Bioprospecting of phosphate-solubilizing bacteria from Isabgol (Plantago ovata Forsk) rhizospheric soils in western arid Rajasthan

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1 Bioprospecting of phosphate-solubilizing bacteria from Isabgol (Plantago ovata Forsk) rhizospheric soils in western arid Rajasthan B. K. Mishra 1*, Ashutosh Sharma 1, P. N. Dubey 1 and Arunabh Joshi 2 1 ICAR-National Research Centre on Seed Spices, Tabiji, Ajmer, India 2 Deparment of Molecular Biology and Biotechnology, RCA, MPUAT, Udaipur, India * bkmmicro@gmail.com ABSTRACT Isabgol (Plantago ovata Forsk) is one of the important cash crops among the cultivated medicinal plants in arid region of India. Composite soil samples from Isabgol growing fields were collected from 13 different sites distributed across western arid Rajasthan of India. The soil sample was collected at the depth of 0 to 15 cm within plots. Soil organic matter was estimated on the basis of per cent organic carbon. The soil samples were analysed for estimation of total viable mesophilic bacterial count and phosphates solubilizing bacterial (PSB) population through serial dilution technique followed by pour plating on nutrient agar medium and National Botanical Research Institute Phosphate (NBRIP) agar medium on Petri-plates under aseptic conditions, respectively. Highest PSB population (2.97 log 10 cfu/g) was recorded with soils having medium organic matter (0.6 %) and high total viable mesophilic bacterial population. Out of seventy rhizobacterial isolates only forty three bacterial cultures were found to have capacity towards phosphate solubilization. The phosphate solubilization index ranged from 1.36 to The maximum tricalcium phosphate solubilization (77.72 µg/ml) was assayed with ISB-49 although it showed low phosphate solubilization index (1.64). The minimum tricalcium phosphate solubilization (6.76 µg/ml) was recorded with ISB-43 though it showed higher phosphate solubilization index (3.89). The results obtained in present investigation clearly indicated that the efficient PSB distribution and occurrence in soil varies with organic matter and other edaphic factors. KEY WORDS: Isabgol, phosphate-solubilizing bacteria, Plantago ovata, rhizosphere INTRODUCTION Isabgol (Plantago ovata Forsk) is one of the most important crops among the cultivated medicinal plants grown in India. It belongs to family Plantaginaceae and genus Plantago having 8 chromosomes (2n=8) and known by different names such as psyllium, spogel seed, ishagola and isaghul. Isabgol is a native of Mediterranean region and West Asia. Of 200 species of genus Plantago, only two, namely P. ovata and P. psyllium have been extensively used for the production of seed, seed husk and mucilage (active substance) which are used in pharmacy and other industries such as pulp and paper production, loom, military and petroleum extraction (Dhar, et al., 2005). Isabgol contains significant amount of proteins and the husk yields colloidal mucilage, which are valued for medicinal application. The husk is the rosy-white membranous covering of the seed which constitutes the drug and is given as a safe laxative, particularly beneficial in habitual constipation, chronic diarrhea and dysentery. India is the largest producer of Isabgol and exports seed and husk to many countries. Isabgol crop is cultivated in Rajasthan, Gujarat, some pockets of Madhya Pradesh and Haryana. In India, maximum cultivation of Isabgol crop is in Rajasthan followed by Gujarat. 9

2 Isabgol is an irrigated winter season (rabi) crop which remains in the field for about 4 months. The crop is grown on marginal, light, well-drained sandy-loam to loamy soils having ph range from 7.2 to 7.9. It requires a cool climate and dry sunny weather as even light showers cause seed shedding. The optimum sowing time is early November; sowing however, is extended till the end of December, but the delayed sowing decreases the yield. The seed rate is 7-8 kg per hectare. Isabgol makes a moderate demand for nutrients. Usually, 25 kg of each N and P per hectare is given at plantation depending upon the fertility of soil and weather conditions (Maiti and Mandal, 2000). Although, Isabgol crop is regarded as moderate nutrient seeker crop, major areas of this crop falls under semiarid and arid region of western Rajasthan and Gujarat in India which are naturally deficient in organic matter resulting poor fertility level having alkalinity and other adverse edaphic stresses. Phosphorus (P) is an essential element in plant development. Although soils generally