Deciphering the antiviral nature of endophytic Bacillus spp. against groundnut bud necrosis virus in cowpea and tomato.

Tomato, the important vegetable crop, is severely affected by Orthotospovirus arachinecrosis which impacts heavy economic losses. The application of insecticide to manage viral diseases is not an environmentally safe approach. In view of these issues, we investigated the antiviral efficacy of 21 bacterial endophytes against GBNV in local lesion host (Cowpea-VBN3). Based on the reduction in lesion number and virus titer as estimated through both DAC ELISA and qPCR in cowpea, the bacterial endophytes viz., Bacillus licheniformis Soya1, Bacillus tequilensis NBL6, and Bacillus velezensis VB7 were selected and further tested in tomato. The study revealed the well-defined antiviral efficacy of these endophytes against GBNV. The percentage of disease incidence ranged from 16 to 24% in endophyte-treated tomato plants compared with untreated plants (88%). In addition, symptom severity was reduced, and the application of endophytes also in promotion of the growth compared with untreated control. DAC ELISA revealed that the tomato plants treated with bacterial endophytes challenged with GBNV showed reduction in the virus titer (0.26-0.39 @ OD 405 nm) at different days of interval after inoculation (0, 5, and 10 days) compared with untreated control (3.475 @ OD 405 nm). Additionally, reduction in the viral copy number in bacterial endophyte-treated plants was evident by real-time PCR. Furthermore, tomato plants bacterized with endophytes depicted significant correlation and reduction in viral load and disease incidence as revealed by the principal-component biplot analysis. Thus, the application of bacterial endophytes has a potential role in reducing the disease incidence, severity, and titer value of GBNV, which will be the promising management approach in future to mitigate the virus infection in tomato plants.

Metagenomic profiling of tomato rhizosphere delineates the diverse nature of uncultured microbes as influenced by Bacillus velezensis VB7 and Trichoderma koningiopsis TK towards the suppression of root-knot nematode under field conditions.

The plant-parasitic Root Knot Nematodes (Meloidogyne spp.,) play a pivotal role to devastate vegetable crops across the globe. Considering the significance of plant-microbe interaction in the suppression of Root Knot Nematode, we investigated the diversity of microbiome associated with bioagents-treated and nematode-infected rhizosphere soil samples through metagenomics approach. The wide variety of organisms spread across different ecosystems showed the highest average abundance within each taxonomic level. In the rhizosphere, Proteobacteria, Firmicutes, and Actinobacteria were the dominant bacterial taxa, while Ascomycota, Basidiomycota, and Mucoromycota were prevalent among the fungal taxa. Regardless of the specific treatments, bacterial genera like Bacillus, Sphingomonas, and Pseudomonas were consistently found in high abundance. Shannon diversity index vividly ensured that, bacterial communities were maximum in B. velezensis VB7-treated soil (1.4-2.4), followed by Root Knot Nematode-associated soils (1.3-2.2), whereas richness was higher with Trichoderma konigiopsis TK drenched soils (1.3-2.0). The predominant occurrence of fungal genera such as Aspergillus Epicoccum, Choanephora, Alternaria and Thanatephorus habituate rhizosphere soils. Shannon index expressed the abundant richness of fungal species in treated samples (1.04-0.90). Further, refraction and species diversity curve also depicted a significant increase with maximum diversity of fungal species in B. velezensis VB7-treated soil than T. koningiopsis and nematode-infested soil. In field trial, bioagents-treated tomato plant (60% reduction of Meloidogyne incognita infection) had reduced gall index along with enhanced plant growth and increased fruit yield in comparison with the untreated plant. Hence, B. velezensis VB7 and T. koingiopsis can be well explored as an antinemic bioagents against RKN. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03851-1.

Molecular Docking of Nimbolide Extracted from Leaves of Azadirachta indica with Protein Targets to Confirm the Antifungal, Antibacterial and Insecticidal Activity.

