Hybrid de novo whole genome assembly of lipopeptide producing novel Bacillus thuringiensis strain NBAIR BtAr exhibiting antagonistic activity against Sclerotium rolfsii.

Bacillus thuringiensis Berliner is recognized as a predominant bioinsecticide but its antifungal potential has been relatively underexplored. A novel B. thuringiensis strain NBAIR BtAr was isolated and morphologically characterized using light and scanning electron microscopy, revealing presence of bipyramidal, cuboidal, and spherical parasporal crystals. The crude form of lipopeptides was extracted from NBAIR BtAr and assessed for its antagonistic activity in vitro, and demonstrated 100 % inhibition of Sclerotium rolfsii Sacc. at a minimum inhibitory concentration of 50 µL of the crude lipopeptide extract per mL of potato dextrose agar. To identify the antagonistic genes responsible, we performed whole genome sequencing of NBAIR BtAr, revealing the presence of circular chromosome of 5,379,913 bp and 175,362 bp plasmid with 36.06 % guanine-cytosine content and 5814 protein-coding sequences. Average nucleotide identity and whole genome phylogenetic analysis delineated the NBAIR BtAr strain as konkukian serovar. Gene ontology analysis revealed associations of 1474, 1323, and 1833 genes with biological processes, molecular function, and cellular components, respectively. Antibiotics & secondary metabolite analysis shell analysis of the whole genome yielded secondary metabolites biosynthetic gene clusters with 100 %, 85 %, 40 %, and 35 % similarity for petrobactin, bacillibactin, fengycin, and paenilamicin, respectively. Also, novel biosynthetic gene clusters, along with antimicrobial genes, including zwittermicin A, chitinase, and phenazines, were identified. Moreover, the presence of eight bacteriophage sequences, 18 genomic islands, insertion sequences, and one CRISPR region indicated prior occurrences of genetic exchange and thus improved competitive fitness of the strain. Overall, the whole genome sequence of NBAIR BtAr is presented, with its taxonomic classification and critical genetic attributes that contribute to its strong antagonistic activity against S. rolfsii.

Molecular characterization of Indian pathotypes of Puccinia striiformis f. sp. tritici and multigene phylogenetic analysis to establish inter- and intraspecific relationships.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most devastating diseases of wheat (Triticum spp.) worldwide. Indian isolates were characterised based on their phenotypic reaction on differential hosts carrying different Yr genes. Based on virulence/avirulence structure, isolates were characterised into ten different pathotypes viz. 70S0-2, 67S64, 70S4, 66S0, 70S64, 66S64-1, 38S102, 47S102, 46S119, and 78S84. These Indian pathotypes of P. striiformis f. sp. tritici and 38 pathotypes of other rust species (P. graminis tritici and P. triticina) were used in this study to analyze their molecular phylogenetic relationship. The nucleotides of rDNA-ITS, partial ß-tubulin and ketopantoate reductase genes of all the pathotypes were sequenced directly after PCR. Based on sequence data of rDNA-ITS and ß-tubulin, three phylogenetic groups corresponding to three different species of Puccinia were obtained. Asian isolates formed a distinct evolutionary lineage than from those derived from USA. The sequence similarity of Indian pathotypes with other Asian (China and Iran) isolates indicated the same origin of pathotypes. The results will allow rapid identification of Indian P.striiformis f. sp. tritici pathotypes causing stripe rust in wheat, assist in making predictions regarding potential rust pathotypes, and identifying sources of resistance to the disease in advance.

Thymol-a plant-based monoterpenoid phenol compound of an essential oil for the management of sheath blight disease of rice.

