An investigation of Sclerotinia sclerotiorum as a containable mycoherbicide

Abstract

A strain of the broad host range phytopathogen Sclerotinia sclerotiorum (S36/0) has been developed as a mycoherbicide for a variety of pasture weeds, in particular Californian thistle. However, the fungus produces sclerotia and ascospores, which enable it to persist and disperse, potentially causing an unacceptable risk of disease to S. sclerotiorum-susceptible crop plants adjacent to the mycoherbicide application site. In developing the mycoherbicide, the risks presented by these structures must be evaluated. This thesis describes the use of molecular fingerprinting to characterise S. sclerotiorum population diversity and to monitor ascospore dispersal of the mycoherbicide strain. In addition, any risk may be minimised by a better knowledge of the processes involved in sclerotium formation. Therefore, two methodologies by which the molecular basis of sclerotium formation may be studied, namely RNA fingerprinting and gene tagging, were also investigated.

Mycelial compatibility, and two molecular fingerprinting methods (amplified fragment length polymorphism (AFLP) and universally primed polymerase chain reaction (UP-PCR)), were used to genotype a population of S. sclerotiorum growing on Californian thistle at Tai Tapu, Canterbury. The former method determined 14 compatibility groups, whilst each primer used for fingerprinting resulted in only 4-5 genotypes. Combining the data from all methods was more sensitive, delimiting 24 groups. Spatial analyses revealed that individual genotypes were clustered within the field, suggesting clonal propagation, whilst novel genotypes at the margins of the paddock suggested recruitment of new strains from external sites.

AFLP, using a single primer (Taq1 + GG), was used to generate a "fingerprint" for S36/0 which was unique when compared to 100 other strains from throughout New Zealand. The fingerprint was then further refined into a diagnostic PCR and RFLP marker, based on a single band from the S36/0 fingerprint. Since this band was shared by several Canterbury strains, the diagnostic PCR/RFLP was used in conjunction with MCG to uniquely identify the mycoherbicide strain. Sequencing of the diagnostic PCR marker band revealed that it encoded part of a translation initiation factor of the eIF-2B type. Field samples were conducted in which ascospores at various points downwind of biocontrol test sites were trapped onto S. sclerotiorum selective agar. The resulting colonies were then genotyped using either the fingerprint or the diagnostic PCR/RFLP. The mycoherbicide dispersed less than 7 m from a biocontrol source in Canterbury sheep pasture, and in a dairy pasture at Takaka, it added few ascospores to those already present in the atmosphere as a result of natural inoculum sources.

The molecular basis of sclerotial formation was investigated by comparing RNA fingerprints of a wild type S. sclerotiorum strain (G1) and a non-sclerotial mutant derived from that strain (G5). Of nine bands present in the wild type but absent in the mutant, six encoded putative genes including transcription activators, a sugar transporter and a Ras homologue. Northern dot blotting confirmed that these genes were differentially expressed in sclerotium-forming mycelia, and that the timing of their expression in the mutant strain was aberrant. Expression of a G protein alpha subunit (Gα), putatively involved in signal transduction during early events in sclerotium formation, was also aberrant suggesting that the genes identified in this study act down stream of a Gα signal pathway.

Gene tagging technology in S. sclerotiorum is limited by the lack of a suitable method of obtaining homokaryotic strains carrying interrupted genes. Mycelial fragments and ascospore germlings produced mainly multinucleate protoplasts, whilst uninucleate microconidia were resistant to lysis enzymes. However, the existence of a strain (GL) that carpogenically germinates within ~30 days growth may provide an efficient way of obtaining homokaryotic mutant strains from wild type/mutant heterokaryons obtained by transformation of mycelia-derived protoplasts.

These studies contribute to several issues in developing S. sclerotiorum as a containable mycoherbicide. Firstly, the molecular fingerprint enabled the mycoherbicide strain to be unambiguously identified in field samples of ascospores. Using this marker, the magnitude of S36/0 dispersal was evaluated; such information could be used in a risk analysis to estimate safety zones around application sites. Secondly, sclerotium formation was studied, leading to the identification of several genes putatively involved in mycelial differentiation. This information may lead to new ways of decreasing the formation and longevity of these structures, either by the gene-interruption technology described in this thesis, or by increasing their rate of degradation.