Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems.

The evolutionary history of crop pathogens is shaped by a complex interaction of natural and anthropogenic factors. The fungus Colletotrichum graminicola causes maize anthracnose which results in significant yield losses worldwide. We conducted a comprehensive investigation into the evolutionary genomics of C. graminicola using a collection of 212 isolates from 17 countries across five continents. Genomic analyses supported the existence of three geographically isolated genetic lineages, with a significant pattern of isolation by distance. We identified two distinct gene flow patterns, driven by short- and long-distance dispersal, likely resulting from the natural spread of the pathogen and the exchange of contaminated seeds. We present evidence of genetic introgression between lineages, suggesting a long history of recombination. We identified significant recombination events coalescing at distinct points in time, with the North American lineage displaying evidence of the most ancient recombination. Demographic modelling has indicated that North America is an intermediate between Brazil, Europe and an ancestral, unsampled source population, which is hypothesised to be Mesoamerican. Our analyses revealed that the global genomic structure of C. graminicola is shaped by geographic differentiation driven by long-distance migration and a long history of recombination and introgression. We show historical relationships amongst these lineages, identifying a potential route for fungal spread, with the North American population emerging ancestrally, followed sequentially by the Brazilian and European populations. Our research indicates that the European lineage is more virulent, which has implications for the potential emergence of new outbreaks of maize anthracnose in Europe.

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide host range. Its natural populations are phenotypically and genetically very diverse. A survey of B. cinerea isolates causing gray mold in the vineyards of Castilla y León, Spain, was carried out and as a result eight non-pathogenic natural variants were identified. Phenotypically these isolates belong to two groups. The first group consists of seven isolates displaying a characteristic mycelial morphotype, which do not sporulate and is unable to produce sclerotia. The second group includes one isolate, which sporulates profusely and does not produce sclerotia. All of them are unresponsive to light. Crosses between a representative mycelial non-pathogenic isolate and a highly aggressive field isolate revealed that the phenotypic differences regarding pathogenicity, sporulation and production of sclerotia cosegregated in the progeny and are determined by a single genetic locus. By applying a bulked segregant analysis strategy based on the comparison of the two parental genomes the locus was mapped to a 110 kb region in chromosome 4. Subcloning and transformation experiments revealed that the polymorphism is an SNP affecting gene Bcin04g03490 in the reference genome of B. cinerea. Genetic complementation analysis and sequencing of the Bcin04g03490 alleles demonstrated that the mutations in the mycelial isolates are allelic and informed about the nature of the alterations causing the phenotypes observed. Integration of the allele of the pathogenic isolate into the non-pathogenic isolate fully restored the ability to infect, to sporulate and to produce sclerotia. Therefore, it is concluded that a major effect gene controlling differentiation and developmental processes as well as pathogenicity has been identified in B. cinerea. It encodes a protein with a GAL4-like Zn(II)2Cys6 binuclear cluster DNA binding domain and an acetyltransferase domain, suggesting a role in regulation of gene expression through a mechanism involving acetylation of specific substrates.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a “biphasic response” to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and ΔBcfhg1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins.

The emergence of new fungal pathogens often depends on the acquisition of complex adaptive traits, yet the mechanisms by which such traits arise remain poorly understood. Here we show that a biosynthetic gene cluster required for pathogenicity in the lupin pathogenic fungus Colletotrichum lupini was acquired within a genomic region derived from a giant Starship transposable element. Comparative and population genomic analyses reveal that the C. lupini genome contains multiple regions derived from ancestrally active Starship elements, enriched in lineage-specific genes and strongly induced during plant infection. One such region harbours a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene cluster that is conserved in pathogenic isolates but absent from closely related non-pathogenic species and from a non-pathogenic strain. Phylogenetic analyses of the PKS-NRPS backbone gene reveal incongruence with species relationships and a distribution across deeply divergent fungal lineages, consistent with horizontal acquisition. Disruption of the PKS-NRPS backbone gene abolishes pathogenicity, demonstrating that this cluster is required for host infection. Phylogenomic analyses further indicate that lupin pathogenicity emerged once within the C. lupini lineage prior to its diversification. Together, these findings identify a Starship -associated virulence determinant and support a model in which giant cargo-mobilizing mobile elements generate genomic novelty by facilitating the acquisition, assembly and integration of adaptive traits during the emergence of fungal pathogenicity.

Colletotrichum species rank among the most important fungal pathogens, threatening food security by infecting nearly all major crops worldwide. Colletotrichum graminicola , the causal agent of maize anthracnose, secretes effector proteins to manipulate host defences and promote colonization. Building on previous work characterizing the nuclear effector CgEP1, we characterized its paralog CgEP4, which is highly conserved across strains of C. graminicola . Phylogenetic analysis of the two genes and their homologs in other species revealed that they originated from a gene duplication event approximately 28 to 18 million years ago, predating the diversification of the Graminicola species complex. This timing aligns with the ecological expansion of C4 grasses, suggesting that the functional divergence of these effectors was an adaptive response to facilitate the colonization of emerging monocot hosts. Functional characterization using gene deletion mutants demonstrated that CgEP4 has a critical role in pathogenicity, characterized by a significant reduction in virulence, delayed penetration, enhanced papilla formation, and decreased fungal biomass. This virulence defect is associated with compromised host colonization and a failure to suppress basal host defences. In the absence of CgEP4 , the pathogen also showed defects in general fungal physiology and stress tolerance. Overall, our findings establish CgEP4 as a new, essential nuclear-localized effector that promotes fungal entry and colonization by manipulating host responses. Our findings demonstrate that evolutionary analysis is a valuable tool for discovering new genes important for host adaptation and pathogen evolution.