Characterization of bacterial strains and their impact on plant growth promotion and yield of wheat and microbial populations of soil

The aim of this research work is to evaluate the potentiality of bacterial strains isolated from rhizosphere of various cropping systems on growth, yield and soil microbial populations at harvest of wheat under pot and field experiments. Bacterial isolates, isolated from rhizosphere of various cropping systems. Morphological, biochemical and molecular characterization of bacterial strains were done. All the bacterial strains were rod shaped and ammonia producers. Most of the strains were Gram’s +ve and showed positive to catalase, VP, MR tests and HCN production. Pseudomonas aeuroginosa showed significantly lowest pH 4.42 of broth and solubilized 160.34 μgml tri-calcium phosphates. Enterobacter sp. produced maximum 27.06 μgml IAA at 100 μgml tryptophan. All the bacterial strains showed >97% similarity with strains already submitted to NCBI Gene bank. Arthrobacter chlorophenolicus and Enterobacter sp. showed maximum and significantly 23.60 and 28.97% plant height in pot and field experiments at 90 days after sowing. Bacillus megaterium caused maximum and significantly 51.45 and 40.47% grain and straw yield, respectively under pot while Serratia marcescens showed 14.78 and 17.04% grain and straw yield in field as compared to control. Most of the strains showed significant effect on microbial populations of post harvest soil under pot and field. It was concluded that A. chlorophenolicus, Enterobacter sp., B. megaterium and S. marcescens are effective strains of plant growth promoting bacterial strains for wheat crop production under IndoGangetic plains of India.


INTRODUCTION
During green revolution, better yielding varieties were introduced to feed the increasing population of world.Enhancing the crop production with the help of synthetic fertilizers and pesticides were used without caring environmental problems and soil health (Elkoca et al., 2010).Consequently, it is great challenge to search for sustainable strategies to alleviate detrimental effects ofintensive farming practices.Soil beneath our foot constitute rich source of different kind of microbes which were affected by both biotic and abiotic factors.The *Corresponding author.E-mail: ashokbhu2010@gmail.com,ashokabt@gmail.complethora of these microbes exists in rhizosphere zone and their size and activities are regulated by physicochemical status of soil and promotion of plant growth by improving the soil health (Aira et al., 2007;Jolly et al., 2010).Plant growth promoting rhizobacteria (PGPR) are soil-borne bacteria that have ability to aggressively colonize the rhizosphere or plant roots or both when applied to seed or crops that enhance the growth and yield of plants (Ashrafuzzaman et al., 2009;Kaymak, 2011).
In India, soils are generally low in available phosphorus due to chemical sorption that renders the application of readily soluble P-fertilizers highly inefficient (Hegde et al., 1999).PGPR comprise different functional and taxonomic groups of bacteria like Pseudomonas, Bacillus, Rhizobium, Azospirillum, Azotobacter, Enterobacter, Arthrobacter and others (Ghosh et al., 2002;Esitken et al., 2010;Kumar et al., 2014).Their efficiency to mobilize either organically or minerally bound nutrients from pedosphere or to fix atmospheric nitrogen and make it available to the plants is an important feature of their application.The direct growth promotion may be due to synthesis of phytohormones (Xie et al., 1996;Ashrafuzzaman et al., 2009), N 2 -fixation (Khan, 2005), synthesis of some enzymes such as ACC deaminase (Bal et al., 2013) that modulate the level of plant hormones (Glick et al., 1998) as well as the solubilization of inorganic phosphate and mineralization of organic phosphate which makes phosphorus available to the plants (Subba Rao, 1982).Pseudomonas sp.(Patten and Glick, 2002b), Arthrobacter sp.(Kumar et al., 2014), Enterobacter sp.(Slininger et al., 2004), Bacillus sp.(Idris et al., 2007) are examples of bacterial species which are capable of producing the indole-3-acetic acid (Marques et al., 2010).
Identification of bacteria in microbiological laboratory is traditionally performed for isolation of the organism and study of their phenotypic characteristics including Gram staining, morphology, culture requirements, and biochemical reactions.However, discovery of polymerase chain reaction (PCR) and Deoxyribonucleic acid (DNA) sequencing, comparison of gene sequences of bacterial species showed that the 16S rRNA is highly conserved within a species and among species of the same genus (Patrick et al., 2003).Hence, 16S ribosomal DNA (rDNA)based molecular characterization will provide authenticity to identify universal distribution of bacteria (Weisburg et al., 1991) and the presence of species-specific variable regions.
This molecular approach has been establishment of large public-domain databases (Maidack et al., 1996) and its application to identification of bacteria (Tang et al., 2000).
In the present study, attempt has been made for biochemical and molecular techniques for identification and characterization of bacteria and their effect on plant growth promotion, yield of wheat and status of microbial population in post harvest soil.