Nimbolide, a tetranortriterpenoid (limonoid) compound isolated from the leaves of Azadirachta indica, was screened both in vitro and in silico for its antimicrobial activity against Fusarium oxysporum f. sp. cubense, Macrophomina phaseolina, Pythium aphanidermatum, Xanthomonas oryzae pv. oryzae, and insecticidal activity against Plutella xylostella. Nimbolide exhibited a concentration-dependent, broad spectrum of antimicrobial and insecticidal activity. P. aphanidermatum (82.77%) was more highly inhibited than F. oxysporum f. sp. cubense (64.46%) and M. phaseolina (43.33%). The bacterium X. oryzae pv. oryzae forms an inhibition zone of about 20.20 mm, and P. xylostella showed about 66.66% mortality against nimbolide. The affinity of nimbolide for different protein targets in bacteria, fungi, and insects was validated by in silico approaches. The 3D structure of chosen protein molecules was built by homology modelling in the SWISS-MODEL server, and molecular docking was performed with the SwissDock server. Docking of homology-modelled protein structures shows most of the chosen target proteins have a higher affinity for the furan ring of nimbolide. Additionally, the stability of the best-docked protein-ligand complex was confirmed using molecular dynamic simulation. Thus, the present in vitro and in silico studies confirm the bioactivity of nimbolide and provide a strong basis for the formulation of nimbolide-based biological pesticides. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01104-6.

Triamcinolone Acetonide Produced by Bacillus velezensis YEBBR6 Exerts Antagonistic Activity Against Fusarium oxysporum f. sp. Cubense: A Computational Analysis.

Fusarium oxysporum f. sp. cubense is one of the most severe and threatening pathogens of bananas, causing "Panama wilt" worldwide. Confrontation assay of Foc antagonistic bacterial endophyte, Bacillus velezensis YEBBR6, with the Foc and GC-MS profiling of excised agar from the zone of inhibition, led to the unveiling of secondary metabolites produced by the endophyte. To refine the probable antifungal compounds among the numerous biomolecules formed during their di-trophic interaction with the pathogen, fungal protein targets were modeled, and docking studies (AutoDock Vina module of the PyRx 0.8 server) were done with all the compounds. Triamcinolone acetonide exhibited the most excellent affinity for the protein targets among the compounds studied. It had a maximum binding affinity of 11.2 kcal/mol for XRN2 (5' → 3'). Further, the protein-ligand complex formation kinetics was done through Molecular Dynamic Simulation studies. Graphs for the RMSD, RMSF, Rg, potential energy, and SASA were generated, and the values during the simulation period suggested the stability of the biomolecule as a complex with the protein. This indicated Triamcinolone acetonide's potential ability to act as a functional disrupter of the target protein and likely an antifungal molecule. Further, the biomolecule was tested for its activity against Foc by screening in the wet lab through the poisoned plate technique, and it was found to be fully inhibitory to the growth of the pathogen at 1000 ppm.

Molecular studies on tobacco streak virus (TSV) infecting cotton in Tamil Nadu, India.

Tobacco streak virus (TSV), the causal agent of cotton necrosis, is of emerging importance in the recent years. Unfortunately, all the cotton varieties and hybrids are susceptible to this virus. Cotton plants cultivated in different districts of Tamil Nadu were surveyed during 2014-2016. Samples collected from different locations confirmed the presence of TSV in cotton. TSV infection was confirmed by direct antigen coating-enzyme linked immuno sorbent assay (DAC-ELISA), dot immuno binding assay (DIBA), and reverse transcriptase polymerase chain reaction (RT-PCR). The virus was morphologically confirmed by transmission electron microscope (TEM). TSV isolate collected from Coimbatore was sequenced to obtain the full-length genome. Full length analysis was done for RNA 1 and RNA 3, whereas there was problem in obtaining few nucleotides in 5' and 3' end in spite of using different primers. Critical domain search in the nucleotide sequences revealed the presence of characteristic viral methyl transferase domain and helicase domain (RNA 1), 'GDD' motif and 'DFSKFD' of viral replicase in RNA 2 and Zinc finger motif in RNA 3. Phylogenetic analysis reveals high nucleotide similarity with TSV isolates of India and USA. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03437-3.

Unraveling the tripartite interaction of volatile compounds of Streptomyces rochei with grain mold pathogens infecting sorghum.

