Germination of conidia of eight botryosphaeriaceous fungi infecting grapevines was evaluated after 2, 4, 6, 12, and 24 h incubation under eight different temperatures (5, 10, 15, 20, 25, 30, 35, and 40°C). The effect of temperature on conidial germination was also evaluated in different stages (hyaline versus pigmented conidia) of the species Lasiodiplodia theobromae. Conidial germination of Botryosphaeriaceae species infecting grapevines was significantly affected by temperature. Overall, conidial germination increased significantly with longer incubation times, especially from 2 to 12 h. In most cases, germination of conidia was not significantly different between 12 and 24 h incubation. Conidia of botryosphaeriaceous species did not germinate (with the exception of Botryosphaeria dothidea and Neofusicoccum parvum) at 5°C, and only B. dothidea, Diplodia seriata, and L. theobromae showed high levels of conidial germination at 40°C. Optimum conidial germination temperatures (defined as the temperature in which germination reached at least 50% in the shortest incubation time) were 25°C for B. dothidea and Dothiorella iberica, 25 to 30°C for Spencermartinsia viticola, 30°C for Diplodia corticola, D. mutila, D. seriata, N. parvum, and hyaline L. theobromae, and 40°C for pigmented L. theobromae conidia. Successful conidial germination of species of Botryosphaeriaceae infecting grapevines was always observed between 10 and 35°C with the exception of Dothiorella iberica and pigmented L. theobromae conidia, neither of which germinated at 35 and 10°C, respectively. Results of this study show conidia of botryosphaeriaceous species infecting grapevines to be capable of germination under a broad range of temperatures including those considered to be extreme, which may explain the success of these species as grapevine pathogens throughout most of the grape-growing areas in both Northern and Southern Hemispheres.

BackgroundPowdery mildew, caused by the obligate biotrophic fungus Erysiphe necator, is an economically important disease of grapevines worldwide. Large quantities of fungicides are used for its control, accelerating the incidence of fungicide-resistance. Copy number variations (CNVs) are unbalanced changes in the structure of the genome that have been associated with complex traits. In addition to providing the first description of the large and highly repetitive genome of E. necator, this study describes the impact of genomic structural variation on fungicide resistance in Erysiphe necator.ResultsA shotgun approach was applied to sequence and assemble the genome of five E. necator isolates, and RNA-seq and comparative genomics were used to predict and annotate protein-coding genes. Our results show that the E. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. Frequent structural variations were found between isolates and included copy number variation in EnCYP51, the target of the commonly used sterol demethylase inhibitor (DMI) fungicides. A panel of 89 additional E. necator isolates collected from diverse vineyard sites was screened for copy number variation in the EnCYP51 gene and for presence/absence of a point mutation (Y136F) known to result in higher fungicide tolerance. We show that an increase in EnCYP51 copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. Increased EnCYP51 copy numbers were detected with the Y136F allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. We also show that EnCYP51 copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a DMI fungicide.ConclusionsTaken together our results show that CNV can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. The results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1081) contains supplementary material, which is available to authorized users.

Botryosphaeria spp. recently have been identified as important grapevine pathogens worldwide. To date, Botryosphaeria rhodina has been the only species associated with cankers on Vitis vinifera in California. A field survey of 166 vineyards in 21 counties was conducted in order to determine the occurrence of other Botryosphaeria spp. in California. In all, 1,735 samples of cankered trunks, cordons, and spurs were collected. Botryosphaeria spp. were the most common fungi isolated from grapevine cankers in California. Morphological identification along with phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA (rDNA) and a partial sequence of the β-tubulin gene showed that at least seven Botryosphaeria spp. occur on grapevines in California: B. australis, B. dothidea, B. lutea, B. obtusa, B. parva, B. rhodina, and B. stevensii. Botryosphaeria spp. were found in grapevine cankers in all grape-growing regions surveyed in California, whereas incidence and distribution varied with location. Grapevine cankers in California have been associated mainly with Eutypa dieback. However, the frequent recovery of Botryosphaeria spp. from cankers in this study indicates that the role of these fungi in grapevine health needs to be more carefully considered.

Fungal species in the family Botryosphaeriaceae have been recently recognized as the most common fungi isolated from grapevine (Vitis vinifera) cankers in California. However, the role of these fungi in causing grapevine dieback as well as their status as canker-causing agents was unknown. Therefore, pathogenicity studies were conducted to determine their importance as grapevine pathogens in California. A total of 72 isolates representing all nine Botryosphaeriaceae species isolated from grapevine cankers from California were used in five different pathogenicity studies. Overall, experiments showed all nine Botryosphaeriaceae species able to infect both young and mature tissues as well as green shoots of the new vegetative growth causing cankers, vascular discoloration, and/or otherwise dark streaking of the wood. However, virulence varied among species. Lasiodiplodia theobromae was the most virulent species followed by Neofusicoccum luteum, N. parvum, and N. australe, all categorized as highly virulent. Botryosphaeria dothidea was considered intermediately virulent and Diplodia mutila, D. seriata, Dothiorella iberica, and D. viticola were shown to be weakly virulent. This study shows species of Botryosphaeriaceae to be much more important pathogens on grapevines than originally thought, and some of them, in view of their virulence, should be considered high risk for causing severe and rapid canker and dieback diseases in the grapevine industry in California.

Perennial cankers and consequent grapevine dieback are a major problem in vineyards of Sonora and Baja California, the most important grape-production areas of Mexico. In order to identify the canker-causing agents, symptomatic arms, cordons, and trunks were collected from 13 and 6 vineyards in Sonora and Baja California, respectively. Two Botryosphaeriaceae spp., Lasiodiplodia theobromae and Diplodia seriata, were isolated frequently from infected wood and identified based on morphological and cultural characters as well as analyses of nucleotide sequences of three genes, the internal transcribed spacer region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). Although both L. theobromae and D. seriata were isolated from grapevine cankers in Baja California, only L. theobromae was found in vines in the Sonora region. Pathogenicity of both species was verified by inoculation of rooted cuttings and green shoots of Thompson Seedless and Chardonnay cultivars. Isolates of L. theobromae were more virulent, based on the extent of spread in the secondary wood and green tissue, than those of D. seriata. These findings confirm L. theobromae and D. seriata as the causal agents of dieback and canker formation of grapevines in northern Mexico.

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Eighteen different fungal species were isolated from symptomatic wood of olive trees (Olea europaea) affected by twig and branch dieback in California and identified by means of morphological characters and multigene sequence analyses of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). These species included Diaporthe viticola, Diatrype oregonensis, Diatrype stigma, Diplodia mutila, Dothiorella iberica, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Phomopsis sp. group 1, Phomopsis sp. group 2, and Schizophyllum commune, which are for the first time reported to occur in olive trees; Eutypa lata, Neofusicoccum luteum, Neofusicoccum vitifusiforme, and Phaeoacremonium aleophilum, which are for the first time reported to occur in olive trees in the United States; and Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum mediterraneum, and Trametes versicolor, which have been previously reported in olive trees in California. Pathogenicity studies conducted in olive cultivars Manzanillo and Sevillano showed N. mediterraneum and Diplodia mutila to be the most virulent species and Diatrype stigma and D. oregonensis the least virulent when inoculated in olive branches. Intermediate virulence was shown for the rest of the taxa. This study demystifies the cause of olive twig and branch dieback and elucidates most of the fungal pathogens responsible for this disease in California.

Eutypa lata is the most common diatrypaceous fungus in grapevine cankers. This study was undertaken to determine the presence and diversity of other members of the Diatrypaceae in grapevines in California. We isolated into pure culture 11 species of Diatrypaceae from the wood of diseased grapevines including Cryptosphaeria pullmanensis, Cryptovalsa ampelina, Diatrype oregonensis, D. stigma, D. whitmanensis, Diatrype sp., Diatrypella verrucaeformis and four putative species of Eutypella. Species were identified or grouped morphologically and by phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) of the rDNA and beta-tubulin gene. Diatrypaceous fungi also occurred frequently on the native flora surrounding vineyards, thus serving as possible inoculum sources for these putative pathogens.