Isolation and purification of bacteria
Soil samples (50 g soil for per samples) were collected from ricewheat, vegetables (cabbage, spinach, ladyfinger, bottle guard), agro-forestry (mango, papaya, guava) and grassland rhizosphere (5 to 20 cm depth) from Indo-Gangetic plains of India (82°59' East, 25°15' North and 82°33' East, 25°8' North) during March to December, 2010.Nitrogen-free Ashby agar medium was used for isolation by serial dilution technique and purification on the same solid medium with repeated plating (Schmidt and Belser, 1982).Thirty-two pure cultures were procured and tested for their morphological and biochemical characteristics.

Genomic DNA extraction
The bacterial strains were grown in nutrient broth at 28 ± 2°C in incubating shaker at 120 rpm for overnight.The method described by Sambrook and Russel (2001) was used with minor modification for Genomic DNA extraction.The 5 ml of overnight grown broth cultures were centrifuged at 10,000 rpm (C-24, Remi) for 10 min and supernatants were decanted.Pellet was resuspend in 0.4 ml TE (0.01 M Tris/HCl, pH 8.0; 0.05 M EDTA, pH 8.0) and added 40 µl of 10% SDS.Suspension was incubated for 30 min at 37°C in water bath with gentle shaking {In case of Gram +ve bacteria, the pellet was resuspend in 0.5 ml SET buffer (57 mM NaCl, 25 mM EDTA pH 8.0, 20 mM Tris-HCl pH 8.0) and added 10 µl lysozyme and incubated for 30 to 60 min at 37°C.After, this added 0.1 volumes of the 10% SDS and 10 µl of Proteinase K (10 mg/ml) and incubated at 55°C for 60 min}.After incubation, 0.4 ml phenol was added with proper mixing and centrifuged for 5 min at 8000 rpm.Then water phase was transferred to in new eppendorf tube and added 0.2 ml phenol and 0.2 ml chloroform with proper mixing.The solution was centrifuged at 8000 rpm for 5 min.Again, water phase was transferred to in new tube and added 0.4 ml chloroform with proper mixing.The solution mixture was centrifuged at 8000 rpm for 5 min.Water phase was transferred to in new tube and estimated volume of solution then added 0.1 volume of 3 M sodium acetate, pH 5.2 and 2 volume of 96% ethanol with proper mixing and kept it at -20°C for 1 h.After that, solution was centrifuged at 8000 rpm for 10 min.The supernatant was discarded and pellet was washed with 70% ethanol (-20°C).The pellet was dissolved in 40 µl of sterile distilled water and kept it at 4°C for further use.

DNA sequencing
Sequencing of 16S rDNA was carried out at Bangalore Genei Pvt. Ltd., Bangalore, India.The 16S rDNA sequences were analyzed with nucleotide database available at the GenBank using BLAST tool at NCBI (www.ncbi.nlm.nih.gov) for identification of bacteria and were submitted at NCBI Gen Bank.