contain a large amount of total P, only a small proportion is immediately available for plant uptake. This explains why P deficiency is a major constraint to crop production in Indian soils. To meet the crop demand, farmers apply up to three- to fourfold the required amount of P to crops, causing a substantial increase in production costs. Due to the high cost of mineral fertilizers, most smallholder farmers opt not to use fertilizers, which ultimately results in poor yields. Microorganisms are involved in a range of processes that affect the transformation of soil P and are thus an integral part of the soil P cycle. Recently, phosphate solubilizing microorganisms have attracted the attention of agriculturists as soil inoculums to improve the plant growth and yield. Microbes play an important role in plant growth and nutrition and reduce the need for chemical fertilizers. Phosphorus being an essential element for plant growth and development remains in insoluble form or in the form of insoluble metal chelates in soil (Vassilev et al., 2006). It is generally accepted that the mechanism of mineral phosphate solubilization by phosphate solubilizing biofertilizer (PSB) strains is associated with the release of low molecular weight organic acids (Mahalakshmi and Reta, 2009), which through their hydroxyl and carboxyl groups chelate the cations bound to phosphate, thereby converting it into soluble forms. With the emphasis on screening for potential PSB from the soil for the agricultural purposes the present experiment was designed to isolate and screen phosphate solubilizing potentials of indigenous rhizospheric bacteria from the Isabgol crop grown under arid regions in Rajasthan state of India. Attempt was also made to establish the relationship of PSB with its soil organic matter and population of P-solubilizing bacteria in soils of different Isabgol cultivating area. MATERIALS AND METHODS The present investigation was carried out at ICAR- National Research Centre on Seed Spices, Tabiji, Ajmer (Rajasthan) in India during rabi season of Isabgol seedling and soil samples were collected from 13 different sites (Jalore, Barmer and Jaiselmer districts) distributed across western arid Rajasthan of India (Table 1). The sites were natural fields under Isabgol cultivation at the time of sampling. At each site, one composite soil sample was collected from 0 to 15 cm soil layer within the plots. Soil organic matter was estimated on the basis of organic carbon as per standard methodology. The soil samples were put in sterile polyethylene bags and immediately used for estimation of total viable mesophilic bacterial count and phosphate solubilizing bacterial population through serial dilution technique. This was followed by pour plating on nutrient agar 10

3 medium and National Botanical Research Institute Phosphate (NBRIP) agar medium on petri-plates under aseptic conditions, respectively as described by Lavania and Nautiyal (2013). For serial dilution of rhizospheric soil samples, 10 grams of soil was suspended in 90 ml sterile physiological saline water (9 g/l NaCl) and mixed thoroughly for 45 min using a mechanical shaker. Serial dilutions were then prepared up to One ml aliquot of each serial dilution was plated evenly on nutrient agar plates (beef extract, 1 g/l; yeast extract, 2 g/l; peptones, 5 g/l; NaCl 5 g/l; agar 15 g/l; adjusted to ph 7.4 before autoclaving) and NBRIP agar plates [glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; MgCl 2 6H 2 O, 0.5 g; MgSO 4 7H 2 O, 0.25 g; KCl,0.2 g; (NH 4 ) 2 SO 4, 0.1 g and agar, 15 g in 1000 ml double distilled water and ph was adjusted to 7.0 using 1 M NaOH before autoclaving]. The samples were incubated for 3 days at 28±2 C. The bacterial populations were expressed as colony forming units (log 10 CFU) per g of soil. The PSB colonies were identified according to their morphology and colony characteristics. Effective PSB were detected by the presence of clear zones of solubilization surrounding or underneath the colony. All of the morphologically different colonies were purified serially in NBRIP agar plates and finally kept in sterile glycerol (20%) under refrigerated condition. All the bacterial strains isolated on NBRIP growth medium were maintained on the nutrient agar slants. Total forty four cultures were isolated based on their colony morphology. These bacterial isolates were designated as ISB 28 to ISB-70. These isolates were further screened for acid production and capacity to solubilise the tricalcium phosphate. All bacterial strains were tested by