Sandra Savocchia

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.

Survey of Botryosphaeriaceae associated with grapevine decline in the Hunter Valley and Mudgee grape growing regions of New South Wales

Species belonging to the fungal family Botryosphaeriaceae are important pathogens of grapevines in Australia. A survey of declining grapevines in the Hunter Valley and Mudgee grape growing regions of New South Wales revealed 36% were infected with species belonging to the Botryosphaeriaceae. The incidence of Diplodia seriata was greatest, followed by Neofusicoccum parvum, Botryosphaeria dothidea and Lasiodiplodia theobromae. These identifications were made using a combination of molecular and morphological characters. Although D. seriata was the most common species found, its role as a primary pathogen of grapevines in Australia is yet to be ascertained. The accuracy of estimating the incidence of species was increased by surveying up to 25 grapevines per vineyard and by sampling both the trunks and cordons. Other pathogens capable of causing trunk diseases were also isolated in this survey, demonstrating that diagnosis based on symptoms alone is not sufficient and isolations on artificial media and sometimes DNA sequencing are required for a definitive diagnosis of the causal organism of decline.

Detection and monitoring of Greeneria uvicola and Colletotrichum acutatum development on grapevines by real-time PCR

Bitter rot (Greeneria uvicola) and ripe rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in subtropical grape-growing regions of Australia, where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute toward their effective management and control, a rapid and reliable species-specific real-time polymerase chain reaction (PCR) method was developed based on the polymorphic portion of the internal transcribed spacer region of the two fungi. It was found that, within 6 to 8 h postinoculation, the assay could detect as little as 20 fg of genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid, and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.

Biology and biocontrol of Sclerotinia sclerotiorum (Lib.) de Bary in oilseed Brassicas

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen infecting over 500 host species including oilseed Brassicas. The fungus forms sclerotia which are the asexual resting structures that can survive in the soil for several years and infect host plants by producing ascospores or mycelium. Therefore, disease management is difficult due to the long term survivability of sclerotia. Biological control with antagonistic fungi, including Coniothyrium minitans and Trichoderma spp, has been reported, however, efficacy of these mycoparasites is not consistent in the field. In contrast, a number of bacterial species, such as Pseudomonas and Bacillus display potential antagonism against S. sclerotiorum. More recently, the sclerotia-inhabiting strain Bacillus cereus SC-1, demonstrated potential in reducing stem rot disease incidence of canola both in controlled and natural field conditions via antibiosis. Therefore, biocontrol agents based on bacteria could pave the way for sustainable management of S. sclerotiorum in oilseed cropping systems.

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.

Botryosphaeria dothidea associated with grapevine trunk disease in south-eastern Australia

Species of Botryosphaeria have been implicated in grapevine decline and dieback symptoms in Australian vineyards. In this study, we report the morphological and molecular identification of Botryosphaeria dothidea isolated from symptomatic grapevines in eastern grape growing regions of New South Wales, Australia. The pathogenicity of B. dothidea towards Vitis vinifera cv. Chardonnay is also confirmed.

Grapevine inflorescences are susceptible to the bunch rot pathogens, Greeneria uvicola (bitter rot) and Colletotrichum acutatum (ripe rot)

Grapevine inflorescences (cv. Chardonnay) were found to be susceptible to infection by the berry rotting pathogens Colletotrichum acutatum and Greeneria uvicola responsible for ripe rot and bitter rot of grapes respectively. Infection of inflorescences on field-grown grapevines at mid-flowering led to subsequent berry rot at veraison. An application of the strobilurin fungicide Cabrio (active ingredient pyraclostrobin) at flowering reduced the incidence of ripe rot and bitter rot at veraison from 88% to 0% and from 86% to 2%, respectively. The infection of detached inflorescences was influenced by temperature and was greatest at 25–30°C for C. acutatum and 30°C for G. uvicola. Our results demonstrate for the first time that grapevine flowers are susceptible to C. acutatum and G. uvicola and that flower infections have the potential to lead to subsequent rotting of the grape berries. The findings have implications for the management of ripe rot and bitter rot of grapes.

Isolation of phytotoxic phenols and characterization of a new 5-hydroxymethyl-2-isopropoxyphenol from Dothiorella vidmadera, a causal agent of grapevine trunk disease

Polyphenols were characterized from Dothiorella vidmadera (DAR78993), which was isolated from a grapevine in Australia. In total, six polyphenols were isolated including a new polyphenol characterized by a spectroscopic method (essentially NMR and HR ESIMS) as 5-hydroxymethyl-2-isopropoxyphenol. Tyrosol, benzene-1,2,4-triol, resorcinol, 3-(hydroxymethyl)phenol, and protocatechuic alcohol, the latter being the main metabolite, were also isolated. Although these are already known as naturally occurring compounds in microorganisms and plants, this is the first time they have been isolated from fungal organisms involved in grapevine trunk disease. When assayed on tomato seedlings, all the compounds show similar phytotoxic effects. However, when assayed on grapevine leaves (Vitis vinifera cv Shiraz), resorcinol was the most toxic compound, followed by protocatechuic alcohol and 5-hydroxymethyl-2-isopropoxyphenol.

Genetic variation of Pleiochaeta setosa from Lupinus albus

A hierarchical collection of isolates of the fungal pathogen Pleiochaeta setosa was made from field-grown Lupinus albus plants across southern NSW, Australia in 2004. This collection, along with some historical reference isolates, was analysed for genetic variation using random amplified polymorphic DNA and enterobacterial repetitive intergenic consensus primer PCR. Anchored inter-simple sequence repeat primers were tested but produced little or no amplification or no detectable polymorphism. The observed overall variation was not explained by the hierarchical structure of the population (individual seedling, host species, host cultivar, regional location, or year). There was significant genetic variation between isolates even at the finest sampling scale (within a single seedling). Some very distinct individual isolates were identified. The high level of scale-independent genetic diversity seen in P. setosa suggests that field-based screening for Pleiochaeta root rotresistant genotypes in a L. albus breeding program at a single location is likely to result in resistance which is effective at multiple and widely separated locations. This resistance would have an increased chance of being polygenically controlled and durable under commercial cultivation conditions.