Control Of Rice Blast Disease Research Articles

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

Exogenous Indole-3-Acetic Acid Suppresses Rice Infection of Magnaporthe oryzae by Affecting Plant Resistance and Fungal Growth.

Auxin is an important phytohormone that regulates diverse biologic processes, including plant growth and immunity. Indole-3-acetic acid (IAA), known as one of the main forms of auxin, is able to activate plant immunity. However, it is unknown whether IAA enhances plant resistance and/or suppresses the growth of the fungal pathogen Magnaporthe oryzae. Here, we found that IAA could induce expression levels of pathogenesis-related genes to enhance disease resistance and could control the development of blast disease through inhibiting M. oryzae infection. Exogenous IAA suppressed mycelial growth and delayed spore germination by inhibiting fungal endogenous IAA biosynthesis and impairing redox homeostasis, respectively. When applied to a field test, two IAA analogues, 1-naphthaleneacetic acid and 2,4-dichlorophenoxy acetic acid, can effectively control rice blast disease. Our study advances the understanding of IAA in controlling rice blast disease through suppressing pathogen growth and enhancing plant resistance.

Antifungal plant flavonoids identified in silico with potential to control rice blast disease caused by Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, poses a severe threat to rice production, particularly in Asia where rice is a staple food. Concerns over fungicide resistance and environmental impact have sparked interest in exploring natural fungicides as potential alternatives. This study aimed to identify highly potent natural fungicides against M. oryzae to combat rice blast disease, using advanced molecular dynamics techniques. Four key proteins (CATALASE PEROXIDASES 2, HYBRID PKS-NRPS SYNTHETASE TAS1, MANGANESE LIPOXYGENASE, and PRE-MRNA-SPLICING FACTOR CEF1) involved in M. oryzae's infection process were identified. A list of 30 plant metabolites with documented antifungal properties was compiled for evaluation as potential fungicides. Molecular docking studies revealed that 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin exhibited superior binding affinities compared to reference fungicides (Azoxystrobin and Tricyclazole). High throughput molecular dynamics simulations were performed, analyzing parameters like RMSD, RMSF, Rg, SASA, hydrogen bonds, contact analysis, Gibbs free energy, and cluster analysis. The results revealed stable interactions between the selected metabolites and the target proteins, involving important hydrogen bonds and contacts. The SwissADME server analysis indicated that the metabolites possess fungicide properties, making them effective and safe fungicides with low toxicity to the environment and living beings. Additionally, bioactivity assays confirmed their biological activity as nuclear receptor ligands and enzyme inhibitors. Overall, this study offers valuable insights into potential natural fungicides for combating rice blast disease, with 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin standing out as promising and environmentally friendly alternatives to conventional fungicides. These findings have significant implications for developing crop protection strategies and enhancing global food security, particularly in rice-dependent regions.

PH-Responsive Pesticide-Loaded Hollow Mesoporous Silica Nanoparticles with ZnO Quantum Dots as a Gatekeeper for Control of Rice Blast Disease.

Nanotechnology-enabled pesticide delivery systems have been widely studied and show great prospects in modern agriculture. Nanodelivery systems not only achieve the controlled release of agrochemicals but also possess many unique characteristics. This study presents the development of a pH-responsive pesticide nanoformulation utilizing hollow mesoporous silica nanoparticles (HMSNs) as a nanocarrier. The nanocarrier was loaded with the photosensitive pesticide prochloraz (Pro) and then combined with ZnO quantum dots (ZnO QDs) through electrostatic interactions. ZnO QDs serve as both the pH-responsive gatekeeper and the enhancer of the pesticide. The results demonstrate that the prepared nanopesticide exhibits high loading efficiency (24.96%) for Pro. Compared with Pro technical, the degradation rate of Pro loaded in HMSNs@Pro@ZnO QDs was reduced by 26.4% after 24 h ultraviolet (UV) exposure, indicating clearly improved photostability. In a weak acidic environment (pH 5.0), the accumulated release of the nanopesticide after 48 h was 2.67-fold higher than that in a neutral environment. This indicates the excellent pH-responsive characteristic of the nanopesticide. The tracking experiments revealed that HMSNs can be absorbed by rice leaves and subsequently transported to other tissues, indicating their potential for effective systemic distribution and targeted delivery. Furthermore, the bioactivity assays confirmed the fungicidal efficacy of the nanopesticide against rice blast disease. Therefore, the constructed nanopesticide holds great prospect in nanoenabled agriculture, offering a novel strategy to enhance pesticide utilization.

