Sheared-root inocula of vesicular-arbuscular mycorrhizal fungi

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 229-232 Vol. 58, No. 1 0099-2240/92/010229-04$02.00/0 Copyright © 1992, American Society for Microbiology Sheared-Root Inocula of Vesicular-Arbuscular Mycorrhizal Fungit D. M. SYLVIA* AND A. G. JARSTFER Soil Science Department, 2171 McCarty Hall, University of Florida, Gainesville, Florida 32611-0151 Received 25 July 1991/Accepted 24 October 1991 Selected vesicular-arbuscular mycorrhizal (VAM) fungi have been shown to enhance the growth of numerous plants of economic importance (18), including vegetables (11, 20, 26), field crops (1, 12, 19), and native plants used for revegetation (5, 28). Nonetheless, VAM fungi are not used widely in crop production, partially because inoculum sources are limited and application technologies are not well developed. The VAM fungi are difficult to culture on a commercial scale because they are obligate symbionts. These fungi can be grown with host plants in pot cultures containing soil (9), sand (31), or expanded clay (1, 6). They have also been grown by using hydroponics (7, 23), aeroponic culture (15), and root organ culture (22, 24). Various strategies have been proposed to apply inocula of VAM fungi (17). Inocula containing soil are considered impractical because of their bulk and the risk of contamination; however, chopped roots in peat blocks (32) and spores within a clay matrix (6) have been proposed for field application. Since the cost of inoculum production and application is high, better methods must be found to process inocula of VAM fungi. For efficient handling, inocula should be processed into small and uniform pieces. Such inocula could then be pelletized (4) or used in fluid-drill systems (3, 25). Roots colonized by Glomus spp. can serve as inocula because of the presence of intraradical vesicles (2) and, for a few species, spores (e.g., Glomus intraradix [10]). Roots colonized by Glomus spp. have been processed by grinding (4), maceration (2), and milling (16). However, these processes usually reduced inoculum density. For example, a drop of nearly 50% in propagule number has been reported when air-dried peat inoculum was milled to a size of 850 ,um (32). In this article we describe the preparation and use of * Corresponding author. t Florida Agricultural Experiment Station Journal Series R-01303. sheared-root inocula of Glomus spp. Our objectives were (i) to determine if root inocula of VAM fungi produced in aeroponic culture would remain viable when sheared, (ii) to quantify the inoculum density of the resulting size fractions, (iii) to ascertain if the sheared inocula could be pelletized or used in a gel carrier, and (iv) to evaluate storage methodology for aeroponic root inocula. MATERIALS AND METHODS Shearing method and particle size distribution. Root samples were removed from eight plants freshly harvested from a 12-week-old aeroponic culture of an undescribed Glomus sp. (isolate S328, INVAM 925) started on nodal stem cuttings of industrial sweet potato (Ipomoea batatas (L.) Lam., cv. White Star). This isolate is similar to Glomus macrocarpum Tul. and Tul. but lacks an expanding outer wall. The aeroponic culture system was described previously (15, 29). The root material was blotted on paper towels and cut into 1-cm lengths with scissors. Separate root samples (5 g [fresh weight]) were then placed in 50 ml of water in the work bowl of a Little Pro food processor (Cuisinart, Norwich, Conn.) and sheared for periods of 0, 5, 10, 20, 40, and 80 s. Processing was interrupted halfway through each run or every 10 s, and roots adhering to the side of the bowl were scraped back into the water. After processing, the contents of the bowl were washed over a stack of four sieves that had openings ranging from 63 to 425 ,um. The smallest fraction was collected on a 5-,um-pore-size nylon membrane (Micron Sep, Honeoye Falls, N.Y.). Each fraction was dried at 65°C and weighed. A U.S. patent application has been filed for this method (30). Propagule density in size fractions. Aeroponically grown roots of sweet potato, colonized with Glomus sp. (isolate S328), were harvested from an 18-week-old aeroponic culture. Roots were blotted on paper towels and cut with scissors into 1-cm lengths, and fresh and dry weights were determined for a portion of the