Gamma-induced mutants of Bacillus and Streptomyces display enhanced antagonistic activities and suppression of the root rot and wilt diseases in pulses.

This study aims to increase Bacillus and Streptomyces antagonistic activity against the root rot and wilt diseases of pulses caused by Macrophomina phaseolina and Fusarium oxysporum f. sp. udum, respectively. To increase antagonistic action, Bacillus subtilis BRBac4, Bacillus siamensis BRBac21, and Streptomyces cavourensis BRAcB10 were subjected to random mutagenesis using varying doses of gamma irradiation (0.5-3.0 kGy). Following the irradiation, 250 bacterial colonies were chosen at random for each antagonistic strain and their effects against pathogens were evaluated in a plate assay. The ERIC, BOX, and random amplified polymorphic studies demonstrated a clear distinction between mutant and wild-type strains. When mutants were compared to wild-type strains, they showed improved plant growth-promoting characteristics and hydrolytic enzyme activity. The disease suppression potential of the selected mutants, B. subtilis BRBac4-M6, B. siamensisi BRBac21-M10, and S. cavourensis BRAcB10-M2, was tested in green gram, black gram, and red gram. The combined inoculation of B. siamensis BRBac21-M10 and S. cavourensis BRAcB10-M2 reduced the incidence of root rot and wilt disease. The same treatment also increased the activity of the defensive enzymes peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase. These findings suggested that gamma-induced mutation can be exploited effectively to improve the biocontrol characteristics of Bacillus and Streptomyces. Following the field testing, a combined bio-formulation of these two bacteria may be utilised to address wilt and root-rot pathogens in pulses.

Induction of moisture stress tolerance by Bacillus and Paenibacillus in pigeon pea (Cajanus cajan. L).

Drought stress is the main growth-limiting factor in pigeon pea production. Plant growth-promoting bacteria (PGPB) induce abiotic stress tolerance in several plants. However, the physiological and molecular changes with PGPB priming are not well understood in pigeon pea. The present study explored the potential of Firmibacteria (Bacillus azotoformans MTCC2953, Bacillus aryabhattai KSBN2K7, and Paenibacillus stellifer M3T4B6) to induce stress tolerance in pigeon pea under pot culture condition. Different physiological and biochemical parameters, including osmolytes, stress enzymes, and antioxidants, were evaluated under two stress conditions (50% and 25% field capacity) and an unstressed condition in pigeon pea. Under moisture stress conditions significant differences were observed in physiological and biochemical parameters between firmibacteria inoculated and control plants.The quantitative real-time polymerase chain reaction was performed to study the bacterial inoculation mediated expression of proline and drought-responsive genes in enhancing the drought tolerance in pigeon pea. Results showed that the inoculation of Bacillus aryabhattai upregulated the expression of drought-responsive genes (C. cajan_29830 and C. cajan_33874) and downregulated the expression of the proline gene by inducing the drought stress tolerance in inoculated plants compared with the uninoculated control plants. Therefore, Bacillus aryabhattai may be recommended for inducing drought stress tolerance and increasing the growth of pigeon pea under moisture stress conditions after field evaluation.

Endomicrobial Community Profiles of Two Different Mealybugs: Paracoccus marginatus and Ferrisia virgata.

Mealybugs (Hemiptera: Coccomorpha: Pseudococcidae) harbour diverse microbial symbionts that play essential roles in host physiology, ecology, and evolution. In this study we aimed to reveal microbial communities associated with two different mealybugs, papaya mealybug (Paracoccus marginatus) and two-tailed mealybug (Ferrisia virgata) collected from the same host plant. Comparative analysis of microbial communities associated with these mealybugs revealed differences that appear to stem from phylogenetic associations and different nutritional requirements. This first report on both bacterial and fungal communities associated with these mealybugs provides a preliminary insight on factors affecting the endomicrobial communities. .

Physiological response of tomato plant to chitosan-immobilized aggregated Methylobacterium oryzae CBMB20 inoculation under salinity stress.