Sorghum is a major grain crop used in traditional meals and health drinks, and as an efficient fuel. However, its productivity, value, germination, and usability are affected by grain mold, which is a severe problem in sorghum production systems, which reduces the yield of harvested grains for consumer use. The organic approach to the management of the disease is essential and will increase consumer demand. Bioactive molecules like mVOC (volatile organic compound) identification are used to unravel the molecules responsible for antifungal activity. The Streptomyces rochei strain (ASH) has been reported to be a potential antagonist to many pathogens, with high levels of VOCs. The present study aimed to study the inhibitory effect of S. rochei on sorghum grain mold pathogens using a dual culture technique and via the production of microbial volatile organic compounds (mVOCs). mVOCs inhibited the mycelial growth of Fusarium moniliforme by 63.75 and Curvularia lunata by 68.52%. mVOCs suppressed mycelial growth and inhibited the production of spores by altering the structure of mycelia in tripartite plate assay. About 45 mVOCs were profiled when Streptomyces rochei interacted with these two pathogens. In the present study, several compounds were upregulated or downregulated by S. rochei, including 2-methyl-1-butanol, methanoazulene, and cedrene. S. rochei emitted novel terpenoid compounds with peak areas, such as myrcene (1.14%), cymene (6.41%), and ç-terpinene (7.32%) upon interaction with F. moniliforme and C. lunata. The peak area of some of the compounds, including furan 2-methyl (0.70%), benzene (1.84%), 1-butanol, 2-methyl-(8.25%), and myrcene (1.12)%, was increased during tripartite interaction with F. moniliforme and C. lunata, which resulted in furan 2-methyl (6.60%), benzene (4.43%), butanol, 2-methyl (18.67%), and myrcene (1.14%). These metabolites were implicated in the sesquiterpenoid and alkane biosynthetic pathways and the oxalic acid degradation pathway. The present study shows how S. rochei exhibits hyperparasitism, competition, and antibiosis via mVOCs. In addition to their antimicrobial functions, these metabolites could also enhance plant growth.

Nematicidal Property of Clindamycin and 5-hydroxy-2-methyl Furfural (HMF) from the Banana Endophyte Bacillus velezensis (YEBBR6) Against Banana Burrowing Nematode Radopholus similis.

Radopholus similis is a burrowing nematode which causes banana toppling disease and is of major economic threat for the banana production. Bacterial endophyte Bacillus velezensis (YEBBR6) produce biomolecules like 5-hydroxy-2-methyl furfural (HMF) and clindamycin in during interaction with Fusarium oxysporum f.sp. cubense. Molecular modelling and docking studies were performed on Radopholus similis protein targets such as calreticulin, cathepsin S-like cysteine proteinase, ß-1,4 -endoglucanase, reticulocalbin, venom allergen-like protein and serine carboxypeptidase to understand the mode of action of HMF and clindamycin against Radopholus similis. Structurally validated protein targets of R. similis were docked with biomolecules through AutoDock Vina module in PyRx 0.8 software to predict the binding energy of ligand and target protein. Among the chosen six targets, docking analysis revealed that clindamycin had the maximum binding affinity for ß-1,4-endoglucanase (- 7.2 kcal/mol), reticulocalbin (- 7.5 kcal/mol) and serine carboxypeptidase (- 6.9 kcal/mol) in comparison with HMF and the nematicide, carbofuran 3G. Besides, clindamycin also had the maximum binding energy for the target sites calreticulin and venom allergen-like protein compared to the small molecule HMF. Novel molecule, clindamycin produced by B. velezensis served as a potential inhibitor of the target sites associated in interrupting the functions of ß-1,4-endoglucanase, reticulocalbin, serine carboxypeptidase, calreticulin, cathepsin S-like cysteine proteinase, and venom allergen-like proteins. Besides, increased binding affinity of clindamycin with the protein target sites facilitated to explore it as a novel nematicidal molecule for the management of banana burrowing nematode R. similis. Thus, present investigation confirmed that, the small molecules clindamycin can be explored for nematicidal activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01011-2.

Flagellin and elongation factor of Bacillus velezensis (VB7) reprogramme the immune response in tomato towards the management of GBNV infection.

The present study investigates the reprogramming of plant defense system, upon interaction with MAMP (Microbe Associated Molecular Pattern) gene products including, flagellin (Flg) and elongation factor (EF-Tu) of Bacillus velezensis (VB7) and groundnut bud necrosis virus (GBNV) in tomato (Shivam). The MAMP gene products induced the plant defense genes including, PAL, PPO, LOX, JAR, MYC2 and PDF 1.2. Secondary metabolites of Bacillus spp. at 1000 parts per million (ppm) concentration effectively reduced GBNV symptom expression in cowpea (CO7) up to 83.1 % compared to untreated, GBNV inoculated, control. The secondary metabolites from B. velezensis (VB7) and B. licheniformis (Soya 1) reduced GBNV symptoms in cowpea (CO7) up to 1.7 lesions/cm2 leaf area compared to 8.6 lesions/cm2 in virus inoculated control. Further, field study revealed that the combined application of B. velezensis (VB7) and B. licheniformis (Soya1) at 1% (10 mL/L) as, soil and foliar application reduced the percent disease incidence (PDI) up to 10.5 (PDI) compared to 28.4 (PDI) in untreated control plants. Besides, it also improved the plant growth and yield up to 23.5 tonnes/ha compared to 13.8 tonnes/ha in untreated control.

Mining the Genome of Bacillus velezensis VB7 (CP047587) for MAMP Genes and Non-Ribosomal Peptide Synthetase Gene Clusters Conferring Antiviral and Antifungal Activity.

Chemical pesticides have an immense role in curbing the infection of plant viruses and soil-borne pathogens of high valued crops. However, the usage of chemical pesticides also contributes to the development of resistance among pathogens. Hence, attempts were made in this study to identify a suitable bacterial antagonist for managing viral and fungal pathogens infecting crop plants. Based on our earlier investigations, we identified Bacillus amyloliquefaciens VB7 as a potential antagonist for managing Sclerotinia sclerotiorum infecting carnation, tobacco streak virus infecting cotton and groundnut bud necrosis infecting tomato. Considering the multifaceted action of B. amyloliquefaciens VB7, attempts were made for whole-genome sequencing to assess the antiviral activity against tomato spotted wilt virus infecting chrysanthemum and antifungal action against Fusarium oxysporum f. sp. cubense (Foc). Genome annotation of the isolate B. amyloliquefaciens VB7 was confirmed as B. velezensis VB7 with accession number CP047587. Genome analysis revealed the presence of 9,231,928 reads with an average read length of 149 bp. Assembled genome had 1 contig, with a total length of 3,021,183 bp and an average G+C content of 46.79%. The protein-coding sequences (CDS) in the genome was 3090, transfer RNA (tRNA) genes were 85 with 29 ribosomal RNA (rRNA) genes and 21 repeat regions. The genome of B. velezensis VB7 had 506 hypothetical proteins and 2584 proteins with functional assignments. VB7 genome had the presence of flagellin protein FlaA with 987 nucleotides and translation elongation factor TU (Ef-Tu) with 1191 nucleotides. The identified ORFs were 3911 with 47.22% GC content. Non ribosomal pepide synthetase cluster (NRPS) gene clusters in the genome of VB7, coded for the anti-microbial peptides surfactin, butirosin A/butirosin B, fengycin, difficidin, bacillibactin, bacilysin, and mersacidin the Ripp lanthipeptide. Antiviral action of VB7 was confirmed by suppression of local lesion formation of TSWV in the local lesion host cowpea (Co-7). Moreover, combined application of B. velezensis VB7 with phyto-antiviral principles M. Jalapa and H. cupanioides increased shoot length, shoot diameter, number of flower buds per plant, flower diameter, and fresh weight of chrysanthemum. Further, screening for antifungal action of VB7 expressed antifungal action against Foc in vitro by producing VOC/NVOC compounds, including hexadecanoic acid, linoelaidic acid, octadecanoic acid, clindamycin, formic acid, succinamide, furanone, 4H-pyran, nonanol and oleic acid, contributing to the total suppression of Foc apart from the presence of NRPS gene clusters. Thus, our study confirmed the scope for exploring B. velezensis VB7 on a commercial scale to manage tomato spotted wilt virus, groundnut bud necrosis virus, tobacco streak virus, S. sclerotiorum, and Foc causing panama wilt of banana.

Thrips diversity of cotton ecosystem and the role of parthenium pollen grains in the transmission of tobacco streak virus (TSV) infection in cotton.

