Advances in Agriculture

Advances in Agriculture / 2014 / Article

Research Article | Open Access

Volume 2014 |Article ID 248591 | 13 pages | https://doi.org/10.1155/2014/248591

Isolation and Molecular Characterization of Potential Plant Growth Promoting Bacillus cereus GGBSTD1 and Pseudomonas spp. GGBSTD3 from Vermisources

Academic Editor: Tibor Janda
Received01 May 2014
Accepted28 Jul 2014
Published01 Sep 2014

Abstract

Vermicompost was prepared from leaf materials of Gliricidia sepium + Cassia auriculata + Leucaena leucocephala with cow dung (1 : 1 : 2) using Eudrilus eugeniae (Kinberg) and Eisenia fetida for 60 days. Nineteen bacterial strains which have the capability to fix nitrogen, solubilize inorganic phosphate, and produce phytohormones were isolated from vermicompost, vermisources, and earthworm (fore, mid, and hind) guts and tested for plant growth studies. Among the bacterial strains only five strains had both activities; among the five Bacillus spp. showed more nitrogen fixing activity and Pseudomonas spp. showed more phosphate solubilizing activity. Hence these bacterial strains were selected for further molecular analysis and identified Bacillus cereus GGBSTD1 and Pseudomonas spp. GGBSTD3. Plant growth studies use these two organisms separately and as consortium (Bacillus cereus + Pseudomonas spp.) in (1 : 1) ratio at different concentrations using Vigna unguiculata (L.) Walp. at different day intervals. The germination percent, shoot length, root length, leaf area, chlorophyll a content of the leaves, chlorophyll b content of the leaves, total chlorophyll content of the leaves, fresh weight of the whole plant, and dry weight of the whole plant were significantly enhanced by the consortium (Bacillus cereus + Pseudomonas spp.) of two organisms at 5 mL concentrations on the 15th day compared to others.

1. Introduction

Nowadays, under the modern agricultural practices, chemical fertilizers are used to boost the crop production. But the application of chemical fertilizers affects the total productivity of the crops and in the long run the soil becomes sterile and unfit for cultivation practices. Hence, in order to enhance the fertility status of the soil, the natural way of feeding the soil with different types of organic inputs (composts, vermicomposts, biofertilizers, farmyard manure, etc.) has been developed so as to ensure sustained productivity [1]. However, a better understanding of nutrient cycling and the factors governing their decomposition in soil is imperative for implementing sustainable management practices. Nutrient cycling in soil involves chemical, biochemical, and physicochemical reactions, with the biochemical reactions being catalyzed by soil enzymes associated with viable cells of microbial origin and plant roots. Therefore, any factor that affects soil microbial population will necessarily alter soil enzyme activity [2]. Sustainability of agricultural systems has become an important issue all over the world. Many of the issues of sustainability are related to soil quality and its change with time. It is well known that intensive cultivation has led to a rapid decline in organic matter and nutrient levels besides affecting soil physical properties. Conversely, management practices with organic materials influence agricultural sustainability by improving physical, chemical, and biological properties of soils [3].

Recently, there is increasing interest in the potential of vermicomposts, as plant growth media and as soil amendments. Vermicomposts are finely divided peat-like materials with high porosity, aeration, drainage, water-holding capacity, and microbial activity, which make them excellent soil amendments or conditioner. It is a sustainable source of macro- and micronutrients, which enlivens the soil through partial substitution of the horticultural container media. Enhancement in plant growth after substitution of soils or greenhouse container media with composts is attributed to modifications in soil structure, change in water availability, increased availability of macro- and micronutrients, stimulation of microbial activity, and augmentation of the activities of critical substances by microorganisms through interactions with earthworms [4]. The vermicompost is rich in available nutrients, soil beneficial microbes, and plant growth promoting substances.

Research during the past few decades has led to the identification of certain biological organisms and their products that could potentially be used as fertilizer sources. This strategy of fertilizing the soil with biological sources has been widely accepted and recognized as a viable alternative to the application of chemical fertilizers. Nitrogen (N) fixing and phosphate (P) solubilizing bacteria may be important for plant nutrition by increasing N and P uptake by the plants and playing a significant role as plant growth promoting bacteria in the biofertilization of crops. Increasing and extending the role of biofertilizers could reduce adverse environmental effects caused by chemical fertilizers. Nitrogen is one of the basic requirements for the growth, productivity, and yield of plants. On a worldwide basis it is estimated that about 175 million tons of nitrogen per year is added to soil through biological nitrogen fixation. Meanwhile superphosphate fertilizer is expensive and is in short supply and hence biofertilizers can bridge the gap. Soluble fertilizer phosphorus (P) is the world’s second largest bulk agricultural chemical under use and as such is absolutely essential for food production. However, most soil phosphorus, approximately 95–99 percent, is present in the form of insoluble phosphates and hence cannot be utilized by plants [5]. To increase the availability of phosphorus to plants, large amounts of fertilizers are used on a regular basis. But, after application, a large proportion of the fertilizer phosphorus is quickly transferred to the insoluble form [6]. Therefore, a very little percentage of the applied phosphorus only is used, making continuous application necessary. It has been reported that many soil fungi and bacteria can solubilize inorganic phosphates [7]. The Bacillus species offer several advantages over the other genera because of their capacity to produce spores in unfavorable environmental conditions. This characteristic facilitates the conversion of spore suspensions to powder formulations without killing bacteria [8]. Vermicompost is rich in NPK, plant growth promoters, and other nutrients and when applied, the soil will be enriched with a variety of nutrients that will become available for the indigenous microflora.

The PGPR have been known to directly enhance plant growth by a variety of mechanisms, namely, fixation of atmospheric nitrogen that is transferred to the plant, production of siderophores that chelate iron and make it available to the plant root, solubilization of minerals such as phosphorus, and synthesis of phytohormones [9]. Plant growth promoting bacteria (PGPB) stimulate plant growth by nitrogen fixation [10], solubilization of nutrients [11], and production of growth hormones and 1-aminocyclopropane-1-carboxylate (ACC). The present paper describes the potential of PGPR isolates from vermisources and their plant growth stimulating activity in pot trials under ambient conditions. Further studies will clarify the potential use of these bacteria used as biofertilizers.

The main objective of this study was to isolate, identify, and characterize the potential plant growth promoting rhizobacteria from vermisources, as well as evaluating their plant growth potential under controlled environment and characterizing the isolated bacteria using various molecular techniques. These bacteria can be considered promising candidates for application in sustainable agricultural management.

2. Materials and Methods

2.1. Vermicompost Preparation

For the present study epigeic earthworms, Eudrilus eugeniae (Kinberg) and Eisenia fetida (Savigny), were collected from the breeding stock of the Department of Biology, Gandhigram Rural Institute-Deemed University, Gandhigram, Tamil Nadu, India, and leaf materials of Gliricidia sepium Jacq, Leucaena leucocephala (Lam.) De Wit, and Cassia auriculata Linn. were collected from Gandhigram campus. The leaf materials were separately subjected to predigestion for 15 days by sprinkling water on the heap and covering it with gunny bag and turning it periodically in order to release out the initial heat produced during decomposition of organic material. The changes in temperature were observed every three days up to 15 days. The vermibeds were prepared in plastic containers of  cm size and the substrate was moistened to hold 60–80 percent moisture and kept for 24 hours stabilization. 20 numbers of healthy clitellate E. eugeniae and 30 numbers of E. fetida were separately introduced in the vermibeds. The vermicomposting trials were carried out in the rearing room with the relative humidity and the temperature of 75–85 percent and 26–28°C, respectively. The substrate was turned (mixed) once in a week and maintained up to 60 days. The experiment was carried out with three replicates for each substrate with proper control [12].

2.2. Microbial Study

The total microbial counts in terms of colony forming units (CFU) of bacteria in the vermicomposts, vermicasts, and earthworm (fore, mid, and hind) guts were determined every 15 days (0, 15, 30, 45, and 60 d) using standard plate count method. From the total colony forming units, only those bacterial colonies (19 bacterial colonies) which showed predominant growth were restreaked onto appropriate agar medium to obtain pure cultures and subjected to characterization and identification.

2.3. Screening for Biofertilization Activities
2.3.1. In Vitro Nitrogen Fixing Activity

The bacterial isolates were screened for nitrogen fixing ability using nitrogen-free liquid medium [13]. Nitrogen-free medium was prepared and sterilized. The strains were inoculated separately on selective nitrogen-free liquid medium. All the test organisms were incubated at 37°C 2°C for 7 days. The nitrogen fixing activities were observed on the basis of the formation of turbidity in the flasks. Among all the nineteen bacterial isolates tested, only five bacterial isolates formed significantly higher rate of turbidity in the nitrogen-free liquid medium and selected for further molecular and plant growth studies.

2.3.2. In Vitro Phosphate Solubilization

All bacterial isolates were screened for inorganic phosphate solubilization using Pikovskaya’s agar medium, the selective medium for phosphate solubilization test [14]. Pikovskaya’s agar medium was prepared and sterilized and the bacterial strains were inoculated separately on the selective medium containing 0.5 percent of (w/v) tricalcium phosphate (Ca3PO4) as complex insoluble phosphate source. All the test plates of the bacteria were incubated at 37°C 2°C for 10 days. After incubation for up to 7 days at 30°C, formation of yellow halos and/or clearing zones was evaluated. The results were expressed as solubilization index (SI) and they were measured using the following formula [15]:

Five bacterial strains which showed phosphate solubilizing activity were identified and grouped as phosphate solubilizing bacteria (PSB). The bacterial strain which showed highest SI was selected for further molecular and plant growth studies.

2.4. Production of Phytohormones (IAA)

The bacterial cultures were inoculated in nutrient broth with tryptophan (5 μg/mL) and incubated at °C for 5 days. After incubation cultures were centrifuged at 3000 rpm for 30 min. Two milliliters of the supernatant was mixed with 2 drops of orthophosphoric acid and 4 mL of Salkowski’s reagent (50 mL of 35% perchloric acid + 1 mL 0.5 FeCl3) and incubated in the dark for 25 minutes. Development of pink colour indicates indole-3-acetic acid (IAA) production. The optical density was measured at 530 nm using Spectronic 200 (India). The quantity of IAA production was estimated using standard IAA graph and expressed as micrograms per milliliter [16, 17].

2.5. Siderophore Production

A qualitative assay of siderophore production was conducted in Chrome Azurol S (CAS) agar medium [18]. CAS agar plates were prepared and spot-inoculated with test organism and incubated at 30°C for 3–5 days. Change of blue color of the medium surrounding the bacterial growth to fluorescent yellow indicated the production of siderophore. Bacillus subtilis was chosen as a positive control [19, 20].

