Population structure of Pyricularia oryzae on rice in Vietnam reveals diversified populations with four pandemic and two endemic clusters.

We characterized the genetic structure of 609 strains of Pyricularia oryzae, the fungal pathogen causing rice blast disease, in three main regions in Vietnam using microsatellites (SSR) markers. From the 447 distinct multilocus genotypes identified, six genetic clusters were defined, all of them showing elevated genetic and genotypic diversities. Four of these clusters were related to rice-attacking lineages already described at the worldwide scale, whereas the two remaining clusters were endemic to Vietnam. Strains were unevenly distributed into the six clusters depending on their groups of rice variety (indica / japonica) or type of varieties (traditional / modern) of origin, but none of the clusters was specifically related to these two factors. The highest diversity of blast population was found in Northern mountainous area, and the lowest in Red River Delta in both terms of genetic diversity and gene diversity. Hierarchical AMOVAs confirmed that all three factors considered (rice variety group, type of variety origin and geography) significantly contributed to the population structure of P. oryzae in Vietnam, with highest contribution from rice variety group. Mating types were unevenly distributed among clusters. Combined with results of female fertility and linkage disequilibirum, we hypothesized that clonal reproduction probably occurred in all clusters, but that sexual reproduction likely took place at least in some restricted areas in the Northern mountainous area for strains belonging to the cluster related to the previously described recombinant lineage (worldwide lineage 1). Our study pictures the genetic diversity, population structure and reproductive mode of the blast fungus in central and north Vietnam, and shows that the observed population structure is explained by several factors, the most important one being the variability of rice variety. All these new information might help for elaborating appropriate strategies to controlling the blast disease.

Evolution of the rice blast pathogen on spatially structured rice landraces maintains multiple generalist fungal lineages.

Traditional agrosystems, where humans, crops and microbes have coevolved over long periods, can serve as models to understand the ecoevolutionary determinants of disease dynamics and help the engineering of durably resistant agrosystems. Here, we investigated the genetic and phenotypic relationship between rice (Oryza sativa) landraces and their rice blast pathogen (Pyricularia oryzae) in the traditional Yuanyang terraces of flooded rice paddies in China, where rice landraces have been grown and bred over centuries without significant disease outbreaks. Analyses of genetic subdivision revealed that indica rice plants clustered according to landrace names. Three new diverse lineages of rice blast specific to the Yuanyang terraces coexisted with lineages previously detected at the worldwide scale. Population subdivision in the pathogen population did not mirror pattern of population subdivision in the host. Measuring the pathogenicity of rice blast isolates on landraces revealed generalist life history traits. Our results suggest that the implementation of disease control strategies based on the emergence or maintenance of a generalist lifestyle in pathogens may sustainably reduce the burden of disease in crops.

Marker-Assisted Pyramiding of Blast-Resistance Genes in a japonica Elite Rice Cultivar through Forward and Background Selection.

Rice blast, caused by Pyricularia oryzae, is one of the main rice diseases worldwide. The pyramiding of blast-resistance (Pi) genes, coupled to Marker-Assisted BackCrossing (MABC), provides broad-spectrum and potentially durable resistance while limiting the donor genome in the background of an elite cultivar. In this work, MABC coupled to foreground and background selections based on KASP marker assays has been applied to introgress four Pi genes (Piz, Pib, Pita, and Pik) in a renowned japonica Italian rice variety, highly susceptible to blast. Molecular analyses on the backcross (BC) lines highlighted the presence of an additional blast-resistance gene, the Pita-linked Pita2/Ptr gene, therefore increasing the number of blast-resistance introgressed genes to five. The recurrent genome was recovered up to 95.65%. Several lines carrying four (including Pita2) Pi genes with high recovery percentage levels were also obtained. Phenotypic evaluations confirmed the effectiveness of the pyramided lines against multivirulent strains, which also had broad patterns of resistance in comparison to those expected based on the pyramided Pi genes. The developed blast-resistant japonica lines represent useful donors of multiple blast-resistance genes for future rice-breeding programs related to the japonica group.

