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.

Detection and characterization of fungus (Magnaporthe oryzae pathotype Triticum) causing wheat blast disease on rain-fed grown wheat (Triticum aestivum L.) in Zambia.

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT) is a threat to wheat production especially in the warmer-humid environments. In Zambia, wheat blast symptoms were observed for the first time on wheat (Triticum aestivum L.) grown in experimental plots and five farmers' fields in Mpika district of Muchinga Province during the 2017-18 rainy season. Infected plants showed the typical wheat blast symptoms with the spike becoming partially or completely bleached with the blackening of the rachis in a short span of time. Incidence of blast symptoms on nearly all wheat heads was high and ranged from 50 to 100%. Examination of diseased plant leaves showed the presence of elliptical, grayish to tan necrotic lesions with dark borders on the leaf often mixed with other foliar diseases. A study was conducted to isolate and identify the causal pathogen(s) using classical and molecular methods and determine the pathogenicity of the detected disease causal agent. Morphobiometrical determination of causal pathogen revealed conidia with characteristic pear shaped 2-septate hyaline spores associated with M. oryzae species. Preliminary polymerase chain reaction screening of six isolates obtained from wheat blast infected samples with diagnostic primers (MoT3F/R) was conducted at ZARI, Zambia, and subsequent analysis of two isolates with MoT3F/R and C17F/R was performed at USDA-ARS, USA. Both experiments confirmed that MoT is the causal agent of wheat blast in Zambia. Further, pathogenicity tests performed with pure culture isolates from samples WS4 and WS5 produced typical blast symptoms on all the six inoculated wheat genotypes. Results of this study indicate that MoT is causing wheat blast in rain-fed wheat grown in Zambia, thus making it the first report of MoT in Zambia and Africa. This inter-continental movement of the pathogen (disease) has serious implication for wheat production and trade that needs to be urgently addressed.

A novel narnavirus from the plant-pathogenic fungus Magnaporthe oryzae.

A novel mycovirus with the proposed name "Magnaporthe oryzae narnavirus virus 1" (MoNV1), was described in the rice blast fungus Magnaporthe oryzae. The virus has a single-stranded (+ss) RNA genome of 2452 nucleotides, contains a single open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RDRP), and is closely related to some viruses of the genus Narnavirus, family Narnaviridae, including Aspergillus fumigatus narnavirus 1 (AfNV1), Neofusicoccum parvum narnavirus 2 (NpNV2) and Alternaria tenuissima narnavirus 1 (AtNV2). Genome sequence comparisons and phylogenetic analysis suggested that MoNV1 is a new member of the genus Narnavirus. The RDRPs of MoNV1 and some closely related narnaviruses do not contain a typical metal-binding "GDD" motif and catalytic site. Further studies are needed to investigate the replication mechanism of these viruses.

Specific Detection of the Wheat Blast Pathogen (Magnaporthe oryzae Triticum) by Loop-Mediated Isothermal Amplification.

Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields.

Vacuole Dynamics in Rice Cells Invaded by the Blast Fungus Magnaporthe oryzae.

We describe a fluorescence imaging method to visualize the dynamics of the central vacuole in rice cells during invasion by the blast fungus Magnaporthe oryzae. This method utilizes the combination of confocal microscopy, rice sheath cells (optically transparent), fluorescently tagged M. oryzae (red fluorescence), and fluorescein diacetate staining (green fluorescence; visualizing vacuole dynamics). Using this method, we demonstrate that the vacuole undergoes progressive shrinkage and collapse during M. oryzae infection.

Coexistence of Multiple Endemic and Pandemic Lineages of the Rice Blast Pathogen.

The rice blast fungus Magnaporthe oryzae (syn., Pyricularia oryzae) is both a threat to global food security and a model for plant pathology. Molecular pathologists need an accurate understanding of the origins and line of descent of M. oryzae populations in order to identify the genetic and functional bases of pathogen adaptation and to guide the development of more effective control strategies. We used a whole-genome sequence analysis of samples from different times and places to infer details about the genetic makeup of M. oryzae from a global collection of isolates. Analyses of population structure identified six lineages within M. oryzae, including two pandemic on japonica and indica rice, respectively, and four lineages with more restricted distributions. Tip-dating calibration indicated that M. oryzae lineages separated about a millennium ago, long after the initial domestication of rice. The major lineage endemic to continental Southeast Asia displayed signatures of sexual recombination and evidence of DNA acquisition from multiple lineages. Tests for weak natural selection revealed that the pandemic spread of clonal lineages entailed an evolutionary "cost," in terms of the accumulation of deleterious mutations. Our findings reveal the coexistence of multiple endemic and pandemic lineages with contrasting population and genetic characteristics within a widely distributed pathogen.IMPORTANCE The rice blast fungus Magnaporthe oryzae (syn., Pyricularia oryzae) is a textbook example of a rapidly adapting pathogen, and it is responsible for one of the most damaging diseases of rice. Improvements in our understanding of Magnaporthe oryzae's diversity and evolution are required to guide the development of more effective control strategies. We used genome sequencing data for samples from around the world to infer the evolutionary history of M. oryzae We found that M. oryzae diversified about 1,000 years ago, separating into six main lineages: two pandemic on japonica and indica rice, respectively, and four with more restricted distributions. We also found that a lineage endemic to continental Southeast Asia displayed signatures of sexual recombination and the acquisition of genetic material from multiple lineages. This work provides a population-level genomic framework for defining molecular markers for the control of rice blast and investigations of the molecular basis of differences in pathogenicity between M. oryzae lineages.