The use of plant growth promoting bacteria as bioinoculant to alleviate salt stress is a sustainable and eco-friendly approach in agriculture. However, the maintenance of the bacterial population in the soil for longer period is a major concern. In the present study, chitosan-immobilized aggregated Methylobacterium oryzae CBMB20 was used as a bioinoculant to improve tomato plant (Solanum lycopersicum Mill.) growth under salt stress. The chitosan-immobilized aggregated M. oryzae CBMB20 was able to enhance plant dry weight, nutrient uptake (N, P, K and Mg2+), photosynthetic efficiency and decrease electrolyte leakage under salt stress conditions. The oxidative stress exerted by elevated levels of salt stress was also alleviated by the formulated bioinoculant, as it up-regulated the antioxidant enzyme activities and enhanced the accumulation of proline which acts as an osmolyte. The chitosan-immobilized aggregated M. oryzae CBMB20 was able to decrease the excess Na+ influx into the plant cells and subsequently decreasing the Na+/K+ ratio to improve tomato plant growth under salt stress conditions. Therefore, it is proposed that the chitosan-immobilized aggregated M. oryzae CBMB20 could be used as a bioinoculant to promote the plant growth under salt stress conditions.

Azospirillum ramasamyi sp. nov., a novel diazotrophic bacterium isolated from fermented bovine products.

A Gram-stain-negative, rod-shaped, aerobic bacterium, designated M2T2B2T, was isolated from fermented bovine products in Suwon, Republic of Korea. The strain displayed growth at 15-45 °C (optimum, 28-30 °C), pH 6.0-10.0 (pH 7.0) and 0-2 % (w/v) NaCl (0 %). Colonies were light pink-coloured, round and convex. The cells were positive for oxidase and weakly positive for catalase. The major fatty acids in whole cells of strain M2T2B2T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), followed by summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), summed feature 2 (C12 : 0 aldehyde/unidentified 10.928/C14 : 0 3-OH/iso-C16 : 1 I), C16 : 0, C18 : 1 2-OH, C16 : 0 3-OH and C17 : 1ω6c. The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, and three unidentified aminolipids. Ubiquinone 10 was the predominant ubiquinone. The DNA G+C content was 68.0 mol%. The strain could fix atmospheric nitrogen, which was evaluated by the acetylene reduction assay. Further, whole genome sequence analysis revealed the presence of a nif gene cluster. Strain M2T2B2T showed the highest 16S rRNA, rpoD and nifH gene sequence similarity to members of the genus Azospirillum, and showed 97.6 % 16S rRNA gene sequence similarity to Azospirillum oryzae COC8T. The phenotypic, phylogenetic and genomic analyses support the proposal of strain M2T2B2T as being a novel species of the genus Azospirillum, for which the name Azospirillumramasamyi sp. nov. is proposed. The type strain is M2T2B2T (=KACC 14063T=NBRC 106460T).

Exploration of Rice Husk Compost as an Alternate Organic Manure to Enhance the Productivity of Blackgram in Typic Haplustalf and Typic Rhodustalf.

The present study was aimed at using cellulolytic bacterium Enhydrobacter and fungi Aspergillus sp. for preparing compost from rice husk (RH). Further, the prepared compost was tested for their effect on blackgram growth promotion along with different levels of recommended dose of fertilizer (RDF) in black soil (typic Haplustalf) and red soil (typic Rhodustalf) soil. The results revealed that, inoculation with lignocellulolytic fungus (LCF) Aspergillus sp. @ 2% was considered as the most efficient method of composting within a short period. Characterization of composted rice husk (CRH) was examined through scanning electron microscope (SEM) for identifying significant structural changes. At the end of composting, N, P and K content increased with decrease in CO2 evolution, C:N and C:P ratios. In comparison to inorganic fertilization, an increase in grain yield of 16% in typic Haplustalf and 17% in typic Rhodustalf soil over 100% RDF was obtained from the integrated application of CRH@ 5 t ha-1 with 50% RDF and biofertilizers. The crude protein content was maximum with the combined application of CRH, 50% RDF and biofertilizers of 20% and 21% in typic Haplustalf and typic Rhodustalf soils, respectively. Nutrient rich CRH has proved its efficiency on crop growth and soil fertility.

