Renaud Travadon

Contrasting patterns of genetic diversity and population structure of Armillaria mellea sensu stricto in the eastern and western United States.

ABSTRACT Armillaria mellea infects hundreds of plant species in natural and managed ecosystems throughout the Northern hemisphere. Previously reported nuclear genetic divergence between eastern and western U.S. isolates is consistent with the disjunct range of A. mellea in North America, which is restricted mainly to both coasts of the United States. We investigated patterns of population structure and genetic diversity of the eastern (northern and southern Appalachians, Ozarks, and western Great Lakes) and western (Berkeley, Los Angeles, St. Helena, and San Jose, CA) regions of the United States. In total, 156 diploid isolates were genotyped using 12 microsatellite loci. Absence of genetic differentiation within either eastern subpopulations (theta(ST) = -0.002, P = 0.5 ) or western subpopulations (theta(ST) = 0.004, P = 0.3 ) suggests that spore dispersal within each region is sufficient to prevent geographic differentiation. In contrast to the western United States, our finding of more than one genetic cluster of isolates within the eastern United States (K = 3), revealed by Bayesian assignment of multilocus genotypes in STRUCTURE and confirmed by genetic multivariate analyses, suggests that eastern subpopulations are derived from multiple founder sources. The existence of amplifiable and nonamplifiable loci and contrasting patterns of genetic diversity between the two regions demonstrate that there are two geographically isolated, divergent genetic pools of A. mellea in the United States.

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens

BackgroundTrunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.ResultsThe integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution.ConclusionsThis study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.

Inferring dispersal patterns of the generalist root fungus Armillaria mellea

Investigating the dispersal of the root-pathogenic fungus Armillaria mellea is necessary to understand its population biology. Such an investigation is complicated by both its subterranean habit and the persistence of genotypes over successive host generations. As such, host colonization by resident mycelia is thought to outcompete spore infections. We evaluated the contributions of mycelium and spores to host colonization by examining a site in which hosts pre-date A. mellea. Golden Gate Park (San Francisco, CA, USA) was established in 1872 primarily on sand dunes that supported no resident mycelia. Genotypes were identified by microsatellite markers and somatic incompatibility pairings. Spatial autocorrelation analyses of kinship coefficients were used to infer spore dispersal distance. The largest genotypes measured 322 and 343 m in length, and 61 of the 90 total genotypes were recovered from only one tree. The absence of multilocus linkage disequilibrium and the high proportion of unique genotypes suggest that spore dispersal is an important part of the ecology and establishment of A. mellea in this ornamental landscape. Spatial autocorrelations indicated a significant spatial population structure consistent with limited spore dispersal. This isolation-by-distance pattern suggests that most spores disperse over a few meters, which is consistent with recent, direct estimates based on spore trapping data.

Cadophora species associated with wood-decay of grapevine in North America

Cadophora species are reported from grapevine (Vitis vinifera L.) in California, South Africa, Spain, Uruguay, and Canada. Frequent isolation from vines co-infected with the Esca pathogens (Togninia minima and Phaeomoniella chlamydospora), and confirmation of its ability to cause wood lesions/discoloration in pathogenicity tests, suggest that C. luteo-olivacea is part of the trunk pathogen complex. In North America, little is known regarding the diversity, geographic distribution, and roles of Cadophora species as trunk pathogens. Accordingly, we characterized 37 Cadophora isolates from ten US states and two Canadian provinces, based on molecular and morphological comparisons, and pathogenicity. Phylogenetic analysis of three loci (ITS, translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (BT)) distinguished two known species (C. luteo-olivacea and Cadophora melinii) and three newly-described species (Cadophora orientoamericana, Cadophora novi-eboraci, and Cadophora spadicis). C. orientoamericana, C. novi-eboraci, and C. spadicis were restricted to the northeastern US, whereas C. luteo-olivacea was only recovered from California. C. melinii was present in California and Ontario, Canada. Morphological characterization was less informative, due to significant overlap in dimensions of conidia, hyphae, conidiophores, and conidiogenous cells. Pathogenicity tests confirmed the presence of wood lesions after 24 m, suggesting that Cadophora species may have a role as grapevine trunk pathogens.

Genes Expressed in Grapevine Leaves Reveal Latent Wood Infection by the Fungal Pathogen Neofusicoccum parvum

Some pathogenic species of the Botryosphaeriaceae have a latent phase, colonizing woody tissues while perennial hosts show no apparent symptoms until conditions for disease development become favorable. Detection of these pathogens is often limited to the later pathogenic phase. The latent phase is poorly characterized, despite the need for non-destructive detection tools and effective quarantine strategies, which would benefit from identification of host-based markers in leaves. Neofusicoccum parvum infects the wood of grapevines and other horticultural crops, killing the fruit-bearing shoots. We used light microscopy and high-resolution computed tomography (HRCT) to examine the spatio-temporal relationship between pathogen colonization and anatomical changes in stem sections. To identify differentially-expressed grape genes, leaves from inoculated and non-inoculated plants were examined using RNA-Seq. The latent phase occurred between 0 and 1.5 months post-inoculation (MPI), during which time the pathogen did not spread significantly beyond the inoculation site nor were there differences in lesion lengths between inoculated and non-inoculated plants. The pathogenic phase occurred between 1.5 and 2 MPI, when recovery beyond the inoculation site increased and lesion lengths of inoculated plants tripled. By 2 MPI, inoculated plants also had decreased starch content in xylem fibers and rays, and increased levels of gel-occluded xylem vessels, the latter of which HRCT revealed at a higher frequency than microscopy. RNA-Seq and screening of 21 grape expression datasets identified 20 candidate genes that were transcriptionally-activated by infection during the latent phase, and confirmed that the four best candidates (galactinol synthase, abscisic acid-induced wheat plasma membrane polypeptide-19 ortholog, embryonic cell protein 63, BURP domain-containing protein) were not affected by a range of common foliar and wood pathogens or abiotic stresses. Assuming such host responses are consistent among cultivars, and do not cross react with other trunk/foliar pathogens, these grape genes may serve as host-based markers of the latent phase of N. parvum infection.

Diversity of Diaporthe species associated with wood cankers of fruit and nut crops in northern California

Diaporthe ampelina, causal agent of Phomopsis cane and leaf spot of grapevine (Vitis vinifera L.) is isolated frequently from grapevine wood cankers, causing Phomopsis dieback. The latter disease is associated with four other Diaporthe species, three of which also are reported from hosts other than grape. To better understand the role of this Diaporthe community in Phomopsis dieback of grapevine and the potential for infection routes among alternate hosts, 76 Diaporthe isolates were recovered from wood cankers of cultivated grape, pear, apricot, almond and the wild host willow in four California counties. Isolates were characterized morphologically and assigned to species based on multigene sequence analyses. This study identified eight Diaporthe species from grapevine and one novel taxon from willow, D. benedicti. We report the first findings of D. australafricana and D. novem in North America. Our findings also expand the host ranges of D. ambigua to apricot and willow, D. australafricana to almond and willow, D. chamaeropis to grapevine and willow, D. foeniculina to willow and D. novem to almond. The generalists D. ambigua and D. eres were the most genetically diverse species, based on high nucleotide and haplotypic diversity, followed by the grapevine specialist D. ampelina. Analyses based on multilocus linkage disequilibrium could not reject the hypothesis of random mating for D. ambigua, which is further supported by relatively high haplotypic diversity, reports of both mating types and reports of successful matings in vitro. Pathogenicity assays revealed that D. ampelina was the most pathogenic species to grapevine wood.

Characterization of Cytospora isolates from wood cankers of declining grapevine in North America, with the descriptions of two new Cytospora species

Cytospora species are ubiquitous pathogens of numerous woody plants, causing dieback and wood cankers in agronomic crops, timber trees, and wildland trees (e.g., Prunus, Eucalyptus, and Salix, respectively). Cytospora chrysosperma, C. cincta, and C. leucostoma have been reported from grapevines in Iran showing symptoms of one or more recognized trunk diseases (Esca, Botryosphaeria-, Eutypa-, and Phomopsis diebacks); however, only C. chrysosperma was shown to be pathogenic to grapevine. To understand the potential role of Cytospora species in the grapevine trunk-disease complex, we examined 21 Cytospora isolates that were recovered from dieback and wood cankers of Vitis vinifera and Vitis interspecific hybrids in seven northeastern U.S. states and two Canadian provinces. Phylogenetic analyses of ITS and translation elongation factor 1-α identified two novel species: Cytospora vinacea sp. nov. and Cytospora viticola sp. nov. Differences in culture morphology and conidial dimensions also distinguished the species. When inoculated to the woody stems of potted V. vinifera ‘Thompson Seedless’ in the greenhouse, both species were pathogenic, based on development of wood lesions and thus fulfillment of Kochs postulates. Cytospora viticola was the most virulent based on lesion length at 12 months post-inoculation. As Cytospora canker shares some of the same general dieback-type symptoms as Botryosphaeria-, Eutypa-, and Phomopsis diebacks, it may be considered part of the grapevine trunk-disease complex in eastern North America. This article is protected by copyright. All rights reserved.

Molecular Polymorphism and Phenotypic Diversity in the Eutypa Dieback Pathogen Eutypa lata

Pathogen adaptation to different hosts can lead to specialization and, when coupled with reproductive isolation, genome-wide differentiation and ecological speciation. We tested the hypothesis of host specialization among California populations of Eutypa lata (causal fungus of Eutypa dieback of grapevine and apricot), which is reported from >90 species. Genetic analyses of nine microsatellite loci in 182 isolates from three hosts (grapevine, apricot, and willow) at three locations were complemented by cross-inoculations on cultivated hosts grapevine and apricot to reveal patterns of host specialization. The cultivated hosts are likely more important sources of inoculum than the wild host willow, based on our findings of higher pathogen prevalence and allelic richness in grapevine and apricot. High levels of gene flow among all three hosts and locations, and no grouping by clustering analyses, suggest neither host nor geographic differentiation. Cross-inoculations revealed diversified phenotypes harboring various performance levels in grapevine and apricot, with no apparent correlation with their host of origin. Such phenotypic diversity may enable this pathogen to persist and reproduce as a generalist. Regular genetic reshuffling through sexual recombination, frequent immigration among hosts, and the lack of habitat choice in this passively dispersed fungus may prevent fixation of alleles controlling host specialization.

Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, with the descriptions of ten new species and one new combination

Cytospora species are destructive canker and dieback pathogens of woody hosts in natural and agroecosystems around the world. In this genus, molecular identification has been limited due to the paucity of multi-locus sequence typing studies and the lack of sequence data from type specimens in public repositories, stalling robust phylogenetic reconstructions. In most cases a morphological species concept could not be applied due to the plasticity of characters and significant overlap of morphological features such as spore dimensions and fruiting body characters. In this study, we employed a molecular phylogenetic framework with the inclusion of four nuclear loci (ITS, translation elongation factor 1-alpha, actin, and beta-tubulin) to unveil the biodiversity and taxonomy of this understudied important genus of plant pathogens. Phylogenetic inferences based on 150 Californian isolates revealed 15 Cytospora species associated with branch and twig cankers and dieback of almond, apricot, cherry, cottonwood, olive, peach, pistachio, plum, pomegranate, and walnut trees in California. Of the 15 species recovered in this study, 10 are newly described and typified, in addition to one new combination. The pathogenic status of the newly described Cytospora species requires further investigation as most species were associated with severe dieback and decline of diverse and economically important fruit and nut crops in California.

Whole-genome resequencing and pan-transcriptome reconstruction highlight the impact of genomic structural variation on secondary metabolite gene clusters in the grapevine esca pathogen phaeoacremonium minimum

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.