Association and pathogenicity of Botryosphaeriaceae fungi on different Vitis vinifera tissues

Botryosphaeriaceae fungi causing Botryosphaeria canker, an internal infection of the wood, which leads to grapevine decline and dieback, are a great concern for the viticulture industry. Primarily regarded as wood pathogens of grapevines in Australia, some Botrysphaeriaceae fungi have been associated with bunch rots in table grapes and wine grapes in the United States and Europe. More recently, species of Botryosphaeriaceae have been isolated from rotten bunches of grapevines in subtropical wine regions of Australia. The role of Botryosphaeriaceae in the infection of the fruit of Australian wine grapes remains unclear. The overall aim of this thesis was to investigate the pathogenicity of Botryosphaeriaceae in grape bunches and whether infection with these fungi could result in bunch rot.

A major part of this study was to survey several different grapevine tissues at various phenological stages for the occurrence of Botryosphaeriaceae. A two year survey of two vineyards in the Hunter Valley (New South Wales, Australia) planted with Chardonnay and Shiraz resulted in a total of 188 isolates conforming to nine different species with Diplodia, Dothiorella and Neofusicoccum anamorphs. These were isolated from dormant buds, flowers, pea-sized berries and mature berries prior to harvest. A further 142 isolates were obtained from the trunks of the same vines. Dothiorella viticola, Diplodia mutila and Neofusicoccum australe were reported for the first time from grapevines in the Hunter Valley. These results provide evidence that grapevine tissue other than wood may act as potential inoculum sources for Botryosphaeriaceae in vineyards.

The second aim was to determine whether species of Botryosphaeriaceae are tissue specific or can infect various tissues, independently from their host tissue origin. A subset of isolates obtained in the survey was assessed for their potential to cross-infect other grapevine tissues. Tests showed that all isolates were able to cause symptoms on one year old canes and mature berries, independently from their tissue of origin and with virulence varying within species. No adverse effects were recorded in dormant buds inoculated with Botryosphaeriaceae.

These findings demonstrate that grapevine wood infected with Botryosphaeriaceae may act as inoculum sources for infection of vegetative tissue and vice versa and therefore Botryosphaeriaceae, which have been considered exclusively as trunk disease pathogens in Vitis vinifera, should be considered as important pathogens of the vegetative tissues of grapevines as well.

The final aim of this study was to determine the genetic diversity of the collection of isolates using amplified fragment length polymorphism (AFLP) analysis. After an initial screening of 24 EcoRI/MseI primer pairs on a subset of the eight most phenotypically diverse isolates, the four primer pairs which showed highest heterozygosity were chosen for the final selective amplifications of 180 isolates from the collection. Capillary electrophoresis was performed to detect AFLP fragments and resulting chromatograms scored for presence and absence of polymorphic peaks, providing individual identity and population structure for isolates.

Isolates showed no meta-population structure according to vineyards, origin tissues or origin plants. The population appeared to be panmictic indicating that asexual reproduction is likely to be the predominant reproductive strategy for these organisms. Population structures also showed that inoculum sources from outside each vineyard were equally important than from within the vineyards, suggesting that there have been multiple introductions of Botryosphaeriaceae and that the infection pathway into reproductive tissues of grapevines is not systemic. The genetic diversity study of these Botryosphaeriaceae populations identified possible gene flow within and between vineyards. Results of this study have provided information on the spread of Botryosphaeriaceae within the vineyards surveyed, as well as within individual plants.

This thesis has developed knowledge on the spread of Botryosphaeriaceae in vineyards; has expanded the known tissue tropism of these fungi; and highlights the importance for management of Botryosphaeriaceae in vineyards beyond the existing control approaches which are limited to their role as trunk disease pathogens.