Genetic diversity and structure of Bipolaris oryzae and Exserohilum rostratum populations causing brown spot of rice in Burkina Faso based on genotyping-by-sequencing.

In recent years, Brown spot disease of rice (BSR) has been observed on leaves and seeds of rice in all rice-growing areas of Burkina Faso. Bipolaris oryzae and Exserohilum rostratum are the main fungal species isolated from BSR infected tissues and they are frequently observed in the same field. However, we are lacking information on the genetic diversity and population structure of these fungi in Burkina Faso. The mode of reproduction is also unknown. The genetic diversity of isolates of B. oryzae (n=61) and E. rostratum (n=151), collected from major rice-growing areas of Burkina Faso, was estimated using genotyping-by-sequencing (GBS). The mean values for nucleotide diversity (π) were 1.9 x10-4 for B. oryzae and 4.8 x10-4 for E. rostratum. There is no genetic differentiation between the geographical populations of each species. The analysis of molecular variance revealed that 89% and 94% of the genetic variances were within the populations of B. oryzae and E. rostratum, respectively. For each species, four genetic clusters were identified by two clustering methods (DAPC and sNMF). The distribution of these genetic groups was independent of the geographical origin of the isolates. Evidence of recombination was detected in the populations of B. oryzae and E. rostratum. For B. oryzae balanced mating type ratios were supporting sexual reproduction. For E. rostratum overrepresentation of MAT1-2 isolates (79%) suggested a predominant asexual reproduction. This study provides important information on the biology and genetics of the two major fungi causing brown spot disease of rice in Burkina Faso.

Correction: Maintenance of divergent lineages of the Rice Blast Fungus Pyricularia oryzae through niche separation, loss of sex and post-mating genetic incompatibilities.

[This corrects the article DOI: 10.1371/journal.ppat.1010687.].

Male fertility in Pyricularia oryzae: Microconidia are spermatia.

Sexual reproduction in Ascomycetes is well described in several model organisms such as Neurospora crassa or Podospora anserina. Deciphering the biological process of sexual reproduction (from the recognition between compatible partners to the formation of zygote) can be a major advantage to better control sexually reproducing pathogenic fungi. In Pyricularia oryzae, the fungal pathogen causing blast diseases on several Poaceae species, the biology of sexual reproduction remains poorly documented. Besides the well-documented production of asexual macroconidia, the production of microconidia was seldom reported in P. oryzae, and their role as male gamete (i.e., spermatia) and in male fertility has never been explored. Here, we characterised the morphological features of microconidia and demonstrated that they are bona fide spermatia. Contrary to macroconidia, microconidia are not able to germinate and seem to be the only male gametes in P. oryzae. We show that fruiting body (perithecium) formation requires microconidia to get in contact with mycelium of strains of opposite mating type, to presumably fertilise the female gametes.

Maintenance of divergent lineages of the Rice Blast Fungus Pyricularia oryzae through niche separation, loss of sex and post-mating genetic incompatibilities.

Many species of fungal plant pathogens coexist as multiple lineages on the same host, but the factors underlying the origin and maintenance of population structure remain largely unknown. The rice blast fungus Pyricularia oryzae is a widespread model plant pathogen displaying population subdivision. However, most studies of natural variation in P. oryzae have been limited in genomic or geographic resolution, and host adaptation is the only factor that has been investigated extensively as a contributor to population subdivision. In an effort to complement previous studies, we analyzed genetic and phenotypic diversity in isolates of the rice blast fungus covering a broad geographical range. Using single-nucleotide polymorphism genotyping data for 886 isolates sampled from 152 sites in 51 countries, we showed that population subdivision of P. oryzae in one recombining and three clonal lineages with broad distributions persisted with deeper sampling. We also extended previous findings by showing further population subdivision of the recombining lineage into one international and three Asian clusters, and by providing evidence that the three clonal lineages of P. oryzae were found in areas with different prevailing environmental conditions, indicating niche separation. Pathogenicity tests and bioinformatic analyses using an extended set of isolates and rice varieties indicated that partial specialization to rice subgroups contributed to niche separation between lineages, and differences in repertoires of putative virulence effectors were consistent with differences in host range. Experimental crosses revealed that female sterility and early post-mating genetic incompatibilities acted as strong additional barriers to gene flow between clonal lineages. Our results demonstrate that the spread of a fungal pathogen across heterogeneous habitats and divergent populations of a crop species can lead to niche separation and reproductive isolation between distinct, widely distributed, lineages.

