Subabul (Leucaena leucocephala L.) is a leguminous species often referred to as the "miracle tree," it provides numerous ecosystem services and exhibits robust ecological characteristics. However, the infection caused by phytopathogenic fungi is poorly understood in Subabul. Therefore, this study provides comprehensive insights into the molecular and genomic characteristics of Fusarium falciforme, the causal agent of wilt disease in Subabul (Leucaena leucocephala). Pathogen isolation from infected samples, followed by morpho-molecular characterization through DNA sequencing of key markers (ITS, LSU, TEF1α) and phylogenetic analysis, confirmed the identity of F. falciforme. Host range analysis demonstrated the pathogen's ability to infect additional leguminous crops, including chickpea (Cicer arietinum) and soybean (Glycine max). A complete genome assembly revealed a genome size of 59.19 Mb, comprising 18,853 protein-coding genes. Comparative genomic analysis elucidated evolutionary relationships with other Fusarium species, while functional annotation identified critical virulence factors, such as polyketide synthases, ABC transporters, and secretory proteins, which facilitate host tissue invasion. These findings enhance the understanding of F. falciforme pathogenicity, enabling improved diagnostic tools and management strategies for controlling wilt disease in Subabul and related legumes.

Stalk rot disease is a major constraint in maize production and till date reported to be caused by two to three species of phytopathogenic fungi but, in our present study, we disclose the first report of stalk rot is caused by complex species of phytopathogens, which belongs to five different genera. Therefore, to substantiate these findings, a total of 105 diseased samples of maize were collected from 21 different locations in six different geographical locations of India from which 48 isolates were used for the research study. Morphological features such as pigmentation, colony color, type of mycelium and pattern of mycelium was examined using macro and microscopic methods. A total of 11 different spp. of pathogens belonging to the five different genera: Fusarium verticillioides (56.25

Post-flowering stalk rot (PFSR) disease drastically affects maize yields, leading to substantial economic losses. Fusarium verticillioides was widely recognized as a major pathogen responsible for maize PFSR disease. Astonishingly, apart from F. verticillioides, another pathogen was also observed infecting the stalk rot and thus, further pathogen purification was initiated. In this study, we revealed the first report of Sarocladium kiliense, as a maize stalk rot pathogen. This research study was validated by isolation, purification, and morphological examination, which revealed the variability among S. kiliense isolates in traits such as pigmentation, colony color, mycelial characteristics, and conidial morphology. Notably, isolates exhibited distinct pigmentation patterns: 3 were orange and 3 were yellow, with consistent white to dirty white colony colors. Pathogenicity assays using toothpick inoculation demonstrated that all the S. kiliense isolates caused significant damage to the maize stalks, with brown discoloration, conversion of pith into a powdery texture, and subsequent stalk lodging. Isolate PKSK55 exhibited the highest pathogenicity, producing the longest lesions among tested isolates. Molecular characterization using ITS rDNA and RPB-2 genes confirmed the identity of S. kiliense across all isolates. Conclusively, this research uncovered and documents the first report of S. kiliense causing stalk rot in maize in India.

To address the fungal wilt of pigeon pea caused by Fusarium oxysporium f. sp. udum, farmers currently rely on chemical fungicides, despite their harmful effects. However, there is a growing need for safer alternatives like green pesticides. Bacterial biocontrol agents and their derivatives serve as potential green pesticides in the management of plant pathogens. In the present study, we aimed to identify indigenous Bacillus subtilis strains effective against F. oxysporium f. sp. udum. We used PCR and MALDI-TOF analysis to identify the active components responsible for the efficiency of efficient strain. Biochemical studies of cell-free extracts extracted from B. subtilis strains demonstrated the highest biosurfactant activity in NBAIR BSWG1, with an oil displacement of 2 cm and an emulsification index of 60%. Molecular characterization confirmed the presence of surfactin, fengycin, and iturin coding genes in the B. subtilis strains, among them, NBAIR BSWG1 showed the highest number of lipopeptide-producing genes. Meanwhile, NBAIR BSWG1 showed inhibition of 79.84 % against F. oxysporium f. sp. udum using cell-free extract. Further metabolite profiling of NBAIR BSWG1 using MALDI-TOF analysis further confirmed surfactin, fengycin, and iturin in the purified cell-free extract of NBAIR BSWG1. Two peaks with m/z of 923.77 and 1149.92 were identified as novel lipopeptide compounds which need further characterization. The present study identified NBAIR BSWG1 as an efficient bacterial strain for the inhibition of F. oxysporium f. sp. udum and its antifungal properties are mainly due to the production of cyclic lipopeptides.

