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Isolation of potash mobilizing microorganisms in tea soil and evaluation of

their efficiency in potash nutrition in tea: a novel approach

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Two and a Bud, 63(1):8-12

RESEARCH PAPER

Isolation of potash mobilizing microorganisms in tea soil and

evaluation of their efficiency in potash nutrition in tea: a novel
approach

P. N. Bhattacharyya1*, P. Dutta1, Mausomi Madhab1, I. K. Phukan2, S. R. Sarmah1, S. K. Pathak1

1
Mycology and Microbiology Department, Tocklai Tea Research Institute, Tea Research Association, Jorhat
– 785 008, Assam, India.
2
Department of Soils, Tocklai Tea Research Institute, Tea Research Association, Jorhat – 785 008, Assam, India

ABSTRACT

Potassium (K) availability is a major problem in tea growing soils of Assam, North-east India. The present
investigation aims at the isolation of potash mobilizing microorganisms in the tea soil and evaluation of their
potential in increasing available potash under in vitro. Ten numbers of potash mobilizing bacteria (KMB) were
isolated from the experimental tea estate of Tocklai Tea Research Institute (TTRI), using a serial dilution plate
method on modified GYCaA media. The isolates were characterized based on their cultural and morphological
characteristics. On screening the isolates, it was found that TKMB11 was significantly superior in mobilizing
soil K; followed by TKMB6, TKMB3 and TKMB8 in same order in efficiency. The present investigation indicates
that the application of KMB might serve as a cost-effective and alternate viable technology to mobilize insoluble
K-source in tea soil for sustainable crop improvement.

INTRODUCTION activity (Parmar and Sindhu, 2013; Sessitsch et al.,

2013).This category of beneficial microorganism has
Potassium is the second most important nutrient in been termed as potash mobilizing bacteria (KMB)
agricultural soil after nitrogen (Ranganathan and by Chandra and Greep (2006). Amino acids,
Natesan, 1985). Sustainable utilization of K as an vitamins and different growth promoting substances
active macronutrient has immense importance, since like indole acetic acids (IAA), gibberellins
K is involved with almost all kinds of biological (Ponmurugan and Gopi, 2006) are usually produced
reactions in plants (Bagyalakshmi et al., 2012). by these beneficial microbes to promote plant
According to Mikkelsen (2007), K is a major and growth. Microorganisms like Acidothiobacillus
essential macronutrient for normal plant growth and ferrooxidans, Bacillus mucilaginosus, B. edaphicus,
development. It can improve the resistance of crops Burkholderia sp., Frateuria sp., Pseudomonas sp.,
in diverse biotic and abiotic stresses and against pests Rhizobium sp., etc. (Lian et al., 2008; Liu et al.,
and diseases. The extent of K released by soils, 2012) are known for their potential in releasing
however, depends on change in different soil insoluble native K-source in soil into a plant
parameters topographical and biogeochemical available nutrient pool. Biodissolution of mineral
characters etc. (Basak and Biswas, 2009). nutrients, thus, becomes an integral part of integrated
nutr ient management (INM) strategies
Microorganisms have the ability to mobilize the (Adesemoye et al., 2008). Information related to
mineral nutrients in soil, thus, making it available potash mobilizers in tea ecosystem and their
for efficient plant uptake. For instance, one of the suitability in increasing the available K in tea soil
possible means of utilizing feldspar, waste mica or are still scarce. The present investigation has been
rock phosphate is by mobilizing their K through initiated to characterize the KMB strains isolated
beneficial microorganisms, where unavailable K is from tea soil and evaluate their potential in nutrient
converted to plant available form through microbial management.
*
Corresponding author: [email protected]