material. Six 5-g (fresh no. 229 Downloaded from http://aem.asm.org/ on January 25, 2016 by guest For efficient handling, vesicular-arbuscular mycorrhizal fungi should be processed into small and uniform inocula; however, processing can reduce the inoculum density. In this article we describe the preparation and use of sheared-root inocula of Glomus spp. in which inoculum densities were increased during processing. Our objectives were to determine inoculum viability and density after shearing and to ascertain if the sheared inocula could be pelietized or used with a gel carrier. Root samples were harvested from aeroponic cultures, blotted dry, cut into 1-cm lengths, and sheared in a food processor for up to 80 s. After shearing, the inoculum was washed over sieves, and the propagule density in each fraction was determined. Sheared inocula were also encapsulated in carrageenan or used in a gel carrier. Shearing aeroponically produced root inocula reduced particle size. Propagule density increased with decreasing size fraction down to a size of 63 ,um, after which propagule density decreased. The weighted-average propagule density of the inoculum was 135,380 propagules g (dry weight) of sheared root material-'. Sheared roots were encapsulated successfully in carrageenan, and the gel served as an effective carrier. Aeroponic root inoculum was stored dry at 4°C for 23 months without significant reduction in propagule density; however, this material was not appropriate for shearing. Moist roots, useful for shearing, began to lose propagule density after 1 month of storage. Shearing proved to be an excellent method to prepare viable root inocula of small and uniform size, allowing for more efficient and effective use of limited inoculum supplies. 230 APPL. ENVIRON. MICROBIOL. SYLVIA AND JARSTFER added to each of five "Pinecell" Conetainers (63-ml capacity; Stuewe and Sons, Inc., Corvallis, Oreg.), five surfacedisinfested (15 min in 25% Clorox bleach) seeds of sea oat (Uniola paniculata L.) were added to each Pinecell, and then the seeds were covered with fine horticultural vermiculite. Plants were grown in a high-intensity-discharge (HID) growth chamber and watered with 0.25x-strength Hoagland's solution (13) for 42 days, at which time complete root systems were harvested and assessed for the presence or C') 5 0 20 PM 40 various sizes of time root inoculum of on the weight) subsamples were processed 40 s with 50 ml of distilled water. was also mixed with 50 ml Size fractionation was accomplished tents of the work bowl over a The of S328). Glomus 28°C scissor-cut water. stack openings of from 63 to 425 jim. The then collected on 33-jim-pore-size mesh Mesh Polyester; Spectrum Medical Angeles, Calif.). The root material collected, and known amounts to the first dilution of a most-probable-number (27) as follows. For the scissor-cut 250 to 425 jim in size, the root vacuum filtration to remove directly for addition to the growth dry-weight determination was fractions. For the fractions of 63 jim in size, the root material filtration and resuspended in addition to the MPN assay and polyester (dry weight fractions jim material excess medium. also 90 to was 50 ml for were removed from dilutions of the stirred processed roots for of dry-weight suspension. were Metro-Mix (W. R. Grace & Co., Fogelsville, of 1:3,529 (wt/vol). Fifty milliliters Pa.) 10000 cog w 1 000 C1) 'i100 C: m a- 10 100 10 SIZE FIG. 2. Propagule densities inoculum (estimated by an after shearing for 40 s. Dotted FRACTION of MPN lines (gim) S328) Downloaded from http://aem.asm.org/ on January 25, 2016 by guest TIME (SEC) FIG. 1. Effect of shearing absence of colonization by VAM fungi. Inoculum encapsulation. Sweet potato roots harvested from a 13-week-old aeroponic culture were stored at 4°C for 51 days and then processed as described above for 40 s. The inoculum was fractionated over standard sieves. The fraction sized between 90 and 250 jim was washed over 0.8-jimpore-size GA4-S Metricel membranes (Gelman Sciences Inc., Ann Arbor, Mich.) with sterile water or a disinfecting solution (2% chloramine T plus 0.02% streptomycin sulfate) (21). This material was then suspended in 2.5% Kappacarrageenan (C-1263; Sigma, St. Louis, Mo.) (8). The carrageenan was prepared by being dissolved in warm water and then being autoclaved at 121°C for 20 min. The solution was in a water bath, and pellets were produced by cooled to extruding the carrageenan suspension through a 16-gauge needle from a 30-ml syringe into 0.3 M KCI at room temperature. Pellets were separated from the KCI by being sieved over a surface-disinfested, 425-jim-pore-size sieve and then were blotted on sterile paper towels. A bioassay for infectivity was conducted in the HID growth chamber. Thirty milliliters of Metro-Mix 200 was placed in each Pinecell. Pellets (5, 10, or 15) were placed on the growth medium, and an additional 20 ml of growth medium was added to five replicates per treatment. Five surface-disinfested seeds of sea oats were placed in each Pinecell and covered with vermiculite. Plants were watered with 0.25xstrength Hoagland's solution for the first 28 days of the assay and with deionized water during the final 14 days. The assay was harvested after 42 days of growth in the HID growth chamber, and the MPN of propagules per pellet was determined. Encapsulation was tested with a second VAM fungus. Sweet potato roots colonized with Glomus etunicatum Becker and Gerdemann (isolate S329, INVAM 906) were grown in an aeroponic culture for 18 weeks, colonized roots were processed as described above for 40 s, and the fraction ,u 90 m was collected on sieves and sized between 63 and washed with water onto a 0.8-jim-pore-size Metricel membrane. This material was then suspended in 2.5% carrageenan. The carrageenan pellets were prepared as described to in a water above except that the solution was cooled 34°C bath. The infectivity assay was conducted in Pinecells by using Zea mays cv. Early Sunglow as the host and pasteurized Arredondo loamy sand (loamy, siliceous, hyperthermic, Grossarenic Paleudult) as the growth medium. Seven replicates of 0, 1, 5, 10, or 20 pellets were placed on 30 ml of soil and covered with an additional 20 ml of soil. Two seeds were placed on the soil surface and covered with vermiculite. The plants were watered with 0.25x-strength Hoagland's solution and harvested after 42 days of growth in the HID growth chamber, at which time the MPN of propagules per pellet was determined. Inoculum in gel carrier. Sweet potato roots colonized by Glomus sp. (isolate S328) were harvested from an 8.5-weekold aeroponic culture and processed as described above for 40 s. The fraction sized between 90 and 425 jim was used to make dilutions in a 2.5% hydroxyethylcellulose carrier (Na- VOL. 58, 1992 INOCULA OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI RESULTS AND DISCUSSION Shearing aeroponically produced root inoculum for up to 20 s reduced the particle size; increasing amounts of inoculum were found on the fine sieves (Fig. 1). By 20 s, the mean length of the root pieces was reduced from 10 to 1 mm. Processing from 20 to 80 s had little effect on the size distribution of the inoculum. Propagule density increased dramatically with decreasing size fraction down to a size of 63 ,um, after which propagule density decreased sharply (Fig. 2). The distribution of total dry weight in each fraction was 17, 63, 11, 3, and 5%, respectively, for the >425-, 250- to 425-, 90- to 250-, 63- to 90-, and 33- to 63-,um-size fractions. The weighted-average propagule density (dry mass of each fraction x propagule density) of the inoculum was 135,380 propagules g (dry mass) of root-1. Our results differ from those of previous studies that show that other types of processing (including grinding, maceration, and milling) reduce inoculum density. Graham and Fardelmann (10) found that the number of root fragments was directly related to propagule density, as determined by an MPN assay. The shearing process cuts roots so cleanly that there is little loss of inoculum viability during processing. By reducing the size of particles, the number of particles per gram of root is greatly increased. Shearing proved to be an excellent method to obtain viable inocula of small and uniform size. Sheared-root inocula from aeroponic culture may provide an economical source of VAM fungi for research and agriculture. With the propagule densities achieved by this process, we have estimated a cost of 2 to 3 cents per 1,000 propagules. To achieve adequate colonization of containergrown plants, we have found that an inoculation rate of approximately 20 propagules per plant is necessary (unpublished data), resulting in an inoculum cost of 5 cents per 