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Black aspergilli are some of the most common mycotoxigenic fungi in vineyards worldwide. The aims of this research were to assess the occurrence of fumonisin-producing black aspergilli in Australian wine grapes and the effects of environmental factors on fumonisin production by A. niger and A. welwitschiae (syn. A. awamori). Thirty-eight Aspergillus isolates (black aspergilli) were collected from six wine grape varieties grown in Australian vineyards. LC-MS/MS analysis of culture extracts revealed that six isolates produced fumonisins FB2 and FB4. Molecular data revealed that all fumonisin-producing isolates were A. niger and A. welwitschiae. None of the reference isolates, A. carbonarius, A. tubingensis, A. japonicus, and A. foetidus, were positive for fumonisin production. The effects of temperature and water activity on the growth and production of fumonisins were studied using two A. niger and an isolate of A. welwitschiae on synthetic grape juice medium (SGJM) at 20 °C, 25 °C, 30 °C, and 35 °C, and 0.92 aw, 0.95 aw, and 0.98 aw levels. All isolates produced FB2 and FB4 at 0.95 aw and 0.98 aw and 20 °C, 25 °C, and 30 °C. The highest growth rate observed was 14.89 mm/day for A. welwitschiae at 0.98 aw and 35 °C, whereas the highest fumonisin production observed was 25.3 mg/kg at 0.98 aw and 20 °C for A. welwitschiae. None of the isolates produced fumonisins at 35 °C at any water activity levels. To our knowledge, this is the first report on the occurrence of fumonisin-positive isolates of Aspergillus from Australian wine grapes and the impact of the environmental factors on fumonisin production by A. welwitschiae.

Grapevine trunk diseases (GTDs) are considered a serious problem to viticulture worldwide. Several GTD fungal pathogens produce phytotoxic metabolites (PMs) that were hypothesized to migrate to the foliage where they cause distinct symptoms. The role of PMs in the expression of Botryosphaeria dieback (BD) symptoms in naturally infected and artificially inoculated wood using molecular and analytical chemistry techniques was investigated. Wood samples from field vines naturally infected with BD and one-year-old vines inoculated with Diplodia seriata, Spencermartinsia viticola and Dothiorella vidmadera were analysed by cultural isolations, quantitative PCR (qPCR) and targeted LC-MS/MS to detect three PMs: (R)-mellein, protocatechuic acid and spencertoxin. (R)-mellein was detected in symptomatic naturally infected wood and vines artificially inoculated with D. seriata but was absent in all non-symptomatic wood. The amount of (R)-mellein detected was correlated with the amount of pathogen DNA detected by qPCR. Protocatechuic acid and spencertoxin were absent in all inoculated wood samples. (R)-mellein may be produced by the pathogen during infection to break down the wood, however it was not translocated into other parts of the vine. The foliar symptoms previously reported in vineyards may be due to a combination of PMs produced and climatic and physiological factors that require further investigation.

Two new phytotoxic 1-substituted ethanones, named luteoethanones A and B, were isolated from

Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 nontarget fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.

Botryosphaeria dieback caused by species of Botryosphaeriaceae is an economically significant disease of grapevine worldwide including in Australia and New Zealand. Symptoms such as dieback of the shoots and cordons, cankers, stunted shoots, bunch rot and bud necrosis are observed, eventually leading to yield loss and reduced grapevine longevity. The disease was first recognised as an important issue in vineyards in Australia and New Zealand in the early 2000s and since then, considerable research has been conducted for better understanding of the causal organisms and their management. However, there is also a need to better understand the role of nursery planting material as a potential source of infection in new or replanted vineyards. This review reports on the current knowledge of Botryosphaeriaceae in grapevines in Australia and New Zealand including their identification, distribution across various wine grape growing regions, symptomatology, pathogenicity, epidemiology and management strategies. Future strategies to increase our knowledge and to better manage these economically important pathogens are discussed.

