Charcterization and screening of beneficial bacteria obtained on King s B agar from tea rhizosphere

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1 Indian Journal of Biotechnology Vol 6, October 2007, pp Charcterization and screening of beneficial bacteria obtained on King s B agar from tea rhizosphere Tanushree Mazumdar 1, C Goswami 1 and N C Talukdar 2 * 1 Soil Microbiology Laboratory, Department of Soil Science, Assam Agricultural University, Jorhat , India 2 Microbial Resources Division, Institute of Bioresources and Sustainable Development Department of Biotechnology (Government of India), Imphal , India Received 13 June 2005; revised 18 December 2006; accepted 22 February 2007 Nine fluorescent Pseudomonas isolates obtained on King s B agar from rhizosphere of tea plants were studied along with a reference strain P. fluorescens MTCC-103 for their biochemical and functional characteristics. They were also tested for their ability to promote growth of tea seedlings. Intrinsic antibiotic resistance profile (IARP) and SDS-PAGE banding pattern indicated that the isolates were distinct from each other and these two properties could be used as marker for their identification. The isolates produced IAA-like substances, siderophores and soluble P in the range of , and µg/ml culture filtrate, respectively. Four isolates were able to utilize cellulose as C-source and another four were capable of inhibiting growth of the saprophytic Rhizoctonia solani in laboratory bioassay. The isolates Psd 11, Psd 12 and Psd 13 were found to enhance total shoot and root biomass of 1-year-old tea seedlings, grown in a fertilizer unamended soil, to significantly greater magnitude. Psd 11 and Psd 13 were identified as two different strains of Bacillus circulans. The growth parameters of tea seedlings in fertilizer P added pot was statistically at par with those of superior strain inoculated seedlings. There was no statistically significant relationship between in vitro determined functional properties observed and the growth promotion ability of the isolates. Although, the three superior isolates were found to produce higher level of soluble P and exhibited maximum in vitro biocontrol activity against R. solani. Keywords: Bacillus, IAA, P solubilization, Pseudomonas, siderophore, tea growth promotion IPC Code: Int. Cl. 8 C12N15/10 Introduction Beneficial bacteria of plant rhizosphere comprise of several genera including Bacillus and Pseudomonas. They are becoming increasingly important component in the management of nutrients and harmful microorganisms in sustainable crop production 1. The mechanisms by which beneficial rhizobacteria promote plant growth may be through production of phytohormone, such as IAA 2, gibberalic acid 3, different types of siderophores 4 and antibiotic like substances 5, and through suppression of deleterious rhizobacteria 6 and solubilization of insoluble P 7. Tea plantation is managed by use of a large amount of chemical fertilizers and toxic pesticidal chemicals. The liberal use of synthetic fertilizers and pesticides has led to a global concern for environmental pollution as well as harmful side-effects created by their excessive use in tea plantation 8,9. These issues *Author for correspondence: Tel: / ; Fax: nctalukdar@yahoo.com are creating a high demand for organic tea. Therefore, characterization of beneficial bacteria from tea rhizosphere soil and adequate strategies to use them as inoculant and enhance their performance hold key to their successful use as bioinoculants in production of organic tea seedlings in nursery. Hence, the present study was carried out with the objective to screen rhizobacteria from tea rhizosphere for their ability to increase growth of tea seedlings in nursery. Materials and Methods Selection of Isolates Nine fluorescent Pseudomonas isolates were taken from the collection of soil microbiology laboratory of Assam Agricultural University, Jorhat for biochemical characterization and testing their effect on tea leaf cuttings. The isolates were arbitrarily designated as Psd 2, 3, 4, 6, 9, 11, 12, 13 and 19. A reference strain P. fluorescens MTCC-103 was also used. The cultures were maintained on King s B medium, since it is a selective medium for pseudomonads.

