Rice Blast Research Articles

Biocontrol potentiality of <i>Burkholderia vietnamiensis</i> nrv12 against the rice blast fungus <i>magnaporthe oryzae</i>

Rice blast disease, caused by the pathogenic fungus Magnaporthe oryzae, is a widespread infection leading to serious crop loss worldwide. In order to achieve sustainable agriculture, root-associated bacteria have been applied to manage fungal diseases and promote growth. The present study aimed to evaluate in vitro the growth-promoting ability and in vivo biocontrol activity against M. oryzae of rice rhizosphere bacterium. Out of sixty-eight isolates recovered from the rhizosphere of blast-infected rice plants, isolate NRV12 exhibited the highest antifungal activity against M. oryzae SH, with an inhibition percentage of 72.7±3.44%. By analysis of 16S rRNA sequence associated with morphology, physiological and biochemical tests, the strain was identified as Burkholderia vietnamiensis. In addition, NRV12 produced hydrolytic enzymes (amylase, cellulase, protease), indole acetic acid (IAA), exhibited nitrogen-fixing potential and the ability to solubilize phosphate and zinc. Innoculation with NRV12 significantly promoted in vivo rice seedling growth to 23.3% as compared to the non-bacteria-treated seedlings. Importantly, infected rice seedlings treated with NRV12 led to a 40% disease reduction in rice blast. These findings suggest that NRV12 is a valuable and promising isolate with biocontrol potential against rice blast caused by M. oryzae.

Multiple Chitin- or Avirulent Strain-Triggered Immunity Induces Microbiome Reassembly in Rice.

Magnaporthe oryzae is one of the most important fungal pathogens of rice. Chitin and avirulent strains can induce two layers of immunity response, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI), in rice with cognate R genes. However, little is known about the assembly of the rice microbiome induced by PTI and ETI in rice. In this study, we investigate the impact of continuous treatment of the avirulent M. oryzae strain with AvrPi9 and chitin on the bacterial endophytic community of rice varieties harboring resistant gene Pi9 and their antagonistic activity against rice blast fungus. Analysis of the 16S rRNA showed a significant increase in the diversity and microbial co-occurrence network complexity and the number of beneficial taxa-Bacillus, Pseudomonas, Microbacterium, and Stenotrophomonas spp.-following the chitin and avirulent strain treatments. The antifungal assay with bacterial endophytes recovered from the leaves showed few bacteria with antagonistic potential in rice treated with avirulent strains, suggesting that the sequential treatment of the avirulent strain decreased the antagonistic bacteria against M. oryzae. Moreover, we identified Bacillus safensis Ch_66 and Bacillus altitudinis Nc_68 with overall antagonistic activities in vivo and in vitro. Our findings provide a novel insight into rice microbiome assembly in response to different innate immunity reactions.

OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

D53 represses rice blast resistance by directly targeting phenylalanine ammonia lyases.

In rice, DWARF 53 directly binds to the promoters of seven phenylalanine ammonia lyase genes, OsPAL1˜OsPAL7, and represses their expression, leading to decreased lignin accumulation and compromised resistance against Magnaporthe oryzae.

The dual-specificity kinase MoLKH1-mediated cell cycle, autophagy, and suppression of plant immunity is critical for development and pathogenicity of Magnaporthe oryzae

Cell cycle progression, autophagic cell death during appressorium development, and ROS degradation at the infection site are important for the development of rice blast disease. However, the association of cell cycle, autophagy and ROS detoxification remains largely unknown in M. oryzae. Here, we identify the dual-specificity kinase MoLKH1, which serves as an important cell cycle regulator required for appressorium formation by regulating cytokinesis and cytoskeleton in M. oryzae. MoLKH1 is transcriptionally activated by H2O2 and required for H2O2-induced autophagic cell death and suppression of ROS-activated plant defense during plant invasion of M. oryzae. In addition, the Molkh1 mutant also showed several phenotypic defects, including delayed growth, abnormal conidiation, damaged cell wall integrity, impaired glycogen and lipid transport, reduced secretion of extracellular enzymes and effectors, and attenuated virulence of M. oryzae. Nuclear localization of MoLKH1 requires the nuclear localization sequence, Lammer motif, as well as the kinase active site and ATP-binding site in this protein. Site-directed mutagenesis showed that each of them plays crucial roles in fungal growth and pathogenicity of M. oryzae. In conclusion, our results demonstrate that MoLKH1-mediated cell cycle, autophagy, and suppression of plant immunity play crucial roles in development and pathogenicity of M. oryzae.

