Characterisation of Botrytis species associated with neck rot of onion in Australia
Neck rot of onion has commonly been attributed to two fungal pathogens, Botrytis aclada (syn. B. allii) and B. byssoidea. However, in other countries, a recently developed PCR technique using sequence characterised amplified region (SCAR) primers (BA2f/BA1r) followed by ApoI digestion of the amplification product, has shown the presence of two groups within B. aclada, B. aclada AI and B. aclada AII syn. B. allii. This method was used to characterise Botrytis spp. of the B. aclada/B. allii complex associated with onion seed and bulbs in Australia. A total of 24 Botrytis isolates, including two from seed and ten from bulbs (Tasmania), five from seed (South Australia), and one from seed and six from a culture collection originally isolated from five bulbs and one stipe (New South Wales), were characterised. Of the 24 isolates, one was identified as B. cinerea, one as B. aclada AI, and 22 as B. aclada All syn. B. allii.
A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues.
Molecular approaches have become an important tool for mycologists studying fungal systematics, molecular evolution, population genetics or plant-fungus interactions. For this purpose the analysis of restriction fragment length polymorphisms (RFLPs), PCR-based techniques like amplification of polymorphic sequences by one random primer (RAPDs) or amplification of defined genomic regions are applied. Prerequisite for such studies is the rapid isolation of pure genomic DNA of high molecular weight not only allowing simultaneous processing of large numbers of samples but also providing reproducibility with regard to restriction, ligation, and PCR. Most common problems encountered in fulfilling these conditions are the rather high production of polysaccharides by some fungal cultures, and extraction of insufficient amounts of DNA from fruit bodies or from lignified and/or infected plant tissues. Few published protocols include steps for efficient removal of polysaccharides (Do and Adams 1991). Moreover, most procedures contain phenol—chloroform treatments (e.g. Garber and Yoder 1983, Blaiseau et al. 1992) and/or more than one precipitation (e.g. Rodriguez and Yoder 1991: four precipitations) — both latter steps are possible causes of shearing. The short protocol given below combines inactivation of proteins by SDS/Proteinase K with precipitation of acidic polysaccharides by hot CTAB in the presence of SDS and high salts (Kim et al. 1990) and only a single selective precipitation of DNA with isopropanol (Marmur 1961, Cryer et al. 1975). The method is appropriate for simultanous processing of many samples because all steps can be done in Eppendorf tubes, but it can also be scaled up easily for bulk preparations. A treatment with methanol containing 0.1 % mercaptoethanol preceding DNA extraction was performed when basidiocarps and plant tissues contained large amounts of reactive compounds (resins, phenolics) interfering with solubilization of DNA. Methanol extraction was found to be superior over other solvents such as acetone (Schneiderbauer et al. 1991). In this way we isolated DNAs from a wide variety of fungi including Fusarium, Pseudocercosporella, Phytophthora, Setosphaeria, Armillaria, Heterobasidion, Boletus, Russula, Lactarius, Suillus, Cortinarius, and from conifer roots and needles (Picea abies, Pinus sylvestris). DNAs were subjected to Pulsed Field Gel Electrophoresis to verify their high molecular weight: the majority of DNA molecules was in the size range of 50-150 kbp and only insignificant amounts of DNA <50 kbp were present (Figure 1, lane 1). DNAs are suited as substrate for restriction, ligation and PCR (Figure 1, lanes 4 6 and 8-10). Yields depend on the species/isolate. For example, of Fusarium spp. yield averaged 100 ng from 30 mg dried mycelium with an average purity measured as A26o/28O °f 1 -78 ± 0.05. Needles and basidiocarps yielded less (10-30 /*g).
Development of a PCR assay to detect Fusarium poae in wheat
Random amplified polymorphic DNA assays were carried out on a range of isolates of F. poae to identify markers common to all isolates. Two fragments were isolated, cloned and used to probe Southern blots of DNA from F. poae and isolates from a range of wheat seed and stem base pathogens. One fragment, which hybridized preferentially to DNA of F. poae was partially sequenced and two pairs of primers (Fp8 F/R and Fp82 F/R) were generated for use in the polymerase chain reaction (PCR). Amplification of target DNA occurred following PCR of all isolates of F. poae but not from any of a range of other fungal species associated with diseases of cereal ears and seed. The primer pair Fp82 F/R was used to detect F. poae in extracts from wheat seed samples contaminated with Fusarium species. This system offers the potential to determine the presence of F. poae in wheat and avoid problems commonly associated with conventional diagnosis of the disease and isolation of the pathogen.