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. 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 al.20. 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. Research Institute’s (Lucknow, India) Phosphate Amplification and sequencing of the 16S rRNA growth medium (NBRIP)22 after incubation for gene was performed at the Institute of Microbial 10 days. Autoclaved un-inoculated medium was used Technology (IMTECH), Chandigarh, India and as a control. The cultures were centrifuged at 10000 GeneOmbio Technologies Pvt. Ltd, Pune, India rpm for 10 min. and the supernatant was decanted according to the direct sequencing method of into a separate vial. P in the supernatant was Hiraishi28 and aligned using the MEGALIGN quantified by the method described by Fiske and program of DNASTAR. The partial 16S rRNA gene Subbrow23. Potash solubilization in the supernatant sequences determined and sequences of strains was tested following the method of Sheng and He24. belonging to the same phylogenetic group retrieved The available potassium was quantified using a from EMBL library were aligned. The 16S rRNA Flame Photometer. Acidification of the medium by sequences of the strains TNCSF KQ29 and TNCSF the inoculated bacteria was observed by measuring 021 were deposited in EMBL under the accession the pH after 10 d using a pH meter. IAA production numbers JN540031 and JQ280393, respectively. An was estimated following the method of Fuentes- evolutionary distance matrix was generated as Ramirez et al.25. described by Jukes and Cantor29. The evolutionary tree for the datasets was constructed using the 30 Identification of isolates neighbor-joining method of Saitou and Nei . The The isolates were observed under a phase- isolate TNCSF KQ29 was deposited as MTCC 9755 contrast microscope to determine their shape and in the IMTECH, Chandigarh, India. The nitrogen- motility. Colony morphology and pigment fixing ability of the representative isolates from production were observed on R2A, PDA, each group (TNCSF KQ29 and TNCSF 021) was Pseudomonas agar and King`s B agar media. confirmed by the presence of the structural gene for Tolerance to 3% NaCl was tested using LGI liquid nitrogenase reductase (nifH) by amplification with medium with yeast extract (100 mg l-1). Utilization genomic DNA extracted from these representative of different carbon sources were assessed on isolates. Amplification with the gene specific solid LGI medium26 in duplicates after 5 days, primers zehrf 5'-TGYGAYCCNAARGCNGA-3' replacing sucrose with respective carbon sources and zehrr 5'-ADNGCCATCATYTCNCC-3' yielded with yeast extract (100 mg L-1). The isolates which an expected 360 bp size product on 2% agarose 31 possessed nitrogen-fixing ability were tested gel . for solubilization of P and K minerals. Ultimately, the isolates possessing nitrogen-fixation, P- and K- Plant-inoculation study solubilization were included for further identification Besides the representative isolates included in the and inoculation experiments. Gluconacetobacter identification experiments (16S rRNA and nifH gene T diazotrophicus (ATCC 49037 ), Burkholderia analysis), two more isolates one from each group T vietnamiensis (LMG 10929 ) Azospirillum sp. [TNCSF 31 - R. terrae and TNCSF 36 - B. gladioli (MTCC 9758) were also used for comparative (Table 1)] along with type strains of studies. G. diazotrophicus (ATCC 49037), B. vietnamiensis (LMG 10929) and Azospirillum sp. (MTCC 9758) 16S rRNA gene sequencing and amplification of nifH gene were used here for comparison. These four isolates One representative isolate from each group showed faster growth in 72 h in N-free liquid medium (TNCSF KQ29 and TNCSF 021) were included for than the other isolates [population was compared sequence analysis. Genomic DNA was isolated using based on the turbidity of the growth medium using a the method of Ausubel et al.27. The cells from culture Spectrophotometer (Nono-Drop) at 540 nm after 72 h plate were scrapped in sterile 0.9% saline water and of growth]. A commercial cultivar of sugarcane pelleted in 2.0 mL centrifuge tube for further (Co 86032) in Tamil Nadu was used for inoculation 164 INDIAN J EXP BIOL, MARCH 2017