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521 bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500 pg/µL. DNA concentration as low as 10 pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100 pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Brown spot (BS) disease causes significant losses to rice productivity. In this study, a roving survey in the Karnataka state of India revealed a wider distribution of BS with a percent disease index range of 20.56–50.74. From the symptomatic geo-distinct samples, pure cultures of 63 isolates were obtained. Based on the conidial morphology, 63 isolates were identified as Bipolaris oryzae (Bo) (n = 40), Curvularia lunata (Cl) (n = 15), and Exserohilum rostratum (Er) (n = 08). The taxonomic identity was further confirmed via ITS-sequencing. A pathogenicity assay on a BS-susceptible rice cultivar GNV-05–01 confirmed the pathogenicity of all three pathogens, which induces typical BS disease on test plants. Further, on PDA media, all isolates of three pathogens showed significant cultural diversity for mycelial color, colony type, and sporulation. We further studied the in-planta distribution of three pathogens on a randomly collected 600 BS spots from 10 different rice fields, which indicated that 77.83

Bacterial leaf blight (BLB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the signi-ficant constraints for rice production. A study was carried out to analyze the virulence, genetic diversity, and population structure of Xoo isolates collected from different rice ecosystems in Karnataka, India. The taxonomic identity of all 54 iso-lates was confirmed using a Xoo-specific PCR assay. The virulence assay on a set of twenty-eight near-isogenic differentials identified nine pathotype groups (KPthX-1 to KPthX-9). Among them, KPthX-6 was highly virulent, whereas KPthx-2 was less virulent. Differentials with four and five resistance (R) genes (Xa4 + xa5 + xa13 + Xa21, Xa4 + Xa7 + xa13 + Xa21, and Xa4 + xa5 + Xa7 + xa13 + Xa21) were effective against all the pathotypes. Further, genetic diversity was deduced using a universal marker set, JEL-1 and JEL-2, complementary to a conserved repetitive sequence IS1112. The amplicon size and numbers varied from 100 bp to 3 Kbp and 1 to 25. Based on the amplicon counts, Jaccard's co-efficient and phylogenetic analyses were carried out and categorized the 54 isolates into three clusters (I to III). Cluster II contains the maximum number of isolates (27), followed by cluster I (23 isolates) and cluster III (one isolate). The structure analysis categorized the isolates into five subgroups, viz SG1, SG2, SG3, SG4, and SG5 indicate the existence of five subpopulations. The present study has identified the genetic and virulence diversity of the Xoo population and reported the effective R gene/s for different rice ecosystems, which can be used in the breeding program for the eco-specific management of BLB of rice.

Rice is the global staple food, contributing over half of the world’s agricultural production. Excessive fungicide use in rice farming poses environmental, fungicide resistance, and beneficial microbial disruption concerns, making biocontrol, particularly bacteria, a promising biocontrol agent due to their rapid growth, ease of handling, and robust colonization attributes. Endophytes are endosymbionts residing within host plants, playing a pivotal role in plant health without inciting any pathogenic effects. In this study, we investigated the antagonistic potential of four characterized bacterial endophytic strains, viz., Bacillus velezensis strains A6 and P42, B. pseudomycoides HP3d and Paenibacillus polymyxa PGSS-1 against major foliar rice pathogens viz., Magnaporthe oryzae and Cochliobolus miyabeanus. Against M. oryzae, all the endophytic strains exhibited significant per cent inhibition (50.00-66.67%) and the highest inhibition of 66.67% was achieved by B. pseudomycoides strain HP3d, while the lowest inhibition of 50 and 51.11% was by B. velezensis strain A6, P42, and P. polymyxa PGSS1, respectively. These endophytes outperformed the positive control, P. fluorescens which showed 44.44% inhibition. Similarly, against C. miyabeanus, 31.25-43.75% inhibition was recorded, of which B. velezensis strain A6 exhibited the highest inhibition (43.75%), while B. velezensis strain P42 showed the lowest (31.25%). Furthermore, a double Petri dish assay was conducted to evaluate the volatile compounds produced by these endophytes against the two rice pathogens. It was observed that the volatile compounds produced by B. pseudomycoides strain HP3d and P. polymyxa strain PGSS1 significantly and effectively inhibited the growth of M. oryzae by 88.89% when compared to P. fluorescens (50.00%), whereas B. velezensis strain A6 showed the lowest inhibition (33.33%). Against the brown spot pathogen, C. miyabeanus, the endophytes demonstrated inhibition ranging from 56.25-87.50%, with B. pseudomycoides strain HP3d and P. fluorescens achieving the highest inhibition (87.50%) and B. velezensis strain P42 showing the lowest (56.25%). These results highlight the significant and varied inhibitory effects of volatile compounds released by these endophytes against fungal pathogens of rice. Overall, our findings highlight the promising biocontrol potential of these endophytic strains under in vitro conditions with B. pseudomycoides strain HP3d and P. polymyxa PGSS1 showing exceptional efficacy and they can be deployed in the field for the management of foliar fungal pathogens in rice.

