The Biology and Epidemiology of the Grapevine Trunk Disease Pathogen Boryosphaeria spp

Grapevine diseases caused by fungal species of Botryosphaeriaceae are known as
Excoriose, Black dead arm, Diplodia cane dieback, bunch rot, Botryosphaeria (‘Bot’) canker or Botryosphaeria dieback. Symptoms include dead arms, dead canes and shoots, delayed bud burst, bud necrosis, bud death, bleached canes and reduced bunch set, which all contribute to a general loss of vine vigour commonly known as grapevine decline. In the past two decades, the importance of species of Botryosphaeriaceae to the grape and wine industry worldwide has gradually become obvious. In Australia, grapevine decline symptoms associated with species of Botryosphaeriaceae have also been reported frequently. In this thesis, a series of studies on the biology and epidemiology of species of Botryosphaeriaceae that cause wood necrosis are presented.

A major survey of grapevine exhibiting decline symptoms was conducted in the Hunter Valley and Mudgee regions in New South Wales, Australia. A hierarchical sampling method was employed in an attempt to unveil the distribution of species of Botryosphaeriaceae on several geographical scales. Twenty-five grapevines per vineyard were surveyed and the accuracy of estimating the incidence of species was increased by sampling both the trunks and cordons of each grapevine. A total of 450 wood samples were obtained from a total of 300 vines. Botryosphaeriaceae were isolated from all 11 vineyards. Isolates of Botryophaeriaceae species were identified using both morphological and molecular methods. Up to 36% of surveyed grapevines were infected with species belonging to the Botryosphaeriaceae. The incidence of Diplodia seriata was greatest, followed by Neofusicoccum parvum, Botryosphaeria
dothidea and Lasiodiplodia theobromae. Other pathogens capable of causing trunk diseases were also, on occasion, isolated in this survey demonstrating that the grapevine decline syndrome is indeed a complex problem.

The second objective of the research was to examine the pathogenicity of the isolates. Even though species belonging to the Botryosphaeriaceae have been subjected to various pathogenicity tests around the world and their ability to induce disease symptoms is no longer in question, factors that contribute to apparent discrepancies in virulence among Botryosphaeriaceae species are still unclear. The effects of a water stressed host and several different incubation temperatures which may have an impact on the virulence of Botryosphaeriaceae species were studied in this project. All four previously isolated species were tested by inoculation of trunks and cordons of mature grapevines (Vitis vinifera, cv. Chardonnay), inoculation of two-year-old potted grapevines in a glasshouse comparing water stressed versus non-water stressed vines, and by inoculation of one-year-old detached canes incubated at 25, 30 and 35 ºC. Significant intra- and inter-species variation in virulence was observed between water stressed and non-water stressed vines. Increased virulence in most isolates was observed
under 30 ºC, while variation in virulence was observed for different isolates of the same species at 35 ºC. In order to better understand the difference in virulence under different temperatures, an optimal growth temperature study was also conducted. L. theobromae was the most rapidly growing species at the optimal growth temperature of 31.3 ºC followed by B. dothidea at 31.6 ºC, N. parvum at 30.2 ºC and D. seriata at 27.6 ºC. At 25 ºC L. theobromae was also the fastest growing species with a growth rate of 1.07 mm/hr followed by D. seriata at 0.87 mm/hr, N. parvum at 0.86 mm/hr and B. dothidea at 0.83 mm/hr. Using data obtained from the optimal growth temperature study and the climatic modelling software, CLIMEX, the potential distribution area of species of
Botryosphaeriaceae in Australia was predicted.

One of the advantages for employing the hierarchical sampling method in the survey was that it enabled a systematic study of population structure of isolates of Botryosphaeriaceae from a large regional scale down to a very small scale (e.g. within a single plant). A range of polymerase chain reaction (PCR) primers, including a selected set of six Inter Simple Sequence Repeat (ISSR) primers, the Intron Splice Junction R1 (ISJ-R1) primer and the M13 core sequence microsatellite primer were used in this study due to their robustness and effectiveness in intra- and inter-specific comparisons in studies of population structure. A total of 171 isolates of Botryosphaeriaceae were assessed, and the results from this study indicate there was detectable variation within
populations of Botryosphaeriaceae even within a vineyard. A total of 280 highly
reproducible polymorphic bands were detected across all isolates following PCR. Intraand inter-specific differences were visualised as a dendrogram and principle coordinates plot. Isolates were separated into four distinguishable groups according to species based on cluster analysis with the integrated locus matrix. Strong evidence suggests that D. seriata was well differentiated regionally. Furthermore, different genotypes of D. seriata between vineyards in several instances were well separated. Greater homogeneity was obvious with isolates of N. parvum between vineyards and regions, while isolates of B. dothidea were homogenous between regions from which they were isolated (Lower and Upper Hunter Valley). The results indicate the degree of variation within populations of Botryosphaeriaceae in vineyards and should be used to guide sampling in future surveys and population studies of the Botryosphaeriaceae.