Stalk rot species diversity and molecular phylogeny associated with diseased maize in India.

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%), F. equiseti (14.5%), F. andiyazi (6.25%), F. solani (2.08%), F. proliferatum (2.08%), F. incarnatum (2.08%), Lasidioplodia theobrame (6.25%), Exserohilum rostrtum (4.16%), Nigrospora spp. (4.16%). and Schizophyllum commune (2.08%) were identified by different housekeeping genes (ITS, TEF-1α, RPB2 and Actin). Fusarium verticillioides, F. equiseti and F. andiyazi were major pathogens involved in stalk rot. This is the first report on F. proliferatum, F. solani, F. incarnatum, Lasidioplodia theobrame, Exserohilum rostrtum, Nigrospora spp. and Schizophyllum commune causing stalk rot of maize and their distribution in the different states of India. Studies on population dynamics of PFSR will enhance the understanding of pathogen behavior, virulence, or its association with different pathogens across India, which will facilitate the development of resistant maize genotypes against the PFSR.

Bacillus velezensis (strains A6 & P42) as a potential biocontrol agent against Klebsiella variicola, a new causal agent of soft rot disease in carrot.

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.

Transcriptomic responses under combined bacterial blight and drought stress in rice reveal potential genes to improve multi-stress tolerance.

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.

Diversity and biopotential of Bacillus velezensis strains A6 and P42 against rice blast and bacterial blight of pomegranate.

Bacillus velezensis is widely known for its inherent biosynthetic potential to produce a wide range of bio-macromolecules and secondary metabolites, including polyketides (PKs) and siderophores, as well as ribosomally and non-ribosomally synthesized peptides. In the present study, we aimed to investigate the bio-macromolecules, such as proteins and peptides of Bacillus velezensis strains, namely A6 and P42 by whole-cell sequencing and highlighted the potential application in controlling phytopathogens. The bioactive compounds, specifically secondary metabolites, were characterized by whole-cell protein profiling, Thin-Layer Chromatography, Infra-Red Spectroscopy, Nuclear Magnetic Resonance, Gas Chromatograph and Electro Spray Liquid Chromatography. Gas Chromatography analysis revealed that the A6 and P42 strains exert different functional groups of compounds, such as aromatic ring, aliphatic, alkene, ketone, amine groups and carboxylic acid. Whole-cell protein profiling of A6 and P42 strains of B. velezensis by nano-ESI LC-MS/MS revealed the presence of 945 and 5303 proteins, respectively. The in vitro evaluation of crude extracts (10%) of A6 and P42 significantly inhibited the rice pathogen, Magnaporthe oryzae (MG01), whereas the cell-free culture filtrate (75%) of strain P42 showed 58.97% inhibition. Similarly, in vitro evaluation of crude extract (10%) of P42 strain inhibited bacterial blight of pomegranate pathogen, Xanthomonas axonopodis pv. punicae, which eventually resulted in a higher inhibition zone of 3 cm, whereas the cell-free extract (75%) of the same strain significantly suppressed the growth of the pathogen with an inhibition zone of 1.48 cm. From the results obtained, the crude secondary metabolites and cell-free filtrates (containing bio-macromolecules) of the strains A6 and P42 of B. velezensis can be employed for controlling the bacterial and fungal pathogens of crop plants.

Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay.

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60 °C for 30 min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100 fg of template DNA compared to 1 ng and 10 pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.

Morpho-molecular and mating-type locus diversity of Ustilaginoidea virens: an incitant of false smut of rice from Southern parts of India.