8
MATERIALS AND METHODS Screening of potash mobilizing activities

Soil sampling The abilities of the isolated bacteria to release K in

GYCaA media were investigated. For this, sterilized
Soil was sampled from Tocklai Experimental Tea GYCaA medium was poured into sterilized
Garden, at Jorhat district (26º75 // N, 94°22// E), petriplates, containing feldspar as the sole source
Assam, Northeast India. Ten different tea growing of potash. After solidification of the media, the plates
locations were selected for the collection of soil were inoculated with the isolated bacterial strain and
samples. Soil was sampled from rhizosphere at 6- incubated at 28-30°C and assayed visually up to 7
10 cm depth, with sterilized hand auger, in aseptic days. Colonies exhibiting clear zone of K release
conditions. Three spots at each location and a total were selected from 104 and105dilutions containing
of 30 sampling points were selected for the plates. A secondary screening was also carried out
collection of soil samples. Randomly collected soil by observing the zone activity of different bacterial
samples were mixed thoroughly, air-dried and passed isolates using Khandeparkar ’s selection ratio
through a 2.0 mm sieve to remove the debris. The (Prajapati and Modi, 2012).
composite sample for each location was brought to
the laboratory and used for the isolation of potash
mobilizing microbes. The soil samples were kept in
a refrigerator at 4±1 °C till isolation procedures were Incubation experiment
completed.
An in vitro study was conducted to evaluate the
Isolation and characterization of potash efficiency of the potent bacterial strains in increasing
mobilizing bacteria the available potash present in the soil. For this, each
isolated strain was inoculated separately in 1.0 kg
In the present investigation, a culture-based of sieved soil at 10% volume/weight (v/w) levels
approach was primarily used to isolate and and incubated at 25°C, with 75% field capacity
characterize the potash mobilizing microbes. 10 g moisture levels for a maximum of 150 days. A
of air-dried and sieved soil sample was suspended constant moisture level (40%) was maintained
in 100 mL of sterile distilled water (SDW) and throughout the experiment by periodic addition of
incubated in an orbital shaking incubator at 28°C water. All the pots were arranged by using the
with periodic shaking at 150 rpm for 30 min. Ten complete randomized design (CRD) with three
fold dilutions were prepared serially, by taking 10 replications (Fig. 1). Soil samples were analyzed at
mL of the soil suspension and dispensing it into 90 periodic intervals like 30, 60, 90, 120 and 150 days
mL of SDW. Soil particles were allowed to settle after incubation for potash availability, following
and the soil suspension of required dilution was the standard procedure of Jackson (1973).
inoculated into modified Glucose Yeast Agar
(GYCaA) medium (glucose 20 g/L, yeast extract 3
g/L, calcium carbonate 5 g/L, agar 18.5 g/L)
supplemented with 5% of feldspar using an
enrichment technique in accordance with Chandra
and Greep, (2006). The plates were incubated at
28±2°C for 7 days for optimum growth of microbes.
Five replicates were maintained in each case.
Colonies surrounded by clear zones were picked up
and streaked onto GYCaA media containing plates.
The plates were again incubated under the same
conditions to confirm their abilities to solubilize
potash. Colony characteristics such as size, shape,
colony type, zone of clearance and consistency were
examined in accordance with Chandra and Greep
(2006) and Cappuccino and Sherman (2004). Pure
colonies were transferred to nutrient agar slants and
stored at 4°C in the Culture Collection Laboratory Fig. 1. KMB strains with well demarcated clearing
(CCL) of the Mycology and Microbiology zones in modified Glucose Yeast Agar (GYEA) media.
Department for further identification. a. TKMB6; b. TKMB11; c. TKMB3; d. TKMB8

9
Data analysis Table 2. Potassium solubilization values of isolated
strains using Khandeparkar’s selection ratio
Standard errors mean (SEM) was calculated at 5%
Zone of Diameter of
level. Duncan Multiple Range Test (DMRT) KMB isolates clearance growth D/d ratio
comparisons were made to analyze available soil K (D) (mm) (d) (mm)
using SPSS 16.0. TKMB-1 2.4 2.1 1.1
TKMB-2 2.5 1.7 1.5
TKMB-3 3.3 2.0 1.7
TKMB-4 3.1 2.2 1.4
RESULTS AND DISCUSSION TKMB-6 2.7 1.3 2.1
TKMB-8 4.1 2.6 1.6
Isolation and characterization of potash TKMB-9 2.1 1.3 1.6
TKMB-10 2.6 1.9 1.4
mobilizing microbes TKMB-11 3.3 1.1 3.0
TKMB-12 2.0 1.5 1.3
Altogether ten isolates were obtained in the present
investigation, on modified GYCaA media. The gram
Table 3. Effect of inoculated KMB strains in
staining reactions, agar slant culture characteristics increasing available soil potash at different
and the cell shape of the isolated strains are shown incubation periods
in Table 1. Most of the microbial isolates were gram
negative, rod-shaped, motile and creamish in Treatments Incubation period (days) Mean
30 60 90 120 150
appearance. The clear-zone in the modified Glucose Available potash
Yeast Agar (GYCaA) media indicated the growth (mg/kg of soil)
and utilization of potassium. The exhibition of Control 90 90 90 91 90 90
potassium releasing zone by the potent isolates are TKMB 3 100 124 127 125 124 120
TKMB 6 101 124 128 125 125 121
shown in Fig. 1. Our results corroborate with the TKMB 8 93 111 115 113 113 109
findings of Chandra and Greep (2006), who too were TKMB 11 118 122 128 127 127 124
able to grow KMB isolated from banana rhizosphere Mean 125.58 142.50 146.92 145.17 144.92
SEM 1.281
on GYCaA media using an enrichment technique. For comparing two at 5%
Clear zones appeared within 72 h of incubation and Strains 1.13
this may have been due to the production of Time 0.90
Strains x time 2.54
antimicrobial substances such as chitinolytic
enzymes, cellulase, antibiotics by potash mobilizing the zone activity using Khandeparkar’s selection
bacteria. ratio were also made by Prajapati and Modi (2012).
Screening of these indigenous and native strains,
Potassium solubilization values of the isolated thus, creates a possibility in utilizing them as
bacterial strains were shown in Table 2. bioinoculants in tea alone or in combination with
Khandeparkar’s selection ratio (zone of clearance low doses of potash supplements.
(D)/diameter of growth (d) ratio) was maximum in
TKMB11 (3.0 mm), followed by TKMB6 (2.1mm). Effect of potash mobilizers in enhancing K
Khandeparkar ’s selection ratio was measured availability
minimum (1.6 mm) in TKMB8. Estimation of
potassium solubilizing activity of bacterial strains, In the present investigation, bacterial inoculation
isolated from ceramic industry soil by examining was found to be a significant approach in improving

