Magnus Karlsson

Modes of reproduction and sexual strategies strongly influence the genetic diversity and evolutionary potential of a species. The ascomycete fungus Clonostachys rosea is reported to be homothallic (sexually self‐fertile), although a rapid decay of genome‐wide linkage disequilibrium is also reported, something that is not in line with an obligate homothallic mode of reproduction. To investigate this phenomenon, we identified the mating‐type ( MAT1 ) locus in 66 genome‐sequenced C. rosea strains under the hypothesis that each strain contains genes from both MAT1 idiomorphs. Eleven strains indeed contained genes from both the MAT1‐1 and MAT1‐2 idiomorphs, suggesting homothallism. However, most strains harboured genes from either the MAT1‐1 or MAT1‐2 idiomorphs and co‐existed in North America, Europe and China, suggesting heterothallism. The MAT1 locus of heterothallic strains was highly conserved, and the linkage disequilibrium half decay distance was 1050 bp, suggesting sexual outcrossing. The presence of conserved MAT1‐1 or MAT1‐2 idiomorphs in strains of other Clonostachys species shows that heterothallism is likely the ancestral state. A phylogenetic analysis of 2800 single‐copy orthologous genes revealed that homothallic and heterothallic strains separated in two well‐supported clades, indicating a single lineage of homothallic C. rosea , likely originating in South America, followed by intercontinental dispersal. Homothallic C. rosea strains displayed higher nucleotide diversity than heterothallic strains, indicating a lack of outcrossing. This unique case of both homothallic and heterothallic lineages within the same species provides an opportunity to study the genomic consequences of selfing in very closely related strains.

Fungal species associated with coffee and their pathogenic potential remain underexplored in Bolivia. This study aimed to investigate the diversity of Fusarium species associated with coffee in the Amazon basin of Caranavi province in the Yungas region of Bolivia and their ability to induce disease symptoms under controlled conditions. A total of 163 putative Fusarium isolates were recovered from symptomatic coffee leaves, fruits, stems and roots. Multilocus DNA barcoding and phylogenetic analyses based on the ITS, TEF1 and RPB2 loci identified seven Fusarium species across six species complexes. Members of the Fusarium oxysporum species complex (FOSC) were the most prevalent, followed by the Neocosmospora solani (synonym: Fusarium solani ) species complex (FSSC). Detached coffee leaves assay under in vitro conditions, and pot assay using coffee seedlings showed that isolates from FSSC and FOSC could induce necrotic lesions on detached leaves and/or root damage in seedlings. Marked variability in virulence was observed among isolates, ranging from highly virulent isolates affecting both leaves and seedlings to isolates with tissue‐specific or low virulence. This study provides the first molecular evidence of diversity within Fusarium species complexes associated with coffee in a previously explored region of Bolivia and demonstrates their potential to induce disease symptoms under controlled conditions. However, further studies under greenhouse and field conditions are required to validate their pathogenic potential in adult coffee plants.

Parasite-host interactions can display a continuum from specialization to generalism. Clonostachys rosea is an ascomycete fungus reported to parasitize range of different hosts, yet our understanding of the evolutionary dynamics shaping its virulence and host range remains limited. In this study, 63 C. rosea strains were assessed for virulence towards host fungi and an oomycete, under the hypothesis that strains differ in virulence and host specialization. Results revealed significant (P ≤ 0.05) variation in virulence between C. rosea strains towards the host fungi Botrytis cinerea, Fusarium graminearum and Rhizoctonia solani. Furthermore, certain C. rosea strains displayed high virulence towards specific hosts, while others intermediate virulence towards all hosts, highlighting variation along the specialist-to-generalist continuum within C. rosea. Genome-wide association analysis identified seven single nucleotide polymorphisms significantly (P ≤ 1.6 × 10−6) segregated with virulence. Different genomic regions segregating with virulence towards different hosts, suggest different underlying virulence determinants. This study provides empirical evidence on evolution of virulence in the mycoparasite C. rosea.

Aphanomyces root rot (ARR), caused by Aphanomyces euteiches , is a major constraint in pea ( Pisum sativum ) production. This study aimed to improve molecular detection of A. euteiches in soil samples, evaluate the susceptibility of selected legume species, and assess their influence on soil inoculum levels. Twenty‐four soil samples were analysed in bioassay 1, which was performed in two batches, using susceptible pea cv. Linnea to determine disease severity index (DSI). DNA analyses employed droplet digital PCR (ddPCR) and quantitative real‐time PCR (qPCR). Susceptibility of 14 legume species was examined by artificial inoculation, and the influence of five species on ARR in a subsequent pea crop was tested in naturally infested field soil. The number of gene copies g ⁻¹ soil differed significantly among soil samples. Soil analysis by ddPCR allowed detection of A. euteiches at a level < 10 gene copies g ⁻¹ soil, demonstrating high sensitivity. In bioassay 1, batch 2, DSI correlated significantly with A. euteiches gene copies g ⁻¹ soil quantified by ddPCR and qPCR. Visual assessment and ddPCR showed that lentil ( Lens culinaris ) and fodder vetch ( Vicia villosa ) were highly susceptible, whereas birdsfoot trefoil ( Lotus corniculatus ), sainfoin ( Onobrychis viciifolia ) and Persian clover ( Trifolium resupinatum ) were not susceptible to A. euteiches infection. Pea plants grown after fodder vetch had significantly higher DSI and number of A . euteiches gene copies. These findings underscore the importance of molecular soil analysis and knowledge of legume species susceptibility for effective ARR management.

Aphanomyces euteiches is an oomycete pathogen causing root rot in pea and other legumes, leading to devastating yield losses in pea production. Previously, three genetic groups of A. euteiches were identified in Europe based on simple sequence repeat markers. In this study, we determined the genome sequences of 68 European A. euteiches strains and performed a comparative genomic analysis with the aim to investigate the population genetic structure of A. euteiches, to delineate species boundaries and to identify gene families evolving under selection for gene gains or losses. Population genetic analysis based on genome-wide single nucleotide polymorphisms identified three genetic groups in Europe, where strains from Italy formed a distinct and genetically isolated group. Genealogical concordance phylogenetic species recognition analysis indicated that strains from Italy may represent a cryptic species. Aphanomyces strains differed in virulence on susceptible and partially resistant pea host genotypes, but virulence did not correlate with genetic groups. Analysis of virulence-related gene family evolution revealed a significant expansion of gene families acting in the detoxification of plant-derived secondary metabolites in plant-pathogenic Aphanomyces species. Moreover, the carbohydrate esterase family 1 (CE1) was significantly expanded in A. euteiches but not in the cryptic species represented by Italian strains. Modular structure analyses revealed exclusive presence of feruloyl esterase domains in CE1 homologs of plant-pathogenic Aphanomyces species, indicating a role in plant cell wall degradation.

Three species of Aphanomyces, A. euteiches, A. cochlioides and A. cladogamus are devastating agricultural plant pathogens causing root rot disease on pea, sugar beet and spinach, respectively. Here we investigated the distribution of the three Aphanomyces species in parts of Sweden, Denmark and Lithuania using climate chamber soil bioassays, and tested for correlations with macronutrient levels in the soil. Aphanomyces euteiches was the most widespread pathogen found in 28% of the tested samples in Sweden and Denmark, while A. cochlioides had a more limited distribution, and was found in 22% of the samples. Aphanomyces cladogamus was only detected in Sweden and was present in 16% of the tested samples. Aphanomyces euteiches only infected pea plants, while A. cochlioides and A. cladogamus infected both sugar beet and spinach. There were significant ( P < 0.001) differences in disease severity index (DSI) between regions, pathogens and for the interaction between regions and crops. Soil calcium (Ca) content ranged from 82 to 3600 mg/100 g dry soil. No species of Aphanomyces was detected in soils with Ca levels above 250 mg/100 g soil, indicating a pathogen-suppressive effect from calcium. Similar results were observed from 1350 soil samples collected from commercial pea growing fields, as DSI values on pea remained very low (below 20 on a 0–100 scale) in soil samples with confirmed presence of A. euteiches and a Ca value above 210 mg/100 g soil. High Ca levels was a significant ( P < 0.05) predictor for low probability of presence of Aphanomyces spp. in soil.

Biological control to manage plant diseases is an environmentally friendly alternative to using chemical pesticides. However, little is known about the role of genetic variation in plants affecting the efficacy of biological control agents (BCAs). The aim of this study was to explore the genetic variation in winter wheat for disease susceptibility to fusarium foot rot caused by Fusarium graminearum and variation in biocontrol efficacy of the fungal BCA Clonostachys rosea to control the disease. In total, 190 winter wheat genotypes were evaluated under controlled conditions in two treatments, i.e. (i) F. graminearum (Fg) and (ii) F. graminearum infection on C. rosea treated seeds (FgCr). Alongside disease severity, plant growth-related traits such as shoot length and root length were also measured. Comparison of genotypes between the two treatments enabled the dissection of genotypic variation for disease resistance and C. rosea efficacy. The study revealed significant variation among plant genotypes for fusarium foot rot susceptibility and other growth traits in treatment Fg. Moreover, significant variation in C. rosea efficacy was also observed in genotype contrasts between the two treatments for all traits. Using a 20K marker array, a genome-wide association study was also performed. We identified a total of 18 significant marker-trait associations for disease resistance and C. rosea efficacy for all the traits. Moreover, the markers associated with disease resistance and C. rosea efficacy were not co-localized, highlighting the independent inheritance of these traits, which can facilitate simultaneous selection for cultivar improvement.

Botrytis cinerea is a notorious pathogen causing pre‐ and post‐harvest spoilage in many economically important crops. Excessive application of site‐specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2 , encoding an ATP‐binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B . cinerea multidrug‐resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen.