V. Govindasamy

Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot Fungus, Rhizoctonia bataticola

A total of 137 bacterial isolates from surface sterilized root, stem, and nodule tissues of soybean were screened for their antifungal activity against major phytopathogens like Rhizoctonia bataticola,Macrophomina phaseolina, Fusarium udam, and Sclerotium rolfsii. Nine bacterial endophytes suppressed the pathogens under in vitro plate assay. These were characterized biochemically and identified at the genus level based on their partial sequence analysis of 16S rDNA. Eight of the isolates belonged to Bacillus and one to Paenibacillus. The phylogenetic relationship among the selected isolates was studied and phylogenetic trees were generated. The selected isolates were screened for biocontrol traits like production of hydrogen cyanide (HCN), siderophore, hydrolytic enzymes, antibiotics, and plant growth promoting traits like indole 3-acetic acid production, phosphate solubilization, and nitrogen fixation. A modified assessment scheme was used to select the most efficient biocontrol isolates Paenibacillus sp. HKA-15 (HKA-15) and Bacillus sp. HKA-121 (HKA-121) as potential candidates for charcoal rot biocontrol as well as soybean plant growth promotion.

Bacillus and Paenibacillus spp.: Potential PGPR for Sustainable Agriculture

The Gram-positive aerobic endospore-forming bacteria (AEFB) belonging to the genus Bacillus and Paenibacillus are essentially ubiquitous and occur abundantly in most rhizospheric soils. In the rhizosphere, species of these two genera are involved in atmospheric nitrogen fixation, solubilization of soil phosphorus and uptake of micronutrients, and production of phytohormones and antimicrobial metabolites. Multiple species of Bacillus and Paenibacillus affect the crop growth and its health by three different ecological mechanisms viz, promotion of host plant nutrition and growth, antagonism against fungal, bacterial, nematode pathogens and insect pests, and stimulation of host defence mechanisms. Specific strains of both Bacillus and Paenibacillus spp. are known to elicit induced systemic resistance (ISR) similar to that of Pseudomonas spp. which leads to the stimulation of host defence mechanisms against multiple pathogens on diverse crop plants. Several species of Bacillus and Paenibacillus are the major source of broad spectrum peptide antibiotics that are active against various microbial and nematode pathogens. Endophytic colonization and biofilm formation by these two genera are also reported. These plant growth promoting abilities of Bacillus and Paenibacillus can make them suitable plant growth promoting rhizobacteria for their application in sustainable agriculture.

Role of Antibiosis in Suppression of Charcoal Rot Disease by Soybean Endophyte Paenibacillus sp. HKA-15

Four defective (AFM−) mutants of Paenibacillus sp. HKA-15 that no longer produced the peptide antifungal metabolites were developed through ethyl methane sulfonate (EMS) mutagenesis and used for in vivo experimentation. Reduced percentage of seed germination by mutants DM1 and DM2 (22.5% and 25%, respectively) and a high percent of disease incidence (69.3% and 67%, respectively) compared to wild-strain HKA-15 (80% seed germination and 27% disease incidence) indirectly indicated the role of peptide metabolite on disease suppression. Plants treated with AFM− clones showed stunted growth and the presence of pepperlike microsclerotia in the stem tissues. Light and scanning electron microscopic studies clearly showed the effect of peptide antibiosis on hyphal morphology. Exposure to crude extracts of antibiotics produced abnormal contraction of fungal cytoplasm, granulation, and fragmentation of hyphal mycelia and cell lysis. The presence of bacterial cells in the lumen of degrading fungal mycelium suggested a direct involvement of Paenibacillus sp. HKA-15 in the lysis of Rhizoctonia bataticola.

Isolation and Characterization of ACC Deaminase Gene from Two Plant Growth-Promoting Rhizobacteria

Lowering of plant ethylene by deamination of its immediate precursor 1-aminocyclopropane-1-carboxylate (ACC) is a key trait found in many rhizobacteria. We isolated and screened bacteria from the rhizosphere of wheat for their ACC-degrading ability. The ACC deaminase gene (acdS) isolated from two bacterial isolates through PCR amplification was cloned and sequenced. Nucleotide sequence alignment of these genes with previously reported genes of Pseudomonas sp. strain ACP and Enterobactercloacae strain UW4 showed variation in their sequences. In the phylogenetic analysis, distinctness of these two genes was observed as a separate cluster. 16S rDNA sequencing of two isolates identified them to be Achromobacter sp. and Pseudomonasstutzeri.

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.

Purification and Partial Characterization of Antifungal Peptides from Soybean Endophyte-Paenibacillus sp strain HKA-15

Culture supernatant of soybean nodule endophytic bacterium Paenibacillus sp strain HKA-15 showed the antifungal activity against Rhizoctonia bataticola, the causative agent of charcoal rot disease in soybean. The activity was detected only during the on set of stationary phase (24h post inoculation) in potato dextrose broth. The culture filtrate was extracted with nbutanol, resolved into two compounds by hydrophobic interaction column (Sephadex LH-20) chromatography and purified by reverse phase HPLC. Bioactive fractions collected from preparative HPLC were characterized as cyclic peptide and depsipeptide. No loss of activity was recorded with these metabolites when exposed to proteinase K, glycerol (50%), sodium dodecyl sulphate (1%), triton X-100 (1%) and wide pH range.

