Puccinia punctiformis as a biocontrol agent for Cirsium arvense: effect of biotic and abiotic factors on success of infection : A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Applied Science at Lincoln University

Californian thistle (Cirsium arvense L. (Scop.)) is a weed in pastures and cropping systems. The fungal biocontrol agent Puccinia punctiformis (F. Strauss) Röhl. performs inconsistently on C. arvense. P. punctiformis establishment and control of C. arvense may be attributable to differing plant endophytic populations in various environments. Therefore, a study on endophytic populations within leaf, stem, and root tissue of C. arvense was undertaken with seasonal dynamics and location considered. Ninety-one endophytic genera, of which 68 not previously known to be associated with C. arvense, were ascertained in this study by culturing methods. To determine the identity of these cultures, PCR with fungal specific primers was performed, followed by sequencing and identification of the PCR products. Endophytic communities of stems and leaves of C. arvense are dynamic, with changes in community composition occurring over seasons. In autumn, a significant increase in leaf endophytic diversity occurred, which may be affected by C. arvense shoots commencing die-off. Root endophytic communities were stable, with no significant changes in endophytic community composition nor diversity occurring. Leaf endophytic diversity was comparable between sites, but community composition differed. This study shows that endophytic communities are dynamic being season, site, and tissue dependent. To enable quantification of P. punctiformis in samples, several comparisons were undertaken to finetune the qPCR assay and determine its robustness. Results were categorised in the following concentration bands: P. punctiformis absent; low concentration present (<5 ng/µL); medium concentration present (5-100 ng/µL); high concentration present (>100 ng/µL). An efficiency correction would be applied to add to the quality of results obtained. To determine the effect of endophytes on P. punctiformis urediniospores infection, two genotypes of C. arvense were grown in either in a controlled environment glasshouse, as potted plants in a potting mix media, or under natural, field conditions in soil. Establishment of P. punctiformis and endophytic communities were compared between genotypes (G27 and F1) and growing conditions (glasshouse and field) by visual estimation as well as quantitative PCR for the presence of P. punctiformis. The number of endophytic genera as well as community composition were determined prior to trial commencement and on completion. Six weeks post-inoculation, G27 plants had higher levels of disease expression in the field compared to plants of the same genotype in the glasshouse (70% vs 20%, 23 vs 10 ng/µL, respectively; P<0.001). Whereas, F1 genotype showed greater infection severity under glasshouse conditions than in the field (46% vs 45%, 75 vs 1 ng/µL, respectively; P<0.001). Endophyte community composition was compared between least and most infected leaves of these plants. In a second study, endophytic populations were compared in symptomatic and asymptomatic shoots from a field with systemic rust infection. From these studies, some genera which may enhance P. punctiformis infection were identified. These included: Bipolaris, Curvularia, Gliocladiopsis, Leptosphaeria, Phoma, Phaebotryosphaeria, Septoria, and Septoriella. To determine the optimal urediniospores concentration and the effect of abiotic factors on P. punctiformis urediniospores infection, four duplicate pot trials were performed. Spore concentrations ranging from 0.1-0.4 mg/mL effectively established P. punctiformis infection. A selection of positively charged compounds (chitosan, and four nitrogen fertilisers) were trialled, with none enhancing P. punctiformis infection. Despite this study not identifying a method to enhance P. punctiformis infection, it has contributed to the knowledge on C. arvense endophytes. Further research could explore if the genera found in highly infected leaves of C. arvense could be used to enhance P. punctiformis infection.