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Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Phytophthora , Phytophthora/genética , Filogenia , Transferência Genética Horizontal , Genoma , Genômica , Plantas/genéticaRESUMO
Powdery mildew is one of the most economically destructive diseases in protected strawberry production. Here we present the first genome assembly for Podosphaera aphanis, the causal agent of powdery mildew on strawberry. This obligate-biotrophic fungal pathogen was sampled from a naturally occurring outbreak on Fragaria × ananassa 'Malling Centenary' plants grown under cover in the United Kingdom. Assembled reads resolved a 55.6 Mb genome, composed of 12,357 contigs whose annotation led to prediction of 17,239 genes encoding 17,328 proteins. The genome is highly-complete, with 97.5% of conserved single-copy Ascomycete genes shown to be present. This annotated P. aphanis genome provides a molecular resource for further investigation into host-pathogen interactions in the strawberry powdery mildew pathosystem.
Assuntos
Ascomicetos , Fragaria , Fragaria/microbiologia , Doenças das Plantas/microbiologia , Ascomicetos/genética , Erysiphe , GenômicaRESUMO
Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.
Assuntos
Genes Bacterianos , Genômica , Prunus avium/microbiologia , Pseudomonas syringae/genética , Alelos , Sistemas de Secreção Bacterianos , Transferência Genética Horizontal/genética , Modelos Biológicos , Filogenia , Doenças das Plantas/microbiologia , Pseudomonas syringae/classificação , Pseudomonas syringae/patogenicidade , Análise de Sequência de DNA , Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Date palm (Phoenixdactylifera) is the most significant crop across North Africa and the Middle East. However, the crop faces a severe threat from Bayoud disease caused by the fungal pathogen Fusarium oxysporum f. sp. albedinis (FOA). FOA is a soil-borne fungus that infects the roots and vascular system of date palms, leading to widespread destruction of date palm plantations in North Africa over the last century. This is considered the most devastating pathogen of oasis agriculture in North Africa and responsible for loss of 13 million trees in Algeria and Morocco alone. In this study, we present a chromosome-scale high-quality genome assembly of the virulent isolate Foa 44, which provides valuable insights into understanding the genetic basis of Bayoud disease. The genome assembly consists of 11 chromosomes and 40 unplaced contigs, totalling 65,971,825 base pairs in size. It exhibits a GC ratio of 47.77% and a TE (transposable element) content of 17.30%. Through prediction and annotation, we identified 20,416 protein-coding genes. By combining gene and repeat densities analysis with alignment to Fusarium oxysporum f. sp. lycopersici (FOL) 4287 isolate genome sequence, we determined the core and lineage-specific compartments in Foa 44, shedding light on the genome structure of this pathogen. Furthermore, a phylogenomic analysis based on the 3,292 BUSCOs core genome revealed a distinct clade of FOA isolates within the Fusarium oxysporum species complex (FOSC). Notably, the genealogies of the five identified Secreted In Xylem (SIX) genes (1, 6, 9, 11 and 14) in FOA displayed a polyphyletic pattern, suggesting a horizontal inheritance of these effectors. These findings provide a valuable genomics toolbox for further research aimed at combatting the serious biotic constraints posed by FOA to date palm. This will pave the way for a deeper understanding of Bayoud disease and facilitate the development of effective diagnostic tools and control measures.
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The evolution of azole resistance in fungal pathogens presents a major challenge in both crop production and human health. Apple orchards across the world are faced with the emergence of azole fungicide resistance in the apple scab pathogen Venturia inaequalis. Target site point mutations observed in this fungus to date cannot fully explain the reduction in sensitivity to azole fungicides. Here, polygenic resistance to tebuconazole was studied across a population of V. inaequalis. Genotyping by sequencing allowed Quantitative Trait Loci (QTLs) mapping to identify the genetic components controlling this fungicide resistance. Dose-dependent genetic resistance was identified, with distinct genetic components contributing to fungicide resistance at different exposure levels. A QTL within linkage group seven explained 65% of the variation in the effective dose required to reduce growth by 50% (ED50). This locus was also involved in resistance at lower fungicide doses (ED10). A second QTL in linkage group one was associated with dose-dependent resistance, explaining 34% of variation at low fungicide doses (ED10), but did not contribute to resistance at higher doses (ED50 and ED90). Within QTL regions, non-synonymous mutations were observed in several ATP-Binding Cassette and Major Facilitator SuperFamily transporter genes. These findings provide insight into the mechanisms of fungicide resistance that have evolved in horticultural pathogens. Identification of resistance gene candidates supports the development of molecular diagnostics to inform management practices.
