton and tepal abscission (Iwaya-Inoue and Tikata, 2001). The vase life and post harvest quality of cut tulip is an important phenomenon of physiological process which depends upon water uptake, transpirational loss of water and water balance. Unlike fruits and vegetables, cut flowers are comprised of many morphological units like- sepal, petal, androcium, gynocium, stem and often leaves. The relationship among these parts determines the water balance and ultimately quality of cut flowers (Khan et al., 2007). The floral preservatives are mainly composed of sugars, biocides and acidifiers. Usually loss of turgor due to depletion of water results in deterioration of quality and vase life of cut flowers. Floral preservatives have been reported to maintain turgor, water balance and thus prolong the cut flower life. These floral preservatives reduce microbial growth, prevent vascular blockage and improve water balance to keep the flower fresh for longer duration (Patil, 2009). When cut flower is detached from the plant, the continuity of water to flower is disrupted. Hence, water relations play an important role in postharvest physiology of cut flowers (Bhaskar et al., 1999). Exogenous supply of sucrose balanced the depletion of carbohydrate and improved the vase life and quality of many cut flowers (Van Doorn, 2004). Chua (1971) suggested that 8-hydroxy quinoline sulphate (8-HQS) has cytokinin like activity in retarding senescence in cut flowers. HQS has also been reported to inhibit ethylene production (Wilkins, 1973). Aluminium sulphate overcome the water stress through its effect as germicides (Mukhophadhyay, 1982) and thereby encouraging continuous water transport through the cut stem. Hence, the present attempt was made to study the role of sucrose, aluminimum sulphate and 8-HQS in maintaining water relations for improving the keeping quality of cut tulips. Materials and methods Tulip cv. Yellow Purissima was grown during 2009-2011 in the experimental farm of CITH, Srinagar using recommended growing practices. Tulip scapes were harvested at bud colour break stage in the morning during third week of March. The flowers were precooled at 5°C for about one hour to remove field heat. Then scapes were sorted to uniform length of 25 cm and lower leaves removed to prevent them touching the preservative solution. After recording initial weight, scapes were placed in conical flask (250 mL) containing vase solution of different floral preservatives. Ten different treatments of floral preservatives were as follows: T1 (sucrose 2 %), T2 (sucrose 4 %), T3 (sucrose 6 %), T4 (aluminium sulphate 100 ppm), T5 (aluminium sulphate 200 ppm), T6 (aluminium sulphate 300 ppm), T7 (8-HQS 100 ppm), T8 (8-HQS 200 ppm), T9 (8-HQS 300 ppm) and T0 control (distilled water). The experiment was laid out in Completely Randomized Design (CRD) with three replications and five scapes constituted one sample unit. The flask were plugged with cotton and covered with aluminium foil to prevent loss of water due to evaporation. The experiment was conducted in the laboratory of Postharvest Technology, CITH, Srinagar at temperature 16 ± 2° with relative humidity 70 ± 5 under natural light. The weight of each container Complementary Copy Key words: Floral preservatives, sucrose, aluminium sulphate, 8-HQS, Tulipa gesneriana, water relations, vase life Enhancing water relations and vase life of cut tulip The difference between the initial and consecutive volume of solution in the flask was recorded as water uptake expressed as g/scape and the difference between consecutive weights of flask with solution plus flower scape recorded as transpirational loss of water (g/scape). The water balance in the flower scape was computed by subtracting the transpirational loss of water from water uptake. The fresh weight change, measured by the difference between initial