Sammonds, Jackie2015-09-232015-09-232015https://hdl.handle.net/10182/6688The Botryosphaeriaceae species are important wound pathogens on grapevines, causing dieback, cankers and ultimately death of plants. This research programme investigated an unexplored aspect of the disease cycle, namely behaviour of conidia during the pre-infection stages. Adhesion properties of conidia were investigated for three species of Botryosphaeriaceae, Botryosphaeria dothidea, Neofusicoccum luteum and N. parvum. The surfaces chosen had different affinities for water, with cellulose being extremely hydrophilic, glass moderately hydrophilic, polystyrene moderately hydrophobic and parafilm (polyolefins +paraffin waxes) extremely hydrophobic. Results showed that surface wettability did notplay a major role in adhesion of the conidia. However, isolates differed in their conidial adhesion properties. For N. luteum isolate MM558, B. dothidea isolate 007 and N. parvum isolate G652, maximum mean percent adhesion on any surface was reached after 5 min (53.1%, 54.0% and 50.6%, respectively) and for N. luteum isolate CC445 after 20 min (61.4%). Overall, fewer conidia of the N. luteum isolates adhered to cellulose than to the other surfaces, namely glass, polystyrene and parafilm. Fewest conidia of B. dothidea 007 adhered to parafilm and fewest conidia of N. parvum G652 adhered to parafilm and polystyrene. Mean percent germination for isolates MM558, CC445, G652 and 007 on the same surfaces was highest on cellulose (72.2%, 76.9%, 52.4% and 74.9%, respectively) but did not differ on the other surfaces tested. Germ tube growth for all isolates was least on cellulose; initial cellulose agar assays indicated that germ tubes might be utilising the cellulose as a food source. Germination assays on surfaces of different hardness showed no discernible patterns of germination or growth related to hardness. Further, isolate MM558 germinated equally well when continuously shaken or when in contact with a surface. Treatment of conidia of isolate MM558 with a protease (pronase E) completely prevented adhesion, indicating that spore surface proteins play a part in adhesion. Treatment with cycloheximide, a protein biosynthesis inhibitor, reduced mean percent adhesion (25.1%) in comparison to the untreated control (72.0%) and heat treatment (110oC for 10 min) reduced the mean percent adhesion (28.0%) in comparison to the non-heated control (85.8%), further indicating a role for proteins in adhesion. Coomassie brilliant blue staining after incubation times conducive to germination was positive for all isolates; a blue ‘halo’, which indicated proteins, was often seen surrounding the conidia or near the germ tube emergence points. This stained matrix material also stained positive for alcian blue (mucopolysaccharides). Since this material was not seen during shorter incubation times, its production appeared to represent a second phase of the adhesion process. Investigations into the volatiles produced upon disruption of grapevine tissues showed increases in volatile compounds known to be produced from wounded tissues. The volatiles produced after wounding stems plus leaves were collected and 13 compounds identified. Of these, nine compounds were emitted in significantly higher amounts upon wounding, with cis-3-hexenal, cis-3-hexen-1-ol and cis-3-henenyl acetate being released at the highest rates (111.52, 96.95 and 46.09 ng/gfw/h, respectively). Collection of volatiles from stems only also identified 13 compounds of which nine were emitted in significantly higher amounts upon wounding, with 2-hexenal emitted at the highest rate (48.74 ng/gfw/h). Exposure of isolate MM558 conidia to volatiles released directly from wounded plant tissue caused a slight reduction in germination (82.9%) and in germ tube growth (33.4 μm) in comparison to the control (85.9% and 41.6 μm). Germination of isolate CC445 was unaffected by exposure to fresh plant volatiles, while mean germ tube growth was 7.3 μm less than for the control (33.8 μm). However, isolate G652 was unaffected. Exposure to wounded stem plus leaf tissue did not affect the germination of isolate MM558 conidia however germ tube growth was reduced by almost 50%. The trapped, eluted and stored liquid volatile mix had little effect on the germination and growth of isolate MM558 conidia. Exposure to 100 μL of some pure volatile compounds reduced germination of isolates MM558 and G652, with almost complete inhibition by trans-2-hexenal and cis-3-hexenyl acetate. Microscope observations of germination indicated a chemotropic response to wounds but attempts to measure the attraction to volatile sources were unsuccessful. The new information in this thesis has shown non-specific adhesion and germination processes for conidia of some Botryosphaeriaceae species and indicated potential for further research directions which may result in innovative control methods.enhttps://researcharchive.lincoln.ac.nz/pages/rightsgerminationhydrophobicityhydrophilicitywoundingvolatilesadhesionmetabolic inhibitorsbotryosphaeriaceous speciesconidiaThesisANZSRC::060505 MycologyQ112910566