study. The soil used was the top layer from a nearby soils, roots and leaves of sugarcane varieties field having a pH of 6.9. Flat plastic containers of (Co 6304, Co 86032 and CoC 671) cultivated in 50 L capacity (60 cm X 40 cm X 30 cm) containing Tamil Nadu, Southern, India (Table 1). Putative 45 kg of thoroughly homogenized soil (dried and N-fixers had to obtain N mainly from air or N traces crushed) were used to grow the plants. There were from medium constituents; P from TCP and K from 8 plant treatments and each treatment had four mica. These eight isolates were representatives of replicates in a Randomized Complete Block micro-flora naturally present in the habitats Design. A control was maintained without the investigated in this study and may have potential application of fertilizers and microbes. Five mL of biotechnological application in future. Colony each inoculant grown in liquid LGI medium with yeast morphology and pigmentation on solid MD plates extract (500 mg L-1) for 48 h (approx. 108 cells mL-1) revealed two distinct groups of isolates. The first was used for inoculation during the time of group was obtained from the dilutions of 10-3 to 10-5 planting. Each container was planted with three and the second group from the dilution of 10-4 to 10-6. single bud stem pieces and after a month, one Cells of the first group were rod shaped, Gram- healthy plant was retained. The plants in the negative and motile; they were able to grow at 37ºC containers were irrigated by natural rain fall and and were tolerant to 3% NaCl. Growth was positive tap water. Leaf chlorophyll contents were analyzed on Pseudomonas agar. They formed translucent on 135th day. Plant total biomass (root and shoot) colonies in R2A, PDA and King`s B agar media, and and N concentration in the leaf were studied to utilized most of the sugars tested (Table 2) and evaluate the efficiency of inoculated bacteria. The closely resembled Roseateles spp. as reported earlier fourth leaf from the top of plants of each treatment by Gomila et al.32. They used different sugars was cut, dried in an oven at 80ºC for 48 h, digested (D-glucose, L-arabinose, D-mannose, sucrose, and analyzed for total N content using Kjel-tech maltose, N-acetyl-D-glucosamine, L-rhamnose, apparatus (Tecator 1002). After 155 days, grown- D-fructose, D-mannitol, L-arabitol, meso-erythritol, up plants were removed from the pots and growth sorbitol, and glycerol) and organic acids (succinic, promotion activity of the inoculants was malic, lactic, adipic, citric and oxalic). The second determined by comparing the leaf N, weight of group of isolates formed a yellow colour on R2A, roots and shoots and total biomass. PDA and King`s B agar, grew well on Pseudomonas agar, tolerated 3% NaCl and closely resembled Statistical analysis Burkholderia spp. These isolates assimilated all the In the present investigation, data for P- and K- sugars (D-glucose, L-arabinose, D-mannitol, solubilization were expressed as the means of maltose, L-arabitol, lactose, rhamnose, erythiritol, quadruplicates. Statistical analysis was performed by fructose, sorbitol, sucrose, meso-erythritol, glycerol) S-N-K procedure using SPSS software (Version 14). and organic acids (malic and succinic - Table 3) Mean differences between the treatments were included in this study unlike other Burkholderia 33-36 compared using the Student – Neuman-Keuls test. species reported so far . Differences were considered significant when P <0.05. The data obtained from pot trial were 16S rRNA gene sequence analysis subjected to Analysis of Variance. Based on 16S rRNA gene sequence similarity (1394 and 1250 nucleotides sequenced respectively Results and Discussion for TNCSF KQ29, (Group I) and TNCSF 021 (Group Screening and identification of isolates II), phylogenetic positions of TNCSF KQ29 and Diazotrophic bacteria possessing P- and K- TNCSF 021 retrieved from the EMBL library solubilization potential were isolated from sugarcane matched most closely with R. terrae (with 99% environments using a novel MD medium (without N, sequence similarity) and B. gladioli (with 99.1% P and K) supplemented with micro nutrients. Out of sequence similarity), respectively. The phylogenetic 720 samples, only 41 isolates were obtained and most position of the isolated strains was inferred from a of them were lost during subsequent sub-culturing neighbor joining phylogenetic tree constructed based using MD liquid medium. Only eight strains were on the 16S rDNA gene sequences. The phylogenetic recovered from rhizosphere and non-rhizosphere position of the strain TNCSF KQ29 (JN40031) was MUTHUKUMARASAMY et al.: PRESENCE OF BACTERIAL STRAINS AND THEIR INOCULATION EFFECTS ON SUGARCANE 165

Table 2—Differential phenotypic characters of Roseateles species versus Roseateles isolates TNCSF KQ29 and TNCSF 31 Phenotypic Roseateles aquatilis Roseateles terrae TNCSF KQ 29 (MTCC characters CCUG 52220* CCUG 48205* CCUG 52222 * 9755) & TNCSF 31 Growth at 10ºC + - V V Growth at 37C + - + + Utilization of sugars D-Glucose + + + + L-Arabinose + - + + D-Mannose + - + + Sucrose - NR NR + Maltose + + + + N-Acetyl-D-glucosamine - - - + L-Rhamnose NR NR NR + D-Fructose + + + + D-Mannitol + - + + L-Arabitol NR NR NR + Meso-erythritol NR NR NR + Sorbitol - NR NR + Glycerol - NR NR + Utilization of organic acids Succinic acid + - + + Malic acid + + V + Lactic acid + + - + Adipic acid V - - + Citric acid + - - + Oxalic acid NR NR NR + + positive; - negative; V-variable; NR- Not Reported, *Reference strains; Data from Gomila et al.26.

Table 3—Differential phenotypic characters of N2-fixing Burkholderia species versus Burholderia isolates TNCSF 021 and TNCSF 36 Phenotypic Bs* Bt* Bu* Bk** Bx* Bv* Bg*** Ba**** TNCSF 021 & characters TNCSF 36 Acetylene Reduction Assay + + + + + + - + + Assimilation of D-Glucose + + + + + + + + + L-Arabinose + + + + - + + + + D- Mannitol + + + + + + + + + Maltose V - - + - - - - + L- Arabitol - + - + NR + - + + Lactose - + - - + - - + Rhamnose + + + + NR - - + + Erythritol + - - + NR - - NR + Fructose + + + + + + + + + Sorbitol + + + + NR + + + + Sucrose + - + - - + - - + Meso-erythritol + - - NR NR - - NR + Glycerol + + + + NR + + NR + Malic acid + + + + + + NR NR + Succinic acid NR NR + + NR NR NR + + [Bs, B. silvatlantica; Bt, B. tropica; Bu, B. unamae; Bk, B. kururiensis; Bx, B. xenovorans; Bv, B. vietnamiensis; Bg, B. gladioli; Ba, B. australis. * Data from Perin et al.35; **Data from Zhang et al.34; ***Data from Viallard et al.33; **** Data from Paungfoo et al.36; For other explanations, please refer Table 1] shown in Fig. 1. The closest known phylogenetic B. gladioli type strain EU02416837 with 99.1% relative of this strain is R. terrae strain AM50144532 sequence similarity. for which the recorded similarity of the 16S rDNA Earlier, R. terrae has been isolated from soil32. genes was 99%. The Fig. 2 demonstrates the This is the first report of association of R. terrae with position of N2-fixing B. gladioli TNCSF 021 sugarcane. Both R. terrae and B. gladioli have been (JQ280393) and its closest phylogenetic relative reported in seeds of hybrid maize38. No other works 166 INDIAN J EXP BIOL, MARCH 2017

Fig. 1—Phylogenetic tree reconstructed using 16S rRNA gene sequence showing the position of the isolated Rosealteles terrae strain TNCSF KQ29 (JN40031) with certain related genera and species. [The number at the nodes reflects the level of the bootstrap support based on neighbor joining analysis of 1000 re-sampled data sets. The 16S rDNA gene sequence of E. coli 0157 EC4115 was used as the out group. Bar indicates 0.02 substitutions per site]

Fig. 2—Phylogenetic position of strain TNCSF 021 (JQ280393) within the genus Burkholderia on the basis of 16S rRNA gene sequences. [The phylogenetic tree was constructed by the neighbor-joining method22, and the 16S rRNA gene sequence of E. coli 0157EC4115 was used as the out group. The numbers at the nodes indicate the levels of the bootstrap support based on a neighbor-joining analysis of 1000 resampled data sets] MUTHUKUMARASAMY et al.: PRESENCE OF BACTERIAL STRAINS AND THEIR INOCULATION EFFECTS ON SUGARCANE 167

reported environmental isolates of R. terrae. endo-rhizosphere of different host plants and different B. gladioli was previously identified as a environs may relate to these abilities. phytopathogen causing leaf and corm diseases on Table 4 shows the extent of macronutrient- onion, gladioli and rice39,40 apart from its association supplementing abilities of R. terrae and B. gladioli with human pulmonary infections41. Castro-González such as N-fixation, P- and K- solubilization and et al.42 reported the predominant association of production of IAA. R. terrae had a lesser AR activity diazotrophic Burkholderia species, particularly and IAA production than B. gladioli isolates. Higher B. unamae and B. tropica, and non-diazotrophic P-solubilization was observed in solid medium as well B. gladioli with sugarcane cultivated in Mexico. as in liquid assay for B. gladioli than R. terrae These species possessed characteristic growth- isolates. Bacterial solubilization of P by the promoting features like biosynthesis of indole microorganisms was reported to be associated with compounds, phosphate solubilization, siderophore the release of organic and inorganic acids44. production and the presence of the acdS gene Girgis et al.45 observed that a relationship seems to be encoding ACC deaminase activity18. established among final pH, total acidity, extra