and the consecutive fresh weight of scape, was expressed as per cent of fresh weight of scape taking original as 100 per cent. The termination of vase life was considered when tepals started wilting, falling and discolouration etc. Data obtained were analyzed statistically by the methods suggested by Gomez and Gomez (1984). Results and discussion Water uptake: Analysis of data revealed that different floral preservatives had significant influence on water relations and vase life of cut tulips. The water uptake by tulip scapes was significantly affected by different preservatives and the increasing concentration of preservatives improved water uptake. On the first day, the water uptake was recorded maximum (Table 1). Among all the treatments, the maximum uptake was found in 8HQS 300 ppm (7.20 g/scape) followed by 8-HQS 200 ppm (6.72 g/scape) and was at par with control. There was no significant difference in the water uptake between 8-HQS 200 ppm and 8-HQS 300 ppm and similar trend was also observed among different concentration of aluminium sulphate. Minimum water uptake was recorded with sucrose 2 % (5.15 g/scape) followed by sucrose 4 % (5.20 g/scape). On the whole, all treatments showed a decreasing trend of water uptake up to 5th day and thereafter a slight increase on 6th day was observed with all the treatments. On day 8th, water uptake increased with different concentrations of aluminium sulphate and 8-HQS. While decreased water uptake was recorded with different concentrations of sucrose and control on 8th day. On day 7th, 9th and 10th, all treatments recorded decreased water uptake. The highest cumulative water uptake was found in 8-HQS 300 ppm (48.61 g/scape) followed by 8-HQS 200 ppm (43.37 g/scape) and minimum in sucrose 2% (28.37 g/scape) (Fig.1). This may be attributed to the fact that 8-HQS and aluminium sulphate acidifies the holding solution and act as biocides and keep it free from micro-organism and thus helps in preventing the plugging of conducting tissues (Bhaskar et al., 1999). Increased water uptake was reported with application of 8-HQS (0.4 %) in daisy by Patil (2009) and 8-HQS (200 ppm) application in gerbera cut flowers by Prasanth et al. (2009). Increased water uptake maintains turgidity, freshness of flowers and thus enhances vase life of cut tulips owing to improved water balance and postharvest physiology. Transpirational loss of water (TLW): The tulip scapes held in different preservatives treatments differed significantly on TLW (Table 2). On each day, among different preservatives, maximum TLW was recorded with 8-HQS followed by aluminium sulphate and sucrose. On first day maximum TLW was recorded with 8-HQS 300 ppm (4.68 g/scape) followed by 8-HQS 200 ppm (4.37 g/scape), which were at par with control and minimum TLW was recorded with sucrose 2% (3.05 g/scape). There were no significant differences among different concentrations of 8-HQS and similar tendency was also reported with different concentration of aluminium sulphate and sucrose. On 2nd day, again maximum TLW was recorded with 8-HQS 300 ppm (4.45 g/scape) followed by 8-HQS 200 ppm (4.26 g/scape) but were differed significantly over control (2.80 g/scape). On 3rd day, maximum TLW was recorded with 8-HQS 300 ppm (4.29 g/ scape) and minimum with control (2.19 g/scape). On 4th and 5th day, maximum TLW was recorded with 8-HQS 300 ppm (3.56 and 3.48 g/scape) and minimum with sucrose 4 % (1.73 and 1.78 g/scape), respectively. On 6th day, maximum TLW was noticed with 8-HQS 300 ppm (4.95 g/scape) followed by 8-HQS 200 ppm Table 1. Effect of floral preservatives on water uptake of tulip cut flower (g/scape) Treatment Days after keeping scape in preservative solution T0-Control 1 6.83 2 4.80 3 3.60 4 3.46 5 2.81 6 3.20 7 1.60 8 1.50 9 1.10 10 0.80 T1-2 % Sucrose 5.15 4.90 4.30 3.20 2.92 3.10 1.70 1.10 1.30 0.70 T2-4 % Sucrose 