Parthiban Subramanian

Arbuscular Mycorrhizal Fungi Community Structure, Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity.

Psychrotolerant Endophytic Pseudomonas sp. Strains OB155 and OS261 Induced Chilling Resistance in Tomato Plants (Solanum lycopersicum Mill.) by Activation of Their Antioxidant Capacity.

Studies on chilling stress damage and its mitigation through microorganisms in members of family Solanaceae is limited, despite their economic importance. We studied chilling stress alleviation in tomato plants colonized by psychrotolerant bacterial strains Pseudomonas vancouverensis OB155-gfp and P. frederiksbergensis OS261-gfp. Log phase cultures of bacterial strains were coated on surface-sterilized seeds (bacterization) before sowing and nonbacterized (control) seeds were coated with sterile bacterial growth medium. All plants were grown at temperatures of 30 and 25°C and at the end of 4 weeks, chilling treatment (12 and 10°C) was imposed for 1 week on half of the bacterized and control plants. Under normal conditions (30 and 25°C), no significant difference was observed in antioxidant activity, proline accumulation, and expression of cold acclimation genes in tomato leaf tissues of both control and bacterized plants. However, plants exposed to temperatures of 12 and 10°C were found to decrease in robustness and nutrient uptake, accompanied by increased membrane damage. Chilling resistance in bacterized plants was evident from reduced membrane damage and reactive oxygen species levels, improved antioxidant activity in leaf tissues, and high expression of cold acclimation genes LeCBF1 and LeCBF3 compared with control plants. Confocal microscopy confirmed effective colonization and intercellular localization of cold-adapted bacterial strains OB155-gfp and OS261-gfp.

Cold Stress Tolerance in Psychrotolerant Soil Bacteria and Their Conferred Chilling Resistance in Tomato (Solanum lycopersicum Mill.) under Low Temperatures

The present work aimed to study the culturable diversity of psychrotolerant bacteria persistent in soil under overwintering conditions, evaluate their ability to sustain plant growth and alleviate chilling stress in tomato. Psychrotolerant bacteria were isolated from agricultural field soil samples colleced during winter and then used to study chilling stress alleviation in tomato plants (Solanum lycopersicum cv Mill). Selective isolation after enrichment at 5°C yielded 40 bacterial isolates. Phylogenetic studies indicated their distribution in genera Arthrobacter, Flavimonas, Flavobacterium, Massilia, Pedobacter and Pseudomonas. Strains OS211, OB146, OB155 and OS261 consistently improved germination and plant growth when a chilling stress of 15°C was imposed and therefore were selected for pot experiments. Tomato plants treated with the selected four isolates exhibited significant tolerance to chilling as observed through reduction in membrane damage and activation of antioxidant enzymes along with proline synthesis in the leaves when exposed to chilling temperature conditions (15°C). Psychrotolerant physiology of the isolated bacteria combined with their ability to improve germination, plant growth and induce antioxidant capacity in tomato plants can be employed to protect plants against chilling stress.

Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in psychrotolerant bacteria modulates ethylene metabolism and cold induced genes in tomato under chilling stress.

The role of stress induced ethylene under low temperature stress has been controversial and hitherto remains unclear. In the present study, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) gene, acdS expressing mutant strains were generated from ACCD negative psychrotolerant bacterial strains Flavobacterium sp. OR306 and Pseudomonas frederiksbergensis OS211, isolated from agricultural soil during late winter. After transformation with plasmid pRKACC which contained the acdS gene, both the strains were able to exhibit ACCD activity in vitro. The effect of this ACCD under chilling stress with regards to ethylene was studied in tomato plants inoculated with both acdS expressing and wild type bacteria. On exposing the plants to one week of chilling treatment at 12/10 °C, it was found that stress ethylene, ACC accumulation and ACO activity which are markers of ethylene stress, were significantly reduced in plants inoculated with the acdS gene transformed mutants. In case of plants inoculated with strain OS211-acdS, ethylene emission, ACC accumulation and ACO activity was significantly reduced by 52%, 75.9% and 23.2% respectively compared to uninoculated control plants. Moreover, expression of cold induced LeCBF1 and LeCBF3 genes showed that these genes were significantly induced by the acdS transformed mutants in addition to reduced expression of ethylene-responsive transcription factor 13 (ETF-13) and ACO genes. Induced expression of LeCBF1 and LeCBF3 in plants inoculated with acdS expressing mutants compared to wild type strains show that physiologically evolved stress ethylene and its transcription factors play a role in regulation of cold induced genes as reported earlier in the literature.

Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp.

Psychrotolerant bacteria isolated from natural and artificially cold environments were screened for synthesis of cold-active protease. The strain IMDY showing the highest protease production at 5°C was selected and phylogenetic analysis revealed that IMDY as novel bacterium with Chryseobacterium soli(T) as its nearest neighbor. Classical optimization enhanced the protease production from 18U/mg to 26U/mg and the enzyme was found to be active at low temperature, activity enhanced by CaCl2, inhibited by PMSF, stable against NaCl, and its activity retained in the presence of surfactants, organic solvents and detergents. On testing, the meat tenderization, myofibril fragmentation, pH, and TBA values were favorable in IMDY-protease treated meat compared to control. SDS profiling and SEM analysis also showed tenderization in meat samples. Hence, this study proposes to consider the cold-active protease from Chryseobacterium sp. IMDY as a pertinent candidate to develop potential applications in food processing industry.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

Stage-Wise Identification and Analysis of miRNA from Root-Knot Nematode Meloidogyne incognita

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita. Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode.

EPS Production, PHB Accumulation and Abiotic Stress Endurance of Plant Growth Promoting Methylobacterium Strains Grown in a High Carbon Concentration

In this study, we compared growth pattern, floc yield, Exo-polysaccharides (EPS) production, Poly-β-hydroxybutyrate (PHB) accumulation, resistance to osmotic and acid stress in Methylobacterium strains CBMB20, CBMB27, CBMB35, and CBMB110. Modified high C:N ratio medium denoted as HCN-AMS medium was used with a C:N ratio of 30:1. The HCN-AMS medium favored increased growth in all the studied strains. All Methylobacterium strains tested positive for EPS production and showed positive fluorescence with calcoflour stain. Elevated levels of EPS production from 4.2 to 75.0% was observed in HCN-AMS medium. Accumulation of PHB in HCN-AMS medium increased by 3.8, 36.7, and 12.0% in strains CBMB27, CBMB35, and CBMB110 respectively. Among the abiotic stresses, osmotic stress-induced growth inhibition of Methylobacterium strains was found to be lowered when grown in HCN-AMS medium. Likewise, growth inhibition due to acid stress at pH 5.0 was lower for strains grown in HCN-AMS medium compared to growth in AMS medium. Enhanced survivability under stress conditions may be attributed to the high EPS and PHB production at increased carbon concentration in the growth medium.

ACC Deaminase Producing Arsenic Tolerant Bacterial Effect on Mitigation of Stress Ethylene Emission in Maize Grown in an Arsenic Polluted Soil

Arsenic is a known hazardous metalloid not only to the animals but also to plants. With high concentrations, it can impede normal plant growth and cause even death of plants at extremely high levels. A known plant response to stress conditions such as toxic levels of metal (loids) is the production of stress ethylene, causing inhibitory effect on root growth in plants. When the effect of various arsenic concentrations was tested to maize plant, the stress ethylene emission proportionately increased with increasing concentration of As(V). The inoculation of two arsenic tolerant bacteria; Pseudomonas grimonti JS126 and Pseudomonas taiwanensis JS238 having respective high and low 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity reduced stress ethylene emission by 59% and 30% in maize grown in arsenic polluted soils. The result suggested the possible use of Pseudomonas grimonti JS126 for phytoremediation of arsenic polluted soils.

Psychrotolerance Mechanisms in Cold-Adapted Bacteria and their Perspectives as Plant Growth-Promoting Bacteria in Temperate Agriculture

Cold-adapted bacteria survive in extremely cold temperature conditions and exhibit various mechanisms of adaptation to sustain their regular metabolic functions. These adaptations include several physiological and metabolic changes that assist growth in a myriad of ways. Successfully sensing of the drop in temperature in these bacteria is followed by responses which include changes in the outer cell membrane to changes in the central nucleoid of the cell. Their survival is facilitated through many ways such as synthesis of cryoprotectants, cold acclimation proteins, cold shock proteins, RNA degradosomes, Antifreeze proteins and ice nucleators. Agricultural productivity in cereals and legumes under low temperature is influenced by several cold adopted bacteria including Pseudomonas, Acinetobacter, Burkholderia, Exiguobacterium, Pantoea, Rahnella, Rhodococcus and Serratia. They use plant growth promotion mechanisms including production of IAA, HCN, and ACC deaminase, phosphate solublization and biocontrol against plant pathogens such as Alternaria, Fusarium, Sclerotium, Rhizoctonia and Pythium.