Rice Blast Research Articles

Improvement of Flowering Stage in Japonica Rice Variety Jiahe212 by Using CRISPR/Cas9 System.

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica-japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica-japonica interspecific hybrid rice breeding with wider adaptability.

Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages.

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

Kosakonia oryziphila NP19 bacterium acts as a plant growth promoter and biopesticide to suppress blast disease in KDML105 rice

This study demonstrates that root-associated Kosakonia oryziphila NP19, isolated from rice roots, is a promising plant growth-promoting bioagent and biopesticide for combating rice blast caused by Pyricularia oryzae. In vitro experiments were conducted on fresh leaves of Khao Dawk Mali 105 (KDML105) jasmine rice seedlings. The results showed that NP19 effectively inhibited the germination of P. oryzae fungal conidia. Fungal infection was suppressed across three different treatment conditions: rice colonized with NP19 and inoculated by fungal conidia, a mix of NP19 and fungal conidia concurrently inoculated on the leaves, and fungal conidia inoculation first followed by NP19 inoculation after 30 h. Additionally, NP19 reduced fungal mycelial growth by 9.9–53.4%. In pot experiments, NP19 enhanced the activities of peroxidase (POD) and superoxide dismutase (SOD) by 6.1–63.0% and 3.0–67.7%, respectively, indicating a boost in the plant’s defense mechanisms. Compared to the uncolonized control, the NP19-colonized rice had 0.3–24.7% more pigment contents, 4.1% more filled grains per panicle, 26.3% greater filled grain yield, 34.4% higher harvest index, and 10.1% more content of the aroma compound 2-acetyl-1-pyrroline (2AP); for rice colonized with NP19 and infected with P. oryzae, these increases were 0.2–49.2%, 4.6%, 9.1%, 54.4%, and 7.5%, respectively. In field experiments, blast-infected rice that was colonized and/or inoculated with NP19 treatments had 15.1–27.2% more filled grains per panicle, 103.6–119.8% greater filled grain yield, and 18.0–35.8% higher 2AP content. A higher SOD activity (6.9–29.5%) was also observed in the above-mentioned rice than in the blast-infected rice that was not colonized and inoculated with NP19. Following blast infection, NP19 applied to leaves decreased blast lesion progression. Therefore, K. oryziphila NP19 was demonstrated to be a potential candidate for use as a plant growth-promoting bioagent and biopesticide for suppressing rice blast.

Analysis of Rice Blast Fungus Genetic Diversity and Identification of a Novel Blast Resistance OsDRq12 Gene.

The rice blast fungus Magnaporthe oryzae poses a significant challenge to maintaining rice production. Developing rice varieties with resistance to this disease is crucial for its effective control. To understand the genetic variability of blast isolates collected between 2015 and 2017, the 27 monogenic rice lines that carry specific resistance genes were used to evaluate blast disease reactions. Based on criteria such as viability, virulence, and reactions to resistance genes, 20 blast isolates were selected as representative strains. To identify novel resistance genes, a quantitative trait locus analysis was carried out utilizing a mixture of the 20 representative rice blast isolates and a rice population derived from crossing the blast-resistant cultivar 'Cheongcheong' with the blast-susceptible cultivar 'Nagdong'. This analysis revealed a significant locus, RM1227-RM1261 on chromosome 12, that is associated with rice blast resistance. Within this locus, 12 disease resistance-associated protein genes were identified. Among them, OsDRq12, a member of the nucleotide-binding, leucine-rich repeat disease resistance family, was chosen as the target gene for additional computational investigation. The findings of this study have significant implications for enhancing rice production and ensuring food security by controlling rice blast and developing resistant rice cultivars.

Nano-enhanced defense: Titanium-enriched Alginate–Bentonite coating augments Bacillus amyloliquefaciens D203 efficacy against Magnaporthe oryzae in Kenyan rice cultivation

Rice blast disease, caused by Magnaporthe oryzae, poses a significant threat to global rice production, necessitating the development of effective and sustainable management strategies. Biological control using beneficial microbes like Bacillus amyloliquefaciens has emerged as a promising approach due to its ability to enhance plant resistance and reduce disease incidence. Nano-encapsulation of bacteria, which involves embedding beneficial microbes within nanomaterials, offers a novel method to improve the stability, survival, and efficacy of these biocontrol agents. This study evaluated the capacity of encapsulated Bacillus amyloliquefaciens D203, embedded within an alginate-bentonite coating infused with titanium nanoparticles (TNs), to stimulate defense responses in rice seedlings challenged by the Magnaporthe oryzae the causal agent of rice blast disease. Encapsulation was achieved using the extrusion technique, with some modifications. Using a completely randomized design, the experiment was conducted in a greenhouse, with four treatments replicated four times. The experiment used the popular Kenyan rice variety "BASMATI 370”. The study investigated the impact of strain D203 on the incidence, severity, and area under disease progress curves related to M. oryzae, as well as the expression of defense-related enzymes. The results demonstrated that rice plants derived from seeds coated with the D203 encapsulated B. amyloliquefaciens strain exhibited higher levels of defense-related enzyme expression, including peroxidase (POD), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD) and catalase (CAT), compared to controls. In addition, the incidence and severity of the disease were markedly lower in plants treated with encapsulated B. amyloliquefaciens compared to controls, sometimes paralleling the efficacy of hexaconazole treatment. These findings suggest that the encapsulation of strain D203 has the potential to enhance resistance against rice blast disease by inducing systemic resistance through the production of antioxidant enzymes.

