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. 2000 Oct;66(10):4475-80.
doi: 10.1128/AEM.66.10.4475-4480.2000.

PCR assays that identify the grapevine dieback fungus Eutypa lata

P Lecomte  1 , J P Péros, D Blancard, N Bastien, C Délye
Affiliations

PCR assays that identify the grapevine dieback fungus Eutypa lata

P Lecomte et al. Appl Environ Microbiol. 2000 Oct.

Abstract

Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.

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Figures

FIG. 1
FIG. 1
PCR products obtained with DNA extracted from E. lata according to a protocol described in reference (top and middle) or by boiling (bottom) were from the following isolates: BX 1-10 (lanes 1, 5, and 9), 8D (lanes 2, 6, and 10), and 8F (lanes 3, 7, and 11). Lanes 4, 8, 12, 16, 20, and 24, H2O negative control (no DNA). Primers used were Lata 1 and Lata 2.1 (lanes 1 to 4), Lata 1 and Lata 2.2 (lanes 5 to 8), Lata 3 and Lata 2.1. (lanes 9 to 12), SCA 10A and SCA 10B (lanes 13 to 16), SCB 02A and SCB 02B (lanes 17 to 20), and SCD 18A and SCD 18B (lanes 21 to 24). Lane M, molecular weight markers (1-kb DNA ladder; Life Technologies).
FIG. 2
FIG. 2
PCR detection of E. lata in wood samples from necrotic lesions. Amplifications were performed from 5-μl aliquots of 10-fold and 100-fold dilutions of supernatants obtained after boiling three wood shavings separately in sterile water (lanes 1 to 2 and 9 to 10, lanes 3 to 4 and 11 to 12, and lanes 5 to 6 and 13 to 14; odd-numbered lanes, 10-fold dilutions; even-numbered lanes, 100-fold dilutions). Primers used were Lata 1 and Lata 2.2 (lanes 1 to 8) and SCA 10A and SCA 10B (lanes 9 to 16). Lanes 7 and 15, H2O negative control (no DNA); lanes 8 and 16, E. lata positive control (DNA extracted from isolate BX 1-10); lane M, molecular weight markers (1-kb DNA ladder; Life Technologies).
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