Draft genome sequences of Neurospora crassa clade B, isolated from burned Cytisus sp. plants in France.

Neurospora crassa clade A is a model system for genetics, biochemistry, molecular biology, and experimental evolution. Here, we present the draft genome sequences of four isolates of N. crassa clade B. These data represent a valuable resource to investigate the population biology and evolutionary history of N. crassa sensu lato.

Adaptive evolution in virulence effectors of the rice blast fungus Pyricularia oryzae.

Plant pathogens secrete proteins called effectors that target host cellular processes to promote disease. Recently, structural genomics has identified several families of fungal effectors that share a similar three-dimensional structure despite remarkably variable amino-acid sequences and surface properties. To explore the selective forces that underlie the sequence variability of structurally-analogous effectors, we focused on MAX effectors, a structural family of effectors that are major determinants of virulence in the rice blast fungus Pyricularia oryzae. Using structure-informed gene annotation, we identified 58 to 78 MAX effector genes per genome in a set of 120 isolates representing seven host-associated lineages. The expression of MAX effector genes was primarily restricted to the early biotrophic phase of infection and strongly influenced by the host plant. Pangenome analyses of MAX effectors demonstrated extensive presence/absence polymorphism and identified gene loss events possibly involved in host range adaptation. However, gene knock-in experiments did not reveal a strong effect on virulence phenotypes suggesting that other evolutionary mechanisms are the main drivers of MAX effector losses. MAX effectors displayed high levels of standing variation and high rates of non-synonymous substitutions, pointing to widespread positive selection shaping the molecular diversity of MAX effectors. The combination of these analyses with structural data revealed that positive selection acts mostly on residues located in particular structural elements and at specific positions. By providing a comprehensive catalog of amino acid polymorphism, and by identifying the structural determinants of the sequence diversity, our work will inform future studies aimed at elucidating the function and mode of action of MAX effectors.

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.

Means, motive and opportunity for biological invasions: Genetic introgression in a fungal pathogen.

Invasions by fungal plant pathogens pose a significant threat to the health of agricultural ecosystems. Despite limited standing genetic variation, many invasive fungal species can adapt and spread rapidly, resulting in significant losses to crop yields. Here, we report on the population genomics of Colletotrichum truncatum, a polyphagous pathogen that can infect more than 460 plant species, and an invasive pathogen of soybean in Brazil. We study the whole-genome sequences of 18 isolates representing 10 fields from two major regions of soybean production. We show that Brazilian C. truncatum is subdivided into three phylogenetically distinct lineages that exchange genetic variation through hybridization. Introgression affects 2%-30% of the nucleotides of genomes and varies widely between the lineages. We find that introgressed regions comprise secreted protein-encoding genes, suggesting possible co-evolutionary targets for selection in those regions. We highlight the inherent vulnerability of genetically uniform crops in the agro-ecological environment, particularly when faced with pathogens that can take full advantage of the opportunities offered by an increasingly globalized world. Finally, we discuss "the means, motive and opportunity" of fungal pathogens and how they can become invasive species of crops. We call for more population genomic studies because such analyses can help identify geographical areas and pathogens that pose a risk, thereby helping to inform control strategies to better protect crops in the future.

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.].

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.

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.

When history repeats itself: exploring the genetic architecture of host-plant adaptation in two closely related lepidopteran species.

The genus Ostrinia includes two allopatric maize pests across Eurasia, namely the European corn borer (ECB, O. nubilalis) and the Asian corn borer (ACB, O. furnacalis). A third species, the Adzuki bean borer (ABB, O. scapulalis), occurs in sympatry with both the ECB and the ACB. The ABB mostly feeds on native dicots, which probably correspond to the ancestral host plant type for the genus Ostrinia. This situation offers the opportunity to characterize the two presumably independent adaptations or preadaptations to maize that occurred in the ECB and ACB. In the present study, we aimed at deciphering the genetic architecture of these two adaptations to maize, a monocot host plant recently introduced into Eurasia. To this end, we performed a genome scan analysis based on 684 AFLP markers in 12 populations of ECB, ACB and ABB. We detected 2 outlier AFLP loci when comparing French populations of the ECB and ABB, and 9 outliers when comparing Chinese populations of the ACB and ABB. These outliers were different in both countries, and we found no evidence of linkage disequilibrium between any two of them. These results suggest that adaptation or preadaptation to maize relies on a different genetic architecture in the ECB and ACB. However, this conclusion must be considered in light of the constraints inherent to genome scan approaches and of the intricate evolution of adaptation and reproductive isolation in the Ostrinia spp. complex.