Bio-efficacy of certain newer insecticides and fungicides both alone and in combination against yellow stem borer and sheath blight in rice

Bio-efficacy of certain newer insecticides and fungicides both alone and in combination against yellow stem borer and sheath blight in rice

A Rapid, Equipment-Free Method for Detecting Avirulence Genes of Pyricularia oryzae Using a Lateral Flow Strip-Based RPA Assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence (Avr) genes in rice planting fields to facilitate the breeding of resistant rice varieties. In this study, we established a rapid recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection system for the identification of AvrPik, Avr-Piz-t, and Avr-Pi9. The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting the three Avr genes exhibited a remarkable specificity at genus-level detection. Under the optimized condition, the lower detected thresholds of AvrPik, Avr-Piz-t, and Avr-Pi9 were 10 fg/μl, 100 fg/μl, and 10 pg/μl, respectively. Notably, the detection sensitivity of the three Avr genes was much higher than that of PCR. In addition, we also successfully detected the presence of AvrPik, Avr-Piz-t, and Avr-Pi9 in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed an RPA detection system for AvrPik, Avr-Pi9, and Avr-Piz-t, combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time, and accurate detection of the three Avr genes in the field, which is helpful to understand the distribution frequency of the three Avr genes in the field and provide theoretical reference for the scientific layout of resistant rice varieties.

A novel Pik allele confers extended resistance to rice blast.

In the ongoing arms race between rice and Magnaporthe oryzae, the pathogen employs effectors to evade the immune response, while the host develops resistance genes to recognise these effectors and confer resistance. In this study, we identified a novel Pik allele, Pik-W25, from wild rice WR25 through bulked-segregant analysis, creating the Pik-W25 NIL (Near-isogenic Lines) named G9. Pik-W25 conferred resistance to isolates expressing AvrPik-C/D/E alleles. CRISPR-Cas9 editing was used to generate transgenic lines with a loss of function in Pik-W25-1 and Pik-W25-2, resulting in loss of resistance in G9 to isolates expressing the three alleles, confirming that Pik-W25-induced immunity required both Pik-W25-1 and Pik-W25-2. Yeast two-hybrid (Y2H) and split luciferase complementation assays showed interactions between Pik-W25-1 and the three alleles, while Pik-W25-2 could not interact with AvrPik-C, -D, and -E alleles with Y2H assay, indicating Pik-W25-1 acts as an adaptor and Pik-W25-2 transduces the signal to trigger resistance. The Pik-W25 NIL exhibited enhanced field resistance to leaf and panicle blast without significant changes in morphology or development compared to the parent variety CO39, suggesting its potential for resistance breeding. These findings advance our knowledge of rice blast resistance mechanisms and offer valuable resources for effective and sustainable control strategies.

TRNA-m1A methylation controls the infection of Magnaporthe oryzae by supporting ergosterol biosynthesis.

Ergosterols are essential components of fungal plasma membranes. Inhibitors targeting ergosterol biosynthesis (ERG) genes are critical for controlling fungal pathogens, including Magnaporthe oryzae, the fungus that causes rice blast. However, the translational mechanisms governing ERG gene expression remain largely unexplored. Here, we show that the Trm6/Trm61 complex catalyzes dynamic N1-methyladenosine at position 58 (m1A58) in 51 transfer RNAs (tRNAs) of M.oryzae, significantly influencing translation at both the initiation and elongation stages. Notably, tRNA m1A58 promotes elongation speed at most cognate codons mainly by enhancing eEF1-tRNA binding rather than affecting tRNA abundance or charging. The absence of m1A58 leads to substantial decreases in the translation of ERG genes, ergosterol production, and, consequently, fungal virulence. Simultaneously targeting the Trm6/Trm61 complex and the ergosterol biosynthesis pathway markedly improves rice blast control. Our findings demonstrate an important role of m1A58-mediated translational regulation in ergosterol production and fungal infection, offering a potential strategy for fungicide development.