2.6. 16S rDNA Gene Amplification and Sequencing

Genomic DNA from each isolate was extracted using the modified method of Smoker and Barnum [21]. The extracted DNA was dissolved in 20 μL TE buffer and used as the template for the PCR reactions. PCR amplifications were performed in a total volume of 50 μL by mixing 20 ng of the template DNA with 2.5 mM concentrations of each deoxynucleotide triphosphate and 1 μm of each universal primer of Bac8uf (5′-AGAGTTTGATCCTGGCTCAG-3′) and Univ1492r (5′-CTACGGCTACCTTGTTACGA-3′) described by Edwars et al. [22]. The thermocycling profile was carried out with an initial denaturation at 95°C (4 min) followed by 30 cycles of denaturation at 95°C (1 min), annealing at 56°C (30 s), extension at 72°C (1 min), and a final extension at 72°C (10 min) in an Eppendorf Gradient thermocycler. The PCR amplified rDNA were purified by using the Quick PCR purification kit (Bangalore Genie, India). 16S rRNA gene sequence of the isolate was compared with 16S rRNA gene sequences available by the BLAST search in the NCBI, GenBank database (http://www.ncbi.nlm.nih.gov). The analysis of alignment and homology of the partial nucleotide sequence of Bacillus sp. was carried out by the basic local alignment search tool (BLAST). The PCR products were stored at 4°C. Aliquots of the PCR products were separated by 2% agarose gel electrophoresis in TAE buffer (pH 8.0). A 100 bp DNA marker (Sigma, Bangalore) was used as a reference. Gels were stained with ethidium bromide (11 g/mL) and visualized under UV light.

2.7. Nucleotide Sequence Accession

The sequence obtained in this study was deposited in the GenBank (USA) nucleotide sequence database under the accession number GQ413962 (B. cereus) and HM753262 (Pseudomonas spp.).

2.8. Phylogenetic Analysis

The 16S rRNA gene sequences were obtained in the present study and the taxonomically related Bacillus spp. were retrieved from the National Center from Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). All the sequences were aligned using multiple sequence alignment program CLUSTAL W developed by Higgins et al. [23]. The pairwise evolutionary distances were computed using the method of Kimura [24]. The multiple distance matrix obtained was then used to construct phylogenetic trees using neighbour joining method of Saitou and Nei [25]. Phylogenetic trees were constructed by using Mega 5 software (Molecular Evolutionary Genetic Analysis).

2.9. Prediction of RNA Secondary Structure

The secondary structure of 16S rDNA of Bacillus spp. was predicted using the bioinformatics Genebee tool.

2.10. Restriction Site Analysis in 16S rDNA

The restriction sites in DNA of Bacillus spp. were analyzed using the NEB Cutter program version 2.0 tools available online at http://tools.neb.com/NEBcutter2/index.php.

2.11. Fermentation Process

For the preparation of inoculum, 50 mL of nutrient broth medium (HiMedia, Mumbai, India) was inoculated with appropriate overnight bacterial culture and incubated at 37°C for 24 h at 120 rpm ( CFU mL−1). The cell-free supernatant was obtained by centrifuging the fermented broth at 10,000 rpm for 10 min. The supernatant was analyzed for plant growth promotion at various concentrations.

2.12. Evaluation of Plant Growth Promoting Activity under Controlled Environment

The nitrogen fixing bacteria (Bacillus spp.) and the phosphate solubilizing bacteria (Pseudomonas spp.) were separately tested for plant growth promoting activity and also as consortium (1 : 1) at 5 different concentrations, that is, 1, 2, 3, 4, and 5 mL at three different day intervals (5, 10, and 15 days) with proper control through Exp1, Exp2, Exp3, and Exp0, respectively, using Vigna unguiculata (L.) Walp. The seeds (30 seeds in each pot) were sown in plastic pots containing sterilized vermiculite and arranged in a completely randomized factorial design. The seedlings were grown in a greenhouse at a temperature of 28–32°C and 85% relative humidity. The pots were watered to 50% water-holding capacity and were maintained at this moisture content by watering to weight every day and plant growth was observed for 15 days. Parameters such as germination percent, shoot length, root length, leaf area, chlorophyll content of the leaves, fresh weight of the whole plant, and dry weight of the whole plant were measured using standard procedure.

2.13. Statistical Analysis

The following statistical tools were used for the analyses and interpretation of the data. The experimental results are presented in the form of tables and graphs using Microsoft Excel 2007. Data obtained from the different treatments were statistically analyzed using the one-way ANOVA and the mean values were compared by the Duncan’s multiple range test for multiple comparisons. Differences were considered significant at the 0.05 level using Origin software (Version 8.5.0) 2010, Origin Lab Corporation.

3. Results

3.1. Isolation and Characterization of Bacterial Isolates

A total of nineteen bacterial strains were isolated from vermicompost, vermicast, and earthworm (fore, mid, and hind) guts (Table 1). The morphological and biochemical characteristics of bacterial isolates were elucidated in Table 1. All the isolates were tested for plant growth promoting activity. Among the 19 bacterial isolates only five bacterial strains showed plant growth promoting activity. Based on colony morphology, microscopic observations, and cultural, biochemical, and physiological properties, the bacterium was given the name Bacillus spp., Alcaligenes spp., Erwinia spp., Serratia spp., or Pseudomonas spp. These five bacterial strains were able to grow in the nitrogen-free medium and they were also identified as phosphate solubilizing microorganisms on the basis of their solubilization index (Table 2). Bacillus spp. have the highest SI (38.55), followed by Pseudomonas spp. (35.22) and Serratia spp. (30.45). All the isolates produced a significant amount of IAA from the medium in the range of 11.3 to 24.2 μg/mL; Bacillus spp. showed higher IAA production (24.2 μg/mL) and Serratia spp. showed lower IAA production (11.3 μg/mL). All the strains (except Alcaligenes spp.) were positive for siderophore production, showing a yellow zone on the CAS agar medium plate (Table 2).


S. number Sample sourceBiochemical characteristicsc  Name of the   strain
Gram’s reactionaCell shapeMotilitybIndole testMethyl Red testVoges Proskauer testCitrate Utilization testCatalase testUrease testGelatin hydrolysis testNitrate reductase testStarch hydrolysis testCasein hydrolysis testGlucose utilization test

1.Vermicompost (T2)+Cocci++++Staphylococcus spp.
2.Vermicompost (T3)+Rod++++Corynebacterium spp.
3.Vermicompost (T5)+Rod+++++++++Bacillus spp.
4.Vermicompost (T6)+Cocci+++++Micrococcus spp.
5.Vermicast (T3)+Cocci++++++Klebsiella spp.
6.Vermicast (T5)+Rod+++++Streptococcus spp.
7.Vermicast (T6)+Cocci++++++Enterobacter spp.
8.Earthworm hind gut (T2)+Cocci++++++Tricoccocus spp.
9.Earthworm hind gut (T3)+Cocci+++++Yersinia spp.
10.Earthworm hind gut (T5)+Cocci++++Micrococcus spp.
11.Vermicompost (T1)Rod+++++Escherichia spp.
12.Vermicompost (T2)Rod+++++Proteus spp.
13.Vermicompost (T4)Rod++++++Paracoccus spp.
14.Vermicompost (T5)Rod++++++Pseudomonas spp.
15.Vermicast (T2)Rod++++++Citrobacter spp.
16.Vermicast (T3)Cocci++++Erwinia spp.
17.Vermicast (T5)Rod+++++++Alcaligenes spp.
18.Vermicast (T6)Cocci++++++Serratia spp.
19.Earthworm mid gut (T2)Rod++Pediococcus spp.

Gram’s staining: (+) positive, (−) negative.
bMotility: (+) motile, (−) nonmotile.
cBiochemical characteristics: (+) positive, (−) negative.

Name of the
organism
Phosphate solubilization index (SI)IAA production
(g/mL)a
Siderophores productionb

Bacillus spp.38.5524.2+++
Alcaligenes spp.29.3014.7
Erwinia spp.28.9011.3+
Serratia spp.30.4516.8++
Pseudomonas spp.35.2219.6+++

Indole acetic acid production in culture media supplemented with tryptophan (5 g/mL) after 5 days.
bIn vitro siderophores production: − represents the absence of siderophores production and +++ represents >10 mm wide yellow-orange zone.

3.2. Molecular Studies

The partial 16S rRNA sequences carried out in the present study for Bacillus spp. and Pseudomonas spp. covered a stretch of approximately 1500 nucleotides for each. About half of the sequences found in the clone library showed only slight relationship to other known sequences, while the other half were highly similar (approximately 95 percent sequence identity) to other database entries for Bacillus spp. and Pseudomonas spp. Less than 0.5 percent of all nucleotides was found to be unique within the conserved regions of the cloned sequence and could almost always be related to reading errors in ambiguous regions of the sequencing gel. On the basis of phylogenetic analysis of 16S rDNA partial sequences, Bacillus spp. GGBSTD1 is identified as Bacillus cereus and Pseudomonas spp. GGBSTD3 is identified as Pseudomonas spp. Phylogenetic analyses of the strains based on the neighbor joining method were represented in Figures 1 and 2. Among eubacteria, the mean guanine and cytosine () content of genomic DNA varies from approximately 25% to 75%. That the bacterial genomic content is somehow related to phylogeny has been suggested [26, 27]. The phylogenetic tree of eubacterial 16S rRNA clearly indicates this relationship. Gram-positive bacteria Bacillus subtilis have genomic , 42%. Gram-negative bacteria with intermediate content, such as Escherichia coli (50%), Serratia marcescens (58%), Salmonella typhimurium (51%), and Pseudomonas fluorescens (60%), belong to the common Gram-negative bacteria [28]. Bacillus cereus have the G  +  C content 53% and A  +  T content 47%, similarly Pseudomonas spp. having content 53% and content 47%. Most functional RNA molecules have characteristic secondary structures that are highly conserved in evolution. In this study RNA secondary structures of Bacillus cereus and the Pseudomonas spp. were predicted, Bacillus cereus have free energy of −294.1 kkal/mol, and Pseudomonas spp. have free energy of −299.8 kkal/mol.

