Cylindrocarpon black foot disease in grapevines: identification and epidemiology
Abstract
This research aimed to improve understanding of the disease development processes in black foot disease on grapevines in New Zealand. The pathogenicity of three isolates from each of Cylindrocarpon liriodendri, C. macrodidymum and C. destructans was investigated by inoculating callused and rooted grapevines from rootstock varieties 101-14 and 5C. Results showed inter- and intra-specific variations, with disease incidences ranging from 32.1% to 82.1%. Callused vines had significantly greater disease incidences than rooted vines (P<0.001), but disease incidences were similar for both rootstock varieties.
Conidia, chlamydospores and mycelium from these species, when added to potting mix, were capable of infecting grapevines through wounded roots and callused basal ends. Conidia caused greater disease incidences than chlamydospores and mycelium, with means of 81.3, 62.5 and 56.3%, respectively, and C. liriodendri was consistently the most pathogenic species. The pathogenicity of the different propagules was confirmed in the field where similar results were observed to those in potting mix. When three propagule concentrations were tested for their ability to cause disease, increasing propagule concentrations resulted in increasing disease incidences. However, even the lowest concentrations of 10² spores /mL and 1 g of mycelium infested wheat grains were capable of infecting grapevines in potting mix.
Two different PCR based methods were developed to identify the different species and to test infested soils using species specific primers from the beta-tubulin 2 gene. The optimised nested PCR had a detection limit of 1 pg of pure DNA for all species, while the quantitative PCR was capable of detecting as little as 11.5, 4.8 and 66.0 pg of C. macrodidymum, C. iriodendri and C. destructans pure DNA, respectively. A method was developed to extract DNA from 10 g of soil. The nested PCR was capable of detecting 1 × 10µ Cylindrocarpon conidia /g of soil for all species while the quantitative PCR was capable of detecting 1.0 × 10³ and 1.9 × 10³ conidia /g for C. liriodendri and C. macrodidymum, respectively. The quantitative PCR was tested with soil which was infested with 10µ conidia /g of soil at 0, 1, 3 and 6 weeks after inoculation. After a week, less than 5% of C. liriodendri propagules were detected, the numbers remaining constant over a period of 6 weeks, while C. macrodidymum propagules were not detected. The rapid reduction of propagule numbers in soil was also observed when conidia and mycelium were buried in mesh bags in soil. The two propagule types were converted into chlamydospores or degraded.
The effects of different stress factors on disease development were investigated. Infection incidence was increased in heavy soil types (P=0.001) and with waterlogging or dry (75% FC) watering regimes (P=0.022) while the effects of prolonged cold storage, wound age (0 to 8 days) and the presence of Phaeomoniella chlamydospora in grapevines did not show an apparent effect on disease development. The presence of wounds on canes, graft-union and trunks was necessary for their infection while disease incidences were similar for wounded or unwounded roots. This study has provided information about the behaviour of propagules of three Cylindrocarpon species in potting mix and in soil and the development of methods to detect these pathogens which could potentially be used to assess disease risks.
