Florent P. Trouillas

A six locus phylogeny reveals high species diversity in Botryosphaeriaceae from California almond

Botryosphaeriaceae are important pathogens on a variety of woody hosts, including almond, a major crop in California. Almond is susceptible to Botryosphaeria dothidea that forms band cankers on almond trunks, and the same fungus was also isolated from cankers of the canopy. To study the diversity and host range of B. dothidea and allied species from almond we used 132 isolates from 36 plant hosts from five continents, including 45 strains from almond in California. Species were identified by comparison to 13 ex-type strains with phylogenetic analyses based on six loci, including the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene repeat and portions of the coding genes elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein, histone-3 and beta-tubulin. Seven species were found from almond: Botryosphaeria dothidea, Neofusicoccum parvum, Neof. mediterraneum, Neof. nonquaesitum, Diplodia seriata and Macrophomina phaseolina were identified from band cankers, and B. dothidea, Neof. mediterraneum, Neof. parvum and Dothiorella sarmentorum from canopy cankers. All were capable of inducing cankers on inoculated almond branches in the field. All species found on almond also occurred on other hosts, suggesting that infected vegetation adjacent to almond orchards could serve as source of inoculum of virulent almond strains. Of the 19 monophyletic groups obtained at the species level, 13 contained ex-type strains, five were morphologically similar to established species and one was morphologically distinct from its closest relatives, Neof. andinum and Neof. arbuti, as well as from the more than 190 described species of Fusicoccum and Neofusicoccum, and thus was described as the new species, Neof. nonquaesitum. Evidence for cryptic speciation was found in B. dothidea, Neof. ribis and Spencermartinsia viticola. Botryosphaeria dothidea and Neof. ribis comprised lineages that formed the morphologically distinct Dichomera anamorph not found in any other isolates recognized as B. dothidea and Neof. ribis. An S. viticola isolate from California was phylogenetically divergent and had conidia that differed morphologically from the type. Neofusicoccum parvum was diverse but lacked any morphological features correlating with molecular diversity. Phylogenetic analyses of combinations of datasets showed that pooled analyses of all six datasets resulted in the highest number of supported branches, suggesting that addition of more data might yet improve phylogenetic resolution.

Diversity of diatrypaceous fungi associated with grapevine canker diseases 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 β-tubulin gene. Diatrypaceous fungi also occurred frequently on the native flora surrounding vineyards, thus serving as possible inoculum sources for these putative pathogens.

Identification and characterization of Eutypa leptoplaca, a new pathogen of grapevine in Northern California.

Although Eutypa lata is the main agent of Eutypa dieback of grapevine, another species of Eutypa has been isolated from diseased grapevines in Northern California. Stromata of this recently discovered Eutypa were also collected from Acer macrophyllum, A. negundo, and Fraxinus latifolia in the vicinity of vineyards, and appeared commonly on Umbellularia californica in some mixed-evergreen forests of Napa and Sonoma counties. This second species of Eutypa was distinguished from E. lata because of the sulcate ostiole of the perithecium and smaller ascospores. A morphological comparison with type specimens revealed identical features between the Californian isolates and E. leptoplaca sensu Rappaz (1987). This identification was confirmed through phylogenetic analyses of Eutypa spp. based on the complete sequence of the internal transcribed spacer (ITS) of the rDNA and partial sequence of the beta-tubulin gene. These analyses also separated collections of E. maura, E. sparsa, E. lejoplaca, E. tetragona, E. leptoplaca and E. lata, confirming the previously proposed species concepts. The pathogenicity of E. leptoplaca on grapevine was established using isolates collected from Vitis vinifera, U. californica, and A. macrophyllum. The importance of E. leptoplaca in relation to Eutypa dieback and its role as a necrotrophic pathogen are discussed.