PH/chitinase dual stimuli-responsive essential oil-delivery system based on mesoporous silica nanoparticles for control of rice blast.

Owing to their surface modifiability, smart mesoporous silica nanoparticles (MSNs) can be designed to respond to plant disease-microenvironmental stimuli, thereby achieving on-demand release of active ingredients to control disease by effectively improving citral (CT) stability. A pH/chitinase dual stimuli-responsive essential oil-delivery system (CT@HMS@CH/TA) was successfully fabricated by encapsulating CT in hollow mesoporous silica (HMS), and coating with tannic acid (TA) and chitosan (CH) within HMS by using the layer-by-layer assembly technique (LbL). CT@HMS@CH/TA with an average particle size of 125.12 ± 0.12 nm and a hollow mesoporous nanostructure showed high CT-loading efficiency (16.58% ± 0.17%). The photodegradation rate of CT@HMS@CH/TA under UV irradiation (48 h) was only 15.31%, indicating a 3.34-fold UV stability improvement. CT@HMS@CH/TA exhibited a higher CT release rate in response to acidic pH and the presence of chitinase, simulating the prevailing conditions as Magnaporthe oryzae infection. Furthermore, CT@HMS@CH/TA exhibited better adhesion without affecting normal rice growth, significantly upregulating chitinase gene expression and enhancing chitinase activity on M. oryzae, thus enhancing CT antifungal activity. CT@HMS@CH/TA improved CT stability and showed intelligent, controlled release-performance and higher antifungal efficacy, thus providing a new strategy for efficient application of essential oils for green control of rice blast disease. © 2024 Society of Chemical Industry.

A smart dual-responsive nanoplatform for delivery of prochloraz for the control of rice blast disease

Nano-controlled release formulations present a promising strategy to mitigate pesticide losses and enhance efficiency. In this study, a pH and GSH-responsive nanoplatform using mesoporous organosilica nanoparticles (MONs) as a carrier and poly(tannic acid) (PTA) as capping agent was established for controlling prochloraz (Pro) release. The obtained Pro@MON@PTA was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA). The results indicate the successful preparation of Pro@MON@PTA nanoparticles, featuring uniform particle size (190 nm), excellent dispersibility, and a prochloraz loading efficiency of 17.2%. Evaluation of contact angle and adhesion work demonstrated superior adhesion of MON@PTA to rice leaves compared to MON. Controlled release studies revealed dual-responsive release properties of Pro@MON@PTA to acid and GSH. Additionally, photostability testing indicated effective ultraviolet light shielding by the carrier, reducing prochloraz degradation under irradiation. Bioassay results indicated equivalent fungicidal activity against Magnaporthe oryzae between Pro@MON@PTA and prochloraz technical and prochloraz EW after a 7-day treatment. However, in vivo experiments demonstrated that Pro@MON@PTA exhibited superior control efficacy compared to prochloraz EW. These findings suggested that MON@PTA holds significant potential for plant disease management.

Antifungal potential of commercial silver nanoparticles against rice blast pathogen Magnaporthe oryzae

The rice blast caused by Magnaporthe oryzae is one of the major and recurrent threats to sustainable rice production in Bangladesh. To mitigate this problem, the present study was undertaken with the aim to investigate the inhibitory effects of commercially available silver nanoparticles (Ag-NPs) on the growth and development of Magnaporthe oryzae, and control of rice blast disease. In vitro experiments demonstrated that Ag-NPs significantly suppressed the radial growth of fungal mycelium, with the highest inhibition (83.54%) at a concentration of 250 ppm, while Trooper 75 WP at 400 ppm completely inhibited the fungal mycelial growth. The production of conidia and colony formation were also reduced significantly by Ag-NPs treatment. Furthermore, Ag-NPs exhibited a substantial inhibitory effect on the germination of M. oryzae conidia, with complete inhibition observed at concentrations between 200 and 250 ppm. Comparative analyses revealed that the fungicidal effect of Ag-NPs was superior to silver nitrate (AgNO3), suggesting the augmented activity of the nano properties. The sensitivity of M. oryzae to Ag-NPs was determined by calculating EC50 and EC95 values, which indicated a mean EC50 value of 131.5 ppm and a mean EC95 value of 276 ppm. Additionally, in vivo experiments showed that preventive and curative treatments with Ag-NPs significantly reduced disease incidence and severity in rice plants infected with M. oryzae, with efficacy comparable to that of the fungicide Trooper 75 WP. At higher concentrations of Ag-NPs, preventive treatment was found to be more effective than curative treatment in disease control. These findings highlighted the potential of Ag-NPs as an effective alternative for the management of rice blast. Ann. Bangladesh Agric. (2023) 27 (1): 17-30