<span>Chemical composition and antifungal activity of <em>Annona reticulata</em> L.</span>

Annona reticulata L. is a small deciduous tree species of Annonaceae, traditionally used for treatment of various ailments. The present study investigated the chemicals of the leaves of Annona reticulate and identified three compounds, including taraxerol (1), fridelin (2), and myricetin-3-O-α-L-rhamnopyranoside (3), using NMR spectroscopy. Among them, fridelin (2) is isolated first time from this species. The extracts and purified compound (3) were subjected to the examination of their effects on fungal activity with two fungi strains (Fusarium oxysporum and Pyricularia oryzae) using disc diffusion method. The results demonstrate beneficial effects of Annona reticulata as the antifungal activity for medicinal usages.

Sheath Blight of rice: A review of host plant interaction and disease management

The devastating pathogen Rhizoctonia solani, which is responsible for significant quality and production losses worldwide, is discussed in this review article along with the symptoms, disease cycle, epidemiology which causes sheath blight disease in rice. R. solani Kuhn, poses a significant threat to tropical Asia's rice production, potentially reducing grain yield by 50%. The article also emphasises the numerous management techniques, such as cultural, biological, and chemical controls. It is a severe production bottleneck in high yielding rice varieties under intensive rice production techniques, monoculture methods, a dense canopy, and extensive nitrogen management. Disease propagation in fields is influenced by air temperature, moisture content, and leaf wetness, with 16-25 C temperature range and 90% humidity favoring growth. Vertical and horizontal spread are influenced by these factors. The article provides comprehensive insight on host plant interaction of pathogen R. solani.

Morphological and molecular identification of fungi isolated from selected brri rice varieties

A total of 19 fungal species were isolated from the seeds of selected rice varieties (BRRI dhan 90 to BRRI dhan 99) following Tissue planting method and Blotter method. The isolated fungi were Aspergillus niger, A. ochraceus, A. oryzae, A. tamarii, A. terreus, Chaetomium globosum, Cladosporium oxysporum, Colletotrichum gloeosporioides, Corynespora cassiicola, Curvularia lunata, Curvularia soli, Daldinia eschscholtzii, Fusarium solani, Penicillium oxalicum,Penicillium sclerotiorum, Pestalotiopsis guepinii, Pyricularia oryzae and Rhizopus stolonifer. Fourteen fungi were selected for molecular identification. Out of the 19 fungal isolates, 14 were confirmed up to species level through ITS sequence based molecular analysis. Among the isolated fungi Pnicillium sclerotiorum and Curvularia soli are the new record for Bangladesh. Association of Daldinia eschscholtzii with rice seeds is also recorded first time from world. Bangladesh J. Plant Taxon. 31(1): 73-82, 2024 (June)

Preparation, antifungal potentiality, and mechanisms of action of agronanofungicides in controlling rice blast disease and enhancing yield of rice

Preparation, antifungal potentiality, and mechanisms of action of agronanofungicides in controlling rice blast disease and enhancing yield of rice

Design and synthesis of bis(indolyl)-hydrazide-hydrazone derivatives and their antifungal activities against plant pathogen fungi

A series of bis(indolyl)-hydrazide-hydrazone derivatives were synthesised, and their structures were characterised using 1H-NMR and HRMS. The antifungal activity of the prepared compounds was evaluated against Pyricularia oryzae Cav., Colletotrichum ­gloeosporioides Penz., Botrytis cinerea Pers.: Fr. and Rhizoctonia solani Kühn using the mycelial growth rate method. The preliminary bioassays revealed that most of the synthesised compounds exhibited antifungal activity against the four tested fungi and displayed a remarkable inhibitory effect on the mycelium growth of R. solani. In particular, compounds 3b, 3c, and 3k demonstrated significant antifungal activity against R. solani, with EC50 values of 26.42, 20.74, and 22.41 μM, respectively, outperforming the positive control shenqinmycin (47.18 μM) and carvacrol (49.13 μM).