Detection of nif genes Presence of nif genes in the representative isolates of these two groups was confirmed by a PCR assay with nifH specific primers which yielded a PCR product at about 360 bp (Fig. 3). It shows that they potentially can fix N. The presence of nif gene has been reported for R. terrae strain (CCUG 52222T) earlier32. Nitrogenase activity in the genus Burkholderia was reported in 9 plant associated species43. The isolates obtained from the present study possessed nitrogen fixation, P- and K- solubilization Fig. 3—Detection of nifH genes from diazotrophic isolates and growth hormone production that may help them obtained from this study. [Lanes: M, Molecular marker; Lane 1, R. terrae (TNCSF KQ 29); Lane 2, B. gladioli (TNCSF 021); to overcome the nutrient stress in plant environments. Lane 3, G. diazotrophicus (ATCC 49037); and Lane N, Negative It is presumed that the survivability and success of control (Sterile nuclease free water was used in place of DNA as a Burkholderia negative control)] species colonizing the rhizosphere and

Table 4—Nitrogen-fixation, solubilization of P and K from insoluble minerals and production of IAA (Means of 4 replicates) by test strains of Roseateles terrae and Burkholderia gladioli Bacterial strains ARA (n moles Phosphate solubilization* Potassium solubilization IAA production -1 -1 -1 C2H4 per vial, h ) using Mica (µg mL ) (g mL ) Solubilization zone in Available P in NBRIP Quantity Final pH*

Pikovskaya plates (mm) broth (µg mL-1) Roseateles terrae TNCSF 23 45.00±1.78b 3.28±08b 15.75±1.11b 105.75±1.49c 4.0 3.93±0.19a TNCSF 24 35.00±1.68a 3.95±0.13b 17.25±0.85b 107.00±2.16c 3.9 6.90±0.12b TNCSF KQ 29 86.50±1.94b 10.26±0.11c 31.75±0.85c 112.33±1.08d 4.00 11.25±1.42b TNCSF 31 66.25±3.54a 7.50±0.16b 19.00±0.71b 106.25±2.63c 3.60 9.23±0.93a Burkholderia gladioli TNCSF 2 66.75±2.39c 16.38±0.26f 45.25±2.06d 6.95±0.13ab 4.6 8.20±0.15bc TNCSF 18 78.75±1.03d 15.08±0.09e 50.50±1.55e 5.98±0.13ab 4.4 7.83±2.25bc TNCSF 021 115.00±2.68c 23.33±0.45d 72.50±1.19d 10.03±0.06b 4.20 13.50±0.58c TNCSF 36 94.00±1.83b 24.03±0.93d 74.75±2.18d 8.55±0.22b 3.15 12.28±0.66b,c Reference strains Gluconacetobacter 109.00±1.08c 22.73±0.25d 73.50±1.32d 7.50±0.18b 2.90 10.38±1.38a,b diazotrophicus (ATCC 49037) B. vietnamiensis 87.00±1.47b 1.50±0.05a 3.49±0.035a 4.20±0.08a 4.20 11.03±0.79a,b (LMG 10929) [* Initial pH was 6.4 and final pH was estimated after 10 days of incubation. Results of the Student-Newman-Keuls test for different treatments are shown as a, b, c, d, e. Means with the same letter are not statistically different according to S-N-K test (P <0.05)] 168 INDIAN J EXP BIOL, MARCH 2017

polysaccharide (EPS) and organic acids produced, in biomass accumulation due to inoculation of depending on the type of bacterial strain and its B. gladioli was high (66%) and this may be due to its solubilizing ability. Solubilization of P may also be multi favorite characters. Studies revealed that due to the phytase activity of microbes46. In the case Burkholderia unamae, B. tropica and B. silvatlantica of K-solubilization, R. terrae isolates released more K exhibited many growth promoting activities such as than B. gladioli. A pH reduction in the medium mineral phosphate solubilization, ACC deaminase (initial pH 6.4) was observed and this may be due to activity18,43, IAA biosynthesis, bio-control and bio- the acid production by the bacterial cultures (Table 4). remediation. Next to B. gladioli, some previously Organic acids such as citric, oxalic, succinic and tested inoculants such as B. vietnamiensis (37%), ά-ketogluconic acids47 and capsular polysaccharides24 Azospirillum sp. (33%), G. diazotrophicus (32%) of microbial origin are capable of mobilizing K from and R. terrae (mean increase of 16 %), showed various K-bearing minerals. a better plant growth promotion than the control

