Detection of black-foot disease pathogens in the grapevine nursery propagation process in Spain

Abstract

Two commercial nurseries located in Comunidad Valenciana region (central-eastern Spain) were sampled in 2010 to evaluate whether the grapevine nursery propagation process could be a source of black-foot disease pathogens. Samples were taken from four sources of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, omega-cut grafting machines, and peat used for callusing. DNA from these samples was extracted and multiplex nested-PCR using primers specific for >Cylindrocarpon> pauciseptatum, Ilyonectria liriodendri and I. macrodidyma-complex (composed of I. alcacerensis, I. estremocensis, I. macrodidyma, I. novozelandica, I. torresensis, and two undescribed species) was used to identify the species present. Ilyonectria liriodendri and I. macrodidyma-complex were detected in hydration tanks, scissors, grafting machines and peat, I. macrodidyma-complex being the most frequent. Additionally, ten grafted cuttings each from five grapevine scion/rootstock combinations were collected from each nursery immediately after callusing, and again after one growing season in a nursery field. Roots of these grafted cuttings and plants were sampled to isolate the fungal pathogens. Only I. torresensis was isolated after callusing, while I. liriodendri, I. novozelandica and I. torresensis were isolated after one growing season, showing the highest incidence at this latter sampling time. Moreover, DNA was extracted from roots and analyzed as described before. Ilyonectria liriodendri and I. macrodidyma-complex were also detected at both sampling times. The use of the multiplex nested-PCR technique improved the detection of I. liriodendri and I. macrodidyma-complex from grafted cuttings and plants in both nurseries. This work shows that the grapevine nursery propagation process should be considered as a potential infection source for black-foot disease pathogens, and confirms that infections caused by Ilyonectria spp. in grapevine planting material increase markedly after one growing season in nursery fields. © 2013 KNPV.