Botryosphaeria dothidea causes diseases in many different hosts worldwide. This species has become the most prominent fungal taxon causing grapevine trunk disease in China, with a recent disease outbreak. To reveal the genetic diversity and explore the origin of B. dothidea , six novel microsatellite markers were developed and used for the genotyping of 151 isolates obtained from China and overseas. The results demonstrated the high genetic diversity of the B. dothidea populations. Bayesian cluster analysis separated the total B. dothidea isolates into five genetic populations. B. dothidea isolates from Chinese grapevines were observed to share alleles with isolates from different hosts within China and from grapevines growing overseas, indicating both endemic host shifts and exotic introduction. In addition, unique pathogen genotypes were identified in Chinese grapevine isolates. Hence, we infer that B. dothidea isolates from multiple origins are contributing to the dieback and canker outbreak currently occurring in China.

Aureobasidium isolated from Vitis vinifera (cv Chardonnay) grapevine tissues were characterised using morphological and molecular techniques. Species level identification of 29 isolates was accomplished by partial amplification and sequencing of the ITS region (ITS1–5.8S–ITS2) using universal primers ITS1 and ITS4. A comparison of nucleotide sequences using BLAST followed by phylogenetic analysis revealed that all isolates examined were Aureobasidium pullulans. Strain level discrimination of a total of 100 epiphytic Aureobasidium isolates including three reference strains was successfully carried out using two inter simple sequence repeat (ISSR) primers, (AAC)5 and (GTG)5 and the Intron Splice Junction R1 (ISJ-R1) primer in which 24, 24 and 15 scorable bands were produced for each primer, respectively. The high level of genetic variation recorded among the isolates further highlighted the high levels of strain diversity among A. pullulans residing on grapevines. Thirty-two epiphytic Aureobasidium isolates were examined for their ability to inhibit the growth of Greeneria uvicola, responsible for bitter rot of grapes. Using an in-vitro dual-culture antagonism assay, all isolates inhibited the growth of G. uvicola (Isolates DAR 77272 and DAR 77273) with inhibition ranging from 15 to 85%. Three Aureobasidium isolates were then examined for their ability to inhibit G. uvicola when co-inoculated onto detached berries, leaves and grape bunches growing on potted vines in a glass house. All isolates reduced the severity of bitter rot infection. The results indicate that A. pullulans has the potential to suppress bitter rot of grapes.

A marker-assisted approach was adopted to search for Bacillus spp. with potential as biocontrol agents against stem rot disease of canola caused by Sclerotinia sclerotiorum . Bacterial strains were isolated from the rhizosphere of canola and screened using multiplex PCR for the presence of surfactin, iturin A and bacillomycin D peptide synthetase biosynthetic genes. Among the 96 isolates screened, only CS-42 harbored all three genes and was subsequently identified as Bacillus cereus using 16S rRNA gene sequencing. This strain was found to be effective in significantly inhibiting the growth of S. sclerotiorum in vitro and in planta . Scanning electron microscopy studies at the dual culture interaction region revealed that mycelial growth was curtailed in the vicinity of bacterial metabolites. Complete destruction of the outmost melanised rind layer of sclerotia was observed when treated with the bacterium. Transmission electron microscopy of ultrathin sections challenged with CS-42 showed partially vacuolated hyphae as well as degradation of organelles in the sclerotial cells. These findings suggested that genetic marker-assisted selection may provide opportunities for rapid and efficient selection of pathogen-suppressing Bacillus strains for the development of microbial biopesticides.

Different phytotoxic metabolites were isolated from the organic extract of

Different phytotoxic metabolites were isolated from the organic extract of Neofusicoccum luteum, Neofusicoccum australe, and Neofusicoccum parvum, causal agents of Botryosphaeria dieback in Australia. N. luteum produced a new disubstituted furo-α-pyrone, a hexasubstituted anthraquinone, and a trisubstituted oxepi-2(7H)-one, luteopyroxin (4), neoanthraquinone (5), and luteoxepinone (7), respectively, together with the known (±)-nigrosporione (6), tyrosol (8), (R)-(-)-mellein (1), and (3R,4S)-(-)- and (3R,4R)-(-)-4-hydroxymellein (2 and 3). The three melleins and tyrosol were also produced by N. parvum, while N. australe produced (R)-(-)-mellein (1), neoanthraquinone (5), tyrosol (8), and p-cresol (9). Luteopryoxin (4), neoanthraquinone (5), and luteoxepinone (7) were characterized by analyses of physical data, essentially one- and two-dimensional nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry. The relative and absolute configurations of luteopyroxin (4) were determined by nuclear Overhauser effect spectroscopy and experimental and calculated electronic circular dichroism data. When assayed on grapevine leaves, neoanthraquinone (5) showed the highest toxic effect, causing severe shriveling and withering. Luteopyroxin (4), nigrosporione (6), and luteoxepinone (7) also showed different degrees of toxicity, while p-cresol (9) displayed low phytotoxicity.