Survey in the cotton fields of Coimbatore and Erode districts confirmed the presence of three different thrips species including, Scirtothrips dorsalis, Scirtothrips oligochaetus, and Frankliniella occidentalis. Their identity was confirmed through morphometric analysis and molecular characterization. Tobacco streak virus (TSV) was detected in the leaves and pollen grains of both parthenium and cotton plants collected from infected cotton fields. The presence of TSV was confirmed through immuno-detection by direct antigen coating enzyme-linked immuno sorbent assay (DAC-ELISA). Further confirmation was accomplished by reverse transcriptase polymerase chain reaction (RT-PCR) using TSV coat protein-specific primers. Other than parthenium, weed plants in the cotton field such as Trianthema portulacastrum, Boerhavia diffusa, and Amaranthus sp. were also confirmed for TSV infection through RT-PCR. Parthenium hysterophorus plants acts as a silent carrier of TSV and they occasionally produced symptoms. Among all the randomly collected thrips samples, two pooled samples were detected positive for TSV through RT-PCR. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02967-6.

Bacterial community analysis on Sclerotium-suppressive soil.

Difficulties in controlling the soil-borne plant pathogenic fungus Sclerotium rolfsii favoured the analysis of its suppressive soil for better understanding. In the present study, culture-independent molecular technique was used to analyse the bacterial communities of suppressive soil and conducive soil. Hence, metagenomic DNAs from both kinds of soils were directly extracted and their sequence polymorphism was analysed by targeting hypervariable domains, V4 + V5, of the 16S rRNA gene. The results of 16S rRNA gene-driven bacterial community diversity analysis along with soil physicochemical and biological properties clearly discriminated S. rolfsii suppressive soil from conducive soil. The dominant phylogenetic group of suppressive soil is Actinobacteria followed by Proteobacteria. The other groups include Acidobacteria, Firmicutes and Cyanobacteria. In contrast, conducive soil had very few Actinobacterial sequences and was dominated by Gamma- and Betaproteobacteria. Based on the relative proportion of different bacterial communities, their diversity and species richness were observed more in suppressive soil than in conducive soil. The present study identifies the dominant bacterial community which shares S. rolfsii suppressiveness.

Comparative study of the ditrophic interaction between Beauveria bassiana and Plutella xylostella.

In the present investigation, hyperparasitic interaction between B. bassiana, TM (MH590235) and P. xylostella was studied through scanning electron microscopy and chromatographic techniques. Dose-mortality responses showed an increase in mortality of larva with an increase in spore concentration. The LC50 value for B. bassiana isolate TM (MH590235) was 2.4 × 107 spores mL-1. The ditrophic interaction between B. bassiana and P. xylostella after 24 h revealed the adherence of conidia on stemmata, sensory setae, maxillary palpi and legs. After 48 h post-infection (hpi), germination of the conidia and appressorium formation was observed. Formation of hyphae and initiation of fungal networking was observed at 72 hpi. Complete ramification by mycelia and conidiogenesis of B. bassiana was observed over the mycosed cadaver after 168 hpi. Subsequently, typical sympodial conidiophores of B. bassiana bearing secondary spores were also observed. The metabolome profile of healthy larvae of P. xylostella revealed the presence of non-volatile organic compounds (NVOCs) like docosene, nonadecene, palmitic acid and heneicosane. However, the NVOC profile was different in the P. xylostella larvae hyperparasitized by B. bassiana. The metabolites present in the infected cadaver were phthalate esters, hydroxyquebrachamine and lactones.

Bacterial endophytome-mediated resistance in banana for the management of Fusarium wilt.