Morphological, biogeographical and molecular evidence of Carpocoris mediterraneus as a valid species (Hemiptera: Pentatomidae).

Carpocoris mediterraneus Tamanini, 1958, synonymized with Carpocoris fuscispinus (Boheman, 1851) by Ribes et al. (2007), is restored to the species level. The shape of the pronotum is a good diagnostic character to distinguish the two species. The existence of two valid species is supported by geographical distribution patterns in Western Europe: Mediterranean-Atlantic for C. mediterraneus, and Continental for C. fuscispinus. In France and Spain, in some areas, the two species are found in sympatry (sometimes even on the same plant). Morphological observations are confirmed at the molecular level by sequencing of the mitochondrial Cytochrome c oxidase I standard barcode fragment. Indeed, inter-specific divergence largely exceeded intra-specific divergence and our phylogenetic reconstructions reveal that Carpocoris mediterraneus and Carpocoris fuscispinus form two reciprocally monophyletic genetic lineages. A morphological identification key is proposed for all the European species of the genus Carpocoris, to facilitate identification. Carpocoris fuscispinus is first time recorded from Portugal.

Scanning the European corn borer (Ostrinia spp.) genome for adaptive divergence between host-affiliated sibling species.

It has recently been shown that the European corn borer, a major pest of maize crops, is actually composed of two genetically differentiated and reproductively isolated taxa, which are found in sympatry over a wide geographical range in Eurasia. Each taxon is adapted to specific host plants: Ostrinia nubilalis feeds mainly on maize, while O. scapulalis feeds mainly on hop or mugwort. Here, we present a genome scan approach as a first step towards an integrated molecular analysis of the adaptive genomic divergence between O. nubilalis and O. scapulalis. We analysed 609 AFLP marker loci in replicate samples of sympatric populations of Ostrinia spp. collected on maize, hop and mugwort, in France. Using two genome scan methods based on the analysis of population differentiation, we found a set of 28 outlier loci that departed from the neutral expectation in one or the other method (of which a subset of 14 loci were common to both methods), which showed a significantly increased differentiation between O. nubilalis and O. scapulalis, when compared to the rest of the genome. A subset of 12 outlier loci were sequenced, of which 7 were successfully re-amplified as target candidate loci. Three of these showed homology with annotated lepidopteran sequences from public nucleotide databases.

Microsatellite markers reveal spatial genetic structure of Tetranychus urticae (Acari: Tetranychidae) populations along a latitudinal gradient in Europe.

The genetic structure of populations of the two-spotted spider mite Tetranychus urticae was investigated along a south-north European transect spanning from southern France to The Netherlands. Mites were collected on Urtica dioica in 6 sampling zones. Microsatellite variation at 5 loci revealed considerable genetic variation with an average heterozygozity of 0.49. Significant heterozygote deficiency was found in 7 populations out of the 18 samples analyzed and one of them was completely monomorphic. Tetranychus urticae populations show some level of genetic structuring. First, genetic differentiation between localities (F (ST) estimates) was significant for all comparisons. Second, the analysis of molecular variance, AMOVA, indicates that there is an effect, albeit low (9%), of the locality in accounting for allele frequency variance. Geographic distance emerges as a factor responsible for this genetic structure. The results are discussed in relation to the biological features of the species and the known patterns of migration. Related agronomical issues are addressed.

The invasive Korea and Japan types of Varroa destructor, ectoparasitic mites of the Western honeybee (Apis mellifera), are two partly isolated clones.

Varroa destructor, now a major pest of the Western honeybee, Apis mellifera, switched from its original host, the Eastern honeybee, A. cerana, ca. 50 years ago. So far, only two out of several known mitochondrial haplotypes of V. destructor have been found to be capable of reproducing on A. mellifera (Korea and Japan). These haplotypes are associated in almost complete cytonuclear disequilibrium to diagnostic alleles at 11 microsatellite loci. By contrast, microsatellite polymorphism within each type is virtually absent, because of a severe bottleneck at the time of host change. Accordingly, 12 mitochondrial sequences of 5185 nucleotides displayed 0.40% of nucleotide divergence between haplotypes and no intra haplotype variation. Hence, each type has a quasi-clonal structure. The nascent intratype variability is subsequent to the clone formation 50 years ago: in both types the variant alleles differ from the most common by one (in 10 cases), two (five cases) or three (one case) repeated motifs. In addition to individuals of the two 'pure' types, five F1 hybrids and 19 recombinant individuals (Japan alleles introgressed into the Korea genetic background) were detected. The existence of F1 and recombinant individuals in admixed populations requires that double infestations of honeybee cells occur in a high proportion but the persistence of pure types suggests a post-zygotic isolation between the two clones.