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.

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.

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.

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.

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.

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.

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.

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.

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.

In planta transcriptome analysis reveals tissue-specific expression of pathogenicity genes and microRNAs during rice-Magnaporthe interactions.

Transcriptional re-programming in host and pathogen upon leaf and neck infection is an evolving area of research for the rice blast community. Analysis of in planta rice transcriptome in leaf and neck tissues revealed tissue-specific and infection-specific expression of rice and Magnaporthe oryzae genes in host and pathogen. The glycosyl hydrolase, isocitrate lyase, cupin domain containing protein, TF2, CMPG1, CHIT17 and OsCML14 genes were uniquely expressed in leaf infection. Genes like cytochrome P450, inhibitor I family protein, GSTU6, abscisic stress ripening, and cupin domain containing protein were up-regulated during neck infection. In our microRNA sequencing study, Osa-miR166n-3p was highly expressed in upon Magnaporthe leaf infection, whereas osa-miR1661-3p, osa-miR166n-3p and osa-miR159b were overexpressed in neck infection. Here we report several transcripts being targeted by up and down regulated microRNAs during infection. The putative genes expressed upon infection in leaf and neck could be used in understanding the dual-epidemics of blast disease.

Comparative analysis of secondary metabolite gene clusters in different strains of Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae continues to be a major constraint in rice production worldwide. Rice is one of the staple crops in India and rice blast causes huge economic losses. Interestingly, the Indian subcontinent is the centre for origin and diversity of rice as well as the Magnaporthe species complex. Secondary metabolites are known to play important role in pathogenesis and M. oryzae has high potential of genes involved in secondary metabolism but, unfortunately most of them remain uncharacterized. In the present study, we analysed the draft genome assemblies of M. oryzae strains isolated from different parts of India, for putative secondary metabolite key gene (SMKG) clusters encoding polyketide synthases, non-ribosomal peptide synthetases, diterpene cyclases and dimethylallyl tryptophan synthase. Based on the complete genome sequence of 70-15 strain and its previous reports of identified SMKGs, we have identified the key genes for the interrogated strains. Expression analysis of these genes amongst different strains indicates how they have evolved depending on the host and environmental conditions. To our knowledge, this study is first of its kind where the secondary metabolism genes and their role in functional adaptation were studied across several strains of M. oryzae.

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.

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.

Genome and Transcriptome sequence of Finger millet (Eleusine coracana (L.) Gaertn.) provides insights into drought tolerance and nutraceutical properties.

BACKGROUND: Finger millet (Eleusine coracana (L.) Gaertn.) is an important staple food crop widely grown in Africa and South Asia. Among the millets, finger millet has high amount of calcium, methionine, tryptophan, fiber, and sulphur containing amino acids. In addition, it has C4 photosynthetic carbon assimilation mechanism, which helps to utilize water and nitrogen efficiently under hot and arid conditions without severely affecting yield. Therefore, development and utilization of genomic resources for genetic improvement of this crop is immensely useful. RESULTS: Experimental results from whole genome sequencing and assembling process of ML-365 finger millet cultivar yielded 1196 Mb covering approximately 82% of total estimated genome size. Genome analysis showed the presence of 85,243 genes and one half of the genome is repetitive in nature. The finger millet genome was found to have higher colinearity with foxtail millet and rice as compared to other Poaceae species. Mining of simple sequence repeats (SSRs) yielded abundance of SSRs within the finger millet genome. Functional annotation and mining of transcription factors revealed finger millet genome harbors large number of drought tolerance related genes. Transcriptome analysis of low moisture stress and non-stress samples revealed the identification of several drought-induced candidate genes, which could be used in drought tolerance breeding. CONCLUSIONS: This genome sequencing effort will strengthen plant breeders for allele discovery, genetic mapping, and identification of candidate genes for agronomically important traits. Availability of genomic resources of finger millet will enhance the novel breeding possibilities to address potential challenges of finger millet improvement.

Genome-Wide Comparison of Magnaporthe Species Reveals a Host-Specific Pattern of Secretory Proteins and Transposable Elements.

Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck), finger millet (leaf and neck), foxtail millet (leaf) and buffel grass (leaf). Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors.

De novo genome assembly and annotation of rice sheath rot fungus Sarocladium oryzae reveals genes involved in Helvolic acid and Cerulenin biosynthesis pathways.

