M. R. Sosnowski

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.

The Influence of Grapevine Cultivar and Isolate of Eutypa lata on Wood and Foliar Symptoms

Grapevine cultivar (Vitis vinifera) and isolate of Eutypa lata influence wood and foliar symptoms of Eutypa dieback. Foliar symptoms of Eutypa dieback developed within 8 months of inoculating young grapevines (cvs. Grenache, Cabernet Sauvignon, and Merlot) in a shadehouse. Isolates of E. lata from various wine regions in southern Australia varied in their ability to colonize inoculated grapevines and induce wood and foliar symptoms. Grapevine cultivars varied for wood and foliar symptom expression but not for mycelial colonization. However, the severity of foliar symptoms was not related to the rate of spread of the fungus in the grapevine. Furthermore, the staining of wood typically attributed to E. lata did not reflect the presence of the fungus because the fungus was detected up to 80 mm beyond the stain. A field trial with mature grapevines revealed significant differences in the rate of spread of wood staining due to E. lata among eight cultivars, with up to 50 mm/year detected in Cabernet Sauvignon and Shiraz grapevines. In the shadehouse, the maximum growth rate of E. lata was recorded to be 115 mm/year for Grenache rootlings. Information from this study may help to optimize management strategies for maintaining productivity of grapevines with Eutypa dieback, thus reducing the economic impact of the disease.

Pathogenic variation of South Australian isolates of Leptosphaeria maculans and interactions with cultivars of canola (Brassica napus)

Cultural characteristics and pathogenicity tests showed that South Australian isolates of Leptosphaeria maculans (Desm.) Ces. et de Not. were highly variable. Isolates were ranked in a continuum based on the severity of disease symptoms on plants, and two of the 40 isolates tested showed very similar characteristics and pathogenicity to a known avirulent isolate. The early stages of infection, on the surface of leaves, through the epidermis and into the mesophyll, were examined histologically, using two canola cultivars (susceptible and resistant) and two isolates (virulent and avirulent). Differences in frequency of penetration into the plant tissue were observed indicating interactions between cultivars and isolates. Up to 6 days after inoculation, hyphae penetrated the stomata more frequently on the susceptible cv. Hyola 42 than on the resistant cv. Dunkeld. This was linked to the lower density of stomata per unit area on Dunkeld compared to Hyola 42.

The influence of climate on foliar symptoms of eutypa dieback in grapevines

ABSTRACT Foliar symptoms of Eutypa dieback, caused by Eutypa lata, in grapevines, cv. Shiraz, varied from year to year in a 6-year study conducted in South Australia and, although trends were similar for vineyards within geographical regions, differences were observed between regions. We attempted to elucidate the causes underlying this variation and hypothesized that it was influenced by climatic factors. A number of possible relationships were identified between climate and symptom expression: (i) increased symptom expression was related to increased winter rainfall 18 months earlier, (ii) decreased disease incidence and prevalence were related to increased temperature in spring, and (iii) a reduction in disease incidence was related to both very high and very low rainfall in October. Theories for these relationships are proposed and require further investigation. A conceptual model was developed which requires validation and has the potential to predict the incidence of foliar symptoms of Eutypa dieback. Information from this study could lead to an improved integrated pest management system to suppress foliar symptoms and sustain productivity of vines infected with E. lata.

Infection of Australian canola cultivars (Brassica napus) by Leptosphaeria maculans is influenced by cultivar and environmental conditions

Infection of canola (Brassica napus) by the blackleg fungus (Leptosphaeria maculans) and subsequent development of leaf and stem lesions was influenced by cultivar resistance and weather conditions. Cultivars Dunkeld, Hyola 42, Karoo, Monty, Pinnacle, Scoop and Mystic showed varying resistance to stem and cotyledon infection, but were susceptible to leaf infection. Fewer lesions were observed on the leaves of the highly resistant cv. Hyola 60 than on other cultivars and scanning electron microscopy revealed that pycnidiospore germination and mycelial growth were inhibited on cv. Hyola 60. Infection by natural ascospore inoculum was favoured by periods of increased rainfall, temperature and wind activity and infection was most prevalent early in the growing season, when plants were most vulnerable to leaf infection. Cultivar Hyola 60 developed very little disease and, hence, was unaffected by environmental conditions. On the intermediate cw. Hyola 42, Monty, Pinnacle and Scoop, disease incidence increased with rainfall pre-sowing, whereas this was not the case for the intermediate cw. Dunkeld, Karoo and Mystic and the susceptible cv. Q2. Disease development on all cultivars except Hyola 60 increased with decreased degree-days during leaf growth. Yield was closely correlated to incidence of stem canker on susceptible cultivars, but not resistant cultivars.