Assessment of Methylobacterium oryzae CBMB20 aggregates for salt tolerance and plant growth promoting characteristics for bio-inoculant development.

Salinity is one of the major factors contributing to the loss of crop productivity and thereby impacting livelihood of people in more than 100 countries of the world and the area of land affected by salinity is increasing day by day. This will worsen due to various factors such as drought that might result in high soil salinity. Use of plant growth promoting rhizobacteria is one of the promising eco-friendly strategies for salinity stress management as part of sustainable agricultural practices. However, it requires selecting rhizobacteria with good survivability and adaptation to salt stress. In this study we report aggregation of Methylobacterium oryzae CBMB20 cells grown in media containing high C/N ratio (30:1) than in media containing low C/N ratio (7:1). Aggregated Methylobacterium oryzae CBMB20 cells exhibited enhanced tolerance to UV irradiation, heat, desiccation, different temperature regimes, oxidative stress, starvation and supported higher population in media. Poly-ß-hydroxybutyrate accumulation, exopolysaccharide production, proline accumulation and biofilm formation were good at 100 mM salt concentration with good microbial cell hydrophobicity at both 50 and 100 mM than other concentrations. Both the aggregated and non-aggregated cells grown under 0-200 mM salt concentrations produced IAA even at 200 mM salt concentration with a peak at 100 mM concentration with aggregated cells producing significantly higher quantities. ACC deaminase activity was observed in all NaCl concentrations studied with gradual and drastic reduction in aggregated and non-aggregated cells over increased salt concentrations.

Control of Wilt and Rot Pathogens of Tomato by Antagonistic Pink Pigmented Facultative Methylotrophic Delftia lacustris and Bacillus spp.

The studies on the biocontrol potential of pink pigmented facultative methylotrophic (PPFM) bacteria other than the genus Methylobacterium are scarce. In the present study, we report three facultative methylotrophic isolates; PPO-1, PPT-1, and PPB-1, respectively, identified as Delftia lacustris, Bacillus subtilis, and Bacillus cereus by 16S rRNA gene sequence analysis. Hemolytic activity was tested to investigate the potential pathogenicity of isolates to plants and humans, the results indicates that the isolates PPO-1, PPT-1, and PPB-1 are not pathogenic strains. Under in vitro conditions, D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 showed direct antagonistic effect by inhibiting the mycelial growth of fungal pathogens; Fusarium oxysporum f. sp. lycopersici (2.15, 2.05, and 1.95 cm), Sclerotium rolfsii (2.14, 2.04, and 1.94 cm), Pythium ultimum (2.12, 2.02, and 1.92 cm), and Rhizoctonia solani (2.18, 2.08, and 1.98 cm) and also produced volatile inhibitory compounds. Under plant growth chamber condition methylotrophic bacterial isolates; D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 significantly reduced the disease incidence of tomato. Under greenhouse condition, D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 inoculated tomato plants, when challenged with F. oxysporum f. sp. lycopersici, S. rolfsii, P. ultimum, and R. solani, increased the pathogenesis related proteins (ß-1,3-glucanase and chitinase) and defense enzymes (phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and catalase) on day 5 after inoculation. In the current study, we first report the facultative methylotrophy in pink pigmented D. lacustris, B. subtilis, and B. cereus and their antagonistic potential against fungal pathogens. Direct antagonistic and ISR effects of these isolates against fungal pathogens of tomato evidenced their possible use as a biocontrol agent.

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.

Mycorrhizal Symbiotic Efficiency on C3 and C4 Plants under Salinity Stress - A Meta-Analysis.