Isolation and Evaluation of Streptomyces melanogenes YBS22 with Potential Application for Biocontrol of Rice Blast Disease.

Plant diseases caused by pathogenic fungi pose a significant threat to agricultural production. This study reports on a strain YBS22 with broad-spectrum antifungal activity that was isolated and identified, and its active metabolites were purified and systematically studied. Based on a whole genome sequence analysis, the new strain YBS22 was identified as Streptomyces melanogenes. Furthermore, eight gene clusters were predicted in YBS22 that are responsible for the synthesis of bioactive secondary metabolites. These clusters have homologous sequences in the MIBiG database with a similarity of 100%. The antifungal effects of YBS22 and its crude extract were evaluated in vivo and vitro. Our findings revealed that treatment with the strain YBS22 and its crude extract significantly reduced the size of necrotic lesions caused by Magnaporthe oryzae on rice leaves. Further analysis led to the isolation and purification of an active compound from the crude extract of the strain YBS22, identified as N-formylantimycin acid methyl ester, an analog of antimycin, characterized by NMR and MS analyses. Consistently, the active compound can significantly inhibit the germination and development of M. oryzae spores in a manner that is both dose- and time-dependent. As a result, we propose that the strain YBS22 could serve as a novel source for the development of biological agents aimed at controlling rice blast disease.

ABCC Transporter Gene MoABC-R1 Is Associated with Pyraclostrobin Tolerance in Magnaporthe oryzae.

Rice blast is a worldwide fungal disease that poses a threat to food security. Fungicide treatment is one of the most effective methods to control rice blast disease. However, the emergence of fungicide tolerance hampers the control efforts against rice blast. ATP-binding cassette (ABC) transporters have been found to be crucial in multidrug tolerance in various phytopathogenic fungi. This study investigated the association between polymorphisms in 50 ABC transporters and pyraclostrobin sensitivity in 90 strains of rice blast fungus. As a result, we identified MoABC-R1, a gene associated with fungicide tolerance. MoABC-R1 belongs to the ABCC-type transporter families. Deletion mutants of MoABC-R1, abc-r1, exhibited high sensitivity to pyraclostrobin at the concentration of 0.01 μg/mL. Furthermore, the pathogenicity of abc-r1 was significantly diminished. These findings indicate that MoABC-R1 not only plays a pivotal role in fungicide tolerance but also regulates the pathogenicity of rice blast. Interestingly, the combination of MoABC-R1 deletion with fungicide treatment resulted in a three-fold increase in control efficiency against rice blast. This discovery highlights MoABC-R1 as a potential target gene for the management of rice blast.

Genetic Variation of Magnaporthe oryzae Population in Hunan Province.

Studies on the population structure and variation of Magnaporthe oryzae in fields are of great significance for the control of rice blast disease. In this study, a total of 462 isolates isolated from different areas of Hunan Province in 2016 and 2018 were analyzed for their population structure and variation tendency. The results showed that from 2016 to 2018, the concentration of fungal races of M. oryzae increased and the diversity decreased; furthermore, 218 isolates in 2016 belonged to ZA, ZB, ZC, ZE, ZF and ZG, with a total of 6 groups and 29 races, in which the dominant-population ZB group accounted for 66.2%; meanwhile, in 2018, 244 isolates were classified into 4 groups and 21 races, including ZA, ZB, ZC and ZG, in which the dominant-population ZB group accounted for 72.54%. In 2018, isolates of ZD, ZE and ZF populations were absent, and the number of total races and isolates of the ZA and ZC groups decreased. Fungal pathogenicity was identified, with 24 monogenic lines (MLs) carrying 24 major R genes. The resistance frequency of R genes to fungal isolates in 2018 decreased significantly, in which except Pikm was 64.5%, the other monogenic lines were less than 50%. Rep-PCR analysis for isolates of Guidong in Hunan also showed that fungal diversity decreased gradually. The influence of R genes on fungal variation was analyzed. The pathogenicity of isolates purified from Xiangwanxian 11 planted with monogenic lines was significantly more enhanced than those without monogenic lines. All the results indicated that in recent years, the fungal abundance in Hunan has decreased while fungal pathogenicity has increased significantly. This study will greatly benefit rice-resistance breeding and the control of rice blast disease in Hunan Province.

