Fungal trunk pathogens in grapevine nurseriescharacterization, potential inoculum sources and management strategies

Resumen

Young vine decline and failure of newly planted vines (1 to 4 years old) are currently common problems in wine-producing countries around the world. The presence of several fungal trunk pathogens in affected plants generated a complex situation which focused the attention of researchers during the late 1990s. The subsequent increase in studies on young vine decline has led to the conclusion that planting material is already infected in young vineyards, either systemically from infected mother vines or by contamination during the propagation process. In this thesis, a characterization study of different fungal trunk pathogens was performed. The evaluation of potential inoculums sources in grapevine nurseries as well as different control strategies to improve disease management was also investigated. Firstly, eight Phaeoacremonium isolates were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the actin and beta-tubulin (BT) genes. Two new species were described. Pm. cinereum was isolated from a young vine in Spain and from older vines in Iran. Pm. hispanicum was isolated only once from a young vine in Spain. In addition, fifty-eight Cadophora luteo-olivacea and three C. melinii isolates were studied by means of phenotypical characterization, DNA analyses and pathogenicity tests. These isolates were obtained from grapevines showing black vascular streaking and decline symptoms characteristic of Petri disease, and from different stages of the grapevine nursery process in Spain: hydration tanks, grafting machines and pruning scissors. Phenotypical data were subjected to cluster analysis, which clearly separated C. luteo-olivacea isolates in four groups. Partial sequences of the nuclear ribosomal internal transcribed spacer, BT and elongation factor were analysed, but no genetic variation occurred within the C. luteo-olivacea and C. melinii isolates in all regions studied. Pathogenicity tests were conducted in 1-year-old grapevine cuttings of four different rootstocks (140 Ruggeri, 161-49 Couderc, 1103 Paulsen and 110 Richter) using four C. luteo-olivacea and one C. melinii isolates. All Cadophora isolates, except the C. melinii isolate, caused lesions in the xylem of grapevine rootstocks that were significantly longer than the controls. The sanitary status of rootstock mother plants in Spain was evaluated in fourteen mother fields in 2006 and 2007. Grapevine shoots were processed and incubated in culture medium for fungal isolation and identification. Additionally, five commercial nurseries were sampled at different stages of the propagation process. Samples were analysed using two different techniques: nested-PCR and fungal isolation by culturing on semi-selective medium. Results showed that grapevine mother plants in Spain are an important inoculum source of fungal trunk disease pathogens, such as Pa. chlamydospora, Pm. aleophilum, Pm. parasiticum, several Botryosphaeriaceae spp., as well as Phomopsis spp. In nurseries, it was shown that potential infection points for plants are: hydration tanks, grafting machines, scissors and peat, where Phaeoacremonium spp. and Pa. chlamydospora were detected. Regarding management strategies, the sensitivity of black foot and Petri disease pathogens to hot-water treatments (HWTs) in vitro was evaluated. Results showed that treatments at 48ºC for 60 min were enough to inhibit completely the germination of conidia and mycelial growth of Cylindrocarpon liriodendri and Cn. macrodidymum. For Ca. luteo-olivacea, Phaeoacremonium spp. and Phaeomoniella chlamydospora, treatments of up to 53-54ºC were necessary to inhibit conidial germination and mycelial growth. The conditions suitable for HWT of dormant rooted rootstocks (41 B Mgt, 161-49 C, 110 R, 140 Ru and 1103 P) and grafting cuttings (Tempranillo/110 R, Merlot/110 R, Bobal/1103 P and Tempranillo/161-49 C) using a broader range of temperatures were also evaluated. Results demonstrated that rootstocks and grafted cuttings can tolerate temperatures from 50°C to 53°C without significant reduction in sprouting or growth. Satisfactory control percentages for Pa. chlamydospora and Pm. aleophilum were obtained at temperatures above 51°C. In vitro evaluation of fungicides showed that azoxystrobin, captan, carbendazim, copper oxychloride, cubiet, didecyldimethylammonium chloride and thiram were the most effective in reducing conidial germination for Pa. chlamydospora and Pm. aleophilum, while carbendazim, flusilazole and tebuconazole were the most effective in reducing mycelial growth. Additionally, an experiment was carried out to determine the effect of fungicides during the hydration stage in the grapevine propagation process. Results showed that non-inoculated cuttings were colonized by Pa. chlamydospora or Pm. aleophilum in the untreated control treatment. The most effective fungicide was didecyldimethylammonium chloride, which reduced the infection by Pm. aleophilum and completely inhibited the infection by Pa. chlamydospora at the highest inoculum concentration. The semi-commercial nursery trial conducted to evaluate the effectiveness of several strategies with selected fungicides at different grapevine nursery stages indicated that soaking planting material in carbendazim during the hydration stage was the most effective treatment since neither Pa. chlamydospora nor Pm. aleophilum were isolated from planting material in two different nurseries. Finally, the effects of artificial inoculations of fungal trunk pathogens associated with Petri disease and esca on the grapevine rootstocks most widely planted in Spain (41 B Mgt, 161-49 C, 110 R, 140 Ru and 1103 P) under field conditions were evaluated. After one growing season, most of the fungal pathogens caused a significant reduction in the percentage of cuttings emerging from dormancy and shoot weight and a significant increase in disease severity in all grapevine rootstocks except 161-49 C. Rootstocks 110 R and 140 Ru were greatly affected by fungi associated with Petri disease and esca. In general, Pa. chlamydospora and Pm. parasiticum caused the greatest reduction in percentage of cuttings emerging from dormancy and shoot weight and the highest increase in disease severity. Regression analyses showed a significant correlation between percentage of cuttings emerging from dormancy and disease severity and between shoot weight and disease severity in almost all rootstocks inoculated with Pa. chlamydospora.