Curvularia lunata causes black kernel disease in rice (Oryza sativa) kernel and enzyme, trihydroxynaphthalene reductase (3HNR) catalyzes a crucial reaction in melanin biosynthesis which aids in appressorium formation. However, the molecular mechanism of pathogenesis and characterization of 3HNR are poorly understood due to the limited research conducted on this pathosystem. Therefore, the C. lunata (GVBR1) was isolated from the diseased rice kernel and identified using microscopic, SEM and molecular methods. To elucidate the molecular mechanism of 3HNR, the C. lunata was treated with different concentrations of systemic fungicide tricyclazole and the gene expression of C. lunata trihydroxynaphthalene reductase (Cl3HNR) analyzed with different concentrations of tricyclazole treated, downregulated the Cl3HNR gene expression as compared to the control. Further, Cl3HNR was cloned into pGEX expression vector, heterologously expressed in Escherichia coli BL21 cells and the expressed protein corresponds to 28 kDa. The molecular docking and dynamic simulation revealed the interaction points within the active site of Cl3HNR. This study highlights the importance of the melanin biosynthetic gene involved in the pathogenesis of the rice fungal pathogen, C. lunata, which paves the way in developing the HNR enzyme inhibitor to control the rice disease.

Red seaweed extracts have been shown to trigger the biotic stress tolerance in several crops. However, reports on transcriptional modifications in plants treated with seaweed biostimulant are limited. To understand the specific response of rice to blast disease in seaweed-biostimulant-primed and non-primed plants, transcriptomics of a susceptible rice cultivar IR-64 was carried out at zero and 48 h post inoculation with Magnaporthe oryzae (strain MG-01). A total of 3498 differentially expressed genes (DEGs) were identified; 1116 DEGs were explicitly regulated in pathogen-inoculated treatments. Functional analysis showed that most DEGs were involved in metabolism, transport, signaling, and defense. In a glass house, artificial inoculation of MG-01 on seaweed-primed plants resulted in the restricted spread of the pathogen leading to the confined blast disease lesions, primarily attributed to reactive oxygen species (ROS) accumulation. The DEGs in the primed plants were defense-related transcription factors, kinases, pathogenesis-related genes, peroxidases, and growth-related genes. The beta-D-xylosidase, a putative gene that helps in secondary cell wall reinforcement, was downregulated in non-primed plants, whereas it upregulated in the primed plants indicating its role in the host defense. Additionally, Phenylalanine ammonia-lyase, pathogenesis-related Bet-v-I family protein, chalcone synthase, chitinases, WRKY, AP2/ERF, and MYB families were upregulated in seaweed and challenge inoculated rice plants. Thus, our study shows that priming rice plants with seaweed bio-stimulants resulted in the induction of the defense in rice against blast disease. This phenomenon is contributed to early protection through ROS, protein kinase, accumulation of secondary metabolites, and cell wall strengthening.

Bacterial soft rot is one of the most devastating diseases and a major constraint encountered during carrot farming. Biological agents are the best eco-friendly alternatives to agrochemicals to manage soft rot disease to ensure environmental sustainability. In this study, about eight isolates of bacterial pathogen causing soft rot in carrots were collected from Karnataka, India. Based on the 16S rRNA sequencing the pathogen isolates causing soft rot of carrot were identified as Klebsiella variicola. The morphological characteristics of K. variicola was investigated under scanning electron microscopy. The pathogenicity assay showed that all eight isolates were pathogenic to the carrot. An in vitro and in planta assay of two novel strains of Bacillus velezensis (A6 and P42) against K. variicola indicated that both strains had strong antagonistic activity against all the pathogen strains. Furthermore, the volatile bioactive compounds produced by A6 and P42 strains were analyzed in GC-MS, which revealed the presence of 10 and 6 bioactive compounds in their culture filtrate, respectively, with antibacterial and antifungal properties. The present study suggests that both A6 and P42 strains of B. velezensis were antagonistic to K. variicola and can be used as biocontrol agents to manage soft rot diseases of carrot under field conditions.