Evaluation of Triazole and Strobilurin Fungicides against Leaf and Panicle Blast of Rice in Farmer's Field

Blast disease of rice caused by the ascomycetes fungus Magnaporthe grisea poses a major threat to rice production from terai to hilly region of Nepal. Field trials were conducted in two successive years, 2021 and 2022 at Bharatpur-5, Chitwan in a farmer's field producing seed of Hardinath-1 variety with the main objective of finding the effective single and/or pre-mix formulation of triazole and strobilurin fungicides against rice blast that could be an alternative to tricyclazole. The trials were conducted in randomized complete block design with three replications and eight treatments viz. azoxystrobin 18.2% + difenoconazole 11.4% SC, propiconazole 13.9% + difenoconazole 13.9% EC, azoxystrobin 11% + tebuconazole 18.3% SC, azoxystrobin 23% SC, propiconazole 25% EC, difenoconazole 25% EC, tebuconazole 25%WDG and a control. The fungicides were applied during tillering and heading stage at the rate of 0.1%. All fungicides significantly reduced the leaf and panicle blast severity resulting in a higher yield. Among the tested fungicides, pre-mix formulation fungicide azoxystrobin 18.2% + difenoconazole 11.4% SC, propiconazole 13.9% + difenoconazole 13.9% EC and azoxystrobin 11% + tebuconazole 18.3% SC gave excellent performance with the lowest leaf and panicle blast severity and higher yield. Hence, these three fungicides could be used for the management of leaf and panicle blast disease for higher yield. However, controlling rice blast disease has become increasingly challenging due to the pathogens' capacity to adapt and evolve into new, more potent strains, making the management and control of this disease more difficult.

Molecular profiling of some blast resistance genes and analysis of physiological variability in rice plants produced by anther culture and hybridization

For genetic improvement of rice plants against blast fungus disease caused by Magnaporthe oryzae, eight rice genotypes including four Egyptian and four foreign genotypes which represented a broad range of variation for blast resistance and other several traits were used in this study through line X tester mating design to produce F1 hybrids used as source of anther culture. Results of analysis of variance showed highly significant differences for all studied traits indicating overall genetic variations among parents and hybrids suggested that specific combining ability referred heavily to the expression of studied traits. Data of anther culture experiment for parents (8 parents) and hybrids (15 crosses) showed highly significant differences for all studied traits which illustrate genetic variations through the used materials. The highest callus induction percentage was found for CR7 (18.3%) and P6 (14.0%) while, the lowest callus induction percentage was found for CR5 (2.0%) and CR1 (4.0%). On the other hand, nine out of 15 hybrids recorded regeneration percentage more than the highest parental genotypes. Anther culture-derived plants and their parental genotypes were evaluated for blast disease resistance according to artificial infection with 10 blast races. The results showed that resistance percentage varied between 10% and 100%. While all anther culture-derived plants showed high level of resistance (100%) against all tested blast races. For enzyme activities for catalase, peroxidase and polyphenol oxidase in anther culture-drived plants and their parent, results showed that line TC9 derived from CR9 gave the highest value for catalase activity (301). Also, CR11 and P4 recorded the high level of peroxidase and polyphenol oxidase activities, respectively. Moreover, genetic diversity among anther culture-derived plants and their parental genotypes using five SSR primers related to Pi5(t), Pikh, Pi-b, Pi-1 and Pi-ta blast resistance genes showed heterozygous bands in all tested genotypes. The highest PIC value recorded for RM 247 and RM 206 markers which had PIC values of 0.778 and 0.761, respectively. Out of the used five SSR markers, RM 247 was the most informative marker with amplicon size ranged from 134 to 242 bp. These markers are therefore useful markers for studying polymorphism concerning blast resistance in rice and hence can be used in the marker-assisted selection for blast resistance. While development of resistant genotypes is the most effective way to control rice blast disease, anther culture technique used in this study helpful in production of resistant double haploid plants used in rice preeding programe in Egypt.