3.3. Evaluation of Biofertilization Abilities under Controlled Conditions

Germination percentage of Vigna unguiculata seeds sown in vermiculite supplemented with Bacillus cereus (Exp1), with Pseudomonas spp. (Exp2), and with consortium (Exp3) (1 : 1 ratio) at five different concentrations (1, 2, 3, 4, and 5 mL) and in the control (Exp0) is shown in Figure 3. The highest germination percentage was observed in Exp3 (consortium) at 5 mL concentration compared to others. The increase in germination percentage and in other growth parameters can be attributed to the combined effect of the nitrogen fixing and phosphate solubilizing microbes which are able to fix atmospheric nitrogen and solubilize P and also produce growth promoting substances. These organisms have been isolated from vermisources and this observation indicates the reason for promotion and enhancement of crop growth and yield when vermicompost is applied. It is a proof that these symbiotic microbes present in vermisources benefit the soil fertility. Observation on growth parameters such as shoot length, root length, and leaf area of V. unguiculata is given in Tables 3 and 4, respectively. The shoot length ( cm), root length ( cm), and leaf area ( cm2) of the plant get significantly () increased in Exp3 (consortium of Bacillus cereus + Pseudomonas spp. [1 : 1]) at 5 mL concentration on the 15th day compared to other experiments. The chlorophyll a, b and total chlorophyll content of the leaves on d 5, d 10, and d 15 are given in Table 5. The chlorophyll a ( mg/g of fresh leaf), chlorophyll b ( mg/g of fresh leaf), and total chlorophyll ( mg/g of fresh leaf) content of leaves were significantly () higher in Exp3 (consortium of Bacillus cereus + Pseudomonas spp. (1 : 1)) at 5 mL concentration on the 15th day compared to other experiments. Observations on the fresh weight of the whole plant are given in Table 6. The fresh weight of the whole plant ( g) was significantly () higher in Exp3 (consortium of Bacillus cereus + Pseudomonas spp. (1 : 1)) at 5 mL concentration on the 15th day compared to other experiments. Observations on the dry weight of the whole plant are given in Table 6. The dry weight of the whole plant ( g) was significantly () higher in Exp3 (consortium of Bacillus cereus + Pseudomonas spp. (1 : 1)) at 5 mL concentration on the 15th day compared to other experiments.


ExperimentsaQuantity of bacterial cultures addedShoot length (cm)bRoot length (cm)b
5th day10th day15th day5th day10th day15th day

Exp01 mL2.97 (ab)3.97 (a)4.97 (ab)1.97 (de)2.83 (ab)3.97 (bcd)
2 mL3.03 (bcd)4.03 (ab)5.00 (ab)2.00 (abc)2.87 (de)4.00 (a)
3 mL3.10 (abc)4.07 (a)5.03 (ab)2.03 (de)2.90 (abc)4.03 (ab)
4 mL3.10 (bcd)4.10 (bcd)5.07 (bcd)2.07 (a)2.97 (de)4.10 (abc)
5 mL3.13 (a)4.13 (ab)5.10 (ab)2.10 (bcd)3.00 (ab)4.13 (de)

Exp11 mL3.40 (ab)4.40 (abc)5.40 (ab)2.33 (abc)3.27 (bcd)4.33 (f)
2 mL4.50 (ab)5.40 (abc)6.47 (ab) 3.33 (ab)4.30 (a)5.40 (abc)
3 mL5.50 (de)6.30 (ab)7.50 (a)4.40 (de)5.50 (abc)6.20 (a)
4 mL6.53 (ab)7.30 (a)8.33 (ab)5.50 (bcd)6.53 (de)7.50 (abc)
5 mL 7.40 (ab)8.40 (abc)9.23 (f)6.40 (abc)7.53 (ab)8.27 (ab)

Exp21 mL2.77 (bcd)3.57 (f)4.40 (de)1.60 (de)2.17 (abc)3.50 (f)
2 mL3.43 (abc)4.47 (ab)5.53 (ab)2.53 (abc)3.40 (bcd)4.27 (bcd)
3 mL4.37 (de)5.43 (abc)6.33 (abc)3.40 (ab)4.43 (f)5.33 (ab)
4 mL5.47 (bcd)6.47 (de)7.33 (a)4.20 (bcd)5.50 (ab)6.33 (ab)
5 mL6.40 (f)7.33 (bcd)8.33 (bcd)5.33 (ab)6.30 (ab)7.30 (bcd)

Exp31 mL5.30 (bcd)6.47 (ab)7.30 (ab)4.33 (de)5.50 (abc)6.20 (ab)
2 mL6.43 (ab)7.40 (bcd)8.27 (f)5.47 (a)6.30 (de)7.33 (ab)
3 mL7.37 (bcd)8.40 (bcd)9.27 (ab)6.40 (ab)7.40 (bcd)8.40 (bcd)
4 mL8.47 (abc)9.30 (a)10.43 (abc)7.40 (f)8.33 (a)9.37 (abc)
5 mL9.57 (ab) 10.30 (f)11.47 (bcd)8.33 (abc)9.40 (ab)10.27 (bcd)

Exp0: nutrient solution (control), Exp1: Bacillus cereus, Exp2: Pseudomonas spp., Exp3: consortium of Bacillus cereus + Pseudomonas spp. (1 : 1).
bMean of two repeated experiments (30 plants). Different letters within parenthesis indicate significant difference between treatments for each growth parameter using Duncan’s multiple range test .