AIMS: To characterize the geo-distinct isolates of Ustilaginoidea virens for morpho-molecular and mating-type locus diversity. METHODS AND RESULTS: Sixty-one isolates of U. virens collected from Southern India exhibited significant diversity in mycelial width (3·45-5·50 µm), colony colour (yellow, pale yellow, and white), and growth pattern (thick leather mat, raised-fluffy, flat-fluffy, and raised). Field-borne chlamydospores of each isolate were significantly smaller in size (3·34-5·26 µm2 ) compared to those formed on culture media (18·6-100·89 µm2 ). The phylogenetic study based on internal transcribed sequences revealed two clusters; however, most isolates (n = 54) were grouped in cluster-I, indicating common ancestral origin. We also identified 42 haplotypes; among them, Hap_3 has the highest number of isolates (n = 19). Mating-type locus (MAT1) analysis revealed all sixty-one isolates as heterothallic, wherein 37 and 24 isolates belonging to MAT1-1-1 and MAT1-2-1 heterothallic mating types, respectively. The microsynteny analysis of MAT1 loci of one of the Indian strain (Uv-Gvt) along with Uv-8b (China) strain revealed synteny conservation at MAT1 locus, which is flanked by conserved genes SLA2 and a hypothetical protein in the upstream and APN2, COX12 and APC5 in the downstream of the locus. CONCLUSIONS: Morpho-molecular study revealed the significant diversity among geo-distinct isolates, and MAT1 loci analysis indicated the distribution of heterothallic mating types in south Indian paddy fields. And also, complete synteny conservation between Indian and Chinese strain was observed at the MAT1 locus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the sexuality of Indian strains of the U. virens, which would help better understand the genetic diversity of the U. virens prevailing in Southern India and aid in developing resistant rice cultivars against this pathogen population.

Identification and characterization of multiple fungal pathogens associated with brown spot disease of rice in India.

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%, 17.33%, and 4.83% of the typical BS were produced by Bo, Cl, and Er, respectively. The ITS region was sequenced for selected 9, 7, and 3 isolates of Bo, Cl, and Er, respectively, and analyzed for their nucleotide and haplotype diversity, and phylogenetic relationships. A phylogenetic study identified the unique clustering patterns, and haplotyping indicated 3, 4, and 6 haplotypes. Tajima's D (D) test showed several rare alleles in the ITS regions. This is the first comprehensive study reporting the three fungal pathogens causing BS of rice and it is useful for re-designing the screening protocol for the host plant resistance breeding program. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04033-3.

Rice-Magnaporthe transcriptomics reveals host defense activation induced by red seaweed-biostimulant in rice plants.

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.

Insights on Novel Effectors and Characterization of Metacaspase (RS107_6) as a Potential Cell Death-Inducing Protein in Rhizoctonia solani.

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.

Spatial distribution and community structure of microbiota associated with cowpea aphid (Aphis craccivora Koch).

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03142-1.

Red-seaweed biostimulants differentially alleviate the impact of fungicidal stress in rice (Oryza sativa L.).

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.

LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed.

Rice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

Comparative Transcriptome Analysis of Rice Resistant and Susceptible Genotypes to Xanthomonas oryzae pv. oryzae Identifies Novel Genes to Control Bacterial Leaf Blight.

The bacterial leaf blight in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) affects crop losses worldwide. In spite of developing resistant varieties by introgressing different Xa genes, the occurrence of diseases is evident. Here we report identification of several genes that are associated with improved plant immunity against Xoo in a resistant genotype BPT-5204 in comparison with susceptible genotype TN-1. The RNA sequencing information was developed to identify the genes that could provide durable resistance in rice. Xoo-resistant rice genotype BPT-5204 with Xa 5, 13 and 21 genes is compared with sensitive Taichung Native 1 (TN-1) to identify the genetic pathways and gene networks involved in resistance mechanisms. The higher levels of salicylic acid resulted in upregulation of many pathogenesis-related (PR) and redox protein encoding transcripts which resulted in higher hypersensitive response in BPT-5204. Many Serine/threonine protein kinase, leucine-rich repeat (LRR) transmembrane protein kinase, protein kinase family genes, Wall-associated kinase (WAK) were upregulated that resulted in activation of bZIP, WRKY, MYB, DOF and HSFs transcription factors that are associated with improved plant immunity. The study provided roles of many genes and their associated plant immunity pathways that can be used for developing resistant rice cultivars.

Comparative metagenomic analysis of rice soil samples revealed the diverse microbial population and biocontrol organisms against plant pathogenic fungus Magnaporthe oryzae.