Detection and Evaluation of Blast Resistance Genes in Backbone Indica Rice Varieties from South China.

Rice blast caused by the pathogenic fungus Magnaporthe oryzae poses a significant threat to rice cultivation. The identification of robust resistance germplasm is crucial for breeding resistant varieties. In this study, we employed functional molecular markers for 10 rice blast resistance genes, namely Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, to assess blast resistance across 91 indica rice backbone varieties in South China. The results showed a spectrum of resistance levels ranging from highly resistant (HR) to highly susceptible (HS), with corresponding frequencies of 0, 19, 40, 27, 5, and 0, respectively. Yearly correlations in blast resistance genes among the 91 key indica rice progenitors revealed Pid2 (60.44%), Pia (50.55%), Pita (45.05%), Pi2 (32.97%), Pikh (4.4%), Pigm (2.2%), Pi9 (2.2%), and Pi1 (1.1%). Significant variations were observed in the distribution frequencies of these 10 resistance genes among these progenitors across different provinces. Furthermore, as the number of aggregated resistance genes increased, parental resistance levels correspondingly improved, though the efficacy of different gene combinations varied significantly. This study provides the initial steps toward strategically distributing varieties of resistant indica rice genotypes across South China.

Cellular changes and PR-1 gene expression accompanying infection-related morphogenesis and cell progression during invasive growth Pyricularia oryzae pathotype Triticum in resistant and susceptible wheat cultivars

Pyricularia oryzae pathotype Triticum (PoT) causes Wheat Blast, a devastating crop disease present in many wheat-producing countries. With the aim to contribute with the understanding of the mechanisms underlying pathogenesis of the fungus on wheat (Triticum aestivum), cytological and molecular studies were performed here.Susceptible and resistant interactions of wheat-PoT in cultivars of different levels of resistance were investigated by cellular changes and the molecular responses of wheat leaves according PR-1 expression. There was significant difference between susceptible (Apogee-PY15-PY34, Buck, MSINTA-PY15) and resistant (Buck-PY34, MSINTA-PY34) interactions in the responses tested. The two isolates of PoT analysed showed different ability to infect wheat plants at the cellular level during early tissue penetration analysed and documented by epi-fluorescence microscopy, patterns of PR-1 genes, and wheat plant defense reaction.Microscopic time-course analysis (24, 48, 72 and 96 h after inoculation) of individual interaction sites per leaf revealed different leaf invasion strategies of the more and less aggressive PoT isolates during the first stages of the course of pathogenesis.The results and conclusions of the present study provides the first evidence that different PR-1 spatial and temporal expression patterns occur in the early infective process of wheat-PoT, underlying two different defence mechanisms in plants according susceptible or resistant interactions. Thus, the results here laid a theoretical foundation for the future control of wheat blast using the different pattern of PR-1 gene as markers for disease resistance during the first stages of infection providing a significant contribution to a more efficient selection of wheat genotypes in breeding studies.

The conserved Xanthomonas effector XopM targets allene oxide synthase OsAOS3 and interferes with jasmonate-mediated defense in rice1

Bacterial blight (BB) of rice caused by the phytopathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is a disease of global importance. Xoo utilizes the type III secretion system (T3SS) and its effectors for virulence, and XopM is a conserved T3SS effector in Xanthomonas spp. However, the virulence function of XopM is largely unknown. In this study, we show that XopM contributes to Xoo virulence in rice. We demonstrate that XopM interacts with allene oxide synthase OsAOS3, a key enzyme involved in jasmonic acid (JA) biosynthesis. The expression levels of OsAOS3 and three homologues of OsAOS were elevated after Xoo infection. Knockout mutants of OsAOS3 exhibited decreased JA accumulation and reduced resistance to Xoo and X. oryzae pv. oryzicola. Moreover, JA-related defense genes were downregulated in osaos3 mutants during Xoo infection. Based on our results, we propose a model showing how XopM hijacks OsAOS3 to interfere with JA-mediated defenses, leading to a suppression of rice immunity. Our findings reveal a novel virulence strategy where Xanthomonas pathogens interfere with the JA pathway and modulate the host defense response.

Re-routing MAP kinase signaling for penetration peg formation in predator yeasts.