Banana (Musa spp.), a major cash and staple fruit crop in many parts of the world, is infected by Fusarium wilt, which contributes up to 100% yield loss and causes social consequences. Race 1 and race 2 of Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) are prevalent worldwide and seriously affect many traditional varieties. The threat of Foc tropical race 4 (Foc TR4) is looming large in African counties. However, its incidence in India has been confined to Bihar (Katihar and Purnea), Uttar Pradesh (Faizabad), Madhya Pradesh (Burhanpur) and Gujarat (Surat). Management of Foc races by employing fungicides is often not a sustainable option as the disease spread is rapid and they negatively alter the biodiversity of beneficial ectophytes and endophytes. Besides, soil drenching with carbendazim/trifloxystrobin + tebuconazole is also not effective in suppressing the Fusarium wilt of banana. Improvement of resistance to Fusarium wilt in susceptible cultivars is being addressed through both conventional and advanced breeding approaches. However, engineering of banana endosphere with bacterial endophytes from resistant genotypes like Pisang lilly and YKM5 will induce the immune response against Foc, irrespective of races. The composition of the bacterial endomicrobiome in different banana cultivars is dominated by the phyla Proteobacteria, Bacteroidetes and Actinobacteria. The major bacterial endophytic genera antagonistic to Foc are Bacillus, Brevibacillus, Paenibacillus, Virgibacillus, Staphylococcus, Cellulomonas, Micrococcus, Corynebacterium, Kocuria spp., Paracoccus sp., Acinetobacter spp. Agrobacterium, Aneurinibacillus, Enterobacter, Klebsiella, Lysinibacillus, Micrococcus, Rhizobium, Sporolactobacillus, Pantoea, Pseudomonas, Serratia, Microbacterium, Rhodococcus, Stenotrophomonas, Pseudoxanthomonas, Luteimonas, Dokdonella, Rhodanobacter, Luteibacter, Steroidobacter, Nevskia, Aquicella, Rickettsiella, Legionella, Tatlockia and Streptomyces. These bacterial endophytes promote the growth of banana plantlets by solubilising phosphate, producing indole acetic acid and siderophores. Application of banana endophytes during the hardening phase of tissue-cultured clones serves as a shield against Foc. Hitherto, MAMP molecules of endophytes including flagellin, liposaccharides, peptidoglycans, elongation factor, cold shock proteins and hairpins induce microbe-associated molecular pattern (MAMP)-triggered immunity to suppress plant pathogens. The cascade of events associated with ISR and SAR is induced through MAPK and transcription factors including WRKY and MYC. Studies are underway to exploit the potential of antagonistic bacterial endophytes against Foc isolates and to develop an understanding of the MAMP-triggered immunity and metabolomics cross talk modulating resistance. This review explores the possibility of harnessing the potential bacterial endomicrobiome against Foc and developing nanoformulations with bacterial endophytes for increased efficacy against lethal pathogenic races of Foc infecting banana. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02833-5.

Metabolites of Trichoderma longibrachiatum EF5 inhibits soil borne pathogen, Macrophomina phaseolina by triggering amino sugar metabolism.

An endophytic fungal antagonist Trichoderma longibrachiatum EF5 exhibited biocontrol activity against a soil-borne fungal pathogen Macrophomina phaseolina. Under dual co-culture, T. longibrachiatum EF5 showed 58% inhibition against M. phaseolina. Crude soluble metabolites (SMs) extracted from EF5 exhibited biocontrol activity (61%), which is more significant than the cell-free extract. Dual culture of both T. longibrachiatum EF5 and M. phaseolina displayed entangled mycelial structures and retarded hyphal growth. The metabolites responsible for antibiosis and pathogenic activity profiled through GC-MS revealed a total of 131 SMs from axenic culture and upon the interaction of T. longibrachiatum EF5 and M. phaseolina. Interestingly, potential plant-growth-promoting and antimicrobial compounds such as 1- pentanol, 1-hexanol, myristonyl pantothenate, bisabolol, d-Alanine, and diethyl trisulphide were unique with T. longibrachiatum EF5. Few compounds that were not observed or produced minimally under axenic culture were increased during their interaction (e.g., 1,6-anhydro-á-d-Glucopyranose and 5-heptyl dihydro-2(3H)-Furanone), suggesting antimicrobial action against the pathogen. This study also unraveled the induction of amino sugar metabolism when T. longibrachiatum EF5 interacts with M. phaseolina, which is responsible for colonization and counterfeiting the pathogen. Hence T. longibrachiatum EF5 could be a potential biocontrol agent employed for defense priming and plant growth promotion.

Understanding the molecular basis on the biological suppression of bacterial leaf blight of anthurium exerted by Bacillus subtilis (BIO3) through proteomic approach.