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Fusarium oxysporum is a soilborne fungal plant pathogen responsible for causing disease in many economically important crops with "special forms" (formae speciales) adapted to infect specific plant hosts. F. oxysporum f. sp. pisi (FOP) is the causal agent of Fusarium wilt disease of pea. It has been reported in every country where peas are grown commercially. Disease is generally controlled using resistant cultivars possessing single major gene resistance and therefore there is a constant risk of breakdown. The main aim of this work was to characterise F. oxysporum isolates collected from diseased peas in the United Kingdom as well as FOP isolates obtained from other researchers representing different races through sequencing of a housekeeping gene and the presence of Secreted In Xylem (SIX) genes, which have previously been associated with pathogenicity in other F. oxysporum f. spp. F. oxysporum isolates from diseased United Kingdom pea plants possessed none or just one or two known SIX genes with no consistent pattern of presence/absence, leading to the conclusion that they were foot-rot causing isolates rather than FOP. In contrast, FOP isolates had different complements of SIX genes with all those identified as race 1 containing SIX1, SIX6, SIX7, SIX9, SIX10, SIX11, SIX12, and SIX14. FOP isolates that were identified as belonging to race 2 through testing on differential pea cultivars, contained either SIX1, SIX6, SIX9, SIX13, SIX14 or SIX1, SIX6, SIX13. Significant upregulation of SIX genes was also observed in planta over the early stages of infection by different FOP races in pea roots. Race specific SIX gene profiling may therefore provide potential targets for molecular identification of FOP races but further research is needed to determine whether variation in complement of SIX genes in FOP race 2 isolates results in differences in virulence across a broader set of pea differential cultivars.
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Phytophthora cactorum is often described as a generalist pathogen, with isolates causing disease in a range of plant species. It is the causative agent of two diseases in the cultivated strawberry, crown rot (CR; causing whole plant collapse) and leather rot (LR; affecting the fruit). In the cultivated apple, P. cactorum causes girdling bark rots on the scion (collar rot) and rootstock (crown rot), as well as necrosis of the fine root system (root rot) and fruit rots. We investigated evidence for host specialisation within P. cactorum through comparative genomic analysis of 18 isolates. Whole genome phylogenetic analysis provided genomic support for discrete lineages within P. cactorum, with well-supported non-recombining clades for strawberry CR and apple infecting isolates specialised to strawberry crowns and apple tissue. Isolates of strawberry CR are genetically similar globally, while there is more diversity in apple-infecting isolates. We sought to identify the genetic basis of host specialisation, demonstrating gain and loss of effector complements within the P. cactorum phylogeny, representing putative determinants of host boundaries. Transcriptomic analysis highlighted that those effectors found to be specific to a single host or expanded in the strawberry lineage are amongst those most highly expressed during infection of strawberry and give a wider insight into the key effectors active during strawberry infection. Many effectors that had homologues in other Phytophthoras that have been characterised as avirulence genes were present but not expressed in our tested isolate. Our results highlight several RxLR-containing effectors that warrant further investigation to determine whether they are indeed virulence factors and host-specificity determinants for strawberry and apple. Furthermore, additional work is required to determine whether these effectors are suitable targets to focus attention on for future resistance breeding efforts.
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The complete mitogenome of Fusarium oxysporum f. sp. albedinis (FOA), the causal agent of the destructive fusarium wilt in date palm, is sequenced and assembled. The circular mitogenome of isolate Foa44 is 51,601 bp in length and contains 26 transfer RNA (tRNA) genes, one ribosomal RNA (rRNA), and 28 protein-coding genes. A mitogenome-based phylogenetic analysis of Fusarium revealed that FOA is congruent with previous nuclear-gene phylogenetic results.
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Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.
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Colletotrichum fructicola is a causal agent of strawberry anthracnose and a major economic pathogen of horticultural and ornamental crops worldwide. Here, we present an annotated draft genome sequence for a C. fructicola isolate previously used for transcriptomic analysis. The assembly totals 58.0 Mb in 477 contigs with 18,143 predicted genes.
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The oomycete Phytophthora fragariae is a highly destructive pathogen of cultivated strawberry (Fragaria × ananassa), causing the root rotting disease, "red core". The host-pathogen interaction has a well described gene-for-gene resistance relationship, but to date neither candidate avirulence nor resistance genes have been identified. We sequenced a set of American, Canadian, and United Kingdom isolates of known race type, along with three representatives of the closely related pathogen of the raspberry (Rubus idaeus), P. rubi, and found a clear population structure, with a high degree of nucleotide divergence seen between some race types and abundant private variation associated with race types 4 and 5. In contrast, between isolates defined as United Kingdom races 1, 2, and 3 (UK1-2-3) there was no evidence of gene loss or gain; or the presence of insertions/deletions (INDELs) or Single Nucleotide Polymorphisms (SNPs) within or in proximity to putative pathogenicity genes could be found associated with race variation. Transcriptomic analysis of representative UK1-2-3 isolates revealed abundant expression variation in key effector family genes associated with pathogen race; however, further long read sequencing did not reveal any long range polymorphisms to be associated with avirulence to race UK2 or UK3 resistance, suggesting either control in trans or other stable forms of epigenetic modification modulating gene expression. This work reveals the combined power of population resequencing to uncover race structure in pathosystems and in planta transcriptomic analysis to identify candidate avirulence genes. This work has implications for the identification of putative avirulence genes in the absence of associated expression data and points toward the need for detailed molecular characterisation of mechanisms of effector regulation and silencing in oomycete plant pathogens.