PGPR-Induced Systemic Resistance (ISR) in Plant Disease Management

Induced resistance is a physiological “state of enhanced defensive capacity” elicited by plant growth promoting rhizobacteria (PGPR) such as Pseudomonas putida, Serratia marcescens, Flavomonas oryzihabitans, Bacillus pumulus, etc. where the plant’s innate defences potentiated against subsequent biotic challenges becomes a popular means of protection of the plant from pathogens through induced systemic resistance (ISR). A number of bacterial determinants act as resistance elicitor compound in the development of resistance in plants such as siderophore, pyoverdin, bacterial SA, fucose, rhamnose (lipopolysaccharide) and flagellins reported so far. The transcriptional activation of PR genes is differentially regulated by NPR1 which is not only required for the SA-dependent expression of PR genes that are activated during SAR, but also for the JA- and ET-dependent activation of defence responses resulting from rhizobacteria-mediated ISR. The enzymes that have been associated with ISR includes phenylalanine ammonia lyase (PAL), chitinase, β-1,3-glucanase, peroxidase (PO), polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT), lipoxygenase (LOX), ascorbate peroxidase (APX) and proteinase inhibitors.

ACC Deaminase Containing PGPR for Potential Exploitation in Agriculture

The beneficial free-living bacteria present in the plant rhizosphere are usually referred to as plant growth promoting rhizobacteria (PGPR). Among the various mechanisms of plant growth promotion, certain PGPR possess the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves plant-produced ACC, the immediate precursor of the stress hormone ethylene. ACC deaminase containing PGPR act as a sink for ACC and protects the developing seedlings from deleterious effects of stress ethylene that is synthesized during various environmental stresses like phytopathogens, flooding, drought, salt, heavy metals, organic contaminants, and high and low temperatures. ACC deaminase is a pyridoxyl 5′ phosphate-dependent enzyme and genes expressing this particular trait have been isolated and characterized from a number of PGPRs of different genera. Some of the rhizobacterial ACC deaminase genes have been already utilized in PGPR strain improvement as well as transgenic crop development. Several studies have reported the potential exploitation of ACC deaminase containing PGPR in improving the crop yields; improving self life and quality of vegetables and ornamental flowers; protecting crop plants against a range of abiotic and biotic stresses; and phytoremediation of organic pollutants and heavy metal contamination in soils.

Diversity and Tissue Preference of Osmotolerant Bacterial Endophytes Associated with Pearl Millet Genotypes Having Differential Drought Susceptibilities

Genetic and functional diversity of osmotolerant bacterial endophytes colonizing the root, stem, and leaf tissues of pearl millet genotypes differing in their drought susceptibility was assessed. Two genotypes of pearl millet, viz., the drought tolerant genotype TT-1 and the drought susceptible genotype PPMI-69, were used in the present study. Diazotrophs were found to be the predominant colonizers, followed by the Gram positive bacteria in most of the tissues of both the genotypes. Higher proportion of bacterial endophytes obtained from the drought tolerant genotype was found to be osmotolerant. Results of 16S rRNA gene-ARDRA analysis grouped 50 of the highly osmotolerant isolates into 16 clusters, out of which nine clusters had only one isolate each, indicating their uniqueness. One cluster had 21 isolates and remaining clusters were represented by isolates ranging from two to four. The representative isolates from each cluster were identified, and Bacillus was found to be the most prevalent osmotolerant genera with many different species. Other endophytic bacteria belonged to Pseudomonas sp., Stenotrophomonas sp., and Macrococcus caseolyticus. High phylogenetic diversity was observed in the roots of the drought tolerant genotype while different tissues of the drought susceptible genotype showed less diversity. Isolates of Bacillus axarquiensis were present in all the tissues of both the genotypes of pearl millet. However, most of the other endophytic bacteria showed tissue/genotype specificity. With the exception of B. axarquiensis and B. thuringiensis, rest all the species of Bacillus were found colonizing only the drought-tolerant genotype; while M. caseolyticus colonized all the tissues of only the drought susceptible genotype. There was high incidence of IAA producers and low incidence of ACC deaminase producers among the isolates from the root tissues of the drought-tolerant genotype while reverse was the case for the drought-susceptible genotype. Thus, host played an important role in the selection of endophytes based on both phylogenetic and functional traits.

Whole genome shotgun sequence of Bacillus paralicheniformis strain KMS 80, a rhizobacterial endophyte isolated from rice (Oryza sativa L.)

Bacillus paralicheniformis strain KMS 80 (MTCC No. 12704) is an isolate from the root tissues of rice (Oryza sativa L.) that displays biological nitrogen fixation and plant growth promoting abilities. Here, we report the complete genome sequence of this strain, which contains 4,566,040 bp, 4424 protein-coding genes, 8692 promoter sequences, 67 tRNAs, 20 rRNA genes with six copies of 5S rRNAs along with a single copy of 16S–23S rRNA and genome average GC-content of 45.50%. Twenty one genes involved in nitrogen metabolism pathway and two main transcriptional factor genes, glnR and tnrA responsible for regulation of nitrogen fixation in Bacillus sp. were predicted from the whole genome of strain KMS 80. Analysis of the ~ 4.57 Mb genome sequence will give support to understand the genetic determinants of host range, endophytic colonization behaviour as well as to enhance endophytic nitrogen fixation and other plant beneficial role of B. paralicheniformis in rice.