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

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.

Brassica napus plants infected by Leptosphaeria maculans after the third to fifth leaf growth stage in south-eastern Australia do not develop blackleg stem canker

Blackleg (Phoma stem canker) caused by Leptosphaeria maculans is the most damaging disease of Brassica napus (canola, rapeseed, colza) worldwide and is controlled by sowing blackleg resistant cultivars and crop management strategies that reduce exposure to inoculum and fungicide application. In experiments in south-eastern Australia, canola cultivars inoculated after the three to five leaf growth stage did not develop stem canker. Although mature canola plants are known to be less susceptible to blackleg than seedlings, this highlights for the first time the specific importance of protecting seedlings up to the three to five leaf growth stage in Australia. This would typically correspond to a period of four to six weeks after emergence. Canola plants are likely to be significantly less vulnerable to infection after this growth stage. However, this timing may vary due to the influence of environmental conditions.

Temperature, wetness period and inoculum concentration influence infection of canola (Brassica napus) by pycnidiospores of Leptosphaeria maculans

Infection of canola (Brassica napus) by pycnidiospores of the blackleg fungus (Leptosphaeria maculans) and subsequent development of leaf and stem infection were influenced by temperature, wetness period and inoculum concentration. On cv. Hyola 42, as temperature decreased the latent period for leaf infection increased exponentially. The greatest number of leaf lesions developed on plants exposed to a day/night temperature of 18/15°C with a 96 h wetness period. Incidence of stem infection was greatest at 23/20°C with a 48–72 h wetness period, whereas no stem infection occurred 42 days after inoculation when plants were incubated at 8/6°C. This is the first report of the effect of temperature and wetness period on infection by pycnidiospores. The number of leaf lesions and incidence of stem cankers were greatest when plants were inoculated with 106 or 107 pycnidiospores/mL, respectively, the highest concentrations used in the experiments. The susceptible cv. Q2 developed significantly more leafand stem lesions than the less susceptible cw. Monty and Dunkeld when inoculated with 107 pycnidiospores/mL, but not at the lower concentrations. The moderately resistant cv. Dunkeld developed significantly fewer stem cankers than the other cultivars when inoculated with pycnidiospores at concentrations greater than 104/mL.

Developing pruning wound protection strategies for managing Eutypa dieback

Background and Aims Eutypa dieback is an economically important disease of grapevines and a major threat to vineyard longevity throughout the world. Developing effective preventative strategies offers the best means of control. In this study, pruning wound protectants and various spray applications were evaluated for their ability to prevent infection by ascospores of Eutypa lata. Methods and Results Fungicides and natural alternative treatments were applied by hand to grapevine pruning wounds in winter prior to inoculation with E. lata. In a series of greenhouse and field experiments, tebuconazole, pyraclostrobin, fluazinam, garlic extract and lactoferrin significantly reduced infection. A range of tractor-driven sprayers was used to apply tebuconazole to pruning wounds, and those which provided good coverage reduced infection by E. lata to a level similar to that when tebuconazole was applied with a paintbrush. Conclusion Eutypa dieback can be controlled with several fungicides and natural treatments. Tractor-driven sprayers, which by design or modification can efficiently deliver maximum possible coverage at high output rates, can be used for application of pruning wound treatments. Significance of the Study Results of this study contributed to registration of fungicides for use as pruning wound treatments to control E. lata in Australia. Demonstrating the ability of tractor-driven sprayers to apply treatments effectively has led to greater industry adoption of dormant treatments and may decrease the future impact of Eutypa dieback.

Calosphaeria Canker of Sweet Cherry Caused by Calosphaeria pulchella in California and South Australia

California is the second largest sweet cherry producer in the United States with annual revenues up to