2 MAZUMDAR et al: CHARACTERIZATION AND SCREENING OF TEA RHIZOBACTERIA 491 Biochemical Characterization Determination of Intrinsic Antibiotic Resistance Profile (IARP) Six antibiotics, viz. kanamycin, streptomycin, rifampicin, gentamycin, ampicillin and chloramphenicol, were used to develop IARP of the isolates. Antibiotics containing King s B broths were inoculated with 50 µl of the culture broth of test bacterial isolates. The test tubes were incubated at 30 C. Appearance of growth of the isolates indicated resistance reaction and inhibition of growth indicated sensitive reaction. Observation was recorded after 48 h of incubation. The resistant populations were then inoculated in the same broth containing higher concentrations of the antibiotics and observations were made. Thus, the pattern of the antibiotics resistance profiles or minimum inhibitory concentration (MIC) value of the six antibiotics for the ten isolates was recorded. Protein Profile Analysis Soluble proteins were extracted from the broth culture of the isolates and electrophoresed in 12% separating gel. Bacterial isolates were grown in 50 ml of King s B broth up to an optical density (OD) 0.6 on an environmental shaker (Rotek-LES) at 180 rpm at 32ºC. The cells were harvested by centrifugation at 10,000 g for 5 min at 4ºC, resuspended in 5 ml of extracting buffer solution and sonicated (model Labsonic (R) U) with micro tip for 30 sec. After sonication, cell debris was precipitated by centrifugation at 10,000 rpm for 20 min at 4ºC. The SDS-PAGE banding pattern was analyzed using SPSS-10 for MS-Windows package to develop similarity matrix and dendrogram. This package uses unweighted average pair group method. Functional Characteristics Determination of IAA-like Substances Production of IAA-like substances was determined by a modified procedure 10. The isolates were inoculated in 50 ml of King s B broth supplemented with 0.1mg/mL and incubated at 32ºC for 96 h at 180 rpm. Bacterial cultures were centrifuged at 10,000 rpm at 4ºC for 15 min. Supernatant acidified to ph 2.5 with 12 N HCl was extracted thrice with 10 ml of ethyl acetate at 30 min interval. Ethyl acetate fraction was evaporated at 40ºC under vacuum and residue was redissolved in 2 ml of absolute methanol and mixed with 2 ml of Fe-HClO 4 reagent (1 ml 0.5 M FeCl 3 & 50 ml 35% HClO 4 ). After 25 min, OD was measured at 530 nm. Determination of Siderophore Production Siderophore production was assayed using modified CAS solution. Cultures were inoculated in 50 ml of modified M9 medium and incubated at 30ºC for 5 d at 180 rpm and centrifuged at 10,000 rpm at 5ºC for 10 min. Supernatant was filtered through 0.45 µm membrane filter. The concentration of siderophore in the filtrate was measured by mixing equal volume of CAS solution and filtrate. After allowing the solutions to equilibrate for 3-4 h, absorbance was measured at 630 nm. A standard curve was prepared by analyzing the absorbance (630 nm) of each standard solution divided by the absorbance (630 nm) of the reference solution (A/A ref) as a function of siderophore concentration 5. Estimation of P Solubilization Soluble P content in cell free extract was determined using Bray s reagent 11. Biocontrol of Rhizoctonia solani by Fluorescent Pseudomonads Biocontrol of Rhizoctonia solani by the isolates was tested by following the dual agar culture 12. A 6 mm diam mycelial disc cut from a 7-d-old culture of R. solani was placed at the center of a fresh Potato Dextrose Agar (PDA) plate. A fluorescent pseudomonad was then placed at c 2 cm away from the agar disc. The diameter of R. solani was measured on the 5 th d after inoculation. Determination of Ability to Utilize Cellulose as C-source and Cellulase Activity of Isolates Isolates were streaked onto Omeliansky s agar media and incubated at 30ºC for 4 d. Their growth on cellulose supplemented media was taken as indication of cellulose degradation ability. Those isolates showing the ability to utilize cellulose were inoculated into 50 ml of Omeliansky broth and incubated at 30 C for 25 d to determine their cellulase activity. These cultures were centrifuged at 2500 g for 10 min and the extracted enzyme (0.5 ml) was added to 32 mg of Whatman No. 1 filter paper. After incubated for 1 h at 50ºC, DNS reagent (0.5 ml) was added and the mixture was heated in a boiling water bath for 5 min. While the tubes were warm, 1 ml of 40% potassium sodium tartarate solution was added. The final volume was made up to 5 ml by adding water and absorbance was measured at 540 nm. Screening of Isolates for Growth Promoting Ability to Tea Cuttings at Callus Initiation Stage A mixture of 500 g surface and sub-surface soil (ph 6.0; O.C., 0.72%; available N, kg/ha;

3 492 INDIAN J BIOTECHNOL, OCTOBER 2007 available P 2 O 5, 40 kg/ha; available K 2 O, kg/ha) was used in each sleeve. The cultures were applied in sterilized compost carrier. Compost with single super phosphate constituted another treatment. Tea cuttings (at callus formation) of clone TV-1 were planted in the pots of respective treatments. The compost-based inoculant of individual isolate was 2 g/pot in a hole and the leaf cutting with callus was inserted. Each treatment was replicated ten times. Growth of the tea plants was recorded after 365 d of planting. Results and Discussion Of the nine bacterial isolates initially identified as P. fluorescence, two were strains of Bacillus circulans (Psd 11 and Psd 13) as identified by MTCC, Institute of Microbial Technology, Chandigarh. Two biochemical markers, i.e. IARP and SDS-PAGE banding pattern of the isolates, were determined in the present study to ascertain that Psd 11 and Psd 13 were different. The antibiotic resistance profile of each of the isolates was unique, except for Psd 11, Psd 12 and Psd 13 showing a degree of similarity. Psd 9 showed the highest level of resistance to all the antibiotics tested, while Psd 4 showed exceptionally higher level of resistance (up to 1600 ppm) to kanamycin and ampicillin (Table 1). Psd 6 also showed resistance to kanamycin up to 1200 ppm concentration. In general, ampicillin and kanamycin were the two antibiotics towards which most of the isolates exhibited higher resistance. The intrinsic levels of resistance pattern for a particular strain to a host of antibiotics can be used to identify the strain. SDS-PAGE banding pattern of soluble proteins also indicated variations among the isolates. The banding patterns were unique for the isolates (Fig. 1A) and they were separated into five clusters based on the comparison of the Jaccard s similarity co-efficient (Fig. 1B). Although Psd 11 and Psd 13 were two strains of B. circulans, they were quite distantly placed on the basis of SDS-PAGE banding pattern. On the contrary, the IARP of the two strains matched to a degree, suggesting the diversity of the two strains lies in property other than IARP. These results suggest that these patterns may be used to confirm the identity of the individual isolates in case of their contamination or otherwise. Presence of a contaminant isolate will obviously provide an IARP and SDS-PAGE pattern different than the ones characteristically seen in case of the pure cultures. The isolates varied in magnitude of five functional traits determined in vitro. They produced higher level of IAA, i.e. in the range µg/ml culture filtrate (Table 2). Earlier reports indicated that isolates of P. fluorescens of peanut rhizosphere produced IAA in the range of ppm 13. Isolate Psd 19 produced highest quantity of IAA-like substances (32.1 µg/ml) and the isolate Psd 6 produced the lowest quantity (8.7 µg/ml). Out of the nine isolates, seven grew on tricalcium phosphate (TCP) supplemented Pikovskaya s agar. Psd 4 and Psd 6 did not develop colonies on Pikovskaya s agar. The soluble P content in TCP supplemented Pikovskaya s broth ranged from 1.38 to 15.7 µg/ml of culture filtrate (Table 2). The maximum amount of soluble P was found in case of Psd 11 and this was also supported by ph drop in the broth. The drop in ph of the Pikovskaya s broth culture is brought about mainly due to production of organic acids 11. The highly significant correlation between ph drop and Isolates Table 1 Intrinsic antibiotic resistance profile of isolates obtained from tea rhizosphere soil Concentration of antibiotics (ppm) Ampicillin Chloramphenicol Kanamycin Rifampicin Gentamycin Streptomycin Psd Psd Psd Psd Psd Psd Psd Psd Psd MTCC Values indicate the minimum inhibitory concentration at which the isolates were found to be sensitive

4 MAZUMDAR et al: CHARACTERIZATION AND SCREENING OF TEA RHIZOBACTERIA 493 Fig. 1 (A): SDS-PAGE banding pattern of the bacterial isolates (lane M, marker PMW-B, Bangalore Genei; lane 1, Psd 2; lane 2, Psd 3; lane 3, Psd 4; lane 4, Psd 6; lane 5, Psd 9; lane 6, Psd 11; lane 7, Psd 12; lane 8, Psd 13 & lane 9, Psd 19), (B): Dendogram showing the relatedness among the bacterial isolates. Table 2 Functional properties of isolates obtained from tea rhizosphere, suppression of growth of R. solani and effect of isolates on growth of tea seedlings Isolates IAA production (µg/ ml) Siderophore production (µmole/ ml) Soluble P in culture filtrate (µg/ ml) ph of the broth culture Growth in Omeliansky s agar Colony diameter of R. solani in presence of isolates (cm) Shoot wt of tea seedlings (mg) Root wt of tea seedlings (mg) Control Psd Psd Psd Psd Psd Psd Psd Psd Psd MTCC LSD soluble P content (-0.921**) in the Pikovskaya s broth is an indication that the production of organic acids by the isolates may be a mechanism of solubilization of TCP. The isolates produced siderophores in the concentration range of 13.6 to µm/ml culture filtrate with the highest production by Psd 2 and lowest by Psd 9 (Table 2). The reported range of siderophore production in CAS assay solution was 130 to 230 µm/ml culture filtrate 4. It has been reported that variation in the siderophore production by P. fluorescens and P. aeruginosa was due to a higher requirement for iron by P. aeruginosa compared to P. fluorescens 14. The variation among the