Bacillus subtilis is a Gram-positive and endospore producing bacterium. Limited studies have shown that lipopeptides produced by B. subtilis can be inhibitory to phytopathogens. Sclerotinia sclerotiorum is a plant pathogenic fungus which causes various diseases like cotton rot, watery soft rot, stem rot, crown rot and blossom blight in vegetable crops. The objective of the study was to isolate lipopeptides from B. subtilis and study their inhibitory potential against S. sclerotiorum. So, the B. subtilis isolates were extracted from the collected soils of Western Ghats of India. They were initially characterized through morphological parameters followed by PCR amplification of the 16S rDNA gene and confirmation through BLAST algorithm in NCBI database. The lipopeptides produced by these isolates were tested against S. sclerotiorum. B. subtilis strains were effective against S. sclerotiorum and exhibited 18.33 to 29.5 % inhibition under dual culture bio-assay. The antagonistic activity of lipopeptides extracted from B. subtilis strains showed 21.56 to 88.89 % inhibition of S. sclerotiorum in the lowest to highest concentration of lipopeptide tested and was found to be significantly higher than the control. The present study has shown that B. subtilis strains vary in the production of lipopeptides and some of them could produce lipopeptides that are highly inhibitory to S. sclerotiorum. B. subtilis strain NBAIR BSWG1 showed the highest inhibition for S. sclerotiorum. Lipopeptide based poison food technique and the dual culture bioassay results showed that B. subtilis strain NBAIR BSWG1 has immense potential for use in the biological control of S. sclerotiorum. Further studies are being carried out in formulating the lipopeptides for field application.

Effectors play an important role in host–pathogen interactions. Though an economically significant disease in rice, knowledge regarding the infection strategy of Rhizoctonia solani is obscure. In this study, we performed a genome-wide identification of the effectors in R. solani based on the characteristics of previously reported effector proteins. A total of seven novel effectors (designated as RS107_1 to RS107_7) in the disease mechanism of R. solani were identified and were predicted to be non-classically secreted proteins with functionally conserved domains. The function, reactivity, and stability of these proteins were evaluated through physiochemical characterization. The target proteins involved in the regulation of rice defense mechanisms were identified. Furthermore, the effector genes were cloned and RS107_6 (metacaspase) was heterologously expressed in Escherichia coli to obtain a purified protein of ~36.5 kDa. The MALD-TOF characterization confirmed that the protein belonged to a metacaspase of the Peptidase_C14 protein family, 906 bp in size, and encoded a polypeptide of 301 amino acids. These findings suggest that the identified effectors can potentially serve as a virulence factor and can be targeted for the management of sheath blight in rice.

Aphid populations were collected on cowpea, dolichos, redgram and black gram from Belagavi and Udupi locations. The samples were shotgun sequenced using the Illumina NovaSeq 6000 system to understand the spatial distribution and community structure of microbiota (especially bacteria) associated with aphids. In the present study, we identified obligatory nutritional symbiont Buchnera aphidicola and facultative symbionts Rickettsia sp. and Bacteroidetes endosymbiont of Geopemphigus sp. in all the aphid samples studied, although in varied abundance. On the other hand, Serratia symbiotica, Arsenophonus sp. and Acinetobacter sp. were only found in aphids on specific host plants, suggesting that host plants might influence the bacterial community structure. Furthermore, our study revealed that microbiota other than bacteria were highly insignificant in the aphid populations. Additionally, functional annotation of aphid metagenomes identified several pathways and enzymes involved in various physiological and ecological functions. Amino acid and vitamin biosynthesis-related pathways were predominant than carbohydrate metabolism, owing to their feeding habit and nutritional requirement. Chaperones related to stress tolerance such as GroEL and DnaK were identified. Enzymes involved in toxic chemical metabolisms such as glutathione transferase, phosphodiesterases and ABC transferases were observed. These enzymes may confer resistance to pesticides in the aphid populations. Overall, our results support the importance of host plants in structuring bacterial communities in aphids and show the functional roles of symbionts in aphid survival and development. Thus, these findings can be the basis for further detailed investigations and devising better strategies to manage the pests in field conditions.

Soft rot disease of carrots is an important limiting factor of carrot production. In this study, carrot roots showing typical soft rot symptoms were identified in the fields, and diseased and healthy root samples were collected for pathogen identification. The pathogen was isolated using an enriched bell pepper method. The bell pepper developed a water-soaked lesion around the pricking region when it was pricked after stabbing the diseased root whereas, no symptoms were produced when bell pepper was pricked after stabbing a healthy carrot root. From samples of the infected roots, circular, whitish, smooth, mucoid, round, convex, and medium-sized colonies were formed on the nutrient agar medium and were morphologically identified as Acinetobacter spp. Pure culture for four isolates was obtained, and one of the isolates (AB1) was further subjected to 16S rDNA sequencing. The BLAST analysis of the 16S rDNA confirmed the identity of AB1 as Acinetobacter baumannii. Pathogenicity test using whole-root assay and slice assay proved AB1 as pathogenic on carrot by producing water-soaked lesion, maceration, and rotting symptoms, whereas water inoculated roots remain healthy. The rotting symptoms on the artificially diseased carrot roots were similar to those caused by Pectobacterium caratovorum and Klebsiella variicola on the carrot. Based on the colony morphology, biochemical tests, and 16S rDNA sequence identity followed by pathogenicity assays, it is evident that A. baumannii causes soft rot disease in carrots. This report is essential for developing specific diagnostics and management against this newly emerging bacterial pathogen of carrot.

Background The unprecedented drought and frequent occurrence of pathogen infection in rice is becoming more due to climate change. Simultaneous occurrence of stresses lead to more crop loss. To cope up multiple stresses, the durable resistant cultivars needs to be developed, by identifying relevant genes from combined biotic and abiotic stress exposed plants. Results We studied the effect of drought stress, bacterial leaf blight disease causing Xanthomonas oryzae pv. oryzae (Xoo) pathogen infection and combined stress in contrasting BPT5204 and TN1 rice genotypes. Mild drought stress increased Xoo infection irrespective of the genotype. To identify relevant genes that could be used to develop multi-stress tolerant rice, RNA sequencing from individual drought, pathogen and combined stresses in contrasting genotypes has been developed. Many important genes are identified from resistant genotype and diverse group of genes are differentially expressed in contrasting genotypes under combined stress. Further, a meta-analysis from individual drought and Xoo pathogen stress from public domain data sets narrowed- down candidate differentially expressed genes. Many translation associated genes are differentially expressed suggesting their extra-ribosomal function in multi-stress adaptation. Overexpression of many of these genes showed their relevance in improving stress tolerance in rice by different scientific groups. In combined stress, many downregulated genes also showed their relevance in stress adaptation when they were over-expressed. Conclusions Our study identifies many important genes, which can be used as molecular markers and targets for genetic manipulation to develop durable resistant rice cultivars. Strategies should be developed to activate downregulated genes, to improve multi-stress tolerance in plants.

Additional file 4. List of DEGs from microarray data set.

Sandalwood is a hemi-parasitic commercially and culturally important tree, mostly valued for the oil and fragrant heartwood. Sandalwood is a forest produce and the second most expensive wood in the world, whereas recent advancements of Next generation sequencing (NGS) technologies considerably promoted forest tree genomics. Genotyping interventions are most important in capturing the natural variability, however draft genome assembly facilitated the sandalwood genomics in developing sequence based markers through GBS, DArT, ddRAD and WGRS which can be utilized in genome-wide association studies, marker assisted selection and genomic selection to develop genetically improved saplings for commercial cultivation.

Red seaweed-derived biostimulants facilitate plant health and impart protection against abiotic stress conditions by their bioactive compounds and plant nutrients. The potency of red seaweed biostimulants (LBS6 and LBD1) on rice cv. IR-64 in response to fungicides induced stress was investigated in this study. Foliar application of LBS6 maintained the stomatal opening and leaf temperature under the fungicidal stress condition. Reactive Oxygen Species (ROS) such as hydrogen peroxide and superoxide radicals were significantly reduced in LBS6-treated stressed plants. After applying seaweed biostimulants, ROS production was stabilized by antioxidants viz., CAT, APX, SOD, POD, and GR. LBS-6 application increased the Ca+ and K+ levels in the stressed plants, which perhaps interacted with ROS and stomatal opening signalling systems, respectively. In the rice plants, fungicidal stress elevated the expression of stress-responsive transcriptional factors (E2F, HSFA2A, HSFB2B, HSFB4C, HSFC1A, and ZIP12). A decline in the transcript levels of stress-responsive genes was recorded in seaweed treated plants. For the first time, we present an integrative investigation of physicochemical and molecular components to describe the mechanism by which seaweed biostimulants in rice improve plant health under fungicidal stress conditions.