We attempted to study the antibacterial activity of rhizospheric Bacillus spp., to curb the bacterial blight of anthurium caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). Twenty-eight bacterial isolates from rhizospheric regions were identified as different Bacillus spp. and Ochrobactrum sp. using 16S rRNA gene sequencing. B. subtilis BIO3 effectively inhibited the growth of Xad up to 1450.7 mm2, and extracted volatile organic metabolites from the isolate BIO3 inhibited the growth of Xad up to 1024 mm2. Tritrophic interaction of anthurium leaves bacterized with B. subtilis BIO3 and challenged with Xad resulted in the expression of 12 unique proteins compared to untreated control. Mascot Peptide Mass Fingerprint-based identification indicated that one was glutathione peroxidase, involved in defence mechanism, other six proteins were identified as leghemoglobin II, CTP synthase-like, predicted protein (Physcomitrella patens), centromere-associated protein E, grain size protein, and five proteins were hypothetical proteins. Foliar application with 1% liquid formulations (108 CFU/ml) of B. subtilis BIO3 significantly suppressed the bacterial leaf blight of anthurium up to 78% over untreated control and also increased the stem length and flower yield.

Flagellin of Bacillus amyloliquefaciens works as a resistance inducer against groundnut bud necrosis virus in chilli (Capsicum annuum L.).

Groundnut bud necrosis virus (GBNV), a member of the genus Tospovirus, has an extensive host range and is associated with necrosis disease of chilli (Capsicum annuum L.), which is a major threat to commercial production. Plant growth promoting rhizobacteria (PGPR) have been investigated for their antiviral activity in several crops and for their potential use in viral disease management. However, the microbial mechanisms associated with PGPR in triggered immunity against plant viruses have rarely been studied. To understand the innate immune responses activated by Bacillus spp. against GBNV, we studied microbe-associated molecular pattern (MAMP) triggered immunity (MTI) in chilli using transient expression of the flagellin gene of Bacillus amyloliquefaciens CRN9 from Agrobacterium clones, which also induced the expression of EAS1 gene transcripts coding for epi-aristolochene synthase, which is responsible for the accumulation of capsidiol phytoalexin. In addition, the transcript levels of WRKY33 transcription factor and salicylic acid (SA)-responsive defense genes such as NPR1, PAL, PO and SAR8.2 were increased. Jasmonate (JA)-responsive genes, viz., PDF, and LOX genes, were also upregulated in chilli plants challenged with GBNV. Further analysis revealed significant induction of these genes in chilli plants treated with B. amyloliquefaciens CRN9 and benzothiadiazole (BTH). The transcript levels of defense response genes and pathogenesis-related proteins were significantly higher in plants treated with Bacillus and BTH and remained significantly higher at 72 h post-inoculation and compared to the inoculated control. The plants treated with flagellin using the agrodrench method and exogenous treatment with B. amyloliquefaciens and BTH showed resistance to GBNV upon mechanical inoculation and a reduced virus titre which was confirmed by qPCR assays. Thus, transient expression of flagellin, a MAMP molecule from B. amyloliquefaciens CRN9, is able to trigger innate immunity and restrain virus growth in chilli via induced systemic resistance (ISR) activated by both the SA and JA/ET signalling pathways.

Induction of in planta resistance by flagellin (Flg) and elongation factor-TU (EF-Tu) of Bacillus amyloliquefaciens (VB7) against groundnut bud necrosis virus in tomato.

Role of plant growth promoting rhizobacteria (PGPR) in growth promotion and induction of resistance against various plant pathogens have been extensively studied. However, MAMP (Microbe Associated Molecular Pattern) triggered immunity (MTI) against plant viruses are not well exploited. The present study enlightens the role of two MAMP genes including, flagellin (Flg) and elongation factor (EF-Tu) in the induction of plant defense against GBNV infecting tomato. Secondary metabolites of Bacillus amyloliquefaciens (VB7), effectively suppressed GBNV symptom expression up to 84% compared to untreated control in cowpea, the indicator host plant. Agrobacterium tumefaciens EHA105 clones expressing the MAMP genes were drenched in the root zone to assess the induction of resistance against GBNV in tomato. Treatment with A. tumefaciens EHA105 clones containing flagellin (Ag- Ba.Flg) and elongation factor-TU (Ag-Ba.EF-Tu) genes as soil drench and foliar spray, reduced virus titre,0.369 OD and 0.379 OD respectively as compared to control 1.249 OD. The disease severity was reduced up to 15% in Ag-Ba.Flg treated plants compared to 88.25% in inoculated control. Further there was an increased expression of defense associated genes including, MAPKK1, WRKY33BB, NPR1 and PR1.The present investigation clearly indicated the efficiency of MAMP genes in triggering defense mechanism in tomato against GBNV.