isolates at similar level of Fe, in the present study, could be their inherent attribute. In the laboratory bioassay, growth of R. solani was inhibited to maximum extent by isolates Psd 9, Psd 11, Psd 12 and Psd 13 as measured by the zone of growth inhibition (Table 2). In an earlier study, it was shown that in vitro siderophore producer PGPR strains could convert a Fusarium-conducive soil into Fusarium-suppressive soil for better plant performance 15. In the present investigation, no relationship was observed between in vitro siderophore level and suppression of R. solani in dual agar culture by the isolates. Production of β-1,4 glucosidase 16, chitinases 17 and antibiotics has been associated with suppression of harmful fungi. Four of the isolates, Psd 9, Psd 11, Psd 12, and Psd 13 grew and developed distinct colonies on cellulose powder supplemented Omeliansky s agar indicating β-1,4 glucosidase activity of these isolates (Table 2). In general, inoculation with the isolates stimulated the growth of the tea seedlings from leaf cuttings except in case of isolate Psd 2. Total shoot and root

5 494 INDIAN J BIOTECHNOL, OCTOBER 2007 biomass of 240-d-old tea seedlings increased to the maximum due to inoculation with Psd 11, followed by Psd 12 and Psd 13 (Table 2). Overall, it is convincing that Psd 11, followed by Psd 12 and Psd 13 are superior strains among the tea rhizosphere isolates screened in the present study. However, it is difficult to ascertain by which mechanisms these strains might have stimulated seedling growth. Higher level of P solubilization, antibiosis against R. solani and cellulose utilization by these isolates in vitro suggested the involvement of all these mechanisms in growth promotion of tea seedlings. However, Patgiri and Bezbaruah proposed enhanced P-availability as the mechanism of growth enhancement of matured tea due to inoculation of Pseudomonas and Bacillus spp 18. The experimental soil used in the present study was from a 48-year-old tea estate, which had a build-up of harmful organisms due to long monoculture and their ability to suppress tea growth has been reported by several earlier studies 19,20. References 1 Chanway C P, Inoculation of tree roots with plant growth promoting soil bacteria: An emerging technology for reforestation, For Sci, 43 (1997) Prikryl Z, Vanchura V & Wurst M, Auxin production by rhizosphere bacteria as a factor of root growth, Biol Plant, 27 (1985) Tien T M, Gaskins M H & Hubbell D H, Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum americanum L.), Appl Environ Microbiol, 37 (1979) Alexander D B & Zuberer D A, Use of Chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria, Biol Fert Soil, 12 (1991) Dileep Kumar B S & Bezbaruah B, Plant growth promotion and fungal pest control through an antibiotic and siderophore producing fluorescent Pseudomonas strain from tea [Camellia sinensis (L.) O. Kuntze] plantations, Indian J Exp Biol, 35 (1997) Upadhyay R S & Jayaswal R K, Pseudomonas cepacia causes mycelial deformities and inhibition of conidiation in phytopathogenic fungi, Curr Microbiol, 24 (1992) Tank N & Saraf M, Phosphate solubilization, exopolysaccharide production and indole acetic acid secretion of rhizobacteria isolated from Trigonella foenum graecum, Indian J Microbiol, 43 (2003) Agnihothrudu V, Potential of using biocontrol agents in tea, Global Adv Tea Sci, (1999) Choudhuri T C, Pesticide residues in tea, in Global advances in tea science, editedby N K Jain [Vedams ebook (P) Ltd.] 1999, Gordon D A & Webber R P, Colorimetric estimation of indole-3-acetic acid, Plant Physiol, 26 (1951) Bray R H & Kurtz L T, Determination of total, organic and available form of phosphorus in soils, Soil Sci, 59 (1945) Utkhede R S & Rahe J E, Interaction of antagonists and pathogen in biological control of white rot, Phytopathology, 73 (1983) Pal K K, Dey R, Bhatt D M & Chauhan S M, Enhancement of groundnut growth and yield by plant growth promoting rhizobacteria, Int Arch Newsl, 19 (1999) Stintzi A & Meyer J M, Search for siderophores in microorganisms, in Microbes for better living, edited by R Sankaran & K S Manja (Conference Secretariate, Defence Food Research Laboratory, Mysore) 1994, Scher F M & Baker R, Effect of Pseudomonas putida and a synthetic iron chelator on induction of suppressiveness to Fusarium wilt pathogens, Phytopathology, 72 (1982) Fridlender M, Inbar J & Chet I, Biological control of soilborne plant pathogens by a β-1-3-glucanase-producing Pseudomonas cepacia, Soil Biol Biochem, 25 (1993) Renwick A, Campbell R & Coe S, Assessment of in vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis, Plant Pathol, 40 (1991) Patgiri I & Bezbaruah B, Strains contributing to phosphorus mobilization in acid soils, Indian J Agric Sci, 60 (1990) Dutta S K, Sarma P C & Barbara B C, Rehabilitation of tea soil before planting, Two Bud, 18 (1971) George U & Singh R S, Biological and chemical factors affecting replanted tea, in Tea research: Global perspactive (Toklai Experimental Station, TRA, Jorhat) 1990,