Intensive cropping degrades soil quality and disrupts the soil microbiome. To understand the effect of rice monocropping on soil-microbiome, we used a comparative 16S rRNA metagenome sequencing method to analyze the diversity of soil microflora at the genomic level. Soil samples were obtained from five locations viz., Chamarajnagara, Davangere, Gangavathi, Mandya, and Hassan of Karnataka, India. Chemical analysis of soil samples from these locations revealed significant variations in pH (6.00-8.38), electrical conductivity (0.17-0.69 dS m-1), organic carbon (0.51-1.29%), available nitrogen (279-551 kg ha-1), phosphorous (57-715 kg ha-1) and available potassium (121-564 kg ha-1). The 16S metagenome analysis revealed that the microbial diversity in Gangavathi soil samples was lower than in other locations. The soil sample of Gangavathi showed a higher abundance of Proteobacteria (85.78%) than Mandya (27.18%). The Firmicutes were more abundant in Chamarajnagar samples (36.01%). Furthermore, the KEGG pathway study revealed enriched nitrogen, phosphorus, and potassium metabolism pathways in all soil samples. In terms of the distribution of beneficial microflora, the decomposers were more predominant than the nutrient recyclers such as nitrogen fixers, phosphorous mineralizers, and nitrifiers. Furthermore, we isolated culturable soil microbes and tested their antagonistic activity in vitro against a fungal pathogen of rice, Magnaporthe oryzae strain MG01. Six Bacillus sp. and two strains of Trichoderma harzianum showed higher antagonistic activity against MG01. Our findings indicate that metagenome sequencing can be used to investigate the diversity, distribution, and abundance of soil microflora in rice-growing areas. The knowledge gathered can be used to develop strategies for maintaining soil quality and crop conservation to increase crop productivity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02783-y.

Genome, Transcriptome, and Germplasm Sequencing Uncovers Functional Variation in the Warm-Season Grain Legume Horsegram Macrotyloma uniflorum (Lam.) Verdc.

Horsegram is a grain legume with excellent nutritional and remedial properties and good climate resilience, able to adapt to harsh environmental conditions. Here, we used a combination of short- and long-read sequencing technologies to generate a genome sequence of 279.12Mb, covering 83.53% of the estimated total size of the horsegram genome, and we annotated 24,521 genes. De novo prediction of DNA repeats showed that approximately 25.04% of the horsegram genome was made up of repetitive sequences, the lowest among the legume genomes sequenced so far. The major transcription factors identified in the horsegram genome were bHLH, ERF, C2H2, WRKY, NAC, MYB, and bZIP, suggesting that horsegram is resistant to drought. Interestingly, the genome is abundant in Bowman-Birk protease inhibitors (BBIs), which can be used as a functional food ingredient. The results of maximum likelihood phylogenetic and estimated synonymous substitution analyses suggested that horsegram is closely related to the common bean and diverged approximately 10.17 million years ago. The double-digested restriction associated DNA (ddRAD) sequencing of 40 germplasms allowed us to identify 3,942 high-quality SNPs in the horsegram genome. A genome-wide association study with powdery mildew identified 10 significant associations similar to the MLO and RPW8.2 genes. The reference genome and other genomic information presented in this study will be of great value to horsegram breeding programs. In addition, keeping the increasing demand for food with nutraceutical values in view, these genomic data provide opportunities to explore the possibility of horsegram for use as a source of food and nutraceuticals.

Metagenome sequencing of fingermillet-associated microbial consortia provides insights into structural and functional diversity of endophytes.

Endophytes confer unique ecological advantages to their host plants. In this study, we have characterized the diversity of endophytic consortia associated with the GPU-28 (GPU) and Udurumallige (UM) finger millet varieties, which are resistant and susceptible to the blast disease, respectively. Whole genome metagenome sequencing of GPU and UM helped to identify 1029 species (includes obligate endophytes) of microbiota. Among them, 385 and 357 species were unique to GPU and UM, respectively. Remaining 287 species were common to both the varieties. Actinobacteria and other plant-growth promoting bacteria were abundant in GPU as compared to UM. Functional annotation of genes predicted from genomes of endophytes associated with GPU variety showed that many genes had functional role in stress response, secondary metabolism, aromatic compounds, glutathione, and cysteine synthesis pathways as compared to UM. Based on in vitro and in planta studies, Bacillus cereus and Paenibacillus spp. were found to be effective in suppressing the growth of blast disease pathogen Magnaporthe grisea (strain MG03). In the future, these strains could serve as potential biocontrol agents to reduce the incidence of blast disease in finger millet crop.