Epidemiology of botryosphaeriaceous species associated with grapevines in New Zealand

Abstract

Botryosphaericeous species affect most grapevines tissues leading to dieback and wood decay. During sampling of vineyards across New Zealand, botryosphaeriaceous species were found in brown necrotic lesions on grapevine trunks (42%), green shoots (20%), canes (17%), plant debris on the ground (7%), weak buds (8%), leaves (3%) and shrivelled flowers (3%). The isolates, identified by morphological and molecular methods as Neofusicoccum australe, N. luteum, N. parvum, Diplodia mutila and D. seriata, were also found in dieback on twigs and branches of non-grapevine woody hosts living around the vineyards. All isolates were pathogenic on wounded grapevine green shoots, except D. seriata. On wounded trunks of potted vines of scion varieties Cabernet Sauvignon, Chardonnay, Pinot noir, Riesling, and Sauvignon blanc, with mycelial or conidial inoculum, the disease developed to a similar extent (P>0.05) with all species except D. seriata, and N. luteum was the most pathogenic. A microscopy investigation of conidium infection showed that 24 h after inoculation, conidia of N. luteum had not germinated and was shed from attached non-wounded leaves and shoots but had germinated and developed mycelium quickly on detached or wounded green leaves and shoots. Longitudinal stem sections showed mycelium within xylem vessels. Buds, green shoots, canes and trunks were susceptible to infection. In trunks of young vines, N. luteum progressed more quickly upward than downward, while bud and shoot infection could progress downward to infect adjacent tissues. As little as two conidia per wound on detached green shoots led to infection. A 12 month investigation of spore dispersal, in a Canterbury vineyard detected conidia of Neofusicoccum spp. (59.8%) and Diplodia spp. (40.2%) in rainwater traps throughout the year. The conidia were identified by molecular methods as N. australe, N. luteum and N. parvum, (the Neofusicoccum spp.) which were most abundant and D. africana, D. olivarum, D. cupressi or D. mutila (the Diplodia spp). No botryosphaeriaceous species conidia were collected on Vaseline®-coated slides set up to trap air-borne spores. When effects of environmental factors on conidial development and viability were investigated, they demonstrated that the conidia were well-adapted to the vineyard environment. The temperature ranges for conidial germination, growth and development varied between N. luteum, N. australe, N. parvum and D. mutila (P<0.001), however all four species germinated after 3 h and grew quickly on PDA at 20-30°C. Exposure to different levels of solar radiation showed no significant difference in germination between N. luteum, N. australe and D. mutila. Conidia exposed to non-filtered sunlight (+UV) had 35% germination after 7 h which reduced to 0% by 56 h, whereas conidia exposed to filtered sunlight (-UV) had 57% germination after 7 h which reduced to 21% after 70 h. However, conidia put under shade had 81% germination after 7 h and 65% after 70 h. Dry conidia placed at 100% RH showed 98% germination in 3 h and had developed into a mycelium by 24 h, however, at 93% RH germination was about 46% and took 24 h, with no mycelium being formed by 48 h. No conidial germination had occurred after 48 h at 84% RH. Survival was also affected by low RH. Conidia incubated at 68% RH and then re-incubated at 100% RH showed levels of mortality that increased with the time of the low RH exposure, reducing from 88% after 7 h and to 26% germination after 70 h at 68% RH. Neofusicoccum luteum conidial suspensions could infect wounds with no continuing surface wetness, although infection and subsequent progression through the plant was significantly greater at 95% than 78% RH. Wounds on trunks of potted vines were susceptible to infection for the first two days and then incidence decreased to 0% by 14 days for conidia and 40% by 30 days for mycelium inoculum. Stress factors such as water stress of 15% or 100% field capacity (FC) had a delayed effect, with significantly increased dieback and more dead buds visible when new growth occurred after winter pruning. In vitro fungicide experiments with 16 products and three isolates each of N. australe, N. luteum and D. mutila showed differences in mycelial growth and conidial germination between isolates and species, and between products with N. luteum being the least sensitive. In vivo evaluations on cane pruning wounds made on potted and field grapevines, subsequently inoculated with N. luteum conidia, showed that flusilazole and carbendazim, were the most effective at protecting pruning wounds from infection.