Sheath blight of rice is a global disease that significantly reduces rice yield. This study reports the antifungal activity of an active compound of essential oil, thymol, at different concentrations against Rhizoctonia solani (strain RS-Gvt). In vitro assay results indicated that thymol concentrations (0.5 mg mL-1 and 0.25 mg mL-1) completely inhibited (100%) the mycelial growth of RS-Gvt (p ≤ 0.01). Microscopic observations of thymol-treated mycelium of RS-Gvt at 0.0312 mg mL-1 and above concentrations, revealed a distorted mycelial morphology with deformed hyphae. Hyphae showed a bead-like appearance, reduction in size, and constriction of the hyphae at uneven points with increased hyphal density often entangling with each other. Further, an on-field experiment was conducted to study the field bio-efficacy of thymol for two consecutive Kharif seasons of 2022 and 2023 using a factorial RCBD design. The disease severity was measured as the percent disease index (PDI), and the results of two seasons were pooled. Pathogen (RS-Gvt) and thymol were inoculated in different combinations/methods as main treatments (M1-M3), and concentrations of thymol (0.0625-1.0 mg ML-1) as sub-treatments. The results indicated that all two factors significantly (P = 0.05) influenced the PDI and grain yield. The pooled data of two seasons indicated a significant difference between the main treatments (M1: RS-Gvt + thymol together; M2: thymol sprayed first followed by RS-Gvt; M3: RS-Gvt first followed by thymol spray) on PDI (53.39-59.67) and grain yield (4.16-4.75 t ha-1). M1 exhibited a lower PDI (53.39) and a higher grain yield (4.75 t ha-1) compared to M2 and M3, indicating a protective mode of action of thymol against sheath blight disease of rice. The sub-treatments have shown significant variation in PDI and grain yield. The PDI and grain yield ranged from 33.70 (at 1 mg mL-1) to 66.21 (at 0.0625 mg mL-1) and 4.18 (at 1 mg mL-1) to 5.26 (at 0.0625 mg mL-1) t ha-1, respectively, among the thymol concentrations. This indicates that increasing concentrations of thymol have negatively influenced the PDI and positively impacted the yield. Therefore, the spray of 1 mg mL-1 of thymol at the potential disease-infection stage is most effective in controlling the sheath blight disease of rice. This study provides an alternative green bioactive compound for controlling the sheath blight disease, and thymol can be included in developing eco-friendly integrated disease management practices. © 2024 Society of Chemical Industry.

Identification of New Source of Resistance to Powdery Mildew of Indian Mustard and Studying Its Inheritance.

Powdery mildew of Indian mustard (Brassica juncea), caused by Erysiphe cruciferarum, is emerging as major problem in India. All the Indian mustard cultivars presently grown in India are highly susceptible to powdery mildew and so far no resistance source has been reported. In this study, with an aim to identify resistant source, 1,020 Indian mustard accessions were evaluated against E. cruciferarum PMN isolate, at Wellington, The Nilgiris, Tamil Nadu, India under natural hot spot conditions. The study identified one accession (RDV 29) with complete resistance against E. cruciferarum PMN isolate for the first time, which was consistent in five independent evaluations. Genetic analysis of F1, F2 and backcross populations obtained from the cross RSEJ 775 (highly susceptible) × RDV 29 (highly resistant) for two season revealed that the resistance is governed by two genes with semi-dominant and gene dosage effect. Further, a new disease rating system using six scales (0, 1, 2, 3, 4, and 5) has also been proposed in this study to score powdery mildew based on progress of fungal growth in different plant parts of the F2 population. The outcome of this study viz. newly identified powdery mildew-resistant Indian mustard accession (RDV 29), information on inheritance of resistance and the newly developed disease rating scale will provide the base for development of powdery mildew-resistant cultivars of Indian mustard.

Comparative genomics of rice false smut fungi Ustilaginoidea virens Uv-Gvt strain from India reveals genetic diversity and phylogenetic divergence.

False smut disease of rice caused by Ustilaginoidea virens, is an emerging threat to rice cultivation worldwide due to its detrimental effects on grain yield and quality. False smut disease severity was 4.44‒17.22% during a roving survey in Kharif 2016 in the four different rice ecosystems of Karnataka, India. Further, 15 pathogen isolates representing four different ecosystems were studied for their virulence and morphometric diversity. Among the 15 strains studied, most virulent strains Uv-Gvt was selected for whole genome sequencing in Illumina NextSeq 500 platform using 2 × 150 bp sequencing chemistry. The total assembled genome of Uv-Gvt was 26.96 Mb, which comprised of 9157 scaffolds with an N50 value of 15,934 bp and 6628 protein-coding genes. Next, the comparative genomic study revealed a similar gene inventory as UV-8b and MAFF 236576 strains reported from China and Japan, respectively. But, 1756 genes were unique to Uv-Gvt strain. The Uv-Gvt genome harbors 422 putative host-pathogen interacting genes compared to 359 and 520 genes in UV-8b and MAFF 236576 strains, respectively. The variant analysis revealed low genetic diversity (0.073‒0.088%) among U. virens strains. Further, phylogenetic analysis using 250 single copy orthologs genes of U. virens revealed a distinct phylogeny and an approximate divergence time. Our study, report the genomic resource of rice false smut pathogen from India, where the disease originated, and this information will have broader applicability in understanding the pathogen population diversity.