100 plants. Q 20 N Z 10 0 0 -i 0 50 -0 ° 40 B z ° 30 y = 1.4882 + 0.82639x RA2 = 0.962 20 ^ z 0 -i 0 0 10 10 100 PROPAGULES / g MEDIA FIG. 3. Effect of propagule density (estimated by an MPN assay) on the infectivity of inoculum encapsulated in carrageenan (A) or in a gel carrier (B). Symbols are the means of five and seven replicates for panels A and B, respectively, and bars represent the standard error of the mean at P s 0.05. Sheared roots were encapsulated successfully in carraFor Glomus sp. (isolate S328), the disinfested inoculum contained 1.2 propagules per pellet, while the nondisinfested inoculum contained 0.5 propagules per pellet. For G. etunicatum, the inoculum contained 0.3 propagules per pellet, although each pellet had an average of 4.0 + 0.85 spores. Increasing the density of propagules encapsulated in carrageenan resulted in improved colonization, demonstrating the viability of this inoculum (Fig. 3A). Natrosol served as an effective carrier of a processed inoculum. The inoculum contained 0.5 propagules ml-', and increasing inoculum density also resulted in increased colonization (Fig. 3B). geenan. 0 100000 0 10000 LLI 1000 uJ Cl) 100 n D 10 0 cr_ 0- 1 0 6 12 18 24 MONTHS OF STORAGE FIG. 4. Propagule densities of Glomus sp. (isolate S328) root inoculum (estimated by an MPN assay) stored dry or moist at 40C in vermiculite. Dotted lines represent 95% confidence limits. Downloaded from http://aem.asm.org/ on January 25, 2016 by guest trosol; Aqualon, Wilmington, Del.). The dilutions resulted in concentrations of 0, 0.1, 0.5, 1, 10, and 100 spores ml-1. A no-gel control was also established. A 42-day bioassay was conducted in the HID growth chamber. Thirty milliliters of growth medium was placed in a Pinecell, 1 ml of an inoculum suspension was added, and this mixture was covered with an additional 20 ml of growth medium. There were seven replicates per treatment. Disinfested seeds of sea oats were placed on the- surface of the medium and then covered with vermiculite. Plants were watered with 0.25 x -strength Hoagland's solution for the first 28 days of the assay and with deionized water during the final 14 days, at which time the MPN of propagules in the initial inoculum was determined. Storage of inocula. Sweet potato roots colonized by Glomus sp. (isolate S328) were harvested from a 13-week-old aeroponic culture, blotted dry with a paper towel, and stored moist in a sealed container at 4°C for 21 days before the experiment was initiated. Roots were then cut into 1-cm-long sections and either air dried for 72 h at 24°C or left moist (moisture content, 92%). Fifty grams (fresh weight basis) of roots was added to 500 ml of oven-dried vermiculite (dry storage) or 500 ml of vermiculite moistened with 100 ml of distilled water (moist storage) and placed in 1-liter screw-cap Nalgene bottles (Nalge Company, Rochester, N.Y.). The bottles were stored in the dark at 4°C. After 0, 1, 3, 6, and 23 months of storage, MPN assays were established to assess propagule density. The MPN assays consisted of three 10-fold dilutions of the initial root-vermiculite mixture. 231 232 APPL. ENVIRON. MICROBIOL. SYLVIA AND JARSTFER This confirms the observation of Hung et al. (14) that Natrosol is a good carrier of VAM fungi. Dry storage was clearly superior to moist storage for aeroponic root inoculum (Fig. 4). Propagule densities of dry roots were nearly constant over the 23-month evaluation period. Hung and Sylvia (15) reported that aeroponic root inocula stored moist at 4°C retained infectivity for at least 9 months; however, our present results indicate that propagule densities of moist roots began to decline after 1 month. Unfortunately, air-dried roots cannot be effectively sheared. Therefore, for maximum inoculum densities, sheared-root inocula should be prepared from moist roots stored at 4°C for less than 3 months. 36:64-67. 17. Jarstfer, A. G., and D. M. Sylvia. Inoculum production and inoculation technologies of vesicular-arbuscular mycorrhizal fungi. In B. Metting (ed.), Soil microbial technologies: applications in agriculture, forestry and environmental management, in press. Marcel Dekker, Inc., New York. 18. Jeifries, P. 1987. Use of mycorrhizae in agriculture. Crit. Rev. Biotechnol. 5:319-357. 19. Medina, 0. A., A. E. Kretschmer, and D. M. Sylvia. 1990. 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