Structure of African Populations of Pyricularia oryzae from Rice.

Rice blast, caused by the filamentous ascomycete Pyricularia oryzae, is one of the most devastating diseases of rice. Four genetic clusters were previously identified, and three have a large geographic distribution. Asia is the center of diversity and the origin of most migrations to other continents, and sexual reproduction persisted only in the South China-Laos-North Thailand region, which was identified as the putative center of origin of all P. oryzae populations on rice. Despite the importance of rice blast disease, little is known about the diversity and the population structure of the pathogen in Africa (including Madagascar). The present study was intended to describe the structure of African populations of P. oryzae and identify the relationship between African and worldwide genetic clusters. A set of 2,057 strains (937 African and 1,120 Madagascan strains) were genotyped with 12 simple sequence repeat markers to assess the diversity and the population structure of P. oryzae. Four genetic clusters were identified in Africa and Madagascar. All four clusters previously identified are present in Africa. Populations from West Africa, East Africa, and Madagascar are highly differentiated. The geographic structure is consistent with limited dispersion and with some migration events between neighboring countries. The two mating types are present in Africa with a dominance of Mat1.2, but no female-fertile strain was detected, supporting the absence of sexual reproduction on this continent. This study showed an unsuspected high level of genetic diversity of P. oryzae in Africa and suggested several independent introductions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tracing the Origin and Evolutionary History of Pyricularia oryzae Infecting Maize and Barnyard Grass.

Blast disease is a notorious fungal disease leading to dramatic yield losses on major food crops such as rice and wheat. The causal agent, Pyricularia oryzae, encompasses different lineages, each having a different host range. Host shifts are suspected to have occurred in this species from Setaria spp. to rice and from Lolium spp. to wheat. The emergence of blast disease on maize in Iran was observed for the first time in the north of the country in 2012. We later identified blast disease in two additional regions of Iran: Gilan in 2013 and Golestan in 2016. Epidemics on the weed barnyard grass (Echinochloa spp.) were also observed in the same maize fields. Here, we showed that P. oryzae is the causal agent of this disease on both hosts. Pathogenicity assays in the greenhouse revealed that strains from maize can infect barnyard grass and conversely. However, genotyping with simple sequence repeat markers and comparative genomics showed that strains causing field epidemics on maize and on barnyard grass are different, although they belong to the same previously undescribed clade of P. oryzae. Phylogenetic analyses including these strains and a maize strain collected in Gabon in 1985 revealed two independent host-range expansion events from barnyard grass to maize. Comparative genomics between maize and barnyard grass strains revealed the presence or absence of five candidate genes associated with host specificity on maize, with the deletion of a small genomic region possibly responsible for adaptation to maize. This recent emergence of P. oryzae on maize provides a case study to understand host range expansion. Epidemics on maize raise concerns about potential yield losses on this crop in Iran and potential geographic expansion of the disease.

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.

Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance.

Crop breeding aims to balance disease resistance with yield; however, single resistance (R) genes can lead to resistance breakdown, and R gene pyramiding may affect growth fitness. Here we report that the rice Pigm locus contains a cluster of genes encoding nucleotide-binding leucine-rich repeat (NLR) receptors that confer durable resistance to the fungus Magnaporthe oryzae without yield penalty. Among these NLR receptors, PigmR confers broad-spectrum resistance, whereas PigmS competitively attenuates PigmR homodimerization to suppress resistance. PigmS expression, and thus PigmR-mediated resistance, are subjected to tight epigenetic regulation. PigmS increases seed production to counteract the yield cost induced by PigmR Therefore, our study reveals a mechanism balancing high disease resistance and yield through epigenetic regulation of paired antagonistic NLR receptors, providing a tool to develop elite crop varieties.

Evolution of Compatibility Range in the Rice-Magnaporthe oryzae System: An Uneven Distribution of R Genes Between Rice Subspecies.

Efficient strategies for limiting the impact of pathogens on crops require a good understanding of the factors underlying the evolution of compatibility range for the pathogens and host plants, i.e., the set of host genotypes that a particular pathogen genotype can infect and the set of pathogen genotypes that can infect a particular host genotype. Until now, little is known about the evolutionary and ecological factors driving compatibility ranges in systems implicating crop plants. We studied the evolution of host and pathogen compatibility ranges for rice blast disease, which is caused by the ascomycete Magnaporthe oryzae. We challenged 61 rice varieties from three rice subspecies with 31 strains of M. oryzae collected worldwide from all major known genetic groups. We determined the compatibility range of each plant variety and pathogen genotype and the severity of each plant-pathogen interaction. Compatibility ranges differed between rice subspecies, with the most resistant subspecies selecting for pathogens with broader compatibility ranges and the least resistant subspecies selecting for pathogens with narrower compatibility ranges. These results are consistent with a nested distribution of R genes between rice subspecies.

South-East Asia is the center of origin, diversity and dispersion of the rice blast fungus, Magnaporthe oryzae.

• Inferring invasion routes and identifying reservoirs of diversity of plant pathogens are essential in proposing new strategies for their control. Magnaporthe oryzae, the fungus responsible for rice blast disease, has invaded all rice growing areas. Virulent genotypes regularly (re)emerge, causing rapid resistance breakdowns. However, the world-wide genetic subdivision of M. oryzae populations on rice and its past history of invasion have never been elucidated. • In order to investigate the centers of diversity, origin and migration of M. oryzae on rice, we analyzed the genetic diversity of 55 populations from 15 countries. • Three genetic clusters were identified world-wide. Asia was the center of diversity and the origin of most migrations to other continents. In Asia, two centers of diversity were revealed in the Himalayan foothills: South China-Laos-North Thailand, and western Nepal. Sexual reproduction persisted only in the South China-Laos-North Thailand region, which was identified as the putative center of origin of all M. oryzae populations on rice. • Our results suggest a scenario of early evolution of M. oryzae on rice that matches the past history of rice domestication. This study confirms that crop domestication may have considerable influence on the pestification process of natural enemies.

The variety mixture strategy assessed in a G × G experiment with rice and the blast fungus Magnaporthe oryzae.

Frequent and devastating epidemics of parasites are one of the major issues encountered by modern agriculture. To manage the impact of pathogens, resistant plant varieties have been selected. However, resistances are overcome by parasites requiring the use of pesticides and causing new economical and food safety issues. A promising strategy to maintain the epidemic at a low level and hamper pathogen's adaptation to varietal resistance is the use of mixtures of varieties such that the mix will form a heterogeneous environment for the parasite. A way to find the good combination of varieties that will actually constitute a heterogeneous environment for pathogens is to look for genotype × genotype (G × G) interactions between pathogens and plant varieties. A pattern in which pathogens have a high fitness on one variety and a poor fitness on other varieties guarantees the efficiency of the mixture strategy. In the present article, we inoculated 18 different genotypes of the fungus Magnaporthe oryzae on three rice plant varieties showing different levels of partial resistance in order to find a variety combination compatible with the requirements of the variety mixture strategy, i.e., showing appropriate G × G interactions. We estimated the success of each plant-fungus interaction by measuring fungal fitness and three fungal life history traits: infection success, within-host growth, sporulation capacity. Our results show the existence of G × G interactions between the two varieties Ariete and CO39 on all measured traits and fungal fitness. We also observed that these varieties have different resistance mechanisms; Ariete is good at controlling infection success of the parasite but is not able to control its growth when inside the leaf, while CO39 shows the opposite pattern. We also found that Maratelli's resistance has been eroded. Finally, correlation analyses demonstrated that not all infectious traits are positively correlated.

Asexual reproduction induces a rapid and permanent loss of sexual reproduction capacity in the rice fungal pathogen Magnaporthe oryzae: results of in vitro experimental evolution assays.

BACKGROUND: Sexual reproduction is common in eukaryotic microorganisms, with few species reproducing exclusively asexually. However, in some organisms, such as fungi, asexual reproduction alternates with episodic sexual reproduction events. Fungi are thus appropriate organisms for studies of the reasons for the selection of sexuality or clonality and of the mechanisms underlying this selection. Magnaporthe oryzae, an Ascomycete causing blast disease on rice, reproduces mostly asexually in natura. Sexual reproduction is possible in vitro and requires (i) two strains of opposite mating types including (ii) at least one female-fertile strain (i.e. a strain able to produce perithecia, the female organs in which meiosis occurs). Female-fertile strains are found only in limited areas of Asia, in which evidence for contemporary recombination has recently been obtained. We induced the forced evolution of four Chinese female-fertile strains in vitro by the weekly transfer of asexual spores (conidia) between Petri dishes. We aimed to determine whether female fertility was rapidly lost in the absence of sexual reproduction and whether this loss was controlled genetically or epigenetically. RESULTS: All the strains became female-sterile after 10 to 19 rounds of selection under asexual conditions. As no single-spore isolation was carried out, the observed decrease in the production of perithecia reflected the emergence and the invasion of female-sterile mutants. The female-sterile phenotype segregated in the offspring of crosses between female-sterile evolved strains and female-fertile wild-type strains. This segregation was maintained in the second generation in backcrosses. Female-sterile evolved strains were subjected to several stresses, but none induced the restoration of female fertility. This loss of fertility was therefore probably due to genetic rather than epigenetic mechanisms. In competition experiments, female-sterile mutants produced similar numbers of viable conidia to wild-type strains, but released them more efficiently. This advantage may account for the invasion of our populations by female-sterile mutants. CONCLUSIONS: We show for the first time that, in the absence of sexual reproduction, female-sterile mutants of M. oryzae rice strains can arise and increase in abundance in asexual generations. This change in phenotype was frequent and probably caused by mutation. These results suggest that female fertility may have been lost rapidly during the dispersion of the fungus from Asia to the rest of the world.

Sex at the origin: an Asian population of the rice blast fungus Magnaporthe oryzae reproduces sexually.

Sexual reproduction may be cryptic or facultative in fungi and therefore difficult to detect. Magnaporthe oryzae, which causes blast, the most damaging fungal disease of rice, is thought to originate from southeast Asia. It reproduces asexually in all rice-growing regions. Sexual reproduction has been suspected in limited areas of southeast Asia, but has never been demonstrated in contemporary populations. We characterized several M. oryzae populations worldwide both biologically and genetically, to identify candidate populations for sexual reproduction. The sexual cycle of M. oryzae requires two strains of opposite mating types, at least one of which is female-fertile, to come into contact. In one Chinese population, the two mating types were found to be present at similar frequencies and almost all strains were female-fertile. Compatible strains from this population completed the sexual cycle in vitro and produced viable progenies. Genotypic richness and linkage disequilibrium data also supported the existence of sexual reproduction in this population. We resampled this population the following year, and the data obtained confirmed the presence of all the biological and genetic characteristics of sexual reproduction. In particular, a considerable genetic reshuffling of alleles was observed between the 2 years. Computer simulations confirmed that the observed genetic characteristics were unlikely to have arisen in the absence of recombination. We therefore concluded that a contemporary population of M. oryzae, pathogenic on rice, reproduces sexually in natura in southeast Asia. Our findings provide evidence for the loss of sexual reproduction by a fungal plant pathogen outside its centre of origin.

The development of simple sequence repeat markers for Magnaporthe grisea and their integration into an established genetic linkage map.

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.