Inoculation effect on sugarcane plants. Increase in leaf chlorophyll content and The inoculation effect of B. gladioli on a sugarcane N concentration in leaves of inoculated plants variety Co 86032 in the pot trial showed a significant was evident of plant growth promoting activity of increase in total chlorophyll content, leaf N, root and inoculated microbes48. Though there is higher shoot weight and total biomass (at P=0.05) over the N content in the inoculated plant tissue, there is no other treatments (Tables 5 and 6). The mean increase direct evidence that the inoculated strains provided

Table 5—Effect of inoculation of four test isolates on Chlorophyll a, b and total Chlorophyll (mg mL-1; mean of triplicates) after 135 days of planting in pots Treatment Chlorophyll a Chlorophyll b Total Chlorophyll Roseateles terrae TNCSF KQ 29 0.2033f 0.1110 0.2201f TNCSF 31 0.8490d 0.07877 0.9278b Burkholderia gladioli TNCSF 021 0.9090b 0.0930 1.0027a TNCSF 36 0.8829c 0.0774 0.9603b Reference strains Gluconacetobacter diazotrophicus (ATCC 49037) 1.8883a 0.1057 0.7024d Burkholderia vietnamiensis (LMG 10929) 0.6912e 0.0983 0.7895c Azospirillum spp.(MTCC 9758) 0.1853 g 0.2970 0.3533e Control 0.2031f 0.0111 0.2142f CD 5%/ LSD p=0.05 0.042 NS 0.347 [Values in the same column followed by the same letter are not significantly different at P = 0.05%; NS, Non–Significant]

Table 6—Effect of four test isolates on leaf N, root wt. shoot wt. and total biomass after 155 d after planting on sugarcane variety Co 86032 cultivated in pots Treatment Leaf N Root weight Shoot weight Total biomass Increase over (mg g-1 dry wt.) (g) (g) (g) control (%) Roseateles terrae TNCSF KQ 29 0.77d 388.33d 857.00g 1246.00g 9 TNCSF 31 0.88d 358.00e 1032.00f 1390.33f 22 Burkholderia gladioli TNCSF 021 1.39a 478.66a 1503.00a 1982.66a 74 TNCSF 36 1.00b 429.00b 1360.00b 1789.00b 57 Reference strains Gluconacetobacter diazotrophicus 0.84d 392.33d 1120.00d 1512.33e 32 (ATCC 49037) Burkholderia vietnamiensis (LMG 10929) 0.94c 434.00b 1127.00c 1561.33c 37 Azospirillum spp.(MTCC 9758) 0.91c 415.33c 1108.00e 1524.00d 33 Control 0.71e 353.66e 788.60h 1142.33h CD 5%/ LSD p=0.05 0.14 7.56 3.00 3.03 [Data are means of triplicates, Values in the same column followed by the same letter are not significantly different at P = 0.05%] MUTHUKUMARASAMY et al.: PRESENCE OF BACTERIAL STRAINS AND THEIR INOCULATION EFFECTS ON SUGARCANE 169

the fixed N to the plants. Isotope dilution studies promoting rhizobacteria (PGPRs) and Sinorhizobium fredii on would be essential to prove this observation. Further, biological nitrogen fixation, nodulation with plant growth of Glycine max cv. Osumi. Plant Soil, 267 (2004) 143. the extent to which each factor (N2-fixation, P- and K- 5 Chennappa G, Naik MK, Adkar-Purushothama CR, solubilization, ACC deaminase activity) contributed Amaresh YS & Sreenivasa MY, PGP potential, abiotic stress to the PGP effect is unclear as we could not perform tolerance and antifungal activity of Azotobacter strains P and K content in the plant samples. Further analysis isolated from paddy soils. Indian J Exp Biol, 54 (2016) 322. of P and K content could potentially clarify this 6 Patel PR, Shaikh SS & Sayyed RZ, Dynamism of PGPR in bioremediation and plant growth promotion in heavy metal ambiguity. In the present study, B. gladioli and contaminated soil. Indian J Exp Biol, 54 (2016) 286. R. terrae have been reported from sugarcane environs 7 Yadav AN , Sachan SG, Verma P & Saxena AK, with a potential for plant growth promotion. Earlier, Bioprospecting of plant growth promoting psychrotrophic inoculation of G. diazotrophicus, A. lipoferum and Bacilli from the cold desert of north western Indian B. vietnamiensis in sugarcane plants was reported to Himalayas. Indian J Exp Biol, 54 (2016) 142. 8 Sharma SB, Sayyed RZ, Sonawane M, Trivedi MH & increase the leaf N content and total biomass under Neurospora 48,49 Thivakaran GA, sp. SR8, a novel phosphate same growth conditions in pot trial experiments . solubiliser from rhizosphere soil of Sorghum in Kachchh, Thus, the inoculation of such potential isolates along Gujarat, India. Indian J Exp Biol, 54 (2016) 644. with application of rock P and K minerals may result 9 Basak BB & Biswas DR, Co-inoculation of potassium in continuous supply of NPK to crops of their solubilizing and nitrogen-fixing bacteria on solubilization of waste mica and their effect on growth promotion and nutrient association. These bacteria can be used to increase Biol Fert Soils 45 acquisition by a forage crop. , 46 (2010) 641. agricultural production as envisaged by Girgis et al. . 10 Pandey P, Kang SC & Maheshwaran DK, Isolation of However, the taxonomical position of these two endophytic plant growth promoting Burkholderia sp. MSSP genera restrains their usage in agriculture until they from root nodules of Mimosa pudica. Curr Sci, 89 (2005) are proved to lack pathogenecity to humans. Though 177. 11 Upadhyay A & Srivastava S, Evaluation of multiple plant these bacterial isolates reported as human pathogens, growth promoting traits of an isolate Pseudomonas these multi-characterized cultures can be tested in flurescens Psd. Indian J Exp Biol, 48 (2010) 601. field crops because even if they lack the 12 Loiret FG, Ortega E, Kleiner D, Ortega-Rodes P, Rodes R & pathogenecity genes, gene transfer is always a Dong Z, A putative new endophytic nitrogen-fixing Pantoea J Appl Microbiol possibility with bacteria. Guaranteeing a perfect lack bacterium sp. from sugarcane. , 97 (2004) 504. of pathogenicity potential may be impossible. 13 da Silva MF, Antonio de Souza S, de Oliveira PJ, Xavier GR, Acknowledgement Rumjanek NG, Soares de Barros LH & Reis VM, Survival of endophytic bacteria in polymer-based inoculants and Authors thank Tamil Nadu Co-operative Union efficiency of their application to sugarcane. Plant Soil, 356 for research grant (Rc.No.1899/05/SF3 dt. 16.2.2006) (2012) 231. to undertake this work. Also, they acknowledge 14 Caballero-Mellado J, Martinez-Aguilar L, Paredes-Valdez G Dr. VS Saravanan, Indira Gandhi Arts and Science & Estrada-de los Santos P, Burkholderia unamae sp nov., an College, Puducherry, India and Mr. Rohinikumar, N2-fixing rhizospheric and endophytic species. Int J Syst Evol Microbiol, 54 (2004) 1165. VIT, Vellore, India for assistance in sequence 15 Reis VM, Estrada-de los Santos P, Tenorio-Salgado S, deposition; and Dr. TS Seshadri, Dr. J Arunkumar and Vogel J, Stoffels M, Guyon S, Mavingui P, Baldani VLD, Dr. S Chandrasekarenthiran, Murugappa Chettiar Schmid M, Baldani JI, Balandreau J, Hartmann A & Research Centre, Chennai, Tamil Nadu, India are Caballero-Mellado J, Burkholderia tropica sp. nov., a novel Int J Syst Evol acknowledged for statistical analysis. nitrogen-fixing, plant-associated bacterium. Microbiol, 54 (2004) 2155. References 16 Fischer D, Pfitzner B, Schmid M, Simoes-Araujo JL, 1 Pariona-Llanos R, de S Ferrara FI, Gonzales HHS & Reis VM, Pereira W, Ormeno-Orrillo E, Hai B, Hofmann A, Barbosa HR, Influence of organic fertilization on the number Schloter M, Martinez-Romero E, Baldani JI & Hartmann A, of culturable diazotrophic endophytic bacteria isolated from Molecular characterization of the bacterial community in sugarcane. Eur J Soil Biol, 46 (2010) 387. uninoculated and inoculated field grown sugarcane 2 Dheeba B, Sampathkumar P & Kannan K, Chromium (Saccharum sp.), Plant Soil, 356 (2012) 83. accumulation potential of Zea mays grown under four 17 Kalita M, Bharadwaz M, Dey T, Gogoi K, Dowarah P, Unni BG, different fertilizers. Indian J Exp Biol, 52 (2014) 1206. Ozah D & Saikia I, Developing novel bacterial based 3 Sayantan D & Shardendu, Phosphorus amendment competitively bioformulation having PGPR properties for enhanced production prevents chromium uptake and mitigates its toxicity in of agricultural crops. Indian J Exp Biol, 53 (2015) 56. Spinacea oleracea L. Indian J Exp Biol, 53 (2015) 395. 18 Caballero-Mellado J, Onofre-Lemus J, Estrada-de los Santos P 4 Lucas Garcia JA, Probanza AB, Ramos B, Barriuso J & & Martinez-Aguilar L, The tomato rhizosphere, an Gutierrez Manero FJ, Effects of inoculation with plant growth environment rich in nitrogen-fixing Burkholderia sp. with 170 INDIAN J EXP BIOL, MARCH 2017

capabilities of interest for agriculture and bioremediation. an aquifer polluted with TCE. Int J Syst Evol Microbiol, 50 Appl Environ Microbiol, 3 (2007) 5308. (2000) 743. 19 Muthukumarasamy R, Kang Ui Gum, Jeon Ottai, Ki-Do 35 Perin L, Martinez-Aguilar L, Paredes G, Baldani JI, Estrada- Park, Weon Tai Jeon, Chang Young Park, Young Son Cho, de los Santos P, Reis VM & Caballero-Mellado J, Jaekyeong Song, Soon-Wo-Kwon, Dong Hyun Roh, & Burkholderia silvatlantica sp. nov., a diazotrophic bacterium Revathi G, Natural association of Gluconacetobacter associated with sugarcane and maize. Int J Syst Evol diazotrophicus and diazotrophic Acetobacter peroxydans Microbiol, 56 (2006) 1931. from wetland rice. Syst Appl Microbiol, 28 (2007) 277. 36 Paungfoo-Lonhienne C, Lonhienne TGA, Yeoh YK, 20 Hardy RWF, Burns RC & Holsten RD, Application of the Webb RI, Lakshmanan P, Chan CX, Phaik-Eem Lim, C2H2 assay for measurement of nitrogen fixation. Soil Biol Ragan MA, Schmidt S & Hugenholtz P, A new species of Biochem, 24 (1973) 47. Burkholderia isolated from sugarcane roots promotes plant 21 Pikovskaya R, Mobilization of P in soil in connection with growth, Microbiol Biotech, 7 (2013) 142. vital activity by some microbial species, Microbiologia, 17 37 Tayeb LA, Lefevre M, Passet V, Diancourt L, Brisse S & (1948) 362. Grimont PA, Comparative phylogenies of Burkholderia, 22 Nautiyal S, An efficient microbiological growth medium for Ralstonia, Comamonas, Brevundimonas and related screening phosphate solubilizing microorganisms. FEMS organisms derived from rpoB, gyrB, and rrs gene sequences. Microbiol Lett, 170 (1999) 265. Res Microbiol, 159 (2008) 169. 23 Fiske CH & Subbarow Y, Colorimetric determination of 38 Liu Y, Zuo S, Xu L, Zou Y & Song W, Study on diversity of Phosphorous. J Biol Chem, 88 (1925) 375. endophytic bacterial communities in seeds of hybrid maize 24 Sheng XF & He LY, Solubilization of potassium minerals by and their parental lines. Arch Microbiol, 194 (2012) 1001. a wild type strain of Bacillus edaphicus and its mutants and 39 Wilsher ML, Kolbe J, Morris AJ & Welch DF, Nosocomial increased potassium uptake by wheat. Can J Microbiol, 52 acquisition of Burkholderia gladioli in patients with cystic (2006) 66. fibrosis. Am J Resp Crit Care Med, 155 (1997) 1436. 25 Fuentez-Ramirez LE, Jimenez-Salgado J, Abarca-Ocampo DR, 40 Bauernfeind A, Schneider I, Jungwirth R & Roller C, Caballero-Mellado J, Acetobacter diazotrophicus, an Discrimination of Burkholderia gladioli from other indoleacetic acid producing bacterium isolated from Burkholderia species detectable in cystic fibrosis patients by sugarcane cultivars of Mexico. Plant Soil, 154 (1993) 145. PCR. J Clin Microbiol, 36 (1998) 2748. 