New insights into the biology, ecology and control of black-foot disease in grapevine stars

Abstract

Grapevine trunk diseases (GTDs) are a growing concern in the wine sector worldwide. Among them, black-foot disease (BFD) has increased its incidence in grapevine nurseries and newly established vineyards. BFD is caused by numerous Cylindrocarpon-like asexual morphs species. This disease has been widely studied in Spain in recent years. However, the constant reclassification and taxonomic expansion of the species associated with BFD, the restriction in the application of fungicides, and the emergence in the use of high-throughput sequencing techniques has made necessary a review and an update of the knowledge obtained so far. In this Ph.D. thesis, the biology and ecology of the disease was studied in detail. The evaluation of different control strategies to improve disease management was also investigated. Firstly, a wide collection of isolates associated to BFD, which were obtained from asymptomatic vines, were characterized. These isolates were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the his3, tef1 and tub2 genes, and the ITS region. Two new species associated with the disease were described: Dactylonectria riojana and Ilyonectria vivaria. Ilyonectria pseudodestructans and Neonectria quercicola were isolated for the first time from grapevine in Spain, raising the total number of fungal species associated to BFD in our country to 17. The development of a semi-selective medium based on previous research allowed the identification and quantification of viable propagules of fungi associated with BFD from soil samples. The presence of BFD inoculum in rotating nursery fields and in mature vineyards was confirmed. Moreover, a positive correlation was established between Colony Forming Units (CFU) of BFD pathogens and the CaCO3 concentration in soil. On the other hand, the fungal and bacterial microbiome of the rhizosphere of 5 rootstocks in two vineyards located in La Rioja and Navarra has been characterized by high-throughput amplicon sequencing (HTAS). The results showed that grapevine rootstock genotype was the most important factor in shaping the microbiome in a mature vineyard (25-year-old), but not in a young vineyard (7-year-old). However, several bacterial and fungal species were found in both vineyards, demonstrating the existence of a “core” microbiome conserved in the vineyard, regardless of the geographic region. In addition, a positive correlation has been observed between the relative abundance of BFD pathogens obtained by HTAS and by qPCR. Moreover, the rhizosphere compartment of the "140 R" and "161-49 C" rootstocks harboured lower number of these pathogens than the "1103 P", "110 R" and "41 B" rootstocks. Finally, regarding control measures, the efficacy of white mustard biofumigation was compared with the use of propamocarb + fosetyl-Al, as well as the effect of Tusal® (Trichoderma atroviride T11 + Trichoderma asperellum T25) application. Biofumigation with Brassica sp. is a promising alternative to the use of chemical fungicides for BFD control, while the application of commercial products based on Trichoderma sp. on the roots before planting resulted ineffective for disease management.