Transcriptome and differential expression analysis revealed the pathogenic-related genes in Magnaporthe oryzae during leaf and panicle infection

Magnaporthe oryzae is one of the most destructive pathogens that threaten rice production around the world. Previous studies mainly focus on pathogenic mechanism of M. oryzae during infection on rice at leaf stage. However, the pathogenic mechanism of M. oryzae infection on panicle tissue is not well understood. In the present study, we performed RNA sequencing (RNA-seq) to study gene expression patterns of M. oryzae during infection at leaf stage and at panicle stage, respectively. The differentially expressed genes (DEGs) of M. oryzae in the infected leaf and panicle tissues were analyzed. Gene ontology (GO) enrichment analysis of DEGs revealed that M. oryzae genes involved in the biological processes were different at leaf and panicle stages. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs indicates that genes related to individual and important pathways may function at different infection stages. In particular, CAZymes carbohydrate esterases (CEs), carbohydrate-binding modules (CBMs), and glycoside hydrolases (GHs) may play important roles during M. oryzae infection on rice leaves, while glycosyltransferases (GTs) and GHs may play important roles during infection at rice panicle stage. Further analysis of effectors (BAS3, BAS113, BAS162, MoCDIP4, and MoHEG13) and their homologous genes suggest that they are involved in host defense suppression. Our findings provide insights into understanding the infection mechanisms of M. oryzae for rice leaf blast and panicle blast disease.

Bacillus amyloliquefaciens LM-1 Affects Multiple Cell Biological Processes in Magnaporthe oryzae to Suppress Rice Blast.

Magnaporthe oryzae, one of the most destructive rice pathogens, causes significant losses during the rice harvest every year. Bacillus amyloliquefaciens has been explored in many crops as a potential biocontrol agent. However, the mechanisms of B. amyloliquefaciens controled rice blast are not fully understood. Here, a biocontrol strain LM-1, isolated from a contaminated medium, was identified as B. amyloliquefaciens using morphological observation, physiological and biochemical tests, and 16S rDNA sequencing. LM-1 inhibited the growth and pathogenicity of M. oryzae and Bipolaris oryzae (Breda de Haan) Shoem. The mycelia of M. oryzae co-cultured with LM-1 were enlarged and broken by fluorescence microscopy using calcofluor white. LM-1 inhibited the mycelia of M. oryzae from producing conidia. Genes itu, srf, and fenB were detected in LM-1. Furthermore, the supernatant of LM-1 interfered with the appressorium formation of M. oryzae, blocked conidial cell death, and reduced autophagy degradation but did not affect the normal germination of rice seeds and seeding growth. Additionally, we observed hypersensitivity reactions, reactive oxygen species, and iron accumulation reduction in rice cells inoculated with supernatant. Our study reveals that LM-1 has a control effect on rice blast and affects cell wall integrity, sporulation, appressorium formation, cell death, and autophagy.

Evolution of wheat blast resistance gene Rmg8 accompanied by differentiation of variants recognizing the powdery mildew fungus.

Wheat blast, a devastating disease having spread recently from South America to Asia and Africa, is caused by Pyricularia oryzae (synonym of Magnaporthe oryzae) pathotype Triticum, which first emerged in Brazil in 1985. Rmg8 and Rmg7, genes for resistance to wheat blast found in common wheat and tetraploid wheat, respectively, recognize the same avirulence gene, AVR-Rmg8. Here we show that an ancestral resistance gene, which had obtained an ability to recognize AVR-Rmg8 before the differentiation of Triticum and Aegilops, has expanded its target pathogens. Molecular cloning revealed that Rmg7 was an allele of Pm4, a gene for resistance to wheat powdery mildew on 2AL, whereas Rmg8 was its homoeologue on 2BL ineffective against wheat powdery mildew. Rmg8 variants with the ability to recognize AVR-Rmg8 were distributed not only in Triticum spp. but also in Aegilops speltoides, Aegilops umbellulata and Aegilops comosa. This result suggests that the origin of resistance gene(s) recognizing AVR-Rmg8 dates back to the time before differentiation of A, B, S, U and M genomes, that is, ~5 Myr before the emergence of its current target, the wheat blast fungus. Phylogenetic analyses suggested that, in the evolutionary process thereafter, some of their variants gained the ability to recognize the wheat powdery mildew fungus and evolved into genes controlling dual resistance to wheat powdery mildew and wheat blast.