Saccharomycopsis yeasts are natural organic sulfur auxotrophs due to lack of genes required for the uptake and assimilation of sulfate/sulfite. Starvation for methionine induces a shift to a predatory, mycoparasitic life strategy that is unique amongst ascomycetous yeasts. Similar to fungal plant pathogens that separated from Saccharomycopsis more than 400 million years ago, a specialized infection structure called penetration peg is used for prey cell invasion. Penetration pegs are highly enriched with chitin. Here we demonstrate that an ancient and conserved MAP kinase signaling pathway regulates penetration peg formation and successful predation in the predator yeast S. schoenii. Deletion of the MAP kinase gene SsKIL1, a homolog of the Saccharomyces cerevisiae ScKSS1/ScFUS3 and the rice blast Magnaporthe oryzae MoPMK1 genes, as well as deletion of the transcription factor SsSTE12 generate non-pathogenic mutants that fail to form penetration pegs. Comparative global transcriptome analyses using RNAseq indicate loss of the SsKil1-SsSte12-dependent predation response in the mutant strains, while a methionine starvation response is still executed. Within the promoter sequences of genes upregulated during predation we identified a cis-regulatory element similar to the ScSte12 pheromone response element. Our results indicate that, re-routing MAP-kinase signaling by re-wiring Ste12 transcriptional control towards predation specific genes contributed to the parallel evolution of this predacious behaviour in predator yeasts. Consequently, we found that SsSTE12 is dispensable for mating.

Genetic studies for blast disease in a biparental RIL population of pearl millet

Genetic studies for blast disease in a biparental RIL population of pearl millet

Breeding of a Near-Isogenic Wheat Line Resistant to Wheat Blast at Both Seedling and Heading Stages Through Incorporation of Rmg8.

Wheat blast caused by Pyricularia oryzae pathotype Triticum (MoT) has been transmitted from South America to Bangladesh and Zambia and is now spreading in these countries. To prepare against its further spread to Asian countries, we introduced Rmg8, a gene for resistance to wheat blast, into a Japanese elite cultivar, Chikugoizumi (ChI), through recurrent backcrosses and established ChI near-isogenic lines, #2-1-10 with the Rmg8/Rmg8 genotype and #4-2-10 with the rmg8/rmg8 genotype. A molecular analysis suggested that at least 96.6% of the #2-1-10 genome was derived from the recurrent parent ChI. The #2-1-10 line was resistant to MoT not only in primary leaves at the seedling stage but also in spikes and flag leaves at the heading stage. The strength of the resistance in spikes of this Rmg8 carrier was comparable to that of a carrier of the 2NS segment, which has been the only genetic resource released to farmers' fields for wheat blast resistance. On the other hand, the 2NS resistance was not expressed on leaves at the seedling stage nor flag leaves at the heading stage. Considering that leaf blast has been increasingly reported and regarded as an important inoculum source for spike blast, Rmg8 expressed at both the seedling and heading stages, or more strictly in both leaves and spikes, is suggested to be useful to prevent the spread of MoT in Asia and Africa.

Morphological, Biochemical and Molecular Characterization of the Causal Agent of Bacterial Panicle Blight Disease of Rice (BPB) In Selected Rice Production Zones of Zamfara State

Morphological, Biochemical and Molecular Characterization of the Causal Agent of Bacterial Panicle Blight Disease of Rice (BPB) In Selected Rice Production Zones of Zamfara State

Understanding rice blast: investigating biotechnological methods to speed up the development of robust rice cultivars

Understanding rice blast: investigating biotechnological methods to speed up the development of robust rice cultivars

K-Nearest neighbour machine method for predicting resistance gene against Magnaporthe oryzae in rice using proteomic markers

k-Nearest neighbour machine method for predicting resistance gene against Magnaporthe oryzae in rice using proteomic markers

Rice Blast Field Assessment in Three Regencies Underlies the Importance of Fungicide Resistance Studies in West Java, Indonesia

Rice Blast Field Assessment in Three Regencies Underlies the Importance of Fungicide Resistance Studies in West Java, Indonesia

Genetic Diversity and Virulence of Phytopathogenic Burkholderia glumae Strains Isolated from Rice Cultivars in Valleys of the High Jungle of Perú.

Burkholderia glumae causes bacterial leaf blight in rice, and its global spread has been exacerbated by climate change. To understand the genetic diversity and virulence of B. glumae strains isolated from rice cultivars in Perú, 47 isolates were obtained from infected rice fields, all belonging to B. glumae, and confirmed by recA and toxB sequences. The BOX-PCR typing group has 38 genomic profiles, and these turn into seven variable number tandem repeats (VNTR) haplotypes. There was no correlation between clustering and geographical origin. Nineteen strains were selected for phenotypic characterization and virulence, using both the maceration level of the onion bulb proxy and inoculation of seeds of two rice cultivars. Several strains produced pigments other than toxoflavin, which correlated with onion bulb maceration. In terms of virulence at the seed level, all strains produced inhibition at the root and coleoptile level, but the severity of symptoms varied significantly between strains, revealing significant differences in pathogenicity. There is no correlation between maceration and virulence scores, probably reflecting different virulence mechanisms depending on the host infection stage. This is the first study to evaluate the VNTR diversity and virulence of Peruvian strains of B. glumae in two commercial cultivars.