Seed bacterization
Wheat seeds var.HUW 234 was taken from Banaras Hindu University, Agriculture farm, Varanasi, Uttar Pradesh, India and were surface sterilized (0.1% HgCl 2 for 2 min and rinsed five times with sterilized water).Bacterial strains, Bacillus megaterium, Paenibacillus polymyxa, Arthrobacter chlorophenolicus, Serratia marcescens, Enterobacter sp., Microbacterium arborescens and Pseudomonas aeuroginosa were grown separately in nutrient broth at 120 rpm in shaking incubator at 30°C for 48 h.P. aeuroginosa BHUPSB02 was obtained from Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Science, Banaras Hindu University (Verma et al., 2013).Healthy wheat seeds were treated with 7 days old broth cultures of each of the bacterial isolate along with 1 ml of sticker solution (2.5 g gum acacia + 5 g sugar in 100 ml sterilized distilled water) such that a population in the range of 10 7 to 10 8 CFU seed −1 could be obtained.

Pot and field experiments
Growth promotion effect of bacterial strains on wheat var.HUW 234 under pot and field experiments were studied during 2011 to 2012 and 2012 to 2013.Sandy loam soil collected from Agricultural Research Farm, BHU was sieved through 10 mesh sieve and filled in 5 kg of earthen pots lined with polythene.Recommended doses of fertilizers for N, P, K (120:60:60) were thoroughly mixed with required quantity of soil.Wheat seeds were treated with B. megaterium, P. polymyxa, A. chlorophenolicus, S. marcescens, Enterobacter sp., M. arborescens and P. aeuroginosa in four replications.Ten bacterial treated seeds were sown in each pot.Three plants after full emergence of the first leaf were maintained in each pot.Uninoculated seeds treated with nutrient broth without any bacterial strain were sown in pot as control.Pots were arranged in a complete randomized design.Necessary agronomical practices were followed.A field experiment with same bacterial strains as in pot culture experiment was conducted following the randomized block design at farmers , field of Bhadohi district, Uttar Pradesh, India with plot size 4 × 3 m (12 m 2 ).Each plot was basely dressed with N, P, K (120:60:60) ha -1 .Bacterial treated seed @ 100 kg ha -1 was sown to each plot in line.Plots were irrigated timely and all other agronomical intercultural processes were followed.Height of plant was measured at 30, 60 and 90 days of sowing.At maturity, crop was harvested, threshed and yield was recorded as described by Iswaran and Marwah (1980).

Enumeration of microbial population of post harvest soil
For enumerating the microbial population of post harvest soil was determined with Kenknight and Munaier's Medium for Actinomycetes, Czapek Dox Agar for Fungi, Pikovskaya agar for phosphate solubilizing bacteria and Soyabean Casein Digest Medium for bacteria via serial dilution techniques as described by Schmidt and Belser (1982).
Ten gram soil sample was taken in 250 ml conical flask containing 95 ml of sterile water and shaken on mechanical shaker to mix soil and water.With the help of sterile pipette 1 ml suspension from conical flask was transferred to culture tube containing 9 ml of sterile water.Culture tube was then shaken using stirrer.Desired level of serial dilution was obtained by adopting the similar procedure.Serial dilution of 10 -5 was made for each microbe. 1 ml of suspension was transferred to sterilized petriplates containing about 20 ml of respective sterilized media (40°C).After solidifying the media, petriplates were inverted and kept in BOD at 28±2°C.Microbial counts were recorded when particular colonies developed.Data were analyzed by one-way analysis of variance (ANOVA).Significant differences between means were compared using Fisher's protected LSD test at P ≤ 0.05.Statistical analysis was performed by using SPSS software version 16.0.