Taxonomy and DNA phylogeny of Diatrypaceae associated with Vitis vinifera and other woody plants in Australia

The Diatrypaceae occur worldwide and comprise a number of pathogens of woody crops, forest and ornamental tree species. Despite the taxonomic difficulties within this family, interest in the Diatrypaceae has increased recently, mainly due to the recent detection of these fungi in the premium grape growing regions of California. In the present study, we investigated the diversity and host range of diatrypaceous fungi from prominent wine grape growing regions in South Australia, New South Wales and Western Australia. Approximately 100 isolates were collected from grapevine and other woody plants and compared with reference collections from the United States and Europe. Phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA and partial sequence of the β-tubulin gene, combined with morphological analyses separated 12 species. These included the previously described species Cryptovalsa ampelina, C. rabenhorstii, Diatrype brunneospora, Eutypa lata, E. leptoplaca, Eutypella australiensis, E. citricola, a Cryptosphaeria sp. and a Diatrype sp., whereas Diatrypella vulgaris, Eutypella cryptovalsoidea and E. microtheca are described as new. Seven species were isolated from grapevine but the prevalence of Diatrypaceae in grapevine cankers varied among the regions surveyed. In many instances in WA and NSW, these newly reported fungi were more widespread and abundant than E. lata. This study provides new information to assist with diagnosis of the causal agents of dieback and canker diseases in Australia and development of management strategies. Further studies to characterize the pathogenicity of diatrypaceous species to grapevines and to elucidate the biology of these fungi are underway.

Pathogenicity of Diatrypaceae species in grapevines in California.

Diatrypaceous fungi have been isolated from cankered wood of grapevines (Vitis vinifera) in California. However, the pathogenicity of these fungi and their potential to cause diseases in grapevine was unknown. A series of pathogenicity tests were conducted to determine if these fungi were grapevine pathogens. In all experiments, species of the family Diatrypaceae were re-isolated from the margins of developing lesions, although frequency of re-isolation and average length of vascular discolorations varied among isolates. Cryptovalsa ampelina, Diatrype stigma, and Eutypa leptoplaca were capable of colonizing dormant canes and causing vascular necrosis. Cryptosphaeria pullmanensis, C. ampelina, D. stigma, Diatrype whitmanensis, and E. leptoplaca infected and caused lesions in green shoots of the new vegetative growth. Diatrype oregonensis and Diatrypella verrucaeformis isolates did not produce lesions that were significantly different from those produced in the control shoots and canes. This suggests that D. oregonensis and D. verrucaeformis fungi may be saprophytic rather than pathogenic to grapevine.

Dothiorella vidmadera, a novel species from grapevines in Australia and notes on Spencermartinsia

Strains of a coelomycete isolated from grapevine cankers in southeastern Australia and identified as Dothiorella iberica in previous studies are redescribed in this study as a novel species based on morphological characters and phylogenetic analyses of DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), and partial sequences of the translation elongation factor 1-α and β-tubulin genes. Dothiorella vidmadera sp. nov. is most closely related to D. iberica, D. americana and D. sarmentorum, but differs in morphological characters and DNA sequences. All four species are known to be associated with Botryosphaeria dieback of grapevines. Additionally, an unidentified ascomycete isolated from dead wood of grapevines in Western Australia is described. Phylogenetically, strains were most closely related to Spencermartinsia viticola, and bore conidia with morphological characters and dimensions consistent with published descriptions. However, ascospores were shorter and narrower than previously reported and lacked the terminal apiculi that typify the genus.

Dothiorella and Spencermartinsia, new species and records from grapevines in Australia

Dothiorella and Spencermartinsia are two Botryosphaeriaceous genera found on various woody hosts that bear two-celled conidia that become brown and 1-septate prior to dehiscence from conidiogenous cells. Based on morphological characters and phylogeny of DNA sequences of the internal transcribed spacer (ITS) region and part of the translation elongation factor (TEF) 1–α gene, three new species, namely Spencermartinsia westrale W.M. Pitt, J.R. Úrbez-Torres & Trouillas, Dothiorella neclivorem W.M. Pitt & J.R. Úrbez-Torres and Dothiorella vinea-gemmae W.M. Pitt & J.R. Úrbez-Torres are described from grapevines in Australia. Additionally, Spencermartinsia plurivora Abdollahz., Javadi & A.J.L. Phillips, and a Dothiorella species, currently unnamed but previously isolated from walnut (Juglans regia L.) in France and Iran and hop hornbeam (Ostrya carpinifolia Scop.) in Italy, are reported for the first time in Australia, the latter also for the first time from grapevines.