ExperimentsaQuantity
of bacterial cultures added
Leaf area (cm2)b
5th day10th day15th day

Exp01 mL0.27 (ab)0.94 (abc)4.07 (b)
2 mL0.35 (ab)1.02 (ab)4.19 (abc)
3 mL0.40 (abc)1.25 (ab)4.71 (bcd)
4 mL0.48 (ab)1.38 (abc)5.10 (ab)
5 mL0.54 (abc)1.61 (b)5.38 (b)

Exp11 mL0.18 (bcd)1.23 (b)6.01 (abc)
2 mL0.25 (ab)1.38 (abc)6.32 (ab)
3 mL0.33 (abc)1.50 (ab)6.84 (b)
4 mL0.38 (ab)1.68 (ab)6.86 (abc)
5 mL0.44 (abc)1.91 (bcd)7.33 (ab)

Exp21 mL0.11 (b)1.07 (ab)5.34 (b)
2 mL0.14 (ab)1.19 (ab)5.59 (ab)
3 mL0.17 (abc)1.27 (ab)5.79 (ab)
4 mL0.21 (ab)1.53 (abc)6.31 (abc)
5 mL0.26 (ab)1.66 (b)6.67 (b)

Exp31 mL0.26 (bcd)2.97 (ab)7.53 (bcd)
2 mL0.33 (b)3.25 (ab)7.87 (ab)
3 mL0.40 (ab)3.50 (bcd)8.11 (bcd)
4 mL0.44 (b)3.84 (b)8.61 (b)
5mL0.62 (ab)4.00 (bcd)9.05 (ab)

Exp0: nutrient solution (control), Exp1: Bacillus cereus, Exp2: Pseudomonas spp., Exp3: consortium of Bacillus  cereus + Pseudomonas spp. (1 : 1).
bMean of two repeated experiments (30 plants). Different letters within parenthesis indicate significant difference between treatments for each growth parameter using Duncan’s multiple range test .

Experimentsa Quantity of bacterial Cultures addedChlorophyll content (mg/g of fresh leaf)b
5th day10th day15th day
Chlorophyll aChlorophyll bTotal
chlorophyll
Chlorophyll aChlorophyll bTotal
chlorophyll
Chlorophyll aChlorophyll bTotal chlorophyll

Exp01  mL0.21 (ab)2.85 (abc)3.07 (a)0.20 (abc)4.81 (a)5.01 (ab)0.15 (bcd)6.12 (abc)6.27 (bcd)
2 mL0.22 (abc)2.93 (ab)3.15 (abc)0.21 (ab)4.89 (ab)5.10 (bcd)0.12 (abc)6.18 (ab)6.30 (ab)
3 mL0.23 (abc)3.01 (ab)3.15 (abc)0.22 (bcd)4.97 (bcd)5.18 (a)0.14 (a)6.33 (cde)6.47 (bcd)
4 mL0.24 (ab)3.08 (f)3.32 (a)0.22 (a)5.04 (b)5.27 (abc)0.15 (bcd)6.41 (cde)6.56 (a)
5 mL0.25 (f)3.16 (abc)3.41 (bcd)0.19 (ab)5.11 (ab)5.30 (abc)0.11 (cde)6.45 (ab)6.50 (abc)

Exp11 mL0.35 (b)4.00 (cde)4.35 (abc)0.11 (b)5.81 (cde)5.93 (cde)0.10 (abc)7.04 (abc)7.15 (f)
2 mL0.26 (abc)4.62 (bcd)4.88 (f)0.11 (abc)6.47 (b)6.58 (abc)0.06 (ab)7.79 (bcd)7.85 (a)
3 mL0.21 (bcd)5.26 (abc)5.47 (ab)0.08 (bcd)7.18 (ab)7.26 (bcd)0.06 (f)8.47 (f)8.53 (ab)
4 mL0.08 (f)5.87 (cde)5.95 (b)0.11 (bcd)7.78 (abc)7.89 (abc)0.04 (bcd)9.33 (b)9.37 (bcd)
5 mL0.26 (abc)6.41 (bcd)6.67 (abc)0.25 (cde)8.34 (cde)8.59 (f)0.07 (abc)9.76 (cde)9.83 (abc)

Exp21 mL0.30 (abc)3.54 (ab)3.84 (b)0.12 (a)5.45 (abc)5.56 (ab)0.07 (bcd)6.81 (abc)6.88 (bcd)
2 mL0.29 (bcd)4.20 (f)4.49 (abc)0.11 (bcd)6.10 (ab)6.21 (abc)0.07 (abc)7.44 (f)7.52 (bcd)
3 mL0.22 (ab)5.04 (abc)5.27 (bcd)0.15 (a)6.77 (cde)6.92 (bcd)0.06 (bcd)8.12 (ab)8.17 (bcd)
4 mL0.10 (f)5.66 (a)5.76 (cde)0.18 (abc)7.44 (f)7.62 (ab)0.05 (ab)8.85 (a)8.90 (abc)
5 mL0.06 (bcd)6.22 (abc)6.28 (abc)0.18 (b)8.10 (abc)8.28 (a)0.04 (cde)9.48 (a)9.52 (f)

Exp31 mL0.27 (ab)4.70 (bcd)4.96 (cde)0.09 (ab)6.68 (b)6.77 (cde)0.10 (a)8.14 (ab)8.25 (a)
2 mL0.20 (ab)5.48 (a)5.68 (f)0.03 (bcd)7.46 (bcd)7.49 (ab)0.09 (ab)8.87 (f)8.97 (ab)
3 mL0.05 (bcd)5.84 (abc)5.89 (ab)0.08 (bcd)8.04 (abc)8.12 (cde)0.10 (ab)9.53 (abc)9.63 (bcd)
4 mL0.23 (f)6.63 (ab)6.86 (cde)0.07 (ab)8.70 (b)8.77 (f)0.10 (abc)10.19 (bcd)10.29 (f)
5 mL0.22 (ab)7.44 (cde)7.66 (a)0.09 (a)9.32 (abc)9.40 (ab)0.10 (a)10.85 (ab)10.95 (abc)

Exp0: nutrient solution (control), Exp1: Bacillus cereus, Exp2: Pseudomonas spp., Exp3: consortium of Bacillus cereus + Pseudomonas spp. (1 : 1).
bMean of two repeated experiments (30 plants). Different letters within parenthesis indicate significant difference between treatments for each growth parameter using Duncan’s multiple range test .

