Eileen S. Scott

Ochratoxin A-producing Aspergilli in Vietnamese green coffee beans

Aims:  To determine the incidence and severity of infection by ochratoxin A (OA)‐producing fungi in Vietnamese green coffee beans.

Trichoderma harzianum T39 and T. virens DAR 74290 as Potential Biological Control Agents for Phytophthora erythroseptica

Trichoderma harzianum isolate T39 and T. virens isolate DAR 74290 were evaluated as potential biological agents for control of pink rot of potato and root and stem rot of tomato caused by Phytophthora erythroseptica. Cell-free metabolites of T. virens DAR 74290 completely inhibited growth of P. erythroseptica in vitro and appeared to be fungicidal. T. virens DAR 74290 and Trichodex, a commercial formulation of T. harzianum T39, were tested for their ability to protect potato and tomato plants from disease caused by P. erythroseptica in glasshouse experiments. Trichodex and T. virens DAR 74290, alone and combined, reduced disease severity in shoots and roots of potatoes 10 weeks after inoculation with the pathogen. The yield of potatoes from plants treated with P. erythroseptica and T. virens DAR 74290 (mean of 12.9 g fresh weight/pot) was significantly greater than in controls inoculated with the pathogen alone (mean of 2.1 g/pot). Treatment with Trichodex alone increased the yield of tubers compared to the uninoculated controls. T. virens DAR 74290 increased the survival of tomato seedlings inoculated with the pathogen, and both this isolate and Trichodex decreased the severity of disease on tomato.

A new species of Phoma causes ascochyta blight symptoms on field peas (Pisum sativum) in South Australia

Phoma koolunga sp. nov. is described, having been isolated from ascochyta blight lesions on field pea (Pisum sativum) in South Australia. The species is described morphologically and sequences of the internal transcribed spacer region compared with those of the accepted pathogens causing ascochyta blight of field peas. P. koolunga was distinct from Mycosphaerella pinodes (anamorph: Ascochyta pinodes), Phoma medicaginis var. pinodella and Ascochyta pisi. Under controlled conditions the symptoms on pea seedlings caused by P. koolunga were indistinguishable from those caused by M. pinodes, other than a 24 h delay in disease development. Isolates of P. koolunga differed in the severity of disease caused on pea seedlings.

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia.

Aims:  The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated.

Influence of the mycorrhizal fungus, Glomus coronatum, and soil phosphorus on infection and disease caused by binucleate Rhizoctonia and Rhizoctonia solani on mung bean (Vigna radiata)

Root-infecting fungal pathogens and also parasites, which do not cause major disease symptoms cause problems of contamination in pot cultures of arbuscular mycorrhizal (AM) fungi. We investigated the effect of the AM fungus, Glomus coronatum Giovannetti on disease caused by binucleate Rhizoctonia sp. (BNR) and R. solani in mung bean in the absence (P0) and presence (P1) of added soil phosphorus (P). When G. coronatum and BNR or R. solani were inoculated at the same time, G. coronatum improved the growth of the plants and reduced colonization of roots by BNR, but not by R. solani. R. solani reduced the growth of non-mycorrhizal mung bean in P0 soil 6 weeks after inoculation, whereas BNR had no effect on growth. G. coronatum reduced the severity of disease caused by BNR or R. solani on mung bean in both soil P treatments. When G. coronatum was established in the roots 3 weeks before BNR or R. solani was added to the potting mix, there was no significant effect of BNR or R. solani on growth of mung bean. Prior colonization by G. coronatum slightly reduced indices of disease caused by BNR or R. solani. In both experiments, addition of P stimulated plant growth and reduced the colonization of roots by BNR, but had little effect on disease severity. We conclude that the reduction of the effect of BNR or R. solani on mung bean could not be explained by improved P nutrition, but could be attributed to the presence of G. coronatum within and among the roots.

An experimental system for characterizing isolates of Uncinula necator

Clonal lines of Uncinula necator were established using conidia from diseased Vitis vinifera leaves or berries collected from various Australian viticultural regions. Techniques for the establishment of single-conidial-chain isolates of U. necator on detached leaves and the subsequent maintenance of clonal lines on micropropagated grapevines in vitro are described. Conidia were successfully mass produced by the detached leaf technique and harvested efficiently using a cyclone separator device. Conidial yields were quantified for 14 clonal lines and ranged from 42 to 112 mg per 20 leaves at the first harvest. Nucleic acid extraction from conidia resulted in high-quality DNA suitable for restriction enzyme digestion and amplification by PCR, with yields ranging from 6 to 9 ng per mg conidia. This is the first report of a DNA extraction procedure for conidia of U. necator. Using the 18 base plant intron splice junction (ISJ) primer, RI, genetic variation among five South Australian clonal lines of U. necator was identified. Three of these clonal lines originated from vines grown within a 0·5 km radius. This preliminary identification of genetic variation in {ilU. necator} and the system for handling different clonal lines provide the essential tools for further development of DNA markers and the molecular characterization of this economically important pathogen.

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.

Chemical and biological induction of resistance to the clover cyst nematode (Heterodera trifolii) in white clover (Trifolium repens)

Possible induction of resistance to the clover cyst nematode, Heterodera trifolii, in white clover, Trifolium repens, by application of two chemical inducers and of soil-borne Pseudomonas-like spp. and Bacillus cereus was investigated. Salicylic acid and benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester, applied as a root drench in growth cabinet soil bioassays, affected development of H. trifolii in cvs Haifa and Grasslands Huia. Treatments reduced fecundity of the nematodes, increasing the proportions of distorted females and of females with fewer eggs compared to water-treated controls. Application of pectinolytic fluorescent pseudomonad strains P29 and P80, and B. cereus strain B1, induced a response similar to that resulting from the chemical induction. Both live and dead cells of P29, but not cell-free culture filtrate, induced these effects on H. trifolii. A non-pectinolytic, fluorescent pseudomonad, strain P37, had no effect. From the nature of the responses, it is concluded that the effects of both the chemical and bacterial agents have similarities to resistance. From the timing of applications and known properties of both agents, these effects seem similar to induced systemic resistance.

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.

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.