Gene Flow between Divergent Cereal- and Grass-Specific Lineages of the Rice Blast Fungus Magnaporthe oryzae.

Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae, including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species (Pyricularia graminis-tritici). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae, each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen.IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.

Genetic Structure of the Rice Blast Pathogen (Magnaporthe oryzae) over a Decade in North Central California Rice Fields.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future.

Nutrients and host attributes modulate the abundance and functional traits of phyllosphere microbiome in rice.

The abundance of phyllosphere bacterial communities of seven genotypes of rice ADT- 38, ADT-43, CR-1009, PB-1, PS-5, P-44, and PB-1509 was investigated, in relation to nutrient dynamics of rhizosphere and leaves. P-44 genotype recorded highest pigment accumulation, while genotypes CR-1009 and P-44 exhibited most number of different bacterial morphotypes, Colony forming units in two media (Nutrient agar and R2A) varied significantly and ranged from 106-107 per g plant tissues. Among the selected 60 distinct morphotypes, IAA and siderophore producers were the dominant functional types. Biocontrol activity against Drechslera oryzae was shown by 38 isolates, while 17 and 9 isolates were potent against Rhizoctonia solani and Magnaporthe oryzae respectively. Principal Component Analysis (PCA) illustrated the significant effects of selected soil and leaf nutrients of seven rice varieties on the culturable phyllospheric population (log CFU), particularly in the R2A medium. Eigen values revealed that 83% of the variance observed could be assigned to Leaf-Fe, Leaf-Mn, chlorophyll b and soil organic carbon (OC). Quantitative PCR analyses of abundance of bacteria, cyanobacteria and archaebacteria revealed a host-specific response, with CR-1009 showing highest number of 16S rRNA copies of bacterial members, while both P-44 and PS-5 had higher cyanobacterial abundance, but lowest number of those belonging to archaebacteria. Nutritional aspects of leaf and soil influenced the abundance of bacteria and their functional attributes; this is of interest for enhancing the efficacy of foliar inoculants, thereby, improving plant growth and disease tolerance.

Avirulence (AVR) Gene-Based Diagnosis Complements Existing Pathogen Surveillance Tools for Effective Deployment of Resistance (R) Genes Against Rice Blast Disease.

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

SCAR marker specific to detect Magnaporthe grisea infecting finger millets (Eleusine coracana).

AIMS: To determine the molecular variability and develop specific Sequence Characterized Amplified Region (SCAR) marker for the detection of Magnaporthe grisea causing blast disease in finger millet. METHODS AND RESULTS: Random amplified polymorphic DNA (RAPD) was performed with 14 isolates of M. grisea using 20 random primers. SCAR marker was developed for accurate and specific detection of M. grisea infecting only finger millets. The genetic similarity coefficient within each group and variation between the groups was observed. Among the primers, OPF-08 generated a RAPD polymorphic profile that showed common fragment of 478 bp in all the isolates. This fragment was cloned and sequenced. SCAR primers, Mg-SCAR-FP and Mg-SCAR-RP, were designed using sequence of the cloned product. The specificity of the SCAR primers was evaluated using purified DNA from M. grisea isolates from finger millets and other pathogens viz., Pyricularia oryzae, Colletotrichum gloeosporioides, Colletotrichum falcatum and Colletotrichum capcisi infecting different crops. The SCAR primers amplified only specific 460 bp fragment from DNA of M. grisea isolates and this fragment was not amplified in other pathogens tested. CONCLUSION: SCAR primers distinguish blast disease of finger millet from rice as there is no amplification in the rice blast pathogen. PCR-based SCAR marker is a convenient tool for specific and rapid detection of M. grisea in finger millets. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic diversity in fungal population helps in developing a suitable SCAR marker to identify the blast pathogen at the early stage of infection.

Directional Selection from Host Plants Is a Major Force Driving Host Specificity in Magnaporthe Species.

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

The Magnaporthe grisea species complex and plant pathogenesis.