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BACKGROUND: Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement and its use has been reported in a growing number of important food crops, including recently Fragaria. In order to inform application of the technology in Fragaria, we targeted the visible endogenous marker gene PDS (phytoene desaturase) in diploid Fragaria vesca ssp. vesca 'Hawaii 4' and octoploid F. × ananassa 'Calypso'. RESULTS: Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% ('Hawaii 4') and 50% ('Calypso') putative transgenic shoot lines (multiple shoots derived from a single callus) exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 75% ('Hawaii 4') and 55% ('Calypso') included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in 'Calypso'. CONCLUSION: We demonstrate that CRISPR/Cas9 may be used to generate biallelic mutants at high frequency within the genomes of diploid and octoploid strawberry. The methodology, observations and comprehensive data set presented will facilitate routine application of this technology in Fragaria to single and multiple gene copy targets where mutant phenotypes cannot be identified visually.
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Fusarium proliferatum is a component of the onion basal rot disease complex. We present an annotated F. proliferatum draft genome sequence, totaling 45.8 Mb in size, assembled into 597 contigs, with a predicted 15,418 genes. The genome contains 58 secondary metabolite clusters and homologs of the Fusarium oxysporum effector SIX2.
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Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics.
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The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.
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Verticillium dahliae is a highly detrimental pathogen of soil cultivated strawberry (Fragaria x ananassa). Breeding of Verticillium wilt resistance into commercially viable strawberry cultivars can help mitigate the impact of the disease. In this study we describe novel sources of resistance identified in multiple strawberry populations, creating a wealth of data for breeders to exploit. Pathogen-informed experiments have allowed the differentiation of subclade-specific resistance responses, through studying V. dahliae subclade II-1 specific resistance in the cultivar "Redgauntlet" and subclade II-2 specific resistance in "Fenella" and "Chandler." A large-scale low-cost phenotyping platform was developed utilizing automated unmanned vehicles and near infrared imaging cameras to assess field-based disease trials. The images were used to calculate disease susceptibility for infected plants through the normalized difference vegetation index score. The automated disease scores showed a strong correlation with the manual scores. A co-dominant resistant QTL; FaRVd3D, present in both "Redgauntlet" and "Hapil" cultivars exhibited a major effect of 18.3% when the two resistance alleles were combined. Another allele, FaRVd5D, identified in the "Emily" cultivar was associated with an increase in Verticillium wilt susceptibility of 17.2%, though whether this allele truly represents a susceptibility factor requires further research, due to the nature of the F1 mapping population. Markers identified in populations were validated across a set of 92 accessions to determine whether they remained closely linked to resistance genes in the wider germplasm. The resistant markers FaRVd2B from "Redgauntlet" and FaRVd6D from "Chandler" were associated with resistance across the wider germplasm. Furthermore, comparison of imaging versus manual phenotyping revealed the automated platform could identify three out of four disease resistance markers. As such, this automated wilt disease phenotyping platform is considered to be a good, time saving, substitute for manual assessment.
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Apple scab is one of the most economically important diseases of apples worldwide. The disease is caused by the haploid ascomycete Venturia inaequalis. We present here an annotated V. inaequalis whole-genome sequence of 72 Mb, assembled into 238 contigs, with 13,761 predicted genes.
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The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of cultivated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large number of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes differed from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phytophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene discovery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops.
Assuntos
Fragaria/microbiologia , Genoma Fúngico/genética , Phytophthora/genética , Sequenciamento Completo do Genoma , Biologia Computacional , Fragaria/genética , Phytophthora/patogenicidade , Melhoramento Vegetal , Doenças das Plantas/genética , Doenças das Plantas/microbiologiaRESUMO
A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.
Assuntos
Fusarium/patogenicidade , Genoma Fúngico/genética , Cebolas/microbiologia , Doenças das Plantas/microbiologia , Virulência/genética , Cromossomos Fúngicos/genética , Produção Agrícola , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Anotação de Sequência Molecular , Filogenia , Raízes de Plantas/microbiologia , Análise de Sequência de DNA , Sintenia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.