5.20 4.98 4.40 3.25 3.00 3.32 1.60 1.20 1.40 0.90 T3-6 % Sucrose 5.80 5.30 4.70 3.65 3.31 3.47 1.75 1.31 1.20 0.95 T4-Alumunium sulphate 100 ppm 6.16 5.80 5.55 4.30 3.77 4.00 2.70 3.70 2.28 2.00 T5-Alumunium sulphate 200 ppm 6.20 5.91 5.68 4.34 3.82 4.03 3.15 3.60 2.40 1.80 T6-Alumunium sulphate 300 ppm 6.25 6.05 5.75 4.40 3.95 4.22 2.81 3.42 2.27 1.90 T7-8-HQS 100 ppm 6.50 6.10 5.92 4.63 4.12 4.52 3.00 3.63 2.10 1.80 T8-8-HQS 200 ppm 6.72 6.32 6.10 4.87 4.31 4.81 2.80 3.50 2.22 1.72 T9-8-HQS 300 ppm 7.20 6.80 6.42 5.40 4.80 5.22 3.32 4.25 2.80 2.40 LSD (P=0.05) 0.62 0.20 0.15 0.62 0.15 0.16 0.10 0.08 0.07 0.11 Complementary Copy with and without flower scape were recorded daily up to 10th day, when more than 90% scapes lost their keeping quality as indicated by tepal fall, discolouration, wilting and scruffy form. Keeping quality of flowers was determined by flower size, shape, condition, longevity, colour, texture, appearance, water balance, fresh weight and wilting. 153 154 Enhancing water relations and vase life of cut tulip Table 2. Effect of floral preservatives on transpirational water loss of tulip cut flower (g/scape) Days after keeping scape in preservative solution 1 2 3 4 5 6 7 8 9 10 T0-Control 4.76 2.80 2.19 3.04 2.91 3.42 1.98 2.80 1.62 1.63 T1-2 % Sucrose 3.05 3.10 2.76 2.00 2.12 2.99 1.78 1.25 1.60 1.11 T2-4 % Sucrose 3.06 2.98 2.78 1.73 1.78 3.11 1.72 1.41 1.72 1.48 T3-6 % Sucrose 3.62 3.26 2.93 1.98 2.01 3.13 1.93 1.55 1.72 1.75 T4-Alumunium sulphate 100 ppm 3.91 3.88 3.85 2.58 2.91 3.90 2.52 3.74 2.38 2.17 T5-Alumunium sulphate 200 ppm 3.89 3.78 3.92 2.46 2.62 3.88 2.89 3.66 2.55 2.01 T6-Alumunium sulphate 300 ppm 3.78 3.80 3.81 2.67 2.68 3.99 2.44 3.51 2.47 2.22 T7-8-HQS 100 ppm 4.20 4.13 4.22 2.88 3.32 4.33 2.77 3.71 2.26 1.99 T8-8-HQS 200 ppm 4.37 4.26 4.27 2.93 3.19 4.53 2.55 3.44 2.34 1.88 T9-8-HQS 300 ppm 4.68 4.45 4.29 3.56 3.48 4.95 2.93 4.12 2.99 2.66 LSD (P=0.05) 0.59 0.15 0.15 0.18 0.09 0.12 0.12 0.11 0.10 0.13 (4.53 g/scape) and minimum with sucrose 2% (2.99 g/scape). On 8th, 9th and 10th day, maximum TLW was recorded with 8-HQS 300 ppm (4.12, 2.99 and 2.66 g/scape) and minimum with sucrose 2% (1.25, 1.60 and 1.11 g/scape), respectively. The highest cumulative TLW found in 8-HQS 300 ppm (38.11 g/scape) followed by 8-HQS 100 ppm (33.81 g/scape) and minimum in sucrose 2% (21.76 g/scape) (Fig.1). Patil (2009) and Prasanth et al. (2009) also obtained increased water loss by 8-HQS (0.4 % and 200 ppm) application in daisy and gerbera cut flowers, respectively. It is apparent from the study that besides increased water uptake, reduction in TLW helps in improving water balance and is essential for extending the vase life of cut flowers. However, water uptake and TLW both were high in 8-HQS but the improved water balance in 8-HQS helped in improving the turgidity and freshness, and ultimately enhanced vase life of cut tulip. Water balance: Among all the treatments, 8-HQS (200 and 300 ppm) maintained positive water balance up to 8th day; aluminium sulphate and 8-HQS (100 ppm) maintained positive water balance up to 7th day, whereas sucrose maintained positive water balance up to 6th day. In control positive water balance was maintained up to 4th day only (Table 3). On first day, maximum water balance was recorded with 8-HQS 300 ppm (2.52 g/scape) followed by alumunium sulphate 300 ppm (2.47 g/scape) and minimum with control (2.06 g/scape). There were no significant differences among the concentrations of each one preservative. On 2nd and 3rd day, maximum water balance was recorded with 8-HQS 300 ppm (2.35 and 2.13 g/scape) followed by alumunium sulphate 300 ppm (2.25 and 1.93 g/scape), respectively. On 4th day, water balance was recorded maximum with 8-HQS 200 ppm (1.94 g/ scape) followed by alumunium sulphate 200 ppm (1.88 g/scape) and minimum with control (0.42 g/scape). All preservatives