TAXONOMY: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Order Magnaporthales; Family Pyriculariaceae (anamorph)/Magnaporthaceae (teleomorph); Genus Pyricularia (anamorph)/Magnaporthe (teleomorph); Species P. grisea (anamorph)/M. grisea (teleomorph). HOST RANGE: Very broad at the species level, including rice, wheat, barley, millet and other species of the Poaceae (Gramineae). DISEASE SYMPTOMS: Can be found on all parts of the plant, including leaves, leaf collars, necks, panicles, pedicels, seeds and even the roots. Initial symptoms are white to grey-green lesions or spots with darker borders, whereas older lesions are elliptical or spindle-shaped and whitish to grey with necrotic borders. Lesions may enlarge and coalesce to eventually destroy the entire leaf. DISEASE CONTROL: Includes cultural strategies, genetic resistance and the application of chemical fungicides. GEOGRAPHICAL DISTRIBUTION: Widespread throughout the rice-growing regions of the globe and has been reported in more than 85 countries. GENOMIC STRUCTURE: Different isolates possess similar genomic sizes and overall genomic structures. For the laboratory strain 70-15: assembly size, 40.98 Mb; number of chromosomes, seven; number of predicted genes, 13 032; G + C composition, 51.6%; average gene contains 451.6 amino acids; mitochondrion genome size, 34.87 kb. USEFUL WEBSITE: http://www.broadinstitute.org/annotation/genome/magnaporthe_comparative/MultiHome.html.

Deciphering Genome Content and Evolutionary Relationships of Isolates from the Fungus Magnaporthe oryzae Attacking Different Host Plants.

Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolates and host-specific lineages, the genome composition in terms of frequencies of genes putatively involved in pathogenicity (effectors, secondary metabolism, cazome) was conserved. However, 529 shared families were found only in nonrice lineages, whereas the rice lineage possessed 86 specific families absent from the nonrice genomes. Our results confirmed that the host specificity of M. oryzae isolates was associated with a divergence between lineages without major gene flow and that, despite the strong conservation of gene families between lineages, adaptation to different hosts, especially to rice, was associated with the presence of a small number of specific gene families. All information was gathered in a public database (http://genome.jouy.inra.fr/gemo).

Early diagnosis of blast fungus, Magnaporthe oryzae, in rice plant by using an ultra-sensitive electrically magnetic-controllable electrochemical biosensor.

As one of the most destructive and widespread disease of rice, Magnaporthe oryzae (also called Magnaporthe grisea) has a significant negative impact on rice production. Therefore, it is still in high demand to develop extremely sensitive and accurate methods for the early diagnosis of Magnaporthe oryzae (M. oryzae). In this study, we developed a novel magnetic-controllable electrochemical biosensor for the ultra sensitive and specific detection of M. oryzae in rice plant by using M. oryzae's chitinases (Mgchi) as biochemical marker and a rice (Oryza sativa) cDNA encoding mannose-binding jacalin-related lectin (Osmbl) as recognition probe. The proposed biosensor combined with the merits of chronoamperometry, electrically magnetic-controllable gold electrode and magnetic beads (MBs)-based palladium nano-particles (PdNPs) catalysis amplification, has an ultra-high sensitivity and specificity for the detection of trace M. oryzae in rice plant. It could be used to detect M. oryzae in rice plant in the initial infection stage (before any symptomatic lesions were observed) to help farmers timely manage the disease. In comparison with previous methods, the proposed method has notable advantages such as higher sensitivity, excellent specificity, short analysis time, robust resistibility to complex matrix and low cost etc. The success in this study provides a reliable approach for the early diagnosis and fast screening of M. oryzae in rice plant.

Effectiveness and durability of the rice pi-ta gene in Yunnan province of China.

Rice blast is one of the most damaging diseases of rice worldwide. In the present study, we analyzed DNA sequence variation of avirulence (AVR) genes of AVR-Pita1 in field isolates of Magnaporthe oryzae in order to understand the effectiveness of the resistance gene Pi-ta in China. Genomic DNA of 366 isolates of M. oryzae collected from Yunnan province of China were used for polymerase chain reaction (PCR) amplification to examine the existence of AVR-Pita1 using gene-specific PCR markers. Results of PCR products revealed that 218 isolates of M. oryzae carry AVR-Pita1. Among of them, 62.5, 56.3, 58.5, 46.7, 72.4, and 57.4% of M. oryzae carry AVR-Pita1 from northeastern, southeast, western, northwest, southwestern, and central Yunnan province, respectively. The detection rate of AVR-Pita1 was, in order: southwestern > northeastern > western > central > southeastern > northwestern Yunnan province. Moreover, in total, 18 AVR-Pita1 haplotypes encoding 13 novel AVR-Pita1 variants were identified among 60 isolates. Most DNA sequence variation was found to occur in the exon region, resulting in amino acid substitution. Six virulent haplotypes of AVR-Pita1 to Pita were identified among 60 field isolates. The AVR-Pita1 has evolved to virulence from avirulent origins via base substitution. These findings demonstrate that AVR-Pita1 is under positive selection and mutations of AVR-Pita1 are responsible for defeating race-specific resistance in nature.