Rmg8 gene against wheat blast.

Rmg8 gene against wheat blast.

Functional genomic regions associated with blast disease resistance in rice predicted syntenic orthologs and potential resistance gene candidates from diverse cereal genomes

Blast disease is a major production constraint in cereal crops worldwide. Genome regions associated with blast resistance in rice were used in homology-based prediction of resistance gene orthologs across cereals. Seventy-four Resistance Gene Analogs (RGAs) that were collocated with rice QTLs (Quantitative Trait Loci) associated with blast resistance, predicted 89 orthologs from cereals,including 61 from finger millet. The RGA orthologs found hits in cereal transcriptomes derived from blast-infected tissues. Over 92% of the putative RGAs encoded NBS-LRR (Nucleotide Binding Site -Leucine Rich Repeat) proteins. Despite conserved primary protein structures, NBS-LRRs were variable in the encoding genes. Primers designed on conserved NBS-LRRs motifs amplified multiple fragments in PCR indicating the presence of highly similar paralogs in genomes. Rice chromosomes 1, 2, 4, 6, and 11 dock clusters of RGAs and syntenic clusters of orthologous RGAs were predicted from other cereal genomes identifying genome hotspots for resistance gene cassettes. RGAs in such cassettes were tightly linked with 50 %–90 % within-cluster similarity. Local finger millet germplasm identified multi-modes of resistance, and lack of correlation between the resistance traits points to independent expression of resistance mechanisms and associated genes in different tissues and development stages. The significant diversity, genomic abundance, and potential functional redundancy complicate use of RGAs for resistance breeding in given host genetic backgrounds, pathogen pools and agro-climates; therefore, robust technology pipelines that enable system-level analysis of expression and effectiveness can increase the practical use of RGAs.

Green synthesis and characterization of silver nanoparticles and its efficacy against Rhizoctonia solani, a fungus causing sheath blight disease in rice.

Rice (Oryza sativa) stands as a crucial staple food worldwide, especially in Bangladesh, where it ranks as the third-largest producer. However, intensified cultivation has made high-yielding rice varieties susceptible to various biotic stresses, notably sheath blight caused by Rhizoctonia solani, which inflicts significant yield losses annually. Traditional fungicides, though effective, pose environmental and health risks. To address this, nanotechnology emerges as a promising avenue, leveraging the antimicrobial properties of nanoparticles like silver nanoparticles (AgNPs). This study explored the green synthesis of AgNPs using Ipomoea carnea leaf extract and silver nitrate (AgNO3), and also examined their efficacy against sheath blight disease in rice. The biosynthesized AgNPs were characterized through various analytical techniques such as UV-vis spectrophotometer, X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Particle size analyzer, Zeta potential, Scanning Electron Microscope (SEM), Field Emission Scanning Electron Microscope (FESEM), Transmission Electron Microscope (TEM) for confirming their successful production and crystalline nature of nanoparticles. The results of UV-visible spectrophotometers revealed an absorption peak ranging from 421 to 434 nm, validated the synthesis of AgNPs in the solution. XRD, DLS, and TEM estimated AgNPs sizes were ~45 nm, 66.2nm, and 46.38 to 73.81 nm, respectively. SEM and FESEM demonstrated that the synthesized AgNPs were spherical in shape. In vitro assays demonstrated the significant inhibitory effects of AgNPs on mycelial growth of Rhizoctonia solani, particularly at higher concentrations and pH levels. Further greenhouse and field experiments validated the antifungal efficacy of AgNPs against sheath blight disease in rice, exhibiting comparable effectiveness to commercial fungicides. The findings highlight the potential of AgNPs as a sustainable and effective alternative for managing rice sheath blight disease, offering a safer solution amidst environmental concerns associated with conventional fungicides.

Various amino acid substitutions in succinate dehydrogenase complex regulating differential resistance to pydiflumetofen in Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is a devastating fungal disease worldwide. Pydiflumetofen (Pyd) is a new succinate dehydrogenase inhibitor (SDHI) that exhibited anti-fungal activity against M. oryzae. However, control of rice blast by Pyd and risk of resistance to Pyd are not well studied in this pathogen. The baseline sensitivity of 109 M. oryzae strains to Pyd was determined using mycelial growth rate assay, with EC50 values ranging from 0.291 to 2.1313 μg/mL, and an average EC50 value of 1.1005 ± 0.3727 μg/mL. Totally 28 Pyd-resistant (PydR) mutants with 15 genotypes of point mutations in succinate dehydrogenase (SDH) complex were obtained, and the resistance level could be divided into three categories of very high resistance (VHR), high resistance (HR) and moderate resistance (MR) with the resistance factors (RFs) of >1000, 105.74–986.13 and 81.92–99.48, respectively. Molecular docking revealed that all 15 mutations decreased the binding-force score for the affinity between Pyd and target subunits, which further confirmed that these 15 genotypes of point mutations were responsible for the resistance to Pyd in M. oryzae. There was positive cross resistance between Pyd and other SDHIs, such as fluxapyroxad, penflufen or carboxin, while there was no cross-resistance between Pyd and carbendazim, prochloraz or azoxystrobin in M. oryzae, however, PydR mutants with SdhBP198Q, SdhCL66F or SdhCL66R genotype were still sensitive to the other 3 SDHIs, indicating lack of cross resistance. The results of fitness study revealed that the point mutations in MoSdhB/C/D genes might reduce the hyphae growth and sporulation, but could improve the pathogenicity in M. oryzae. Taken together, the risk of resistance to Pyd might be moderate to high, and it should be used as tank-mixtures with other classes of fungicides to delay resistance development when it is used for the control of rice blast in the field.

Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae.

Filamentous plant pathogens deliver effector proteins into host cells to suppress host defence responses and manipulate metabolic processes to support colonization. Understanding the evolution and molecular function of these effectors provides knowledge about pathogenesis and can suggest novel strategies to reduce damage caused by pathogens. However, effector proteins are highly variable, share weak sequence similarity and, although they can be grouped according to their structure, only a few structurally conserved effector families have been functionally characterized to date. Here, we demonstrate that Zinc-finger fold (ZiF) secreted proteins form a functionally diverse effector family in the blast fungus Magnaporthe oryzae. This family relies on the Zinc-finger motif for protein stability and is ubiquitously present in blast fungus lineages infecting 13 different host species, forming different effector tribes. Homologs of the canonical ZiF effector, AVR-Pii, from rice infecting isolates are present in multiple M. oryzae lineages. Wheat infecting strains of the fungus also possess an AVR-Pii like allele that binds host Exo70 proteins and activates the immune receptor Pii. Furthermore, ZiF tribes may vary in the proteins they bind to, indicating functional diversification and an intricate effector/host interactome. Altogether, we uncovered a new effector family with a common protein fold that has functionally diversified in lineages of M. oryzae. This work expands our understanding of the diversity of M. oryzae effectors, the molecular basis of plant pathogenesis and may ultimately facilitate the development of new sources for pathogen resistance.

Biocontrol of blast disease in KDML105 rice by root-associated bacteria

Biocontrol of blast disease in KDML105 rice by root-associated bacteria

Biotechnological Approaches toward Blast Disease Resistance in Finger Millet

Plant disease globally causes significant losses in crop production that hindering the urgent need for a 60% increase in food demand. The main impact is reduced food quantity and quality leading to compromised food safety from pesticides and toxins. Biotechnology is essential for protecting crop yield and enhancing sustainability in agriculture. Agricultural biotechnology has advanced offering options for understanding plant molecular mechanisms and breeding. The devastating finger millet blast disease caused by the fungus Magnaporthe oryzae affects crop growth and yield leading to instability due to breakdown of blast resistance. Novel strategies are essential for sustainable finger millet production focusing on breeding techniques such as molecular markers, expressed sequence tags (ESTs), gene expression profiling, genome-wide association studies, and genetic transformations to develop new disease-resistance varieties. This comprehensive review explores current breeding strategies and future directions that may offers insights for effective disease control and optimizing finger millet productivity.