RESULTS AND DISCUSSION
Biochemical characteristics such as catalase, MR, VP test, NH 3 , HCN production, gram's straining and shape have been given in Table 1.Bacterial strains; S. marcescens, Enterobacter sp. and P. aeuroginosa show Gram negative reaction.All strains are rod in shape.All bacterial strains show positive catalase test and ammonia production except Enterobacter sp which showed negative catalase test.Most of the strains show positive methyl red, Voges -Proskauer and HCN production.Biochemical characters were showed by bacterial strains play very crucial role in plant growth promotion activity (Aneja, 2003;Kumar et al., 2014).Various studies have indicated a disease protective effect to HCN as in the suppression of "root-knot" and black rot in tomato and tobacco root caused by the nematodes Meloidogyne javanica and Thielaviopsis basicota, respectively (Siddiqui et al., 2006).To date, different bacterial genera Alcaligenes, Aeromonas, Bacillus, Pseudomonas and Rhizobium have shown their capacity to produce HCN (Ahmad et al., 2008).
The maximum and significant solubilization of tricalcium phosphate was recorded by P. aeuroginosa (160.34 µgml -1 ) followed by B. megaterium (153.60 µgml - 1 ) over control at 5 days after inoculation.Except Enterobacter sp.all strains changed pH of broth culture and also solubilized tri-calcium phosphate.Change in pH of broth culture varied from 4.42 to 5.12 in vitro conditions.P. aeuroginosa showed maximum and significant decrease in pH of broth followed by B. megaterium over most of the bacterial strains.The low availability of P to plants is because the conversion of soluble form of P to insoluble forms of P as plants can absorb P in forms of H 2 PO 4 − and HPO 4 2− (Glass, 1989).Most soil bacteria solubilize insoluble phosphates; particularly active are those that belong to the genera Pseudomonas, Enterobacter and Bacillus (Whitelaw, 2000).Bacterial strains name and their accession numbers were showed in Table 2 and also present BLAST search admirable agreement for such close 16S rDNA database similarity.The 16S rDNA gene characterization confirmed the identification of plant growth promoting rhizobacteria.

Effect of bacterial strains on periodic growth of wheat plant under pot and field experiments
Height of plant under pot culture experiment after seed inoculation with different strains increased with successive periods (Table 3). A. chlorophenolicus showed significant and maximum plant height at 30, 60 and 90 days after seed inoculation over uninoculated control.P. polymyxa gave same plant height as A. chlorophenolicus at 30 days but lesser height of plant at advanced period of wheat that might be due to delay acclimatization of P. polymyxa in soil than A. chlorophenolicus.Besides plant height, also other factors like efficiency and time taken in acclimatization of bacterial strains in soil.More plant heights were recorded at 60 days after sowing of bacterial strains.Increase in plant height from 4.70 to 22.20%, 4.30 to 19.50% and 3.60 to 23.60%, respectively as compared to uninoculated control at 30, 60 and 90 days after sowing due to various bacterial strains under pot experiment.Cakmakci et al. (2007) reported that 2.2 to 24.6% plant height was recorded in green house experiment with Bacillus sp., Pseudomonas putida and P. polymyxa as compared to the uninoculated control.Kumar et al (2014) reported almost similar finding in plant height of wheat with inoculation of different PGPR.
A. chlorophenolicus and M. arborescens showed significant and maximum 42% increase in plant height at 30 days while M. arborescens caused significantly 27% increase in plant height at 60 days after sowing over uninoculated control under field experiment (Table 3).However at 90 days, Enterobacter sp. could lead up to 29% height of plant as compared to control.The increase in plant height by various strains in filed at successive period was due to efficiency of strains to enhance the uptake of essential nutrients required to plants and efficacy of their acclimatization in soil.In general strains which performed better in pot experiment, also contributed significantly under field experiment.Higher plant height was observed under pot than field due to better management which helped in checking the loss of nutrients in pot culture experiment and consequently greater nutrient acquisition positively influenced the height of plant (Kumar et al., 2014).Ali et al. (2011) has reported that inoculation of wheat with thermotolerant P. putida significantly increased 1.12 fold more shoot height than uninoculated control.Bacillus, Arthrobacter and Enterobacter were evaluated for their effect on growth of wheat (Zhang et al., 2012;Kumar et al., 2014).

Effect of bacterial strains on yield of wheat plant under pot and field experiments
B. megaterium showed maximum and significant 51.45% grain and 40.47% straw yield followed by A. chlorophenolicus as compared to unninoculated control under pot (Table 4).All strains except P. polymyxa, P. aeuroginosa and S. marcescens caused significant enhancement in grain yield over uninoculated control.Bacterial strains increased from 9.77 to 51.45% and 6.50 to 40.47% grain and straw yield, respectively as over control pot.Almost, similar result was also recorded with inoculation of B. polymyxa and E. cloacae on winter wheat that increase grain yield by 17.56% and 4.65%, respectively over control under growth chamber (Renato de Freitas, 2000).Significant increase in yields due to seed bacterization of P. aeruginosa, S. proteamaculans, Arthrobacter, Enterobacter and Bacillus sp. also have been reported (Zahir et al., 2009;Kumar et al., 2014).S. marcescens showed maximum and significant 14.78% grain and 17.04% straw yield followed by Enterobacter sp. and P. polymyxa as compared to uninoculated control in field experiment.All strains except M. arborescens showed significantly greater yield as compared to uninoculated control in field.However, yield depends on efficiency and performance of strains, how they can easily acclimate in soil environmental conditions.Turan et al. (2012) have reported that inoculation of Bacillus sp. and Azospirillum brasilense increased grain yield by 24 and 19% of wheat crop.Almost similar result was reported on wheat crop by inoculation of various PGPR strains (Rodríguez Cáceresa et al., 2009;Kumar et al., 2014).

Microbial population of post harvest soil under pot and field experiments
Microbial populations of bacteria, Actinomycetes, fungi and phosphate solubilizing bacteria (PSB) under pot have been depicted in Figure 1.As compared to uninoculated control, significant population of bacteria and PSB were recorded with inoculation of P. aeuroginosa while maximum and significant population of fungi and actinomycetes were caused by M. arborescens of post harvest soil.In general, post harvest soil having maximum population of bacteria followed actinomycetes, fungi and least by PSB.Analyzed post harvest soil increased population up to 58, 50, 87 and 114% of bacteria, Actinomycetes, fungi and PSB as compared to uninoculated control in pot experiment.Increases in soil microbial population due to seeds were treated with different bacterial strain which enhanced the microbial population in soil.Almost similar results have also been reported by Kalaigandhi et al. (2010) and Shinde and Latake (2009).
In field experiment, all the bacterial strains except P. polymyxa showed significantly greater bacterial population in soil as compared to uninoculated control (Figure 2).All inoculated strains gave significantly higher population of bacteria, fungi, actinomycetes and PSB those ranged from 4.17 to 58.33%, 7.14 to 85.71%, 10 to 50% and 14.29 to 114.29%, respectively over uninoculated control.In comparison to pot, higher population of various microorganisms recorded in field soil due to better acclimatized and natural environment to flourish the microflora.Maurya et al. (2012) also reported higher population of Azotobacter and Azospirillum in field condition.

Conclusions
These studies show that single inoculation of S. marcescens, A. chlorophenolicus, Enterobacter and B. megaterium enhance growth and yield of wheat.These  strains may be used as efficient PGPR for wheat production in farmer's fields.It is an environmentally friendly and cost effective technology.

Table 1 .
Biochemical characteristics of plant growth promoting bacterial strains.
VP', Voges -Proskauer; 'MR', Methyl Red; Data are average of four replicates ± SD.Mean with different letters in the same column differ significantly at P ≤ 0.05 (Fisher's protected LSD).

Table 2 .
Plant growth promoting activities of bacterial strains in vitro.

Table 3 .
Effect of bacterial strains on periodic growth of wheat under pot and field experiments.Days after sowing, 'cm', centimeter; Data are average of four replicates ± SD.Mean with different letters in the same column differ significantly at P ≤ 0.05 (Fisher's protected LSD).

Table 4 .
Effect of bacterial strains on yield of wheat under pot and field experiments.