Structural and functional study in the rhizosphere of Oryza sativa L. plants growing under biotic and abiotic stress.

AIMS: A structural and functional study has been carried out in the rice production area of the Guadalquivir marshes in southern Spain aiming to increase knowledge of rice rhizosphere structure and function for further application on integrated management practices. METHODS AND RESULTS: Rhizosphere bacterial structure (analysis of 16S rRNA partial sequences from total soil DNA), metabolic diversity (analysed by Biolog FF for fungal community and GN for microbial community) and a screening for putative plant growth-promoting rhizobacteria (PGPR) to identify potential isolates for development of local biofertilizers, and biodiversity of culturable micro-organisms (analysis of 16S rRNA partial sequences) from four areas differing in salinity and Magnaporthe oryzae incidence in two moments of the crop cycle were studied. Results indicate that the dominant taxon in libraries from the four areas was Proteobacteria. Metabolic diversity was higher in areas affected only by salinity or incidence of Magnaporthe than in the control or area affected by both stresses. It seems that rice plants selected, in their rhizosphere, micro-organisms able to affect plant hormonal balance under all conditions, and this activity relied in different bacterial genera depending on the environmental stress. CONCLUSIONS: Bacterial genera for each stress, as well as generalist strains, were found present in all the studied areas. Potential molecular markers and taxonomic markers (Sphingobacteria for salt and Thermococci for Magnaporthe) of the different stress situations have been highlighted, and Class Verrucomicrobiae could be a marker for nonstressed areas. In addition, putative PGPR strains isolated in this study could be used as biofertilizers. SIGNIFICANCE AND IMPACT OF THE STUDY: Rice paddies are great ecologically important ecosystems. The results are very relevant as they may be included in the process of rice production, improving crop conditions with less environmental impact.

MoMon1 is required for vacuolar assembly, conidiogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

Mon1 protein is involved in cytoplasm-to-vacuole trafficking, vacuolar morphology and autophagy, and is required for homotypic vacuole fusion in Saccharomyces cerevisiae. Here we identify MoMON1 from Magnaporthe oryzae as an ortholog of S. cerevisiae MON1, essential for the morphology of the vacuole and vesicle fusion. Target gene deletion of MoMON1 resulted in accumulation of small punctuate vacuoles in the hypha and hypersensitivity to monensin, an antibiotic that blocks intracellular protein transport. The ΔMomon1 mutant exhibited significantly reduced aerial hyphal development and poor conidiation. Conidia of ΔMomon1 were able to differentiate appressoria. However, ΔMomon1 was non-pathogenic on rice leaves, even after wound inoculation. In addition, ΔMomon1 was slightly hypersensitive to Congo red and SDS, but not to cell wall degrading enzymes, suggesting significant alterations in its cell wall. The autophagy process was blocked in the ΔMomon1 mutant. Taken together, our results suggest that MoMON1 has an essential function in vacuolar assembly, autophagy, fungal development and pathogenicity in M. oryzae.

The early diagnosis and fast detection of blast fungus, Magnaporthe grisea, in rice plant by using its chitinase as biochemical marker and a rice cDNA encoding mannose-binding lectin as recognition probe.

As one of the most destructive and widespread disease of rice, Magnaporthe grisea (M. grisea) has a significant negative impact on rice production. Therefore, it is significant to develop a method for sensitive and high-throughput detection of M. grisea in order to help farmers to find and control blast in the early stage. We herein first cloned and expressed the M. grisea's chitinases (Mgchi) and a rice cDNA encoding mannose-binding jacalin-related lectin (Osmbl) with Mgchi-related gene. We demonstrated that Mgchi could be used as a biochemical marker for the detection of M. grisea and there was a specific interaction between Osmbl and Mgchi. Using Mgchi as biochemical marker and Osmbl as recognition probe, we developed a visual method for the specific and sensitive detection of M. grisea based on the PdNPs-catalyzed TMB/H(2)O(2) system. The proposed method could be used to detect Mgchi as low as 7.5×10(-9) M by naked eye observation and 2.5×10(-11) M Mgchi by the micro-plate reader. With the help of the method, we had successfully detected M. grisea in the real M. grisea-infected rice plant with recoveries of 88-109% and RSD<7%. The proposed method was sensitive, specific, potentially high-throughput and cost-effective. The success in this study provides a promising substitution for the early diagnosis and fast screening of M. grisea in rice plant.

Comparative analysis of the genomes of two field isolates of the rice blast fungus Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus.