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.

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.

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.

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) x Azucena (susceptible) and Azucena x Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [ Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F(2) populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.

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.

Understanding how fungi specialize on their plant host is crucial for developing sustainable disease control. A traditional, centuries-old rice agro-system of the Yuanyang terraces was used as a model to show that virulence effectors of the rice blast fungus Magnaporthe oryzaeh play a key role in its specialization on locally grown indica or japonica local rice subspecies. Our results have indicated that major differences in several components of basal immunity and effector-triggered immunity of the japonica and indica rice varieties are associated with specialization of M. oryzae. These differences thus play a key role in determining M. oryzae host specificity and may limit the spread of the pathogen within the Yuanyang agro-system. Specifically, the AVR-Pia effector has been identified as a possible determinant of the specialization of M. oryzae to local japonica rice.DOI: http://dx.doi.org/10.7554/eLife.19377.001

BackgroundSexual 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.ResultsAll 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.ConclusionsWe 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.

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.