improved water balance over control but there were no significant differences among different preservative treatments except 8HQS 200 ppm which was statistically superior from sucrose 2 %. On 5th day, maximum water balance was recorded with 8-HQS 300 ppm (1.32 g/scape) followed by alumunium sulphate 300 ppm (1.27 g/scape) and minimum with control (-0.10 g/scape). On 6th day, maximum water balance was recorded with sucrose 6 % (0.34 g/scape) followed by 8-HQS 200 ppm (0.28 g/scape) and minimum with control (-0.22 g/scape). On 7th and 8th day maximum water balance was recorded with 8-HQS 300 ppm (0.39 and 0.13 g/scape) and minimum with control (-0.38 and -1.30 g/scape), respectively. On 9th and 10th day, all the treatments maintained negative water balance. Table 3. Effect of floral preservatives on water balance of tulip cut flower (g/scape) Treatment Days after keeping scape in preservative solution 1 2 3 4 5 6 7 8 9 10 T0-Control 2.06 2.00 1.40 0.42 -0.10 -0.22 -0.38 -1.30 -0.52 -0.83 T1-2 % Sucrose 2.10 1.80 1.53 1.20 0.80 0.11 -0.08 -0.15 -0.24 -0.41 T2-4 % Sucrose 2.14 2.00 1.61 1.52 1.22 0.21 -0.12 -0.21 -0.28 -0.58 T3-6 % Sucrose 2.18 2.04 1.76 1.67 1.30 0.34 -0.18 -0.24 -0.45 -0.80 T4-Alumunium sulphate 100 ppm 2.25 1.92 1.69 1.72 0.86 0.10 0.18 -0.04 -0.07 -0.17 T5-Alumunium sulphate 200 ppm 2.31 2.13 1.75 1.88 1.20 0.15 0.26 -0.06 -0.13 -0.21 T6-Alumunium sulphate 300 ppm 2.47 2.25 1.93 1.73 1.27 0.23 0.37 -0.09 -0.17 -0.32 T7-8-HQS 100 ppm 2.30 1.97 1.70 1.75 0.80 0.19 0.23 -0.08 -0.16 -0.19 T8-8-HQS 200 ppm 2.35 2.06 1.83 1.94 1.12 0.28 0.25 0.06 -0.12 -0.16 T9-8-HQS 300 ppm 2.52 2.35 2.13 1.84 1.32 0.27 0.39 0.13 -0.19 -0.26 LSD (P=0.05) 0.25 0.28 0.21 0.70 0.16 0.17 0.13 0.15 0.17 0.18 Complementary Copy Treatment Enhancing water relations and vase life of cut tulip 155 The cumulative water balance was recorded highest with 8-HQS 300 ppm (10.50 g/scape) followed by alumunium sulphate 300 ppm (9.67 g/scape) and minimum with control (2.53 g/scape) (Fig. 1). The variation in water balance might be on account of different water uptake and water loss behaviour under treatments. Active role of sugar in improving the water uptake and restricting the stomatal closure might have resulted in improved water balance. Antimicrobial agent (8-HQS and aluminium sulphate) improved water balance by inhibiting vescular blockage (Wani et al., 2010). Aluminium sulphate reduces transpiration and improves water balance due to stomatal closure, thus keep flowers afresh for a longer duration (Patil, 2009). Fresh weight change: Change in fresh weight differed significantly among the different treatments (Table 4). Increase in fresh weight change was found up to 6th day and then decreased sharply during 7th to 10th day in different treatments. On the whole, tulip scapes kept in solution of 8-HQS and aluminium sulphate maintained increased fresh weight over initial up to 9th day, while sucrose maintained increased fresh weight over initial up to 7th day as compared to control (up to 6th day). On 6th day, fresh weight change was found maximum with 8-HQS 300 ppm (129.28 %) followed by aluminium sulphate 300 ppm (126.29 %) and minimum with control (108.97 %). Treatments 8-HQS (200 and 300 ppm) and aluminium sulphate (200 and 300 ppm) maintained increased fresh weight over initial on 10th day also, while other treatments showed decreased fresh weight over initial on 10th day. On tenth day, maximum fresh weight change was recorded in 8-HQS 300 ppm (105.13%) followed by aluminium sulphate 300 ppm (103.75%) in comparison to control (89.91%). Stimart (1983) reported that there was initial increase in fresh weight changes followed by decline and increase being larger in flower kept in preservatives than those kept in de ionised water. Decline in fresh weight of scapes may be attributed to poorer water relation parameters. 8-HQS might have reduced the physiological stem plugging, whereas aluminium sulphate acted as antimicrobial agent (Mukhopadhyay, 1982) and improved water uptake and thereby maintained the improved fresh weight of scapes over control. Decrease in pool of dry matter and respiratory substrate especially in tepal might be considered as other important factors responsible for decline in fresh weight of cut tulip. A significant improvement in vase life of cut tulip was observed due to various preservative treatments (Fig. 2). Floral preservative showed their superiority in enhancing vase life and maximum vase life was recorded in 8-HQS 300 ppm (10.1 days) followed by aluminium sulphate 300 ppm (8.9 days), 8-HQS 200 ppm (8.8 days), aluminium sulphate 200 ppm (8.6 days) as compared to control (5.4 days). Table 4. Effect of floral preservative on fresh weight change (%) of tulip cut flower Treatment T0-Control 1 110.55 2 111.66 Days after keeping scape in preservative solution 3 4 5 6 7 8 113.03 116.13 119.64 108.97 97.20 95.02 9 93.59 10 89.91 T1-2 % Sucrose 109.66 112.12 113.25 116.46 120.16 122.67 114.36 99.64 97.73 96.43 T2-4 % Sucrose 109.83 112.50 113.42 116.62 120.74 123.81 115.00 104.97 99.55 97.73 T3-6 % Sucrose 110.02 113.10 113.86 117.45 121.98 124.75 114.97 108.06 99.44 99.83 T4-Alumunium sulphate 100 ppm 110.35 113.29 114.69 118.37 121.60 124.58 111.62 107.90 103.5 97.73 T5-Alumunium sulphate 200 ppm 110.63 113.57 116.18 119.11 122.04 125.49 115.24 111.35 106.00 102.37 T6-Alumunium sulphate 300 ppm 111.35 114.74 116.35 119.55 123.53 126.29 115.55 110.77 106.13 103.75 T7-8-HQS 100 ppm 114.30 116.28 116.90 120.33 122.65 125.30 117.15 109.88 104.47 99.66 T8-8-HQS 200 ppm 114.97 116.41 117.45 120.80 123.09 126.04 119.47 111.38 109.11 103.45 T9-8-HQS 300 ppm 115.19 116.40 117.48 123.81 124.91 129.28 119.66 114.36 106.35 105.13 4.25 2.71 0.32 0.45 0.52 0.41 0.38 1.37 0.48 0.41 LSD (P=0.05) Complementary Copy Fig. 1. Effect of floral preservatives on water uptake, TLW and water balance of cut tulip 156 Enhancing water relations and vase life of cut tulip Fig. 2. Effect of floral preservatives on vase life of cut tulip Depletion in sugar pool, plugging of vascular tissue by micro organism and damage by ethylene have been identified as the major cause of poor keeping quality of many cut flowers (Qadri et al., 2001 and Khan et al., 2007). Applied sugar might have countered the depleted sugar and improved vase life over control. While aluminium sulphate and 8-HQS improved the water balance and protected the flower from microbial vascular blockage and thus improved vase life of cut tulips. The fact of 8-HQS in the improving the vase life of cut flowers may be attributed to its nature as broad specturum bactericide and fungicide that reduce physiological stem blockage by chelating metal ions of enzymes active in creating the stem blockage (Marousky, 1972). It is, therefore, concluded that improved water relations due to low microbial activity in tulip scapes held in 8-HQS and aluminium sulphate solutions lead to higher water potential in comparison to control. The floral preservatives delayed the senescence of cut tulip by maintaining turgidity and improving postharvest physiology of cut tulips. The preservatives improved water uptake and thereby maintained better water balance leading to improved fresh weight and vase life of cut tulips. Among all the treatments, the greatest cumulative water balance and maximum vase life were recorded in 8-HQS 300 ppm (10.5 g/scape and 10.1 days) followed by aluminium sulphate 300 ppm (9.67 g/scape and 8.9 days) over control (2.53 g/scape and 5.4 days), respectively. Hence, enhanced water relations and vase life of tulip can be achieved through the use of 300 ppm aluminium sulphate and 8-HQS. Bhaskar, V.V., P.V. Rao and Y.N. Reddy, 1999. 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