BACKGROUND: Sheath rot disease caused by Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth. RESULTS: The genome size of S. oryzae was 32.78 Mb with contig N50 18.07 Kb and 10526 protein coding genes. The functional annotation of protein coding genes revealed that S. oryzae genome has evolved with many expanded gene families of major super family, proteinases, zinc finger proteins, sugar transporters, dehydrogenases/reductases, cytochrome P450, WD domain G-beta repeat and FAD-binding proteins. Gene orthology analysis showed that around 79.80 % of S. oryzae genes were orthologous to other Ascomycetes fungi. The polyketide synthase dehydratase, ATP-binding cassette (ABC) transporters, amine oxidases, and aldehyde dehydrogenase family proteins were duplicated in larger proportion specifying the adaptive gene duplications to varying environmental conditions. Thirty-nine secondary metabolite gene clusters encoded for polyketide synthases, nonribosomal peptide synthase, and terpene cyclases. Protein homology based analysis indicated that nine putative candidate genes were found to be involved in helvolic acid biosynthesis pathway. The genes were arranged in cluster and structural organization of gene cluster was similar to helvolic acid biosynthesis cluster in Metarhizium anisophilae. Around 9.37 % of S. oryzae genes were identified as pathogenicity genes, which are experimentally proven in other phytopathogenic fungi and enlisted in pathogen-host interaction database. In addition, we also report 13212 simple sequences repeats (SSRs) which can be deployed in pathogen identification and population dynamic studies in near future. CONCLUSIONS: Large set of pathogenicity determinants and putative genes involved in helvolic acid and cerulenin biosynthesis will have broader implications with respect to Sarocladium disease biology. This is the first genome sequencing report globally and the genomic resources developed from this study will have wider impact worldwide to understand Rice-Sarocladium interaction.

Indica rice genome assembly, annotation and mining of blast disease resistance genes.

BACKGROUND: Rice is a major staple food crop in the world. Over 80% of rice cultivation area is under indica rice. Currently, genomic resources are lacking for indica as compared to japonica rice. In this study, we generated deep-sequencing data (Illumina and Pacific Biosciences sequencing) for one of the indica rice cultivars, HR-12 from India. RESULTS: We assembled over 86% (389 Mb) of rice genome and annotated 56,284 protein-coding genes from HR-12 genome using Illumina and PacBio sequencing. Comprehensive comparative analyses between indica and japonica subspecies genomes revealed a large number of indica specific variants including SSRs, SNPs and InDels. To mine disease resistance genes, we sequenced few indica rice cultivars that are reported to be highly resistant (Tetep and Tadukan) and susceptible (HR-12 and Co-39) against blast fungal isolates in many countries including India. Whole genome sequencing of rice genotypes revealed high rate of mutations in defense related genes (NB-ARC, LRR and PK domains) in resistant cultivars as compared to susceptible. This study has identified R-genes Pi-ta and Pi54 from durable indica resistant cultivars; Tetep and Tadukan, which can be used in marker assisted selection in rice breeding program. CONCLUSIONS: This is the first report of whole genome sequencing approach to characterize Indian rice germplasm. The genomic resources from our work will have a greater impact in understanding global rice diversity, genetics and molecular breeding.

Genome analysis of rice-blast fungus Magnaporthe oryzae field isolates from southern India.

The Indian subcontinent is the center of origin and diversity for rice (Oryza sativa L.). The O. sativa ssp. indica is a major food crop grown in India, which occupies the first and second position in area and production, respectively. Blast disease caused by Magnaporthe oryzae is a major constraint to rice production. Here, we report the analysis of genome architecture and sequence variation of two field isolates, B157 and MG01, of the blast fungus from southern India. The 40 Mb genome of B157 and 43 Mb genome of MG01 contained 11,344 and 11,733 predicted genes, respectively. Genomic comparisons unveiled a large set of SNPs and several isolate specific genes in the Indian blast isolates. Avr genes were analyzed in several sequenced Magnaporthe strains; this analysis revealed the presence of Avr-Pizt and Avr-Ace1 genes in all the sequenced isolates. Availability of whole genomes of field isolates from India will contribute to global efforts to understand genetic diversity of M. oryzae population and to track the emergence of virulent pathotypes.

Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree.

Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC-600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways.