The molecular identification and characterisation of Eutypa dieback and a PCR-based assay for the detection of Eutypa and Botryosphaeriaceae species from grapevine in South Africa

[摘要] ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for theslow decline of vines. In particular, Eutypa dieback of grapevine has had a devastatingimpact on vineyards worldwide, reducing growth and yield, eventually killing thegrapevine. The causal organism of Eutypa dieback was first described as Eutypaarmeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul(anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least twospecies that are capable of infecting grapevines are responsible for Eutypa dieback.Consequently, the molecular identification and characterisation of Eutypa dieback wasused to delineate the species occurring on infected grapevines in South Africa. Thisinvolved the molecular analyses of three molecular markers, namely, the internaltranscribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon,and the -tubulin gene. The results obtained revealed the presence of a second species,namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata oninfected grapevines.Also co-habiting with these pathogens were related fungi form the Diatrypaceae family,Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart.Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca,and E. vitis revealed that all were pathogenic to grapevine. Several species ofBotryosphaeriaceae that commonly invade the woody tissue of grapevines are alsopathogenic to grapevine. The symptoms in grapevine commonly associated withBotryosphaeriaceae are easily confused with the symptoms produced by Eutypa diebackwhich prompted the need for the development of a detection method that can correctlyidentify the presence of multiple pathogens.A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide arapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypadisease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplifyand label the regions of DNA that are used as pathogen specific probes. Consequently,membrane immobilised species-specific oligonucleotides synthesised from the ITS, -tubulin and LSU molecular data were evaluated during the application of this diagnosticmethod to detect Eutypa species. It was found that the species-specific oligonucleotides,designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca.The application of the RDBH method for the detection of these Eutypa species, based on-tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, aRDBH method, utilising species-specific oligonucleotides designed from elongationfactor-1α sequence data, was successfully applied for the detection of Botyrosphaeriadothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels)Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels)Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.)Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for thedetection of Diplodia seriata De Not.In addition to the above-mentioned shortcomings, the RDBH was not amenable to thedetection of pathogens directly from field or environmental samples, but requiredpreparation of DNA from pure cultures. The method, however, allows for theidentification of multiple pathogens in a single assay. As DNA extraction methods areamended, improved and honed to obtain DNA from environmental samples, so would itincrease the usefulness of RDBH.

[发布机构] Stellenbosch University