5.5.18
PCR-BASED DETECTION OF TBZ RESISTANCE IN HELMINTHOSPORIUM

LR COOKE1,2 and GJ McKAYl

lDept. of Applied Plant Science, The Queen's University of Belfast, Newforge Lane, Belfast; 2Applied Plant Science Division, Dept. Agriculture N. Ireland, Newforge Lane, Belfast, BT9 5PX, UK

Background and objectives
The fungus Helminthosporium solani causes blemishing lesions (silver scurf) on the surface of potato tubers. The benzimidazole fungicide thiabendazole (TB2) has been used in the UK since the mid-1970s, but over the last ten years control of H. solani has been impaired by selection of TBZ-resistant strains [1]. Currently, these are detected by culturing the pathogen on fungicideamended agar, which may take several weeks. Benzimidazole resistance in field isolates of phytopathogenic fungi usually results from a point mutation in the fungal P-tubulin gene at codons 198 or 200. This study aimed to identify the mutations associated with TBZ resistance in H. solani and to develop a rapid molecular detection technique to identify resistant isolates of H. solani from the field.

Materials and methods
Seventy-nine isolates of H. solani from trials and advisory samples were tested for resistance by growing them on TBZ-amended agar. Isolates were classified as sensitive if they grew only on unamended agar, intermediate if they grew on 5 mg, but not 100 mg TBZ 1-1 and resistant if they grew on 5 and 100 mg TBZ 1-1 . DNA was extracted from 14 isolates, amplified by PCR using primers P-tubfl and P-tubrl, designed from conserved regions within the Ptubulin gene, and sequenced using a PE Applied Biosystems Model 373A DNA Sequencer. Primers SS-for and SS-rev, specific to H. solani, were designed from two regions within the P-tubulin gene identified as unique from previously published P-tubulin sequence data and tested against fungal DNA from 27 other species [2]. DNA was amplified directly from conidia from agar cultures of the 79 H. solani isolates and from infected tubers using the specific primers. After digestion with the restriction enzyme Bsa 1, the products were electrophesed and visualised.

Results and conclusions
Of the 14 sequenced isolates of H. solani, five TBZ-sensftive isolates all had the sequence GAG (Glu) at codon position 198 of the P-tubulin gene. A point mutation was detected at codon 198 in the other nine isolates. In three intermediate and three resistant isolates, codon 198 was converted to CAG (Gin) and in the other three resistant isolates to GCG (Ala) due to single base transversion mutations. Apart from codon 198, DNA sequences for this region of the P-tubulin gene were identical in all H. solani isolates. Primers SS-for and SS-rev amplified a product of the expected size (870 bp) from all isolates of H. solani and failed to amplify genomic DNA from other fungal species. The DNA nucleotide sequence at codon 198/199 of the TBZ-sensitive isolates equated to a restriction site for Bsa 1, which should thus cleave DNA at this site from sensitive isolates only. As predicted, Bsa 1 digestion of the PCR product from TBZ-sensitive isolates generated three sub-fragments, whilst resistant isolates generated two sub-fragments. Twenty-two isolates of H. solani designated TBZ-sensftive in vitro were classified as sensitive by the PCR-based test. Of 18 isolates designated intermediate in vitro, 13 were classified as resistant by PCR and five as sensitive. Of 39 isolates designated resistant in vitro, 37 were classified as resistant and two as sensitive by PCR; the anomalous results may have been from mixed cultures. DNA directly amplified from infected potato tubers gave results consistent with previous findings, but banding patterns were less intense. The overall time taken from sample preparation to visualising the agarose gel results was approximately 4 h. Thus, use of species-specific PCR primers, designed to amplify a specific region of the fungal P-tubulin gene, in conjunction with an appropriate restriction endonuclease, provides a rapid diagnostic test to differentiate field isolates of H. solani.

References
1. Hide, GA, Hall, SM, Boorer, KJ, 1988. Plant Pathology 37, 377-380.
2. McKay, GJ, Cooke. LR, 1997. FEMS Microbiology Letters 152, 371-378.