an agar assay using NBRIP medium supplemented with 1.5% Bacto-agar. Four bacterial strains per plate were stabbed in triplicate using sterile toothpicks. The halo and colony diameters were measured after 14 days of incubation of the plates at 25 C. The ability of the bacteria to solubilize insoluble phosphate was described by the solubilization index [= the ratio of the total diameter (colony + halo zone) to the colony diameter]. Phosphorus solubilizing ability of bacterial strains were tested in Pikovskaya s broth with composition of yeast extract 0.5 g; dextrose 10.0 g, calcium phosphate 5.0g; ammonium sulphate 0.5 g; potassium chloride 0.2g; magnesium sulphate 0.1g; manganese sulphate 0.1 mg and ferrous sulphate 0.1mg in 1000 ml double distilled water. Erlenmeyer flasks containing 50ml Pikovskaya s broth were inoculated with 8% (v/v) bacterial growth suspension and incubated on shaker at 28C for 7 days. After centrifuging at 10,000 rpm for 15 min, bacterial growth cultures were separated from Pikovskaya s broth for estimation of concentration of P released in the medium. Dissolved phosphate concentration in the culture filtrate was determined by vanado-molybdate method as described by Kaushik et al. (2004) and expressed in terms of µg /ml phosphorus released in culture broth. RESULTS AND DISCUSSION After nitrogen, phosphorus (P) is second most important nutrient, limiting the agricultural production. Soils are often abundant in insoluble P, either in organic or inorganic forms, but deficient in soluble phosphates essential for growth of most plants and microorganisms. Soluble forms of phosphate fertilizers are widely applied to agricultural soils in order to circumvent P-deficiency but 75 to 90% of added P is rapidly precipitated as insoluble forms and becomes unavailable to plants. Viewing these, an attempt has been made to 11

4 evaluate efficiency of P solubilising activity of isolated bacteria and their occurrence in Isabgol fields in arid region of India. The thirteen soil samples from Isabgol fields were analyzed for total organic matter, total viable mesophilic bacterial population and total phosphate solubilizing bacterial population. Maximum organic matter (0.604 %) was recorded in samples of Kharwa, Bhinmal- B in Jalore district of Rajasthan and minimum organic matter ( 0.137%) was recorded for Fatehgarh, Sam in Jaiselmer district of Rajasthan and mean of soil organic matter in collected Isabgol fields samples was found to be low (0.421 %). Table 1: Soil organic matter, total viable mesophilic bacterial population and phosphate solubilizing bacterial population (Log 10 CFUs/g) in Isabgol growing soils Soil sample District Village/location Soil organic matter (%) Total viable mesophilic bacterial population P-1 Jalore Kharwa, Bhinmal-A Total phosphate solubilizing bacterial population P-2 Jalore Kharwa, Bhinmal-B P-3 Jalore Kharwa, Bhinmal-C P-4 Jalore Ghaseri, Bhinmal P-5 Jalore Hadetar, Sanchoor-A P-6 Jalore Hadetar, Sanchoor-B P-7 Jalore Hadetar, Sanchoor-C P-8 Barmer Sajitada-A P-9 Barmer Sajitada-B P-10 Barmer Nimbli P-11 Barmer Bhukha P-12 Jaiselmer Fatehgarh, Sam P-13 Jaiselmer Pokharana Mean SD Total viable mesophilic bacterial population ranged between from to log 10 CFUs/g in Isabgol soil samples and mean of soil bacterial population in collected Isabgol fields samples was found to be low (7.111 log 10 CFUs/g). Maximum mesophilic bacterial population (7.995 log 10 CFUs/g) was recorded with Sajitada-B in Barmer district having lower organic matter (0.364 %) and that of minimum bacterial population (5.977 log 10 CFUs/g) was observed with Kharwa, Bhinmal- B in Jalore district with soil samples (Table 1). This reduced microbial population in soil samples of high organic matter may be due to some other edaphic factors such as alkaline ph and other elemental composition and previous crop due to allelopathic effect. Soil microorganisms, just like higher plants depends entirely on soil for their nutrition, growth and activity. Total PSB population was maximum (2.973 log 10 CFUs/g) for samples from Nimbli in Barmer district where as minimum PSB population (2.041 higher organic matter (0.604) content in 12

