Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus Verticillium dahliae by Agrobacterium tumefaciens-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacteriumtumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae

A rapid genetic assay for the identification of the most common Pocillopora damicornis genetic lineages on the Great Barrier Reef

Pocillopora damicornis (Linnaeus, 1758; Scleractinia, Pocilloporidae) has recently been found to comprise at least five distinct genetic lineages in Eastern Australia, some of which likely represent cryptic species. Due to similar and plastic gross morphology of these lineages, field identification is often difficult. Here we present a quick, cost effective genetic assay as well as three novel microsatellite markers that distinguish the two most common lineages found on the Great Barrier Reef. The assay is based on PCR amplification of two regions within the mitochondrial putative control region, which show consistent and easily identifiable fragment size differences for the two genetic lineages after Alu1 restriction enzyme digestion of the amplicons

Ochratoxin A and related mycotoxins

Ochratoxins are toxins produced by fungal species belonging not only to Aspergilli but also to Penicillia with ochratoxin A (OTA) being the main toxin of the group followed by ochratoxin B (OTB) and ochratoxin C (OTC). These mycotoxins can occurare formed in a variety of plant products as well as in some animal-derived products following fungal invasion. Human exposure to OTA is mainly due to contaminated cereal products, coffee and red wine. OTA it is continuously studied for its occurrence in different foods and feeds and for its harmfulness for human and animal health. OTA is immunotoxic, nephrotoxic and potentially carcinogenic in animals. Kidneys are its target organs and its level in foods is strictly regulated by laws with OTA being classified as a member of group 2B of carcinogens. According to the molecular structure, several enzymatic activities in addition to the primary pathway for phenylalanine biosynthesis are required for the biosynthesis of ochratoxin A: these are a polyketide synthase for the synthesis of the polyketide dihydroisocoumarin, a chlorinating enzyme, a methylase, an esterase, and a peptide synthetase for ligation of the phenylalanine to the dihydroisocoumarin