Evidence of seed transmission of dolichos yellow mosaic virus, a begomovirus infecting lablab-bean in India.

Yellow mosaic disease in field bean caused by begomoviruses belonging to the family Geminiviridae is a major threat to the cultivation of crop in South India. Appearance of bright yellow mosaic symptom in emerging seedlings in farmers field was suggestive of seed transmission of the begomovirus associated with the disease which was investigated in the present study. The begomovirus causing yellow mosaic disease was identified as dolichos yellow mosaic virus (DoYMV) and the presence of DoYMV in matured seeds was confirmed by DAS-ELISA with OD value up to 3.268. In PCR with DoYMV specific primer (DoYMV-CP) the virus was detected in different parts of the seeds viz., seed coat, endosperm and embryo. In embryo the virus was detectable up to 100 per cent followed by endosperm (69.23%). When the non symptomatic leaves of 30 days old grow-out test plants were subjected to DAS-ELISA, the virus was detected up to 46.6%. This is the first evidence of seed transmission of DoYMV.

Documentation of virulence and races of Xanthomonas citri pv. malvacearum in India and its correlation with genetic diversity revealed by repetitive elements (REP, ERIC, and BOX) and ISSR markers.

Thirty-four Xanthomonas citri pv. malvacearum (Xcm) isolates collected from three cotton-growing zones of India were subjected for virulence and race documentation and further correlated with genetic diversity as revealed by repetitive elements [repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX elements] and intersimple sequence repeat (ISSR)-PCR analyses. Among the 34 isolates tested for virulence on susceptible cultivar LRA 5166, 7 were recorded as highly virulent (HV), 16 were moderately virulent (MV) and 11 were less virulent (LV). Eight different races were recorded by using ten cotton host differentials. Twenty-two isolates (65%) belonged to race 18. Twelve isolates (35%) pertained to races 3, 5, 6, 7, 8, 11 and 13. REP, ERIC, BOX, combined repetitive elements, and ISSR analyses revealed the presence of 7, 10, 9, 11, and 8 clusters, respectively, at similarity coefficient of 0.70 in dendrograms. Principal coordinate analysis (PCoA) exhibited 76.4% and 77.5% cumulative variability for combined repetitive elements and ISSR analyses. ERIC produced the highest polymorphic information content (PIC) value (0.928). A lot of intra-pathovar variability was observed in virulence and genomic fingerprinting among Xcm isolates. Many of the isolates grouped based on geographical origin irrespective of virulence or race. The spread of the pathogen races in India might be due to the transport of germplasm lines and seed materials from one place to others.

Biocontrol Potentials of Antimicrobial Peptide Producing Bacillus Species: Multifaceted Antagonists for the Management of Stem Rot of Carnation Caused by Sclerotinia sclerotiorum.

Bacillus species are widely exploited as biocontrol agents because of their efficiency in impeding various plant pathogens with multifaceted approach. In this study, Bacillus species were isolated from rhizosphere of various plants viz., carnations, cotton, turmeric, and bananas in Tamil Nadu state of India. Their potential to control the mycelial growth of Sclerotinia sclerotiorum was assessed in vitro by dual plate and partition plate techniques. B. amyloliquefaciens strain VB7 was much effective in inhibiting mycelial growth (45% inhibition of over control) and sclerotial production (100%). PCR detection of AMP genes revealed that B. amyloliquefaciens (VB7) had a maximum of 10 diverse antibiotic biosynthesis genes, namely, ituD, ipa14, bacA, bacD, bamC, sfP, spaC, spaS, alba, and albF, that resulted in production of the antibiotics iturin, bacilysin, bacillomycin, surfactin, subtilin, and subtilosin. Further, metabolites from B. amyloliquefaciens strains VB2 and VB7, associated with inhibition of S. sclerotiorum, were identified as phenols and fatty acids by gas chromatography mass spectrometry (GC-MS). Delivery of bacterial suspension of the effective strains of Bacillus spp. as root dip was found promising for the management of stem rot of cultivated carnations. Minimal percent disease incidence (4.6%) and maximum plant growth promotion was observed in the plants treated with B. amyloliquefaciens (VB7).