6.141
CLADOBOTRYUM
  • SPECIES CAUSING COBWEB DISEASE OF MUSHROOMS
    CLADOBOTRYUM
  • SPECIES CAUSING COBWEB DISEASE OF MUSHROOMS GJ MCKAY 1 , D EGAN2, E MORRI S2 and AE BROWNl lDepartment of Applied Plant Science, The Queen's University of Belfast, Belfast, BT9 5PX, UK; 2Mushroom Research Group, NAVBC, University College Dublin, Ireland. Background and objectives The fungus Ciadobotryum dendroides (Cobweb mould) rapidly colonises the casing surface of mushroom compost, causing infected mushrooms to turn brown and rot and is a major cause of concern within the mushroom industry. Current fungicide control measures are limited due to the increased incidence of resistance to benzimidazole (MBC) fungicides, which have been associated with a mutation within the P-tubulin gene [1]. The aims of this study were to identify such a mutation linked with MBC-resistance in C. dendroides, develop a rapid PCR-based diagnostic test to identify resistant isolates and to investigate the taxonomy of the causal agent concerned. Matedals and methods Sixty isolates of Cobweb mould from three continents were analysed using both molecular and morphological techniques. Poison plate assays were used to determine MBC tolerance levels. P-tubulin nucleotide sequence vvas ascertained (using primers designed from within conserved regions of the gene from other filamentous fungi) for both MBC-sensitive and resistant isolates. A phylogenetic investigation of nucleotide sequence data from the ITS region of the RDNA and RAPD analysis of total genomic DNA was carded out. Microsatellite markers were used as characters to differentiate isolate groupings based on geographical location. The morpholgical characteristics of the isolates were noted and compared to previously published descriptions of the species [2]. Results and conclusions Nucleotide sequence analysis of the P-tubulin gene revealed that resistance to MBC fungicides was associated with a point mutation at codon 50 within the gene, producing a tyrosine to cysteine amino acid shift. All isolates designated resistant by poison plate assay contained the point mutation, while sensitive isolates did not. A PCR-based diagnostic test using species-specific primers and the restriction enzyme Acc 1, was able to differentiate between MBC-resistant and sensitive isolates. Phylogenetic analysis of all isolates formed three distinct clades. Nucleotide sequence analysis of the ITS region revealed that all MBC-resistant isolates showed identical sequence data different to that of MBC-sensitive isolates, which formed 2 separate clades. Variation at nucleotide sequence level was very small suggesting the close relationship between the isolates concerned. RAPD analysis also produced 3 clades as before, but with greater variation within each clade based on total genomic DNA analysis. All resistant isolates clustered strongly together, whereas sensitive isolates were observed to be more diverse. Microsatellite analysis confirmed these findings and was able to define isolates from different geographic locations based on the length of a simple variable GA repeat. All resistant isolates showed no vadation and formed a single clade, unlike sensitive isolates which displayed specific length variation dependent on isolate origin. Morphological characteristics of conidia, phialides, microscierotia, sporulation and growth rates were examined according to Gams' description [2]. Morphological variation between MBC-resistant and sensitive isolates vvas insignificant, however, all sensitive isolates produced microscierotia while only some resistant isolates did likewise. In conclusion, resistance to the control of Cobweb disease by MBC fungicides continues to pose a major economic threat to the mushroom industry. The disease appears to be caused by two closely related taxa of Ciadobotryum
  • , with resistant isolates constituting a distinct taxon of their own. A rapid PCR-based diagnostic test can identify fungicide resistant isolates and may facilitate effective fungicide treatment strategies in the future. This project was funded by the International Fund for Ireland. References 1. McKay, GJ, Egan, D, Morris, E and Brown, AE, 1998. Mycological Research, 102, 000-000. 2. Gams, W, Hoozemans, ACM, 1970. Persoonia, 6, 95-110.