ExperimentsaQuantity of bacterial cultures addedFresh weight of the whole plant (g)bDry weight of the whole plant (g)b
5th day10th day15th day5th day10th day15th day

Exp01 mL0.58 (bcd)0.61 (abc)0.64 (ab)0.06 (a)0.06 (abc)0.06 (ab)
2 mL0.58 (ab)0.61 (a)0.64 (abc)0.06 (bcd)0.06 (ab)0.06 (ab)
3 mL0.58 (abc)0.61 (ab)0.64 (bcd)0.06 (abc)0.06 (bcd)0.06 (b)
4 mL0.41 (abc)0.61 (ab)0.64 (ab)0.06 (abc)0.06 (ab)0.06 (abc)
5 mL0.58 (abc)0.61 (abc)0.64 (bcd)0.06 (a)0.06 (bcd)0.06 (def)

Exp11 mL0.62 (abc)0.65 (abc)0.68 (ab)0.06 (bcd)0.06 (ab)0.07 (bcd)
2 mL0.65 (bcd)0.68 (bcd)0.71 (def)0.06 (abc)0.07 (a)0.07 (ab)
3 mL0.68 (abc)0.71 (def)0.74 (bcd)0.07 (abc)0.07 (ab)0.07 (abc)
4 mL0.71 (ab)0.74 (abc)0.77 (abc)0.07 (bcd)0.07 (b)0.08 (bcd)
5 mL0.74 (abc)0.77 (def)0.80 (ab)0.07 (def)0.08 (a)0.08 (b)

Exp21 mL0.56 (ab)0.59 (bcd)0.62 (ab)0.06 (ab)0.06 (ab)0.06 (abc)
2 mL0.59 (abc)0.62 (abc)0.65 (def)0.06 (ab)0.06 (abc)0.06 (bcd)
3 mL0.62 (bcd)0.65 (a)0.68 (bcd)0.06 (abc)0.06 (bcd)0.07 (abc)
4 mL0.65 (abc)0.68 (ab)0.71 (b)0.06 (ab)0.07 (abc)0.07 (b)
5 mL0.68 (abc)0.71 (b)0.74 (a)0.07 (def)0.07 (abc)0.07 (bcd)

Exp31 mL0.74 (ab)0.77 (ab)0.80 (abc)0.07 (a) 0.08 (bcd)0.08 (ab)
2 mL0.77 (bcd)0.80 (a)0.83 (bcd)0.08 (a)0.08 (bcd)0.08 (abc)
3 mL0.80 (bcd)0.83 (bcd)0.86 (abc)0.08 (ab)0.08 (b)0.09 (b)
4 mL0.83 (bcd)0.86 (abc)0.89 (ab)0.08 (abc)0.09 (ab)0.09 (bcd)
5 mL0.86 (ab)0.89 (b)0.92 (def)0.09 (bcd)0.09 (bcd0.09 (b)

Exp0: nutrient solution (control), Exp1: Bacillus cereus, Exp2: Pseudomonas spp., Exp3: consortium of Bacillus cereus + Pseudomonas spp. (1 : 1).
bMean of two repeated experiments (30 plants). Different letters within parenthesis indicate significant difference between treatments for each growth parameter using Duncan’s multiple range test .