26 Cavalcante VA & Dobereiner J, A new acid-tolerant 41 Bernhardt SA, Spilker T, Coffey T & Lipuma, JJ, nitrogen-fixing bacterium associated with sugarcane, Plant Burkholderia cepacia complex in cystic fibrosis: Frequency Soil, 108 (1998) 23. of strain replacement during chronic infection. Clin Infect 27 Ausubel FM, Brent R, Kington RE, More DD, Seidman JG, Dis, 37 (2003) 780. Smith JA & Struhi K, Current protocols in molecular 42 Castro-González R, Martínez-Aguilar L, Ramírez-Trujillo A, biology, (Greene/Wiley Inter science, New York), 1987. Estrada-de los Santos P & Caballero-Mellado J, High 28 Hiraishi A, Direct automated sequencing of 16S rDNA diversity of culturable Burkholderia species associated with amplified by polymerase chain reaction from bacterial sugarcane. Plant Soil, 345 (2011) 155. cultures with DNA purification, Lett Appl Microbiol, 15 43 Onofre-Lemus J, Hernandez-Lucas I, Girard L & Caballero- (1992) 210. Mellado J, ACC (1-Aminocyclopropane-1-Carboxylate) 29 Jukes TH & Cantor RR, Evolution of protein molecules. In: deaminase activity, a wide spread trait in Burkholderia Mammalian protein metabolism, (Ed. Munro HN, Academic species and its growth promoting effect on tomato plants. Press, New York), 1969, 21. Appl Environ Microbiol, 75 (2009) 6581. 30 Saitou N & Nei M, Universal chemical assay for detection 44 Richardson AE, Prospects for using soil microorganisms to and determination of siderophores, Anal Biochem, 160 improve the acquisition of phosphorous by plants. Aust J (1987) 47. Plant Physiol, 28 (2001) 897. 31 Peng GX, Wang HR, Xhang GA, Hou W, Liu Y, Wang ET 45 Girgis MGZ, Khalil MA & Sharaf MS, In vitro evaluation of & Tan ZY, Azospirillum melinis sp. nov., a group of rock phosphate and potassium solubilizing potential of some diazotrophs isolated from tropical molasses grass. Int J Syst Bacillus strains. Aust J Basic Appl Sci, 2 (2008) 68. Evol Microbiol, 56 (2006) 1263. 46 Li X, Yang SH, Yu XC, Jin ZX, Li WD, Li L & Li MG, 32 Gomila M, Bowien B, Falsen E, Moore ERB & Lalucat J, Construction of transgenic Bacillus mucilaginosus strain with Description of Roseateles aquatilis sp. nov. and Roseateles improved phytase secretion. J Appl Microbiol, 99 (2005) terrae sp. nov., the class , and emended 878. description of the genus Roseateles. Int J Syst Evol 47 Welch SA & Vandevivere P, Effects of microbial and other Microbiol, 58 (2008) 6. naturally occurring polymers on mineral dissolution. 33 Viallard V, Poirier I, Cournoyer B, Haurat J, Wiebkin S, Geomicrobiol J, 12 (1994) 227. Ophel-Keller K & Balandreau J, Burkholderia graminis sp. 48 Muthukumarasamy R, Revathi G & Lakshminarasimhan C, nov., a rhizospheric Burkholderia species, and reassessment Diazotrophic associations in sugarcane cultivation in South of [Pseudomonas] phenazinium, [Pseudomonas] pyrrocinia India. Trop Agric, 76 (1999) 171. and [Pseudomonas] glathei as Burkholderia. Int J Syst Evol 49 Govindarajan M, Balandreau J, Muthukumarasamy R, Microbiol, 48 (1998) 549. Revathi, G & Lakshminarasimhan C, Improved yield of 34 Zhang H, Hanada S, Shigematsu T, Shibuya K, Kamagata Y micro-propagated sugarcane following inoculation by & Kurane R, Burkholderia kururiensis sp. with TCE. nov., a endophytic Burkholderia vietnamiensis. Plant Soil, 280 trichloroethylene (TCE)-degrading bacterium isolated from (2006) 239.