Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice.

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H2O2-containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.

A comprehensive review of the spoilage of shrimp and advances in various indicators/sensors for shrimp spoilage monitoring.

Shrimp is a popular internationally traded shellfish due to its unique taste, texture, and nutritional value. Shrimp is highly perishable because it has enough free amino acids, high moisture levels, non-nitrogenous compounds used for microbial growth, and melanosis. Shrimp spoilage after death is caused by various reasons, like autolysis (endogenous proteinases actions during shrimp storage), growth of spoilage microorganisms, ATP degradation, melanin formation, and lipid peroxidation. A microbial byproduct, total volatile basic nitrogen, is one of the major reasons for the generation of foul odors from shrimp spoilage. Shrimp freshness monitoring is crucial for market sellers and exporters. Traditional methods for estimating shrimp freshness are expensive and inaccessible to the general public. Sensors are rapid, sensitive, selective, and portable food toxins' detection tools, devoid of expensive instruments, skilled people, sample pretreatment, and a long detection time. This review addresses shrimp spoilage causes. The mechanisms of different stages of shrimp spoilage after death, like rigor mortis, dissolution of rigor mortis, autolysis, and microbial spoilage mechanisms, are discussed. This review highlights the last five years' advances in shrimp freshness detection sensors and indicators like colorimetric pH indicators, fluorescence sensors, electronic noses, and biosensors, their working principles, and their sensitivities. Commercially available indicators and sensors for shrimp spoilage monitoring are also discussed. A review highlighting the applications of the different sensors and indicators for monitoring shrimp freshness is unavailable to date. Challenges and future perspectives in this field are explained at the end.

Nicotinic Acid Catabolism Modulates Bacterial Mycophagy in Burkholderia gladioli Strain NGJ1.

Burkholderia gladioli strain NGJ1 exhibits mycophagous activity on a broad range of fungi, including Rhizoctonia solani, a devastating plant pathogen. Here, we demonstrate that the nicotinic acid (NA) catabolic pathway in NGJ1 is required for mycophagy. NGJ1 is auxotrophic to NA and it potentially senses R. solani as a NA source. Mutation in the nicC and nicX genes involved in NA catabolism renders defects in mycophagy and the mutant bacteria are unable to utilize R. solani extract as the sole nutrient source. As supplementation of NA, but not FA (fumaric acid, the end product of NA catabolism) restores the mycophagous ability of ΔnicC/ΔnicX mutants, we anticipate that NA is not required as a carbon source for the bacterium during mycophagy. Notably, nicR, a MarR-type of transcriptional regulator that functions as a negative regulator of the NA catabolic pathway is upregulated in ΔnicC/ΔnicX mutant and upon NA supplementation the nicR expression is reduced to the basal level in both the mutants. The ΔnicR mutant produces excessive biofilm and is completely defective in swimming motility. On the other hand, ΔnicC/ΔnicX mutants are compromised in swimming motility as well as biofilm formation, potentially due to the upregulation of nicR. Our data suggest that a defect in NA catabolism alters the NA pool in the bacterium and upregulates nicR which in turn suppresses bacterial motility as well as biofilm formation, leading to mycophagy defects. IMPORTANCE Mycophagy is an important trait through which certain bacteria forage over fungal mycelia and utilize fungal biomass as a nutrient source to thrive in hostile environments. The present study emphasizes that nicotinic acid (NA) is important for bacterial motility and biofilm formation during mycophagy by Burkholderia gladioli strain NGJ1. Defects in NA catabolism potentially alter the cellular NA pool, upregulate the expression of nicR, a negative regulator of biofilm, and therefore suppress bacterial motility as well as biofilm formation, leading to mycophagy defects.

The alternative sigma factors, rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1.

Bacteria utilize RpoN, an alternative sigma factor (σ54) to grow in diverse habitats, including nitrogen-limiting conditions. Here, we report that a rice-associated mycophagous bacterium Burkholderia gladioli strain NGJ1 encodes two paralogues of rpoN viz. rpoN1 and rpoN2. Both of them are upregulated during 24 h of mycophagous interaction with Rhizoctonia solani, a polyphagous fungal pathogen. Disruption of either one of rpoNs renders the mutant NGJ1 bacterium defective in mycophagy, whereas ectopic expression of respective rpoN genes restores mycophagy in the complementing strains. NGJ1 requires rpoN1 and rpoN2 for efficient biocontrol to prevent R. solani to establish disease in rice and tomato. Further, we have identified 17 genes having RpoN regulatory motif in NGJ1, majority of them encode potential type III secretion system (T3SS) effectors, nitrogen assimilation, and cellular transport-related functions. Several of these RpoN regulated genes as well as certain previously reported T3SS apparatus (hrcC and hrcN) and effector (Bg_9562 and endo-ß-1,3-glucanase) encoding genes are upregulated in NGJ1 but not in ΔrpoN1 or ΔrpoN2 mutant bacterium, during mycophagous interaction with R. solani. This highlights that RpoN1 and RpoN2 modulate T3SS, nitrogen assimilation as well as cellular transport systems in NGJ1 and thereby promote bacterial mycophagy.

Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors.

Bacteria utilize type VI secretion system (T6SS) to deliver antibacterial toxins to target co-habiting bacteria. Here, we report that Burkholderia gladioli strain NGJ1 deploys certain T6SS effectors (TseTBg), having both DNase and RNase activities to kill target bacteria. RNase activity is prominent on NGJ1 as well as other bacterial RNA while DNase activity is pertinent to only other bacteria. The associated immunity (TsiTBg) proteins harbor non-canonical helix-turn-helix motifs and demonstrate transcriptional repression activity, similar to the antitoxins of type II toxin-antitoxin (TA) systems. Genome analysis reveals that homologs of TseTBg are either encoded as TA or T6SS effectors in diverse bacteria. Our results indicate that a new ORF (encoding a hypothetical protein) has evolved as a result of operonic fusion of TA type TseTBg homolog with certain T6SS-related genes by the action of IS3 transposable elements. This has potentially led to the conversion of a TA into T6SS effector in Burkholderia. Our study exemplifies that bacteria can recruit toxins of TA systems as T6SS weapons to diversify its arsenal to dominate during inter-bacterial competitions.

Enzymatic and non-enzymatic functional attributes of plant microbiome.

Microbiome plays an important role in plant growth and adaptation to various environmental conditions. The cross-talk between host plant and microbes (including microbe-microbe interactions) plays a crucial role in shaping the microbiome. Recent studies have highlighted that plant microbiome is enriched in genes encoding enzymes and natural products. Several novel antimicrobial compounds, bioactive natural products and lytic/degrading enzymes with industrial implications are being identified from the microbiome. Moreover, advancements in metagenomics and culture techniques are facilitating the development of synthetic microbial communities to promote sustainable agriculture. We discuss the recent advancements, opportunities and challenges in harnessing the full potential of plant microbiome.

Calcium regulates the mycophagous ability of Burkholderia gladioli strain NGJ1 in a type III secretion system-dependent manner.

BACKGROUND: A rice associated bacterium Burkholderia gladioli strain NGJ1 demonstrates mycophagy, a phenomenon wherein bacteria feed on fungi. Previously, we have reported that NGJ1 utilizes type III secretion system (T3SS) to deliver a prophage tail-like protein (Bg_9562) into fungal cells to establish mycophagy. RESULTS: In this study, we report that calcium ion concentration influences the mycophagous ability of NGJ1 on Rhizoctonia solani, an important fungal pathogen. The calcium limiting condition promotes mycophagy while high calcium environment prevents it. The expression of various T3SS apparatus encoding genes of NGJ1 was induced and secretion of several potential T3SS effector proteins (including Bg_9562) into extracellular milieu was triggered under calcium limiting condition. Using LC-MS/MS proteome analysis, we identified several calcium regulated T3SS effector proteins of NGJ1. The expression of genes encoding some of these effector proteins was upregulated during mycophagous interaction of NGJ1 with R. solani. Further, mutation of one of these genes (endo-ß-1, 3- glucanase) rendered the mutant NGJ1 bacterium defective in mycophagy while complementation with full length copy of the gene restored its mycophagous activity. CONCLUSION: Our study provides evidence that low calcium environment triggers secretion of various T3SS effectors proteins into the extracellular milieu and suggests the importance of cocktail of these proteins in promoting mycophagy.

Remediation of arsenic in mung bean (Vigna radiata) with growth enhancement by unique arsenic-resistant bacterium Acinetobacter lwoffii.