4. Discussion

Inoculation of these newly identified nitrogen fixing and phosphate solubilizing microbes in the plant growth medium enhanced various physiological activities culminating in higher biomass production in V. unguiculata. Since this is a nonhazardous way of fertilization of crop plants, it is very relevant to a developing country like India. This technique can save the farmers of India from the problem posed by high cost of fertilizers by offering a comparatively inexpensive alternative (biofertilizers), while additionally preventing the degradation of the soil and thereby ensuring sustainable agriculture. Bess (1999) reported that the content in composting needs to be determined through the concentration of six functional groups of microorganisms such as aerobic bacteria, anaerobic bacteria, fungi, actinomycetes, Pseudomonas, and nitrogen fixing bacteria [29]. Now there are ways to evaluate the concentrations of these organisms in the finished compost and that can serve as an interpretation guide to determine the quality of the compost as an inoculant of soil microorganisms. Grafff (1981) and Atlavinyte have reported a higher P content in the cast than in the surrounding soil [30]. Satchell (1983) has linked the phosphatase activity in the gut of worms to the availability of bound P in soils. The bacterial strains which have the capability to fix nitrogen only can grow in the nitrogen-free medium [31]. Hence the nitrogen fixing ability was tested in the present study based on the growth of bacterial strain in nitrogen-free medium. Five bacteria strains showed more nitrogen fixation; among all the five bacterial strains tested only one strain (i.e., Bacillus spp.) showed significantly more turbidity compared to the other strains. The ultimate source of the nitrogen used by plants is N2 gas, which constitutes 78% of the Earth’s atmosphere. Unfortunately higher plants cannot metabolize N2 directly into protein. N2 gas must be converted to a plant available form [32] isolated many species of nitrogen fixing Bacillus (B. megaterium, B. cereus, B. subtilis, B. licheniformis, and B. azotoformans) from the rhizospheric zone of rice fields in Central China. The widely studied Bacillus genus represents one of the most diverse genera in the bacilli group. Numerous Bacillus and Paenibacillus strains express plant growth promoting (PGP) activities and a number of these strains have already been commercially developed as biological fungicides, insecticides, and nematicides or generic plant growth promoters. The use of these strains in agriculture has recently been reviewed [33]. Those strains besides having several PGP properties can also fix nitrogen and also do phosphate solubilization. The symbiotic fixation of nitrogen through inoculation of legume crops with effective rhizobia is well known [34]. Asymbiotic nitrogen fixing bacteria, which live in the rhizosphere and/or endophytically, often increase yields of crops. Many bacterial species have nitrogen fixing properties, including Bacillus spp., Azotobacter spp., Azospirillum spp., Beijerinckia spp., and Pseudomonas spp. [35]. Nitrogen fixing bacteria have been used in foliar applications in mulberry. The solubilization index (SI) was formed due to solubilization of insoluble phosphates by organic acid secretion [36]. El-Komy (2005) indicated that Pseudomonas fluorescence and Bacillus megaterium strains were the most powerful phosphate solubilizers on PVK plates as well as on Pikovskaya’s broth [37]. The P concentration in Pikovskaya’s broth increased gradually, achieving a peak on the sixth day and declined slowly during the late days. In this study, pH values decreased gradually in PVK broth during the early incubation days and no revival was observed during later days for all the tested bacterial strains. Several bacteria, particularly those belonging to the genus Bacillus spp., convert insoluble phosphate into soluble forms by secreting organic acids such as formic acid, acetic acid, propionic acid, citric acid, fumaric acid, gluconic acid, glyoxylic acid, ketobutyric acid, malonic acid, succinic acid, and tartaric acid. These acids lower the pH and bring about the dissolution of bound forms of phosphate. Some of the hydroxyl acids may chelate with calcium and iron resulting in effective solubilization and utilization of phosphates [38].

Beneduzi et al. (2008) had isolated the plant growth promoting strain SVPR30 and identified by 16S rRNA gene sequence as Bacillus spp. and they had tested it for plant growth through in vivo experiments [39]. This strain was characterized as a high IAA producer, able to solubilize phosphate and also fix a considerably high amount of nitrogen. The inoculation of rice with Bacillus spp. SVPR30 strain showed a significant increase in the root and shoot parts when compared with the controls within 15 and 30 days after sprouting. Numerous Bacillus and Paenibacillus strains expressed plant growth promoting (PGP) activities and a number of these strains have been commercially developed as generic plant growth promoters. The use of these strains in agriculture has recently been reviewed [33].

Various reports indicate that coinoculation of beneficial organisms generally increased plant growth relative to single inoculation with a sole beneficial organism [40]. Most of the effects of the individual microorganisms coinoculation are additive, although a synergistic effect has been reported in some cases. The average root to shoot ratio was higher in B. subtilis treated yam minisetts in comparison to those not treated with the bacterial culture [41]. In earlier reports, root elongation was found to occur in Sesbania aculeata by inoculation with Azotobacter spp. and Pseudomonas spp. in Vigna radiata by Pseudomonas putida [16] and in Pennisetum americanum by Azospirillum brasilense [42]. Esitken et al. (2006) had found out that Bacillus OSU-142 and Pseudomonas BA-8 alone or in combination had a great potential to increase the growth, yield, and nutrition of sweet cherry plant [43]. Bacillus M3, Bacillus OSU-142, and Micobacterium FS01 separately or in combination were found to have great potential for use as plant growth promoting rhizobacteria to increase production in apples and in many other crops [44]. These phosphate solubilizing microbes consist predominantly of fungal, bacterial, and actinomycetes species, collectively called phosphate solubilizing microorganisms—PSM [45]. There are several microorganisms which can also solubilize the cheaper sources of phosphorous such as rock phosphate. Bacteria such as Bacillus are widely used in plant production system and are important phosphorus solubilizing microorganisms, resulting in improved growth, yield of crops, and metabolic activities, especially in synthesis of protein [34, 46].

5. Conclusions

This study illustrates the isolation of plant growth promoting bacteria from vermisources, screening under in vitro conditions for multiple PGPR traits and their evaluation under controlled conditions in a pot experiment. The findings of this study indicated that the consortium of B. cereus and Pseudomonas spp. acts as a potential biofertilizer, due to its various plant growth promotion abilities such as solubilization of phosphate and production of IAA and siderophore; it can be developed as potential microbial inoculant for various crops.

Conflict of Interests

The authors declared that there is no conflict of interests regarding this paper.

Acknowledgments

The authors are thankful to the Department of Biology, Gandhigram Rural Institute—Deemed University, Gandhigram. This research was funded by University Grants Commission under UGC Research Fellowship in Science for Meritorious Students (BSR) Grant no. F.4-1/2008.

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Copyright © 2014 Balayogan Sivasankari and Marimuthu Anandharaj. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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