A wide range of C3 and C4 plant species could acclimatize and grow under the impact of salinity stress. Symbiotic relationship between plant roots and arbuscular mycorrhizal fungi (AMF) are widespread and are well known to ameliorate the influence of salinity stress on agro-ecosystem. In the present study, we sought to understand the phenomenon of variability on AMF symbiotic relationship on saline stress amelioration in C3 and C4 plants. Thus, the objective was to compare varied mycorrhizal symbiotic relationship between C3 and C4 plants in saline conditions. To accomplish the above mentioned objective, we conducted a random effects models meta-analysis across 60 published studies. An effect size was calculated as the difference in mycorrhizal responses between the AMF inoculated plants and its corresponding control under saline conditions. Responses were compared between (i) identity of AMF species and AMF inoculation, (ii) identity of host plants (C3 vs. C4) and plant functional groups, (iii) soil texture and level of salinity and (iv) experimental condition (greenhouse vs. field). Results indicate that both C3 and C4 plants under saline condition responded positively to AMF inoculation, thereby overcoming the predicted effects of symbiotic efficiency. Although C3 and C4 plants showed positive effects under low (EC < 4 ds/m) and high (>8 ds/m) saline conditions, C3 plants showed significant effects for mycorrhizal inoculation over C4 plants. Among the plant types, C4 annual and perennial plants, C4 herbs and C4 dicot had a significant effect over other counterparts. Between single and mixed AMF inoculants, single inoculants Rhizophagus irregularis had a positive effect on C3 plants whereas Funneliformis mosseae had a positive effect on C4 plants than other species. In all of the observed studies, mycorrhizal inoculation showed positive effects on shoot, root and total biomass, and in nitrogen, phosphorous and potassium (K) uptake. However, it showed negative effects in sodium (Na) uptake in both C3 and C4 plants. This influence, owing to mycorrhizal inoculation, was significantly higher in K uptake in C4 plants. For our analysis, we concluded that AMF-inoculated C4 plants showed more competitive K(+) ions uptake than C3 plants. Therefore, maintenance of high cytosolic K(+)/Na(+) ratio is a key feature of plant salt tolerance. Studies on the detailed mechanism for the selective transport of K in C3 and C4 mycorrhizal plants under salt stress is lacking, and this needs to be explored.

Genetic Diversity and Association Characters of Bacteria Isolated from Arbuscular Mycorrhizal Fungal Spore Walls.

Association between arbuscular mycorrhizal fungi (AMF) and bacteria has long been studied. However, the factors influencing their association in the natural environment is still unknown. This study aimed to isolate bacteria associated with spore walls of AMF and identify their potential characters for association. Spores collected from coastal reclamation land were differentiated based on their morphology and identified by 18S rDNA sequencing as Funneliformis caledonium, Racocetra alborosea and Funneliformis mosseae. Bacteria associated with AMF spore walls were isolated after treating them with disinfection solution at different time intervals. After 0, 10 and 20 min of spore disinfection, 86, 24 and 10 spore associated bacteria (SAB) were isolated, respectively. BOX-PCR fingerprinting analysis showed that diverse bacterial communities were associated to AMF spores. Bacteria belonging to the same genera could associate with different AMF spores. Gram positive bacteria were more closely associated with AMF spores. Isolated SAB were characterized and tested for spore association characters such as chitinase, protease, cellulase enzymes and exopolysaccharide production (EPS). Among the 120 SAB, 113 SAB were able to show one or more characters for association and seven SAB did not show any association characters. The 16S rDNA sequence of SAB revealed that bacteria belonging to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bactereiodes were associated with AMF spore walls.

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.

CIMIDx: Prototype for a Cloud-Based System to Support Intelligent Medical Image Diagnosis With Efficiency.

BACKGROUND: The Internet has greatly enhanced health care, helping patients stay up-to-date on medical issues and general knowledge. Many cancer patients use the Internet for cancer diagnosis and related information. Recently, cloud computing has emerged as a new way of delivering health services but currently, there is no generic and fully automated cloud-based self-management intervention for breast cancer patients, as practical guidelines are lacking. OBJECTIVE: We investigated the prevalence and predictors of cloud use for medical diagnosis among women with breast cancer to gain insight into meaningful usage parameters to evaluate the use of generic, fully automated cloud-based self-intervention, by assessing how breast cancer survivors use a generic self-management model. The goal of this study was implemented and evaluated with a new prototype called "CIMIDx", based on representative association rules that support the diagnosis of medical images (mammograms). METHODS: The proposed Cloud-Based System Support Intelligent Medical Image Diagnosis (CIMIDx) prototype includes two modules. The first is the design and development of the CIMIDx training and test cloud services. Deployed in the cloud, the prototype can be used for diagnosis and screening mammography by assessing the cancers detected, tumor sizes, histology, and stage of classification accuracy. To analyze the prototype's classification accuracy, we conducted an experiment with data provided by clients. Second, by monitoring cloud server requests, the CIMIDx usage statistics were recorded for the cloud-based self-intervention groups. We conducted an evaluation of the CIMIDx cloud service usage, in which browsing functionalities were evaluated from the end-user's perspective. RESULTS: We performed several experiments to validate the CIMIDx prototype for breast health issues. The first set of experiments evaluated the diagnostic performance of the CIMIDx framework. We collected medical information from 150 breast cancer survivors from hospitals and health centers. The CIMIDx prototype achieved high sensitivity of up to 99.29%, and accuracy of up to 98%. The second set of experiments evaluated CIMIDx use for breast health issues, using t tests and Pearson chi-square tests to assess differences, and binary logistic regression to estimate the odds ratio (OR) for the predictors' use of CIMIDx. For the prototype usage statistics for the same 150 breast cancer survivors, we interviewed 114 (76.0%), through self-report questionnaires from CIMIDx blogs. The frequency of log-ins/person ranged from 0 to 30, total duration/person from 0 to 1500 minutes (25 hours). The 114 participants continued logging in to all phases, resulting in an intervention adherence rate of 44.3% (95% CI 33.2-55.9). The overall performance of the prototype for the good category, reported usefulness of the prototype (P=.77), overall satisfaction of the prototype (P=.31), ease of navigation (P=.89), user friendliness evaluation (P=.31), and overall satisfaction (P=.31). Positive evaluations given by 100 participants via a Web-based questionnaire supported our hypothesis. CONCLUSIONS: The present study shows that women felt favorably about the use of a generic fully automated cloud-based self- management prototype. The study also demonstrated that the CIMIDx prototype resulted in the detection of more cancers in screening and diagnosing patients, with an increased accuracy rate.

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.

Arsenic-tolerant plant-growth-promoting bacteria isolated from arsenic-polluted soils in South Korea.

The Janghang smelter in Chungnam, South Korea started in 1936 was subsequently shutdown in 1989 due to heavy metal (loid) pollution concerns in the vicinity. Thus, there is a need for the soil in the area to be remediated to make it usable again especially for agricultural purposes. The present study was conducted to exploit the potential of arsenic (As)-tolerant bacteria thriving in the vicinity of the smelter-polluted soils to enhance phytoremediation of hazardous As. We studied the genetic and taxonomic diversity of 21 As-tolerant bacteria isolated from soils nearer to and away from the smelter. These isolates belonging to the genera Brevibacterium, Pseudomonas, Microbacterium, Rhodococcus, Rahnella, and Paenibacillus, could tolerate high concentrations of arsenite (As(III)) and arsenate (As(V)) with the minimum inhibitory concentration ranging from 3 to >20 mM for NaAsO2 and 140 to 310 mM NaH2AsO4 · 7H2O, respectively. All isolates exhibited As(V) reduction except Pseudomonas koreensis JS123, which exhibited both oxidation and reduction of As. Moreover, all the 21 isolates produced indole acetic acid (IAA), 13 isolates exhibited 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, 12 produced siderophore, 17 solubilized phosphate, and 13 were putative nitrogen fixers under in vitro conditions. Particularly, Rhodococcus aetherivorans JS2210, P. koreensis JS2214, and Pseudomonas sp. JS238 consistently increased root length of maize in the presence of 100 and 200 µM As(V). Possible utilization of these As-tolerant plant-growth-promoting bacteria can be a potential strategy in increasing the efficiency of phytoremediation in As-polluted soils.