Richard Lardner

Phenolic and heterocyclic metabolite profiles of the grapevine pathogen Eutypa lata

The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound.

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.

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.

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.

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.