First Report of Twig and Branch Dieback of English Walnut (Juglans regia) Caused by Neofusicoccum mediterraneum in California

California produces 99% of the U.S. English walnut crop with more than 30 cultivars on ~89,000 ha. Production for 2008 was ~436,000 tons with a value of

Efficacy of fungicides on mycelial growth of diatrypaceous fungi associated with grapevine trunk disease

527 million. In early summer of 2009 and 2010, branch and twig dieback of English walnut (Juglans regia L.) was detected in orchards in Yolo County and submitted to our diagnostic laboratory. Disease symptoms included death of twig tips, branch dieback, wood lesions, and canker formation. Pycnidia were embedded within the bark of dead twigs. Conidia from pycnidia were hyaline, fusoid-ellipsoidal, widest usually in the middle, and 21 to 24 (-27) × 5 to 7 μm (n = 30). Isolations from cankers yielded the fungus Neofusicoccum mediterraneum Crous, M.J. Wingf. & A.J.L. Phillips (1). Fungal colonies of N. mediterraneum grew light olive green to gray on potato dextrose agar, becoming dark olive green with age. Identification of fungal isolates was confirmed by sequence comparison of Californian isolates with ex-type (CBS 121558) sequences in GenBank (3) using the internal transcribed spacer region of the rDNA, a portion of the β-tubulin gene, and part of the translation elongation factor. Sequences of Californian isolates (GenBank HM443604-HM443609) were identical to the ex-type sequences for all three genes. Previous studies in California reported the occurrence and pathogenicity of N. mediterraneum into grapevine (Vitis vinifera L.) (3) and almond trees (Prunis dulcis L.) (2). Inderbitzin et al (2) investigated the host range of N. mediterraneum in California and reported the occurrence of pycnidia on English walnut trees. However, this study did not investigate the pathogenicity of N. mediterraneum on this host. In the current study, the pathogenicity of N. mediterraneum in J. regia cvs. Hartley and Chandler was investigated in an orchard at UC Davis using two fungal isolates. Pathogenicity tests were performed by inoculating eight 2- to 4-year-old branches of mature J. regia trees. Inoculations were made in June 2009 with a 5-mm cork borer to remove bark and placing an 8-day-old 5-mm-diameter agar plug bearing fresh mycelium of the fungal isolates directly into the fresh wound, mycelium side down. An additional eight branches of each cultivar were inoculated with noncolonized agar plugs to serve as controls. Inoculated wounds were covered with petroleum jelly and wrapped with Parafilm to retain moisture. Branches were harvested after 10 months of incubation and checked for canker development. The extent of vascular discoloration was measured in each branch and isolations were made from the edge of discolored tissue to confirm Kochs postulates. Statistical analyses were performed with analysis of variance and Dunnetts t-test to assess significant differences in the extent of vascular discoloration between inoculations with N. mediterraneum and the control. Necrosis length for the two isolates averaged 131.5 mm in Hartley branches and 110 mm in the Chandler branches. Average necrosis lengths in the control branches were 18.5 mm and 16.7 mm, respectively, significantly lower (P < 0.05) than the average necrosis length found in branches inoculated with N. mediterraneum. Fungal recovery was 75% in both varieties. To our knowledge, this study is the first report of N. mediterraneum as a pathogen of J. regia trees in California. References: (1) P. W. Crous et al. Fungal Planet 19, 2007. (2) P. Inderbitzin et al. Mycologia. Online publication. doi:10.3852/10-006, 2010. (3) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

Evidence that Eutypa lata and other diatrypaceous species occur in New South Wales vineyards.

Several species of Diatrypaceae have been recently isolated from the wood of cankered grapevines in several regions of the world and shown to be pathogenic with varying degrees of virulence when inoculated in stems of potted vines. Existing management strategies have focussed on the development of procedures and products to prevent or reduce Eutypa lata infection. The best method to prevent infection is by applying fungicides to wounds, but there are few registered chemicals for any of the diatrypaceous fungi in Australia. Six selected fungicides were evaluated in vitro for their efficacy in reducing mycelial growth of Eutypa lata, Cryptovalsa ampelina, Diatrypella vulgaris, Eutypa leptoplaca, Eutypella citricola and Eutypella microtheca. Carbendazim, fluazinam, tebuconazole, and prothioconazole + tebuconazole were effective at inhibiting mycelial growth of all Diatrypaceae spp. tested. Pyraclostrobin reduced colony diameter of most of the fungal species by 50% or more. Pyrimethanil was ineffective at reducing mycelial growth of these pathogens. Five of the six fungicides evaluated in this study have shown efficacy in vitro against Diatrypaceae spp. and require further evaluation in the field. This study represents the first approach for fungicide evaluation against mycelial growth of diatrypaceous fungi, other than Ea. lata. It contributes to the development of integrated management strategies for grapevine trunk diseases.