3.3.13
DETECTION OF HELMINTHOSPORIUM SOLANI WITH SPECIES-SPECIFIC PCR PRIMERS

C OLIVIER and R LORIA

Cornell University, Dept. of Plant Pathology, Ithaca, NY 14853, USA

Background and objectives
Helminthosporium solani is the cause of silver scurf of potato tubers, a tuber blemish disease that has gained great economic importance. The pathogen recently developed resistance to the postharvest fungicide thiabendazole (TBZ) and, at present, disease control strategies are lacking. H. solani is very slow growing and cannot be isolated from soil. Therefore, little is known about its ecology. It was our objective to develop species-specific primers for detection of H. solani by PCR in plant tissue and soil samples; techniques for detection of this pathogen are necessary in order to test hypotheses about soil survival, alternate hosts and saprophytic activity.

Materials and methods
The internal transcribed spacer (ITS) regions and 5.8S ribosomal DNA (rDNA) of six strains of H. solani (3-SS5 (New York), 12-SS2 (New York), 17-SS8 (Pennsylvania), HSWS04 (Wisconsin), YG21A (British Columbia)) were amplified by PCR and sequenced, using the universal primers ITS5 and ITS4 [1]. After database comparisons to published ITS sequences (BLAST, FASTA), a pair of primers specific for H. solani (HSF19-HSR447) was designed and tested by PCR for amplification from DNA of 30 isolates of H. solani from nine locations in North America, and from DNA of 35 other fungal species, including potato pathogens. The primer pair was also tested for amplification from DNA extracted from artificially and naturally infested soil. Included in these experiments were sterile soil (350 mg/sample) infested with H. solani mycelium (lyophilized, 0.5-10 mg/sample) or spores (2x104/sample), and soil (500 mg/sample) collected from potato fields with a history of silver scurf, which was assumed to be naturally infected. The specificity of the primers was also examined using plant tissue, since H. solani has been reported to have saprophytic ability [2]. DNA was extracted from the periderm of potato tubers (greenhouse-grown tubers inoculated with H. solani, field-grown tubers with silver scurf lesions) and amplified by PCR with HSF19-HSR447. Controls consisted of disease-free, greenhouse-grown potato tubers and tubers infected with Colletotrichum sp. Alfalfa, barley, buckwheat, canola, corn, mustard, oat, rye, sorghum, snap bean and wheat were grown in the greenhouse. Leaf and stem pieces (10 mm length) were surface-sterilized with 70% ethanol, air-dried and vacuum-infiltrated with spore suspensions of H. solani (105 spores/ml). Plant tissue was air-dried immediately or after four weeks of incubation in moist chambers. DNA was extracted from dried leaf or stem tissue (100 mg tissue/sample) and amplified by PCR. Controls consisted of non-inoculated plant tissue.

Results and conclusions
DNA of all strains of H. solani amplified a single product (447 bp) with HSF19-HSR447, whereas DNA from other fungi did not amplify with this primer pair. The 447 bp product was amplified from DNA extracted from soil samples infested with mycelium or spores of H. solani and from soil samples collected from potato fields with a history of silver scurf. No product was amplified from sterile soil. Primer pair HSF19-HSF447 also amplified a single product (447 bp) from potato periderm samples with silver scurf symptoms; disease-free tubers or tubers infected with Colletotrichum sp. did not yield an amplification product. DNA extracted from plants infiltrated with H. solani amplified a single product (447 bp), whereas no product was amplified from non-infiltrated plant tissue. Amplification results were confirmed by Southern hybridization, using the purified PCR product (447 bp) as a probe. We conclude that primers HSF19 and HSF447 are specific for H. solani and can be used to detect the fungus in soil or plant tissue, thereby aiding research on the ecology and epidemiology of this pathogen.

References
1. White TJ, Bruns T, Lee S, Taylor, J, 1990. Pp. 315-322 in: Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR protocols. Academic Press, 482 pp.
2. Merida CL, Loria R, 1994. American Potato Journal 71, 591-598.