Belinda Stummer

Genetic diversity in populations of Uncinula necator : comparison of RFLP- and PCR-based approaches

Clonal isolates of Uncinula necator , the grapevine powdery mildew pathogen, were characterized by mating type, RFLP and PCR analyses. Five random genomic clones, consisting of three multi-copy and two low-copy sequences, were used as probes for hybridization. The oligonucleotides (CAC) 5 , (GACA) 4 , the core sequence of the M-13 minisatellite region and the consensus region from a plant intron splice junction (R1), were used as primers in PCR reactions. The DNA probes detected high levels of genetic diversity in U. necator , identifying 34 unique haplotypes among the 81 isolates analysed, with a Shannon diversity index of D ’ = 0·85. In comparison, 15 haplotypes were resolved in PCR reactions with the four primers, resulting in a diversity index of D ’ = 0·59. The overall diversity within the collection, by combining RFLP and PCR data, was D ’ = 0·88, distinguishing 37 haplotypes. The RFLP analysis provided greater discrimination of individual haplotypes than did PCR. Phenetic analysis of the RFLP and PCR data separately and in combination revealed two broad phenetic groups. The average overall similarity between the two groups was 40%. Mating of isolates from the two phenetic groups resulted in viable ascospores. The combination of RFLP, PCR and mating techniques was effective in determining levels of diversity and provided insight into the genetic relatedness of U. necator isolates.

Molecular identification and detection of Eutypa lata in grapevine

Eutypa lata, the causal agent of Eutypa dieback of grapevines, is difficult to identify on the basis of colony morphology and is often out-competed by other fungi when isolated from wood. To facilitate diagnosis of the pathogen, we designed SCAR primers capable of amplifying DNA of E. lata and constructed a genomic DNA library from which DNA sequences specific to E. lata were identified and sequenced. SCAR primers were used to identify E. lata directly from culture without the requirement for DNA extraction or prolonged incubation periods and could also detect the pathogen in DNA isolated from grapevine wood. RFLP probes were used in slot-blot assays to detect the pathogen in DNA isolated from 1 yr old cane as well as from mature grapevine trunks. The markers developed in this study have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata.

Reduced sensitivity of Uncinula necator to sterol demethylation inhibiting fungicides in southern Australian vineyards

Isolates of Uncinula necator with reduced sensitivity to triadimenol and, to a lesser extent, fenarimol have been detected in Australian vineyards. Grapevine leaves and berries affected by powdery mildew were collected from a home garden and 13 vineyards in nine viticultural regions in Australia between 1993 and 1998. Single-spore isolates of U. necator established from vines, either not exposed to DMIs (‘unexposed’ collection) or treated with DMIs (‘selected’ collection), were maintained on micro propagated grapevines in vitro. A bioassay for fungicide sensitivity was used to test 60 single-spore isolates of U. necator for sensitivity to triadimenol. Of these, 34 were tested for sensitivity to fenarimol. Mean EC50 values for the 12 unexposed isolates were 0.07 and 0.08 mg/L for triadimenol and fenarimol, respectively. For the selected isolates, mean EC50 values were 0.83 mg/L for triadimenol and 0.19 mg/L for fenarimol. In comparison with the unexposed population, there appeared to have been a shift in the selected population towards reduced sensitivity. Examination of more isolates from vineyards not exposed to DMIs may strengthen this conclusion. Cut-off EC50 values, used to define individual isolates as having reduced sensitivity, were 0.42 mg/L (resistance factor, RF = 6) for triadimenol and 0.12 mg/L (RF = 1.5) for fenarimol. The findings were consistent with the existence of cross-resistance between triadimenol and fenarimol.

DNA markers identify variation in Australian populations of Uncinula necator

Restriction fragment length polymorphisms (RFLPs) were identified among total DNA from clonal lines of Uncinula necator when cloned sequences of total U. necator DNA were used as probes. Four probes, pUnP14, pUnP27, pUnE21 and pUnE4, hybridized to multiple-copy sequences and, with the exception of pUnE4, detected genetic variation among clonal lines of U. necator. Clones pUnP14, pUnP27 and pUnE4 produced banding patterns that were stable for DNA extracted from different asexual generations of U. necator clonal lines over at least 15 months. In addition, clones were evaluated for species specificity. Clones pUnP27 and pUnE4 detected only U. necator sequences in total DNA from infected grapevine leaves. Clones pUnP14 and pUnE4 did not hybridize to total DNA from a range of fungi. Genetic diversity in a sample of the Australian U. necator population was investigated; 15 genotypes were identified among 29 U. necator clonal lines examined. Genetic variation was detected in samples collected within micro-geographical areas, for example, different genotypes representing both mating types of U. necator were detected on a single plant of Vitis amurensis. RFLP analysis of the banding patterns produced using pUnP14, pUnP27 and pUnE21, identified two broad genetic groups, designated A and B. Analysis of DNA fragment patterns obtained using the polymerase chain reaction (PCR) and the plant intron splice junction (ISJ) primer R1 also supported the allocation of U. necator clonal lines into groups A and B.

Detection of novel genotypes in progeny from a controlled cross between isolates of Uncinula necator belonging to distinct phenetic groups

Sexual recombination and segregation in 18 ascospore progeny of Uncinula necator were analysed by mating type (Mat locus), RFLP and PCR markers. Progeny were analysed from a cross of parental isolates representing the genetically distinct phenetic Groups I (AHd2) and II (BNb2). A total of 27 markers was analysed, consisting of four single loci (Mat locus and three RFLP probes), 16 multi-loci (three multi-copy fingerprinting probes) and seven PCR loci. Novel hybrid genotypes were detected in 28% of the ascospore-derived progeny using the multi-copy RFLP probes, pUnl22-11 and pUnP27, and the PCR primers (CAC)5 and R1, confirming that recombination can occur between isolates representative of the two phenetic groups. This study provides evidence that out-crossing between isolates of U. necator generates novel genotypes and, as such, may have important implications for disease management practices, such as the durability of fungicides and host resistance.

Diaporthe perjuncta Does Not Cause Phomopsis Cane and Leaf Spot Disease of Grapevine in Australia

In Australia, Diaporthe perjuncta (formerly known as Phomopsis taxon 1) and Phomopsis viticola (Phomopsis taxon 2) have been associated with Phomopsis cane and leaf spot of grapevine. Although P. viticola causes distinct leaf spots, as well as lesions on shoots and canes, the pathogenicity of D. perjuncta is relatively unknown. The pathogenicity of D. perjuncta and P. viticola was studied in relation to symptom expression and bud loss. Only P. viticola caused brown-black, longitudinal, necrotic lesions on stem tissue and leaf spots characteristic of the disease, whereas both D. perjuncta and P. viticola induced bleaching of dormant canes. Inoculation of dormant buds with D. perjuncta did not cause bud death. D. perjuncta and P. viticola were reisolated from inoculated tissue and into pure culture. D. perjuncta colonized the epidermis and cortex of the grapevine shoot but not the vascular tissue. D. perjuncta appears to be an endophyte, rather than a pathogen of grapevine.

Genetic variation in Australian isolates of the grapevine pathogen Eutypa lata

Eutypa lata is an ascomycete fungus that causes eutypa dieback of grapevines, a disease which threatens vine productivity and longevity throughout the world. We assessed genetic variation among 35 isolates received as E. lata from Australia, Europe, California and South Africa using restriction fragment length polymorphism analysis. A subset of 10 isolates previously analysed for secondary metabolite production was also analysed using randomly amplified polymorphic DNA markers. Molecular analyses showed a high level of genetic diversity, with each isolate having a unique haplotype. Phylogenetic analysis did not allow separation of isolates based on geographic location or host species from which isolateswere obtained. Therewas no apparent correlation betweenDNAprofile and secondary metabolite production by E. lata. The results of this study showed a degree of genetic diversity similar to that reported for European and North American populations of E. lata, and are consistent with the hypothesis that the sole means of dispersal of the pathogen is by ascospores.

Powdery mildew and wine quality

Powdery mildew, caused by the fungus Uncinula necator, is a widespread and economically important disease of grapevines. The fungus grows superficially on green tissues of grapevines and other members of the Vitaceae. Powdery mildew costs the Australian grape and wine industry approximately A

The role of Diaporthe perjuncta in delayed budburst and death of grapevine buds

30 million per year in terms of lost yield, reduced quality and disease management.

Effects of powdery mildew on the sensory properties and composition of Chardonnay juice and wine when grape sugar ripeness is standardised

Diaporthe perjuncta has been associated with Phomopsis cane and leaf spot disease of grapevine in Australia. Both D. perjuncta and Phomopsis viticola cause bleaching of cane and, for this reason, diagnosis of the two fungi is often confused in the vineyard. P. viticola causes leaf spots and shoot lesions in the growing season, whereas more recent studies have indicated that D. perjuncta might not be a pathogen. Field studies were conducted to assess the role of D. perjuncta in delayed budburst and death of grapevine buds. Over three seasons (1999–2001), vines were assessed at four vineyards in South Australia for percentage of budburst and incidence of D. perjuncta. A D. perjuncta-specific probe, pT1P180, was used to detect D. perjuncta in unburst buds, cane and shoots on spurs having buds that failed to burst. D. perjuncta was detected in both bleached and non-bleached cane. Although there was a significant association between the incidence of D. perjuncta and buds that failed to burst in 1999 only, overall bud burst was normal in all vineyards and bud loss was not a concern. Bunch number and shoot growth was not affected. Mites were considered as a possible factor in damage to unburst buds, but it was more likely that failure of buds to burst was related to physiological or environmental effects. D. perjuncta did not adversely affect grapevine productivity and cannot be regarded as a pathogen of grapevine.