Wayne Pitt

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.

Evaluation of Fungicides for the Management of Botryosphaeria Canker of Grapevines

The family Botryosphaeriaceae comprises a number of species that are associated with the dieback disease of grapevine (Vitis vinifera), referred to as Botryosphaeria canker. To date, there are few effective agents available for the management of this disease. In this study, fungicides were evaluated for controlling the disease using a combination of in vitro tests and field trials. Twenty fungicides registered for use on other diseases in Australian viticulture were tested in vitro for their effect on mycelial growth of four species within the Botryosphaeriaceae. The concentrations of fungicide at which 50% of mycelial growth is inhibited (EC50 values) were significantly affected both by fungicide and isolate (P < 0.001). Differences in sensitivities of the four species to the fungicides were negligible (0.41 to 0.59 mg/liter). The most effective fungicides were fludioxonil, carbendazim, fluazinam, tebuconazole, flusilazole, penconazole, procymidone, iprodione, myclobutanil, and pyraclostrobin, for which EC50 values were <1.0 mg/liter. These fungicides were evaluated under field conditions, in addition to the pruning wound protectants Bacseal Super, Garrison, and ATCS tree wound dressing, as well as the biological control agent Vinevax. In field trials, carbendazim (Bavistin), fluazinam (Shirlan), tebuconazole (Folicur), Garrison, and ATCS tree wound dressing applied to freshly cut pruning wounds were the most effective and reduced infection by Diplodia seriata and D. mutila by 41 to 65%. These results suggest that the occurrence of Botryosphaeria canker on grapevines may be reduced via treatment of pruning wounds with selected fungicides as soon as possible after pruning.

Plectosporium alismatis comb. nov. a new placement for the Alismataceae pathogen Rhynchosporium alismatis

The phytopathogen Rhynchosporium alismatis, occurring on Alisma, Sagittaria and other genera in the Alismataceae, is under investigation as a mycoherbicide for alismataceous weeds in Australian rice crops. The type species of Rhynchosporium, R. secalis, belongs in the Helotiales while the phylogenetic relationships of R. alismatis were unknown. To identify fungi related to R. alismatis, the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1, 5.8S rRNA gene, ITS2) of 56 isolates was sequenced and compared to those available in databases. Analysis of ITS sequences revealed close relationships between R. alismatis and the teleomorph genus Plectosphaerella, as well as several anamorphic fungi which were primarily species of Verticillium. Rhynchosporium alismatis and Plectosphaerella cucumerina clustered together with 98 % bootstrap support. Morphological comparisons supported this relationship indicating that R. alismatis and the anamorphic genus Plectosporium are congeneric. Rhynchosporium alismatis is transferred to Plectosporium, a name proposed for conservation.

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.

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

Eutypa dieback, caused by the fungus Eutypa lata is a serious disease of grapevines that affects vineyard productivity and longevity. Grapevines displaying foliar symptoms typical of Eutypa dieback or evidence of dead spurs, cankers, or discoloured vascular tissue, were surveyed from 77 vineyards throughout New South Wales (NSW), Australia. Fungal cultures were tentatively identified based on cultural morphology, before further identification using sequence analysis of rDNA internal transcribed spacer regions. E. lata and several other species from the Diatrypaceae including Cryptovalsa ampelina, and species of Eutypella and Diatrypella were isolated from diseased grapevines. Eutypa dieback was found to be more widespread in NSW than first thought, with confirmation that the disease is present both in the Central Ranges and southern NSW districts, regions recognised for their cooler climates and higher annual rainfall, both of which favour the growth of E. lata.

Infection process of Plectosporium alismatis on host and non-host species in the Alismataceae

In Australia, the endemic fungus Plectosporium alismatis (syn. Rhynchosporium alismatis) has potential use as a mycoherbicide for several species in the Alismataceae, a family of aquatic and semi-aquatic marsh herbs, which are considered to be important weeds in rice crops. Of five species identified in south-eastern Australia where rice is grown, two species, Sagittaria graminea and Sagittaria montevidensis are resistant (non-hosts), and no records of P. alismatis on these species have been reported. To better understand the interactions that lead to resistance in these pathosystems, the infection process of the fungus was studied on these species and also on the host Alisma plantago-aquatica, using light, fluorescent and scanning electron microscopy. On all three species both conidial germination and appressorium formation commenced within 6 h of inoculation with greater than 50 % of conidia elongating to form germ tube structures and associated appressoria 12-18 h post inoculation. Germ tube elongation and appressorium formation occurred randomly over the leaf surface. Direct host penetration was facilitated by the production of penetration hyphae that emerged from beneath appressoria. Penetration sites were clearly identified by the presence of spherical holes 0.25-0.5 microm in diam, and were frequently accompanied by resistance reactions in non-host species. Visible symptoms of disease occurred 4-6 d after inoculation of susceptible (host) species.