Arsenic, a carcinogenic and toxic contaminant of soil and water, affects human health adversely. During last few decades, it has been an important global environmental issue. Among several arsenic detoxification methods remediation using arsenic resistant microbes is proved to be environment-friendly and cost-effective. This study aimed to test the effects of arsenic utilizing bacterial strain Acinetobacter lwoffii (RJB-2) on arsenic uptake and growth of mung bean plants (Vigna radiata). RJB-2 exhibited tolerance up to 125mM of arsenic (V) and 50mM of arsenic (III). RJB-2 produced plant growth promoting substances e.g. indole acetic acid (IAA), siderophores, exopolysaccharide (EPS) and phosphate solubilization in the absence and in presence of arsenic. Pot experiments were used to scrutinize the role of RJB-2 on arsenic uptake and growth of mung bean plants grown in soil amended with 22.5mgkg-1 of sodium arsenate (Na2HAsO4·7H2O). RJB-2 could arrest arsenic uptake in just 7days and increase plant growth, number of plants per pot, chlorophyll and carotenoid content of the mung bean plants. RJB-2 formed biofilm and its root-association helped to abate arsenic uptake in mung bean. Confocal and light microscopic studies also revealed the abatement of arsenic uptake and increase in chlorophyll content in mung bean plants in presence of RJB-2. RJB-2 was also responsible for less production of reactive oxygen species (ROS) in mung bean plants reducing the oxidative damage caused by arsenic. The lower percentage of electrolytic leakage (EL) in RJB-2 inoculated mung bean plants proved arsenic abatement. The study also reported the distribution of arsenic in various parts of mung bean plant. RJB-2 owing to its intrinsic abilities of plant growth promotion even in presence of high concentrations of arsenic could inhibit arsenic uptake completely and therefore it could be used in large-scale cultivation for phytostabilization of plants.

Bacteria-fungal Confrontation and Fungal Growth Prevention Assay.

There are some bacteria which can grow and multiply at the cost of living fungal biomass. They can potentially utilize fungi as a source of nutrients to forage over them. Such phenomenon is known as bacterial mycophagy, however, its mechanistic insights need to be explored to identify the molecules involved in mycophagy for potential utilization in controlling various fungal diseases. Recently we have demonstrated that a rice-associated bacteria Burkholderia gladioli strain NGJ1 exhibits mycophagous ability on several fungi, including Rhizoctonia solani, the necrotrophic fungal pathogen causing sheath blight disease in rice. We hereby describe our validated and efficient methods used to study B. gladioli strain NGJ1-R. solani interactions. These methodologies would be useful for designing assays to study the confrontation between bacteria and fungi which in turn enable discovery of novel antifungal molecules from such bacteria.

A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi.

Some bacteria can feed on fungi, a phenomenon known as mycophagy. Here we show that a prophage tail-like protein (Bg_9562) is essential for mycophagy in Burkholderia gladioli strain NGJ1. The purified protein causes hyphal disintegration and inhibits growth of several fungal species. Disruption of the Bg_9562 gene abolishes mycophagy. Bg_9562 is a potential effector secreted by a type III secretion system (T3SS) and is translocated into fungal mycelia during confrontation. Heterologous expression of Bg_9562 in another bacterial species, Ralstonia solanacearum, confers mycophagous ability in a T3SS-dependent manner. We propose that the ability to feed on fungi conferred by Bg_9562 may help the bacteria to survive in certain ecological niches. Furthermore, considering its broad-spectrum antifungal activity, the protein may be potentially useful in biotechnological applications to control fungal diseases.Some bacteria can feed on live fungi through unclear mechanisms. Here, the authors show that a T3SS-secreted protein, which is homologous to phage tail proteins, allows a Burkholderia gladioli strain to kill and feed on various fungal species.

Low-cost field test kits for arsenic detection in water.

Arsenic, a common contaminant of groundwater, affects human health adversely. According to the World Health Organization (WHO), the maximum recommended contamination level of arsenic in drinking water is 10 µg/L. The purpose of this research was to develop user-friendly kits for detection of arsenic to measure at least up to 10 µg/L in drinking water, so that a preventive measure could be taken. Two different kits for detection of total arsenic in water are reported here. First, the arsenic in drinking water was converted to arsine gas by a strong reducing agent. The arsine produced was then detected by paper strips via generation of color due to reaction with either mercuric bromide (KIT-1) or silver nitrate (KIT-2). These were previously immobilized on the detector strip. The first one gave a yellow color and the second one grey. Both of these kits could detect arsenic contamination within a range of 10 µg/L-250 µg/L. The detection time for both the kits was only 7 min. The kits exhibited excellent performance compared to other kits available in the market with respect to detection time, ease of operation, cost and could be easily handled by a layman. The field trials with these kits gave very satisfactory results. A study on interference revealed that these kits could be used in the presence of 24 common ions present in the arsenic contaminated water. Though the kits were meant for qualitative assay, the results with unknown concentrations of real samples, when compared with atomic absorption spectrophotometer (AAS) were in good agreement as revealed by the t-test.