5 log 10 CFUs/g) for samples from Bhukha in Barmer district of Rajasthan with low average of PSB population in comparison to total mesophilic bacterial population in different Isabgol field soil samples (Table 1). This may be one of probable reasons for arid soils being deficient in available phosphorus. Similar observations were made by Ndung u-magiroi et al. (2012) who had determined the phosphorus (P)- solubilizing efficiency of native populations of phosphate-solubilizing bacteria (PSB) in 13 Kenyan soils with differing chemical characteristics and reported that PSB populations varied among the sites tested and had a positive and significant correlation (p 0.05) with organic carbon (r =0.76), exchangeable calcium (r =0.93) and exchangeable magnesium (r =0.92) and among the total 150 PSB isolates only 5% of the total isolates were efficient in terms of phosphate-solubilizing efficiency. Phosphate solubilizing bacteria (PSB) are heterotrophic bacteria which depend on external C sources to solubilize P. They also require energy and essential nutrients, including Ca, to grow and reproduce. The C in these soils is mainly associated with decomposing organic matter and higher the soil content of decomposing organic matter, the more active and prolific the soil microbes are. Vikram et al. (2007) also reported high populations of heterotrophic PSB in Vertisols with high organic C, demonstrating the need to improve soil organic matter in soils through improved soil fertility management. No significant correlations between PSB populations and total bacterial population were observed; most likely due to the low range of organic matter levels in those soils, since the soils were collected from cultivated field which had not received high organic manures during the current seasons due to less availability of organic manures as well as the soil of western arid region of Rajasthan are prone to fast mineralization of organic matter due to prevailing high temperature and dry climate. Out of 70 rhizobacterial isolates only forty three bacterial cultures were found to have capacity for phosphate solubilization. The maximum tricalcium phosphate solubilization (77.72 µg /ml) was assayed with ISB-49 although it was showing low (1.64) phosphate solubilization index The minimum tricalcium phosphate solubilization (6.76 µg /ml) was assayed with ISB-43 though it was showing higher (3.89) phosphate solubilization index (Table 2). Similar observations were made by Mishra et al. (2015) in case of phosphate solubilization potential of native PSB isolates from fennel crop rhizosphere soil of Rajasthan state in India. This clearly indicates that qualitative screening for phosphate solubilization based on halo zone around bacterial isolates is not a desirable method instead qualitative estimation of tricalcium phosphate solubilization under liquid growth medium should be considered for screening of PSB isolates. These results indicates that PSB exists in the rhizosphere of Isabgol grown in Rajasthan and might help plants to take up phosphorus by roots. Similarly, a total of 40 isolates were isolated from different rhizospheric soil and roots of cauliflower from the vicinity of three districts (Kangra, Hamirpur and Bilaspur) of Himachal Pradesh belonging to subtemperate Himalayan location. Out of 40, only 5 isolates (MK 2, MK 4, MK 5, MK 7 and MK 9 ) showed maximum plant growth promoting attributes like P- solubilization, N- fixation etc. All the five isolates solubilized tricalcium phosphate under in vitro conditions however, MK5 and MK7 had highest phosphate solubilizing efficiency of and % were recorded (Kaushal and Kaushal, 2013). 13

6 Table 2: Screening of Phosphate solubilization capacity of Isabgol rhizospheric bacterial isolates S. No. Soil sample Bacterial Isolate Tricalcium phosphate solubilization (µg /ml) 1 P-1 ISB P-1 ISB P-1 ISB P-1 ISB P-1 ISB P-1 ISB P-2 ISB P-2 ISB P-2 ISB P-2 ISB P-2 ISB P-2 ISB P-2 ISB P-2 ISB P-2 ISB P-3 ISB P-3 ISB P-3 ISB P-3 ISB P-3 ISB P-3 ISB P-3 ISB P-3 ISB P-4 ISB P-4 ISB P-4 ISB P-4 ISB P-4 ISB P-5 ISB P-6 ISB P-7 ISB P-7 ISB P-8 ISB P-9 ISB P-10 ISB P-10 ISB P-10 ISB P-10 ISB P-11 ISB P-11 ISB P-12 ISB P-13 ISB P-13 ISB Mean SD

7 Although the mechanism of the bacterial phosphate solubilizing activity observed in the current study is not clear at present, all of the isolates slightly decreased the ph value of the culture medium suggesting that organic acid secretion by the bacteria might play a role in phosphate solubilizing activity. Several lines of evidence suggest that the principal mechanism for mineral phosphate solubilization is the production of organic acids, and acid Phosphatases by PSB which play a major role in the mineralization of organic phosphorous in soils (Vessey, 2003). During the past couple of decades, the use of microbial inoculants for sustainable agriculture has increased tremendously in various parts of the world. Significant increases in