Table 1. Culture characteristics of the KMB strains

Sl. no. KMB isolates Colony morphology Cell shape Gram reaction
1. TKMB-1 Creamish white, irregular margin, powdery surface Rod Gram (- )
2. TKMB-2 Creamish white, regular margin, slimy surface Rod Gram (-)
3. TKMB-3 Creamish white, serrated margin, slimy surface Rod Gram (-)
4. TKMB-4 Creamish yellow, irregular margin, slimy surface Rod Gram (+)
5. TKMB-6 White, furrowed margin, slimy surface Rod Gram (-)
6. TKMB-8 Creamy white, irregular margin, slightly elevated, slimy surface Rod Gram (-)
7. TKMB-9 Yellow, irregular margin, sticky elevated surface Rod Gram (+)
8. TKMB-10 Greyish white, irregular and furrowed margin, slimy surface. Cocci Gram (+)
9. TKMB-11 Creamy white, irregular margin, sticky surface Rod Gram (-)
10. TKMB-12 Creamish white, irregular margin, slimy surface Rod Gram (-)

10
the soil nutrient availability. Release of K content Basak, B.B. and Biswas, D,R. (2009). Influence of
by inoculated KMB strains in soil is the sole reason potassium solubilizing microorganism
here. Inoculation of maize and wheat plants with (Bacillus mucilaginosus) and waste mica on
different potash mobilizers resulted in higher K potassium uptake dynamics by sudan grass
mobilization (Singh et al., 2010). The isolated KMB (Sorghum vulgare Pers.) grown under two
strains, when tested for their efficiency in increasing Alfisols. Plant Soil, 317:235-55.
available potash in tea soil, four strains TKMB11,
TKMB6, TKMB3 and TKMB8 were found to be Cappucino, J.G. and Sherman, N. (2004). In
effective in increasing available potash in the soil Microbiology – A Laboratory Manual. 7th
(Table 3). The effect was more with strain TKMB11, edition. Pearson Education, Dor ling
followed by TKMB6, TKMB3 and TKMB8, Kindersley (India) Pvt. Ltd.
respectively. The bacteria may produce bacterial
acids, alkalis or chelants to enhance the release of Chandra, K. and Greep, S. (2006). Potash mobilizing
potassium containing minerals in soil (Sugumaran bacteria (Frateuria aurentia). Regional
and Janarthanam, 2007). The present findings Centre of Organic Farming, No. 34, 5th Main
corroborate with Basak and Biswas (2009) who Road, Hebbal, Bangalore. 24.
studied the influence of potash solubilising microbes
in increasing K-dynamics under two alfisols. Jackson, M. (1973). In Soil chemical analysis.
Prentice Hall of India. Pvt. Ltd., New Delhi,
pp. 1-498.
CONCLUSION
Lian, B., Wang, B., Pan, M., Liu, C. and Teng, H.H.
The present research work focused on the (2008). Microbial release of potassium from
exploitation of potash mobilizing microorganisms K-bearing minerals by thermophilic
for their significant potentiality in improving fungus Aspergillus fumigatus. Geochimica
available K status in soil. Fur ther field et Cosmochimica Acta, 72(1):87-98.
experimentation will fix the dose, method of
application etc. With the existing economic crisis Liu, D., Lian, B. and Dong, H. (2012). Isolation of
of tea growers, potash mobilizers may be an effective Paenibacillus sp. and assessment of its
biofertilize,r integrated with reduced level of K potential for enhancing mineral weathering.
fertilizers to reduce cost and support eco-friendly, Geomicrobiology Journal, 29:413-21. doi:
quality tea production. 10.1080/ 01490451.2011.576602

Mikkelsen, R. (2007). Managing potassium for

ACKNOWLEDGEMENT organic crop production. In: Western
Nutrient Management Conference, pp 111-
The authors are thankful to the Director, TTRI, TRA, 116, Salt Lake City, UT.
Jorhat, Assam, India for providing the necessary
facilities to undertake the study. Parmar, P.  and  Sindhu,  S.S.  (2013).  Potassium
solubilization by rhizosphere bacteria:
Influence of nutritional and environmental
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