The regulatory subunit MoB56 of PP2A phosphatase regulates pathogenicity, growth and development in a protein complex with the atypical catalytic subunit Ppg1 in the rice blast fungus Magnaporthe oryzae

Protein phosphatase 2A (PP2A) is usually a heterotrimeric enzyme, consisting of a catalytic subunit (C) and a scaffolding subunit (A) associated with a third, variable regulatory subunit (B). Fungi usually carry a single gene for A and C subunits, and three genes for the B subunit. In addition, fungi contain a conserved atypical C subunit named Ppg1, which is essential to the pathogenicity of the rice blast fungus Magnaporthe oryzae. However, it remains largely unknown how the B subunit combinatorically assembles with the A and C subunits or Ppg1 to regulate fungal growth, development and pathogenicity. Here we report and functionally characterize one regulatory subunit of PP2A, named MoB56, in M. oryzae. We generated a MoB56 deletion mutant Δmob56, which was severely defective in vegetative growth, conidiation and septum formation, and had lost pathogenicity. The defects of Δmob56 could be rescued by introducing MoB56 fused with GFP (MoB56-GFP) at its C terminus. Fluorescence microscopic observations revealed that the MoB56-GFP signals were widely distributed in the cytoplasm and formed a dot-like structure at the center of the septum in conidia, appressoria and infection hyphae, supporting its function in septation. Further, we performed co-immunoprecipitation and pull-down assays, indicating that MoB56 forms a protein complex with the A subunit and Ppg1 in mycelial cells. The yeast two-hybrid assay showed that MoB56 could interact with the A subunit of PP2A but not with Ppg1, while Ppg1 could interact with the A subunit, suggesting that the A subunit ties MoB56 with Ppg1 for the protein complex formation. In addition, we revealed that MoB56 has multiple isoforms, which are likely originated from alternative splicing and sumoylation. This is the first report revealing that the regulatory subunit B56 is associated with the PP2A-like phosphatase Ppg1 in fungi. Importantly, this study showed that B56, like Ppg1, is essential to the pathogenicity of M. oryzae, offering a potential new lead to control this devastating fungal pathogen by targeting specific PP2A-like phosphatase. Together, this study provides important information for understanding how the regulatory subunit B56 of PP2A regulates fungal pathogenicity and for the control of rice blast disease.

Effects of rice blast biocontrol strain Pseudomonas alcaliphila Ej2 on the endophytic microbiome and proteome of rice under salt stress.

Soil salinity is a prevalent environmental stress in agricultural production. Microbial inoculants could effectively help plants to alleviate salt stress. However, there is little knowledge of the biocontrol strain Pseudomonas alcaliphila Ej2 mechanisms aiding rice plants to reduce the adverse effects caused by salt stress. We performed integrated field and greenhouse experiments, microbial community profiling, and rice proteomic analysis to systematically investigate the Ej2 mechanism of action. The results displayed that biocontrol strain Ej2 increased shoot/root length and fresh/dry weight compared with control under salt stress. Meanwhile, strain Ej2 has the ability to control rice blast disease and promote rice growth. Furthermore, the microbial community analysis revealed that the alpha-diversity of Ej2-inoculated plants was higher than the control plants, expect the Shannon index of the bacterial microbiome and the Ej2-inoculated samples clustered and separated from the control samples based on beta-diversity analysis. Importantly, the enriched and specific OTUs after Ej2 inoculation at the genus level were Streptomyces, Pseudomonas, Flavobacterium, and Bacillus. Moreover, we observed that Ej2 inoculation influenced the rice proteomic profile, including metabolism, plant-pathogen interactions, and biosynthesis of unsaturated fatty acids. These results provide comprehensive evidence that Ej2 inoculation induced the rice endophytic microbiome and proteomic profiles to promote plant growth under salt stress. Understanding the biocontrol strain effects on the endophytic microbiome and rice proteomics will help us better understand the complex interactions between plants and microorganisms under salt stress. Furthermore, unraveling the mechanisms underlying salt tolerance will help us more efficiently ameliorate saline soils.

Synthesis of Nickel-Chitosan Nanoparticles for Controlling Blast Diseases in Asian Rice.

Rice blast caused by Pyricularia oryzae is one of most devastating fungal diseases in rice, reducing the annual yield of rice worldwide. As an alternative to fungicide for curbing rice blast, synthesis of nickel-chitosan nanoparticles (Ni-Ch NPs) was performed with nickel chloride and assessed its efficacy in inflating plant growth and hindrance of Pyricularia oryzae (blast pathogen). Characterization of Ni-Ch NPs from SEM, TEM, and DLS analyses showed smooth- and spherical-shaped nanoparticles in the range of 20-70nm. Colloidal stability of NPs was revealed from Zeta potential exhibiting polydispersity index of 0.22. EDX spectroscopy corroborated the presence of nickel (14.05%) in synthesized Ni-Ch NPs. A significant increase in germination and growth attributes in terms of shoot and root length and number of lateral roots over control was observed in paddy seeds on the treatment with Ni-Ch NPs. Furthermore, the application of NPs in paddy plants under glasshouse condition demonstrated a remarkable improvement in plant growth. Protein profiling of NP-treated plants revealed new polypeptides (Rubisco units) enlightening the enhanced photosynthetic rate. Also, Asian rice exhibited reduced blast symptoms on leaves treated with NPs under glasshouse condition while displaying 64% mycelia inhibition in Petri plates. All these results suggest that nickel-chitosan nanoparticles could be exploited as an effective plant growth promoter cohort in controlling rice blast disease.

Bacillus subtilis KLBMPGC81 suppresses appressorium-mediated plant infection by altering the cell wall integrity signaling pathway and multiple cell biological processes in Magnaporthe oryzae.

Magnaporthe oryzae is one of the most destructive crop pathogens in the world, causing huge losses in rice harvest every year. Bacillus subtilis is a potential biocontrol agent that has been explored in many crop systems because it is a potent producer of bioactive compounds. However, the mechanisms by which these agents control rice blasts are not fully understood. We show that B. subtilis KLBMPGC81 (KC81) and its supernatant (SUP) have high antimicrobial activity against M. oryzae strain Guy11. To better exploit KC81 as a biocontrol agent, the mechanism by which KC81 suppresses rice blast pathogens was investigated. This study shows that KC81 SUP is effective in controlling rice blast disease. The SUP has a significant effect on suppressing the growth of M. oryzae and appressorium-mediated plant infection. KC81 SUP compromises cell wall integrity, microtubules and actin cytoskeleton, mitosis, and autophagy, all of which are required for M. oryzae growth, appressorium development, and host infection. We further show that the SUP reduces the activity of the cyclin-dependent kinase Cdc2 by enhancing the phosphorylation of Cdc2 Tyr 15, thereby impairing mitosis in M. oryzae cells. SUP induces the cell wall sensor MoWsc1 to activate the cell wall integrity pathway and Mps1 and Pmk1 mitogen-activated protein kinases. Taken together, our findings reveal that KC81 is an effective fungicide that suppresses M. oryzae growth, appressorium formation, and host infection by abnormally activating the cell wall integrity pathway, disrupting the cytoskeleton, mitosis, and autophagy.

Host induced gene silencing of Magnaporthe oryzae by targeting pathogenicity and development genes to control rice blast disease.

Rice blast disease caused by the hemi-biotrophic fungus Magnaporthe oryzae is the most destructive disease of rice world-wide. Traditional disease resistance strategies for the control of rice blast disease have not proved durable. HIGS (host induced gene silencing) is being developed as an alternative strategy. Six genes (CRZ1, PMC1, MAGB, LHS1, CYP51A, CYP51B) that play important roles in pathogenicity and development of M. oryzae were chosen for HIGS. HIGS vectors were transformed into rice calli through Agrobacterium-mediated transformation and T0, T1 and T2 generations of transgenic rice plants were generated. Except for PMC1 and LHS1, HIGS transgenic rice plants challenged with M. oryzae showed significantly reduced disease compared with non-silenced control plants. Following infection with M. oryzae of HIGS transgenic plants, expression levels of target genes were reduced as demonstrated by Quantitative RT-PCR. In addition, treating M. oryzae with small RNA derived from the target genes inhibited fungal growth. These findings suggest RNA silencing signals can be transferred from host to an invasive fungus and that HIGS has potential to generate resistant rice against M. oryzae.

Biocontrol potential of Burkholderia sp. BV6 against the rice blast fungus Magnaporthe oryzae.

To investigate the broad-spectrum antifungal activity of Burkholderia sp. BV6, that is isolated from rice roots and its biocontrol potential against rice blast caused by Magnaporthe oryzae. We evaluated the ability to isolate BV6 in the biological control of rice blast disease and investigated its antifungal mechanisms. BV6 strongly inhibited the hyphal growth of M. oryzae Guy11 and other plant pathogenic fungi, and pot experiments showed that BV6 significantly decreases the disease index of rice blast from 47.5 to 24.6. The secreted small-molecule secondary metabolites were regarded as weapons during the antifungal process by inhibiting the germination of M. oryzae conidia and mycelial growth, and thereby prevent the following infection. Liquid chromatography-mass spectrometry analysis of the metabolites from the supernatant of isolate BV6 showed that the antifungal weapons of isolate BV6 are novel, small, molecular hydrophilic compounds that are different from reported antifungal compounds. The isolate BV6 inhibits the M. oryzae infection by the production of small-molecule antifungal compounds. The current study discovers the role of the Burkholderia sp. BV6 in the biocontrol of plant pathogenic fungi. Therefore, isolate BV6 is a potential candidate for developing a microbial formulation for the biocontrol of the most common disease of rice blast.

RNA-Seq Analysis of Magnaporthe grisea Transcriptome Reveals the High Potential of ZnO Nanoparticles as a Nanofungicide.

Magnaporthe grisea is one of the most destructive pathogen that encounters a challenge to rice production around the worldwide. The unique properties of ZnO nanoparticles (NPs), have high attractiveness as nanofungicide. In the present study, the response of fungi to ZnO NPs was evaluated using RNA sequencing (RNA-seq). Two different aligners (STAR and Hisat2) were used for aligning the clean reads, and the DEseq2 package was used to identify the differentially expressed genes (DEGs). In total, 1,438 and 761 fungal genes were significantly up- and down-regulated in response to ZnO NPs, respectively. The DEGs were subjected to functional enrichment analysis to identify significantly enriched biological pathways. Functional enrichment analysis revealed that “cell membrane components,” “ion (calcium) transmembrane transporter activity,” “steroid biosynthesis pathway” and “catalytic activity” were the contributed terms to fungal response mechanisms. The genes involved in aflatoxin efflux pumps and ribosome maturation were among the genes showing significant up- and down-regulation after ZnO NPs application. To confirm the obtained RNA-seq results, the expression of six randomly selected genes were evaluated using q-RT-PCR. Overall, the RNA-seq results suggest that ZnO NPs primarily act on the fungal cell membrane, but accumulation of ROS inside the cell induces oxidative stress, the fungal catalytic system is disrupted, resulting into the inhibition of ROS scavenging and eventually, to the death of fungal cells. Our findings provide novel insights into the effect of ZnO NPs as a promising nanofungicide for effective control of rice blast disease.

Identification of Scytalone Dehydratase Inhibitors Effective against Melanin Biosynthesis Dehydratase Inhibitor-Resistant Pyricularia oryzae.

Melanin is a secondary metabolite required for the infection of the rice blast fungus Pyricularia oryzae. Melanin biosynthesis enzymes are targets for controlling rice blast disease, and three types of commercial melanin biosynthesis inhibitors (MBIs) including MBI-R, MBI-D, and MBI-P have been developed. However, the occurrence of MBI-D-resistant strains containing scytalone dehydratase (SDH1/RSY1) with V75M mutations has been recently reported. In this study, we aimed to identify inhibitors of SDH1-V75M. We screened the RIKEN Natural Products Depository chemical library using chemical array technology and evaluated the inhibition of SDH1-V75M by candidate compounds. NPD13731 strongly inhibited the activity of wild-type and mutant SDH1. The structure-activity relationship data were used to create a more potent inhibitor 16, which controlled rice blast disease in rice plants infected with MBI-D-resistant P. oryzae. Compound 16, which we named melabiostin, may be used to develop fungicides for controlling rice blast infections.