growth and yield of agronomical important crops in response to inoculation with b/iofertilizers have been repeatedly reported (Kumar et al., 2013; Mishra et al., 2013). Use of microorganisms for plant growth promotion and disease control is well recognized. There is huge potential for the use of PSB as biofertilizing agents for a wide variety of crop plants in a wide range of climatic and edaphic conditions. To enhance sustainability of agroecological system, isolation of native strains adapted to the environment and their study may contribute to the formulation of PSB inoculants to be used in region specific crops. CONCLUSION The results of present study indicated that there were many PSB strains in the soils tested, but only few were effective in terms of their phosphatesolubilizing ability. It is therefore unlikely that native PSB contribute significantly to solubilizing phosphate in the soils tested, which would ultimately benefit plant growth. Therefore, inoculation with effective strains with a high P solubilization potential is necessary. Thus it can be concluded that the diverse agroclimatic conditions of arid Rajasthan offers a great potential for microbiological exploration for novel microbes and especially phosphate solubilizing bacteria for application in Isabgol and other agricultural crops. ACKNOWLEDGEMENTS: Department of Biotechnology (DBT), Ministry of Science & Technology, Govt. of India is duly acknowledged for funding this research project. Authors are also thankful to the Director, ICAR-NRC on Seed Spices (Indian Council of Agricultural Research), Ajmer, for providing necessary facilities during the present investigation. REFERENCES Dhar, M.K., Kaul S., Sareen S. and Koul A.K Plantago ovata: Genetic diversity, cultivation, utilization and chemistry. Plant Genetic Resources, 3(2): Kaushal, M. and Kaushal, R Screening and characterization of rhizobacterial strains of Bacillus spp. isolated from rhizosphere of cauliflower (Brassica oleracea var. botrytis L.). African Journal of Microbiology Research, 7: Kaushik, B.D., Saxena, A.K. and Prasanna, R Techniques in Microbiology: A laboratory manual for post graduate students, Indian Agricultural Research Institute, New Delhi, India, pp60. Kumar, M., Prasanna, R., Bidyarani, N., S., Mishra, B. K., Kumar, A., Adak, A., Jauhari, S., Yadav Kuldeep, Singh Rajendra, Saxena, A. K Evaluating the plant 15

8 growth promoting ability of thermotolerant bacteria and cyanobacteria and their interactions with seed spice crops. Scientia Horticulturae, 164: Lavania, M. and Nautiyal, C. S Solubilization of tricalcium phosphate by temperature and salt tolerant Serratia marcescens NBRI1213 isolated from alkaline soils. African Journal of Microbiology Research, 7: Mahalakshmi, S. and Reta, D Assessment of plant growth promoting activities of bacterial isolates from the rhizophere of tomato (Lycopersicon esculentum L.). Recent Research in Science and Technology, 1: Maiti, S. and Mandal, K Cultivation of Isabgol. National Research Center for Medicinal and Aromatic Plants, Anand, India, Bulletin, pp1-8. Mishra, B. K., Sharma Ashutosh, Aishwath, O.P., Sharma, Y.K., Kant Krishna, Vishal, M.K., Saxena, S.N. and Ranjan, J.K Microbiological profile of coriander (Coriandrum sativum L.) crop rhizosphere in Rajasthan and screening for auxin producing rhizobacteria. International Journal of Seed Spices, 3(2): Mishra, B. K., Singh, B., Singh, P., Rathore, S. S., Aishwath, O. P., Kant, K. and Dubey, P. N Isolation and evaluation of phosphate solubilizing microorganisms from fennel (Foeniculum vulgare Mill.) rhizospheric soils of Rajasthan. International Journal of Seed Spices, 5: Ndung u-magiroi, K. W., Herrmann, L., Okalebo, J. R., Othieno, C. O., Pypers, P. and Lesueur, D Occurrence and genetic diversity of phosphate-solubilizing bacteria in soils of differing chemical characteristics in Kenya. Annals of Microbiology, 62: Vessey, J. K Plant growthpromoting rhizobacteria as biofertilizers. Plant Soil, 255: Vikram, A., Krishnaraj, P.U., Hamzezarghani, H. and Jagadeesh, K.S Growth promotional potential of Pseudomonas fluorescens FPD 10 and its interaction with Bradyrhizobium sp. Research Journal of Microbiology, 2: [MS received 2 April 2015; MS accepted 11 June 2015] Disclaimer: Statements, information, scientific names, spellings, inferences, products, style, etc. mentioned in Current Biotica are attributed to the authors and do in no way imply endorsement/concurrence by Current Biotica. Queries related to articles should be directed to authors and not to editorial board. 16