Laccases are one of many groups of inducible enzymes produced by the filamentous fungus, Botrytis cinerea during colonisation of host plant tissues. While the processes involved in laccase induction are not fully understood, Cupric ions (e.g. CuSO4) and gallic acid (GA) have been reported as laccase inducers. This study investigates laccases activities and the expression of three laccase genes (BcLCC1, BcLCC2, BcLCC3) in three B. cinerea isolates grown in laccase-inducing medium (LIM) supplemented with CuSO4 and GA. Laccase activity in culture filtrates with CuSO4 increased after 48 h of growth in LIM at 24°C. The induction of BcLCC2 transcription was greatest at a concentration of 0.6 mM CuSO4, concentrations greater than 0.6 mM inhibited fungal growth. In contrast, no laccase induction was observed in the presence of GA. Liquid chromatography-mass spectroscopy (NanoLC ESI MS/MS) analysis confirmed the presence of a 63.4 kDa protein, the BcLCC2 isoform in the culture filtrate with 0.6 mM CuSO4. Analysis of mRNA transcripts further showed BcLCC3 was also inducible and the expression of BcLCC2 and BcLCC3 was isolate-dependent. In conclusion, CuSO4 induces a 63.4 kDa laccase in B. cinerea by induced transcription of the BcLCC2 gene.

Two new phytotoxic 1-substituted ethanones, named luteoethanones A and B, were isolated from Neofusicoccum luteum, the causal agents of Botryosphaeria dieback in Australia. Luteoethanones A and B were characterized, by spectroscopic methods (essentially 1 D and 2 D NMR and HR ESIMS), as 1-(8-Methoxy-2,4-methyl-1-oxa-4-aza-spiro[2.5]octa-5,7-dien-6-yl)-ethanone and its 2-demethyl analogue. When assayed on detached grapevine leaves (Vitis vinifera cv. Shiraz) both the compounds showed phytotoxic activity.

Endophytic strains of Pseudomonas were isolated from grapevine tissues and exhibited antagonistic activity against several grapevine trunk disease pathogens. The draft genome sequences of the four strains revealed the presence of putative gene clusters that may impart biocontrol activity against plant pathogens.

Pitt, W. M., Sosnowski, M. R., Huang, R., Qiu, Y., Steel, C. C., and Savocchia, S. 2012. Evaluation of fungicides for the management of Botryosphaeria canker of grapevines. Plant Dis. 96:1303-1308. 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

Pathogenicity and virulence of the Botryosphaeriaceous fungi Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae and Neofusicoccum parvum were confirmed by inoculation of trunks and cordons on mature grapevines (Vitis vinifera 'Chardonnay'), inoculation of two-year-old potted grapevines in a glasshouse comparing water stressed versus non-water stressed vines, and by inoculation of one-year-old detached canes incubated at 25, 30 and 35°C. All four species were pathogenic with N. parvum the most virulent species followed by L. theobromae, B. dothidea and D. seriata in all pathogenicity assays. Incubation at 30°C resulted in increased virulence in most isolates. However, variation in virulence was observed for different isolates of the same species at 35°C. Significant intra- and inter-species variation in virulence was also observed between water stressed and non-water stressed vines. Optimal temperatures for colony growth and growth rate were also studied in an attempt to understand the variability observed in the pathogenicity tests. L. theobromae was the most rapidly growing species at the optimal growth temperature of 31.3°C followed by B. dothidea at 31.6, N. parvum at 30.2 and D. seriata at 27.6°C. At 25°C L. theobromae was also the fastest growing species with a growth rate of 1.07 mm h-1 followed by D. seriata at 0.87 mm h-1, N. parvum at 0.86 mm h-1 and B. dothidea at 0.83 mm h-1. Using these data the climatic prediction software, CLIMEX, was used to study the possible distribution of the four species in Australia.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

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.

