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Isolation of Bacterial Strains Possessing Nitrogen-Fixation, Phosphate and Potassium-Solubilization and Their Inoculation Effects on Sugarcane

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Isolation of Bacterial Strains Possessing Nitrogen-Fixation, Phosphate and Potassium-Solubilization and Their Inoculation Effects on Sugarcane Indian Journal of Experimental Biology Vol. 55, March 2017, pp. 161-170 Isolation of bacterial strains possessing nitrogen-fixation, phosphate and potassium-solubilization and their inoculation effects on sugarcane Ramachandran Muthukumarasamy*,·Gopalakrishnan Revathi, Muthiayan Vadivelu & Kannan Arun Main Bio-control Research Laboratory (Unit of Tamil Nadu Co-operative Sugar Federation), Good Will Avenue, Venpakkam, Chengalpattu-603 111, Tamil Nadu, India Received 29 June 2015; revised 07 September 2016 Inorganic nitrogen (N), phosphate (P) and potash (K) are the most influencing macro-nutrients for plant growth and microbial supplementation of these minerals through N2-fixation, P- and K-solubilization is gaining importance. In the present study, a macronutrient deficient (MD), N-free novel medium, supplemented with tri calcium phosphate (TCP as P- source) and Mica (as K- source) was used for isolation of microbes possessing nitrogen fixing, P- & K solubilizing abilities. Samples of rhizosphere and non-rhizosphere soils, roots and leaves of sugarcane varieties (viz., Co 6304, Co 86032 and CoC 671) collected from Tamil Nadu, India were used for isolation. Totally, 8 individual nitrogen-fixing, phosphate- and potash-solubilizing bacterial strains were obtained. Nitrogen-fixing abilities of these isolates were confirmed by analyzing acetylene reduction (AR) activity and the presence of nif genes. P- and K- solubilizing activities were confirmed by cultivating these isolates in solid/liquid medium supplemented with insoluble forms of P and K. These isolates which produced growth hormone IAA, were in two groups as Roseateles terrae and Burkholderia gladioli, respectively based on the morphological, physiological, biochemical and 16S rDNA gene sequence analysis. Association between diazotrophic, P- and K-solubilizing R. terrae and B. gladioli with sugarcane has not been reported earlier. These isolates were tested for their growth-promoting abilities in sugarcane cultivated in pots, and the results showed that these isolates were able to increase the leaf chlorophyll, N content and total biomass. This study may encourage farmers to use single microbe for microbial supplementation of N, P and K instead of consortium of microbes wherein the compatibility between different microbes is often compromised. Keywords: Burkholderia gladioli, Macronutrients, Roseateles terrae, Saccharum officinarum L. Nitrogen (N), phosphorus (P) and potassium (K) are Rhizobium, Burkholderia, Acetobacter, Azotobacter, major nutrients for plant growth and development, and Pseudomonas and Gluconacetobacter. Bacteria of the are supplied to plants as fertilizers through organic and genus Bacillus, Neurospora, etc. are common soil inorganic sources1-3. However, chemical fertilizers are microbes playing important role in P- and K- not only expensive but also have undesirable side solubilization4,5. Sharma et al., have reported a novel effects in terms of environment, food chain, etc. phosphate solubiliser, Neurospora sp. SR8, from A potential option for decreasing the use of chemical rhizosphere soil of Sorghum in Kachchh, Gujarat, 8 fertilizers is exploitation of plant growth promoting India . Co-inoculation of N2-fixing Azotobacter (PGP) bacteria4-7. These bacteria may provide a natural chroococcum and K-solubilizing B. mucilaginosus and eco-friendly means to improve growth and yield of enhanced growth and nutrient acquisition in a forage crops via various mechanisms like nitrogen-fixation, crop9. The genus Burkholderia is reported to possessing solubilization of insoluble P and K minerals, nitrogen-fixation, potential ACC (1-amino- phytohormone production, control of pests and diseases, cyclopropane–carboxylate) deaminase enzyme activity, bioremediation, etc., thereby minimizing the use of solubilization of phosphate, antagonism to phyto- agrochemicals1,4-6. Cereals associating with different pathogens and excretion of phytohormones10. Later, types of diazotrophs can obtain up to 30% of N through multiple plant growth promoting pseudomonas biological nitrogen fixation1. Some species of the major fluorescens strains were reported from India possessing bacterial genera with this ability include Spirillum, phosphate solubilization, production of antimicrobial 11 —————— compounds and indole acetic acid (IAA) . * Correspondence: Many species of diazotrophic bacteria have been Fax: +91 44 24348024 E-mail: [email protected] isolated from sugarcane, most notably the members of 162 INDIAN J EXP BIOL, MARCH 2017 the genera Azospirillum, Pantoea, Gluconacetobacter, Surface sterilization and