Among the diseases caused by Sclerotinia in Australia, lettuce drop, caused by the fungal pathogens Sclerotinia minor and Sclerotinia sclerotiorum, was considered as a model in this study as it poses a major threat to lettuce production in Australia,. The management of this disease with synthetic fungicides is strategic and the presence of fungicide residues in the consumable parts of lettuce is a continuing concern to human health. To address this challenge, Bacillus cereus SC-1 was chosen from a previous study, for biological control of S. sclerotiorum induced lettuce drop. Soil drenching with B. cereus SC-1 applied at 1×108 cfu mL-1 in a glasshouse trial completely restricted the pathogen, and no disease incidence was observed (P=0.05). Sclerotial colonization was tested and it was found that 6-8 log cfu per sclerotium of B. cereus resulted in a reduction of sclerotial viability to 1.58% compared to the control (P=0.05). Volatile organic compounds (VOC) produced by the bacteria increased root length, shoot length, and seedling fresh weight of the lettuce seedlings by 46.6, 35.4, and 32% respectively when compared with the control (P=0.05). In addition to VOC induced growth promotion, B. cereus SC-1 enhanced lettuce growth, resulting in increased root length, shoot length, head weight, and biomass weight by 34.8, 21.5, 19.4, and 24.8%, respectively when compared with untreated control plants (P=0.05). The bacteria were able to survive in the rhizosphere of lettuce plants for up to 30 days reaching populations of 7 log cfu g-1 of root-adhering soil. These results indicate that biological control of lettuce drop with B. cereus SC-1 could be feasible when used either alone or integrated into IPM and best management practice programs for sustainable management of lettuce drop and other Sclerotinia diseases.

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.

Background and Aims: Species of Botryosphaeriaceae have been reported in association with various grapevine diseases, such as trunk diseases and fruit rots worldwide. Their distribution, however, has not been thoroughly studied at the genetic level. The aim of this study was to evaluate genetic variation within four species of otryosphaeriaceae isolated from south-eastern Australian vineyards at several geographical scales. Methods and Results: Fungal isolates were taken using a hierarchical methodology. A range of polymerase chain reaction (PCR) primers, including a selected set of six inter-simple sequence repeat primers, the intron splice junction R1 primer and the M13 core sequence microsatellite primer, were selected after preliminary screening. A total of 171 isolates of the Botryosphaeriaceae was assessed, comprising 127 Diplodia seriata, 23 Neofusicoccum parvum, 18 Botryosphaeria dothidea and 3 Lasiodiplodia theobromae isolates from three closely located regions. From these, 280 highly reproducible polymorphic bands were detected across all isolates following PCR. Intra- and inter-specific differences were visualised as a dendrogram and a principle coordinates plot. Cluster analysis with the integrated locus matrix separated the isolates into four distinguishable groups according to species (cophenetic correlation = 0.97). Conclusions: Our study indicates that genetic differentiation by region was evident within D. seriata, and there was variation in genotype between vineyards in several instances. Isolates of N. parvum showed greater homogeneity between vineyards and regions, while B. dothidea showed homogeneity between regions from which they were isolated. Significance of the Study: The degree of variation in populations of Botryosphaeriaceae in vineyards indicated in this study can be helpful in the future for controlling the spread of the pathogen in Australian vineyards and can be used to guide sampling in future surveys and population studies of the genus. The unique banding patterns of each species detected in this study can be utilised in the future for designing species-specific primers, which would enable in-field real-time detection of Botryosphaeriaceae without destructive sampling.

Stem rot caused by Sclerotinia sclerotiorum is a major fungal disease of canola worldwide. In Australia the management of stem rot relies primarily on strategic application of synthetic fungicides. In an attempt to find alternative strategies for the management of the disease, 514 naturally occurring bacterial isolates were screened for antagonism to S. sclerotiorum. Antifungal activity against mycelial growth of the fungus was exhibited by three isolates of bacteria. The bacteria were identified as Bacillus cereus (SC-1 and P-1) and Bacillus subtilis (W-67) via 16S rRNA sequencing. In vitro antagonism assays using these isolates resulted in significant inhibition of mycelial elongation and complete inhibition of sclerotial germination by both non-volatile and volatile metabolites. The antagonistic strains caused a significant reduction in the viability of sclerotia when tested in a greenhouse pot trial with soil collected from the field. Spray treatments of bacterial strains reduced disease incidence and yielded higher control efficacy both on inoculated cotyledons and stems. Application of SC-1 and W-67 in the field at 10% flowering stage (growth stage 4�00) of canola demonstrated that control efficacy of SC-1 was significantly higher in all three trials (over 2 years) when sprayed twice at 7-day intervals. The greatest control of disease was observed with the fungicide Prosaro� 420SC or with two applications of SC-1. The results demonstrated that, in the light of environmental concerns and increasing cost of fungicides, B. cereus SC-1 may have potential as a biological control agent of sclerotinia stem rot of canola in Australia.

Two new phytotoxic tetrasubstituted 1,4-naphthoquinones, named diploquinones A and B, were isolated together with vanillic acid from Diplodia mutila (DAR78993), a grapevine pathogen involved in Botryosphaeria dieback in Australia. Diploquinones A and B were characterized as 6,7-dihydroxy-2-methoxy-5-methylnaphthalene-1,4-dione and 3,5,7-trihydroxy-2-methoxynaphthalene-1,4-dione using spectroscopic methods (essentially 1D and 2D 1H and 13C NMR and HR ESIMS). The already known vanillic acid was isolated for the first time as fungal phytotoxin and as metabolite of D. mutila. The three compounds were assayed on detached grapevine leaves ( Vitis vinifera cv. Shiraz) at concentrations of 10-3 M and 2.5 × 10-3 M. Vanillic acid showed the highest phytotoxic effect on grapevine leaves irrespective of the tested concentration, while diploquinones A and B showed varying degrees of toxicity.

In addition to Eutypa lata, which causes Eutypa dieback, numerous other fungi in the Diatrypaceae family have been isolated from diseased grapevines (Vitis vinifera) and other woody hosts. Pathogenicity trials comprising 70 strains of diatrypaceous fungi representing nine species in six genera were conducted to determine whether these fungi, collected in Australia, were pathogenic to grapevines. When inoculated into wounded trunks of ‘Cabernet Sauvignon’, eight species, including E. lata, E. leptoplaca, Cryptovalsa ampelina, C. rabenhorstii, Eutypella citricola, E. microtheca, Diatrypella vulgaris, and a Diatrype sp. produced necrotic lesions significantly longer than on controls. In addition, all nine species (including a Cryptosphaeria sp.) were reisolated from the margins of developing lesions and at varying distances above and below the point of inoculation. Diatrypaceous fungi were frequently isolated from asymptomatic or otherwise healthy tissue several centimeters ahead of the disease margin. Methods to control diseases associated with diatrypaceous fungi must take into account their propensity to colonize woody tissues ahead of or in the absence of visible symptoms. Current recommendations for the management of Eutypa dieback using remedial surgery and pruning wound protection appear sufficient for the control of the other diatrypaceous fungi included in this study.

Botryosphaeriaceae species are among the most common fungi isolated from grapevine (Vitis vinifera) cankers in Australia. Thirty-eight isolates comprising eight Botryosphaeriaceae species isolated from grapevine cankers throughout New South Wales and South Australia were used in a pathogenicity study on mature wood of 15-year-old Chardonnay grapevines. Experiments showed that all eight species were able to infect grapevines under field conditions causing vascular discoloration and/or staining of the wood (lesions). However, differences in pathogenicity were evident among strains and species (P

Greeneria uvicola causes bitter rot on Vitis vinifera (bunch grapes) and Muscadinia rotundifolia (muscadine grapes) in warm moist temperate and subtropical regions. This study investigated the phylogenetic relationship of G. uvicola representatives from Australia (67 isolates), the USA (31 isolates), India (1 isolate) and Costa Rica (1 isolate) and compared their pathogenicity and fungicide sensitivity. Differences in cultural and conidial morphology were observed between the isolates from Australia and the USA. Phylogenetic relationships were determined based on three gene regions: the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1–5∙8S–ITS2), 28S large subunit (LSU) nuclear rDNA and β-tubulin-2. Greeneria uvicola isolates were clearly differentiated into four groups: isolates from Australia and India; USA isolates from V. vinifera; USA isolates from M. rotundifolia; and the isolate from Costa Rica. All isolates were pathogenic on V. vinifera (cv. Chardonnay) berries although those originating from M. rotundifolia were not as aggressive as isolates from V. vinifera, irrespective of geographical origin. Sensitivity to pyraclostrobin and salicylhydroxamic acid (SHAM) was studied. Despite differences in fungicide applications, hyphal growth inhibition was not significantly different for geographical location, cultivar, tissue, year of collection or different spray regimes. For the Australian and USA isolates, fungal growth inhibition was significantly greater for pyraclostrobin than for SHAM, and was significantly greater for the combined treatment than for each of the fungicides applied singly. The aetiological and epidemiological knowledge of bitter rot collected through this study will aid better prediction and management strategies of this pathogen.

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.

Botryosphaeriaceae isolates of six species were assessed for their potential to infect grapevine tissues other than their tissues of isolation, primarily to determine sources of inocula that could contribute to bunch rot. Pathogenicity tests were conducted in vitro on berries and wood and in vivo on dormant buds of cultivars Chardonnay and Shiraz in glasshouse and field experiments. Tissue specificity and variation in virulence for different isolates was assessed. All isolates were able to infect and cause symptoms on detached 1-year-old canes and mature berries. Virulence was not affected by origin tissue and varied between isolates and within species. Inoculation of dormant buds did not affect bud burst or further development of shoots and fruit, however, a small number of Botryosphaeriaceae were reisolated from bunches at later growth stages. We conclude that Botryosphaeriaceae species are important pathogens of both the vegetative tissues and wood of grapevines. Grapevine wood infected with Botryosphaeriaceae could act as a source of inoculum for reproductive and vegetative tissue. Equally, the vegetative and reproductive tissues can also act as inoculum sources for wood infection. Therefore, all sources of inocula should be taken into consideration when developing management strategies for Botryosphaeria bunch rot and Botryosphaeria canker diseases.

Background and Aims: Ripe rot of grapes is widespread in subtropical vineyards of south-eastern Australia. We aimed to re-evaluate the causative agent(s), berry susceptibility, fungicide sensitivity and differences in histopathology. Methods and Results: Both Colletotrichum acutatum and C. gloeosporioides were shown to be responsible for ripe rot in three vineyards surveyed in the Hastings Valley, NSW in 2007 and 2009. Observation on detached berries of Vitis vinifera (cv. Cabernet Sauvignon and Chardonnay) revealed that C. acutatum had a faster infection rate than C. gloeosporioides. C. acutatum also formed appressoria and penetrated grape tissue faster than C. gloeosporioides, which produced longer hyphae on the berry surface before penetration. Both C. acutatum and C. gloeosporioides produced acervuli 48 h after inoculation. In contrast, spore germination rates and growth rates on potato dextrose agar were greater for C. gloeosporioides. The two species differed in their sensitivity to the fungicides benomyl, captan and triadimenol. C. acutatum was significantly more sensitive to captan and triadimenol, whereas C. gloeosporioides was more sensitive to benomyl. Conclusions: Both C. acutatum and C. gloeosporioides cause ripe rot of wine grapes in Australia. Subtle differences in the infection process may explain the relative prevalence of the two species. Significance of the Study: This study confirms the involvement of C. acutatum and C. gloeosporioides in ripe rot of grapes in Australia. Knowledge of growth characteristics and fungicide sensitivity of ripe rot pathogens should aid disease management strategies.

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.

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.

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.

Background and Aims: Botryosphaeriaceae species are recognised as important pathogens of grapevines both in Australia and overseas. The identity, prevalence and distribution of Botryosphaeriaceae species in vineyards throughout the major winegrowing regions of New South Wales (NSW) and South Australia (SA) was determined to provide a foundation for improved disease prevention and management. Methods and Results: Field surveys from 91 vineyards across NSW and SA resulted in the collection of 2239 diseased wood samples and subsequent isolation of 1258 Botryosphaeriaceae isolates. Morphological identification along with phylogenetic analysis of ribosomal DNA internal transcribed spacer regions (ITS1-5.8S-ITS2) and partial sequences of the translation elongation factor 1-α gene (EF1-α) showed that eight Botryosphaeriaceae species from four phylogenetic lineages occur on grapevines in eastern Australia, including Diplodia seriata, Diplodia mutila, Lasiodiplodia theobromae, Neofusicoccum parvum, Neofusicoccum australe, Botryosphaeria dothidea, Dothiorella viticola and Dothiorella iberica. Conclusions: The prevalence of individual species varied according to geography and climate. Species of Diplodia and Dothiorella, characterised by thick-walled, pigmented conidia were the most prevalent and were distributed widely throughout both NSW and SA. Species with hyaline conidia, such as Neofusicoccum and Fusicoccum, were isolated less frequently and displayed more limited geographic ranges, whilst only a single isolate of Lasiodiplodia was recovered, this being from the northern most region of NSW. Significance of the Study: The identification of eight taxa within the Botryosphaeriaceae, and their distributions throughout south-eastern Australia was established and discussed in context with climate, reported optimum growth temperatures, and more recent taxonomic and nomenclatural revisions. We established a sound base for control strategies based on the prevailing species in Australian viticultural regions.

Detached Vitis vinifera 'Cabernet Sauvignon' berries (12.5° Bé) were inoculated either singularly or in combination with spore suspensions of Botrytis cinerea, Colletotrichum acutatum and Greeneria uvicola and the degree of disease expression examined at either 20 or 27 °C. Berries were more susceptible to B. cinerea at 20 °C and to G. uvicola at 27 °C but were highly susceptible to C. acutatum at either temperature. In experiments involving inoculation of berries with mixtures of fungal organisms, B. cinerea infection was diminished at 27 °C by either C. acutatum or G. uvicola but only by C. acutatum at 20 °C. G. uvicola infection was diminished by C. acutatum at both temperatures investigated. B. cinerea reduced the level of infection of both C. acutatum and G. uvicola at 20 °C. The findings have implications for seasonal bunch rot management of grapes in relation to predicted changes in global temperature., VITIS - Journal of Grapevine Research, Vol. 50 No. 2 (2011): Vitis

A simple system for in vitro dual culture of grapevine (Vitis spp.) plantlets and root-knot nematode (Meloidogyne javanica (Treub) Chitwood) is described. Based on the presence or absence of mature females, or the total number of nematodes in the roots after 36-day co-culture, the system reliably discriminated resistant (cv. Ramsey) and susceptible (cv. Chardonnay) grapevines. The system was sensitive enough to differentiate between infestation levels of cvs Börner and Chardonnay, both susceptible in the in vitro conditions. A modification of the system to use plantlets from rooted petioles has reduced labour and space requirements and would suit mass screening of grapevine genotypes in traditional or genetic engineering-based breeding programmes. In both systems, nematodes in roots of cv. Ramsey tended to be associated with brown tissue and, compared with those in roots of cv. Chardonnay, were more likely to be confined to tips rather than be distributed along the root after co-culture for 11 or 36 days.

Spraying aqueous solutions of ethanol (between 2.5 and 10%, v/v) 1 month before harvest has been shown to increase berry weight of Cabernet Sauvignon by about 10% at harvest compared with controls sprayed with water. These observations were made in France and in Australia. The increase in berry weight was not associated with significant changes in total soluble solids. Additional measurements showed that spraying aqueous solutions of ethanol onto the berry surface 1 month before harvest had only small effects on the colour of berry skin extracts. In another trial, spraying ethanol at night 1 month earlier than other trials lead to a slight increase in the colour of the skin extracts. Optimisation of the spraying conditions is discussed.

Three different ethanol solutions were sprayed onto Cabernet Sauvignon (Vitis vinifera L.) clusters during the ripening period: 2.5, 5 and 10% by volume in water. Controls were sprayed with water alone. Three different times of spraying were also tested: 8, 10 and 13 weeks post-flowering. One of the observed changes was a lower titratable acidity in grape samples at harvest, when the clusters were sprayed with ethanol at 10 weeks, in comparison with controls. The wines made with grapes treated with ethanol after mid-veraison, had higher ODs at 520 nm than did the controls. This may due to a combined effect of red pigment levels and acidity. In addition, following malolactic fermentation, the acidity levels of wines made with ethanoltreated grapes were slightly higher than those made with the control grapes. Spraying ethanol at 13 weeks post-flowering increased the berry weight by 10% at harvest without decreasing the °Brix value. The corresponding wines had similar degrees of alcohol. This observation was made for the first time in 2001.

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.

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.

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.