isolation of bacteria from Herbaspirillum, Burkholderia, etc.12,13. Bacteria samples were performed following the method of belonging to Burkholderia cepacia complex (Bcc), Muthukumarasamy et al.19. One gram each of shade- B. unamae14 and B. tropica15 were reported to be dried, ground and sieved rhizosphere and non- associated with sugarcane. Fischer et al.16 rhizosphere soil samples, homogenized roots and leaf demonstrated nifH expression of Bradyrhizobium sp. samples were used for 10-fold serial dilutions in and Rhizobium sp. in sugarcane roots. At present, sterile water. Aliquots (0.1 mL) were added into 20 mL various microbes are being used as consortia in crop penicillin vials containing 10 mL of MD liquid cultivation to help in supplementing N, P and K. medium. The composition of MD medium includes: If an inoculant provides these three major (g-1) sodium sulphate 0.3; magnesium sulphate 0.5; nutrients when applied along with cheap insoluble P sodium chloride 0.02; calcium carbonate 0.5; sucrose and K sources, then it could be a boon to sustainable 10; tri calcium phosphate (tcp) 1.0; mica powder farming. Recently, kalita et al.17 reported that a (k- mineral source) 2.0; bromothymol blue (0.5% in consortia of Bacillus ceres and two strains of 0.2 n koh) 2 mL; micronutrient solution 1.0 mL Pseudomonas rhodesiae (MTCC 8297 and MTCC (sodium molybdate 0.2 g; manganese sulphate 0.235 g; 8300) enhanced the productivity of tomato, boric acid 0.280 g; copper sulphate 0.008 g; zinc cauliflower, chili and brinjal by a variety of sulphate 0.024 g; water 200 mL); vitamin solution mechanisms. Yet, thousands of bacterial species 1.0 mL (biotin 10 mg, pyridoxin HCL 20 mg, water occurring in agricultural soils and plant 100 mL); water 1000 mL (HPLC grade, SISSCO environments remain un-cultivable using standard Research Laboratories, India). pH and incubation methods18. In this study, we tried to (i) isolate temperature were maintained at 6.4 and 32ºC, individual bacterial strains possessing N2-fixation, respectively in all experiments unless specified. Vials solubilization of P and K minerals using a medium were incubated for 5 days and tested for nitrogen- lacking N, P and K; and (ii) test their inoculation fixation potential. Acetylene reduction (AR) activity effect on sugarcane growth. was performed by the method followed by Hardy et 20 al. This assay was carried out by injecting 10% Materials and Methods (v/v) acetylene in the head space above the MD liquid Sample collection and isolation of diazotrophic, P- and K- medium (after 48 h of inoculation) and incubated for solubilizing bacteria 1 h. Ethylene production was measured using a Two samples from each location (Mamundur, Systronics Gas Chromatograph with a Poropak Q Venpakkam, Cheyyar, Sethiathope and Karunkuzhi - column and a flame ionization detector connected to a 10 sites in total) were collected at random in chromatography data computer system. Bacteria triplicates from rhizosphere, non-rhizosphere soils, showing growth in vials lacking N, P and K were roots and leaves from three different sugarcane purified using solid MD agar plates. Purity was checked on potato dextrose agar. varieties (CoC 671, Co 6304 and Co 86032) cultivated in Tamil Nadu, India (Table 1). Totally, Solubilization of TCP by the isolates was tested 720 samples were collected for the present study. using Pikovskaya agar plates21 with four replicates Table 1—Origin of diazotrophic isolates obtained from various sugarcane environments Isolation Strain code Origin dilution First group of isolates (Roseateles terrae) -3 TNCSF 23 10 Co 6304, Sugarcane root, Mamundur, Maduranthagam Sugar Mills, Tamil Nadu -4 TNCSF 24 10 Co 86032, Sugarcane non-rhizosphere soil, Venpakkam, Chengalpattu, Tamil Nadu -4 TNCSF KQ 29 10 Co 86032, Sugarcane root, Cheyyar, Cheyyar Sugar mills, Tamil Nadu -5 TNCSF 31 10 Co 86032, Sugarcane non-rhizosphere soil, Venpakkam, Chengalpattu,Tamil Nadu Second group of isolates (Burkholderia gladioli) TNCSF 2 10-4 CoC 671, Sugarcane leaf, Mamundur, Maduranthagam Sugar Mills, Tamil Nadu TNCSF 18 10-5 Co 86032, Sugarcane root, Sethiathope Sugar mills, Mill site, Chidambaram, Tamil Nadu TNCSF 021 10-5 Co 86032, Sugarcane rhizosphere soil, Mamundur, Maduranthagam Sugar Mills, Tamil Nadu TNCSF 36 10-6 Co 86032, Sugarcane root, Karunkuzhi, Sethiathope Sugar Mills, Sethiathope, Tamil Nadu MUTHUKUMARASAMY et al.: PRESENCE OF BACTERIAL STRAINS AND THEIR INOCULATION EFFECTS ON SUGARCANE 163 after 10 days. The halo zone and colony diameters processing of genomic DNA isolation. Quality were measured and solubilization ability was assessment of genomic DNA was performed by 1% calculated by subtracting colony diameter from the agarose gel electrophoresis as well as DNA was total diameter. Available phosphorus in broth culture quantified using Qubit TM Fluorometer (Invitrogen, was estimated using liquid National Botanical USA) for measurement of DNA concentration.
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