Determining the key pathogenicity factors in Sclerotinia sclerotiorum to improve its potential as a mycoherbicide

The objective of this research project was to provide a basis for enhancing the storage and field performance of a S. sclerotiorum-based mycoherbicide intended for control of Ranunculus acris in dairy pastures. The approach was to determine the key factors regulating the germination, infection, growth and in vitro survival of the pathogenic fungus. Histopathology, biochemical and molecular studies were used to determine the response of the pathogen-host interaction to varying treatments to define the optimal conditions for disease development. Moisture had a significant effect on pathogenicity of S. sclerotiorum in detached leaf and whole plant tissue. In the leaf experiments, the humid treatment produced 78, 93 and 95% more disease than 12 h humid/dry, 4 h humid/dry and continuous dry treatments, respectively. Histopathology assessments showed that under humid conditions, surface hyphae, infection cushions and infection hyphae were produced at 2, 6 and 12 h after inoculation (HAl), respectively. In contrast, under the dry treatment surface hyphae and infection cushion were first observed at 6 and 12 h, respectively, while no infection hyphae were observed at any assessment time. In whole plant experiments, significantly more disease was produced under the continuous humid treatment compared to dry, combination of 6 or 12 h humid/dry and 6 h humid/dry/humid treatments. Pathology, biochemical and molecular experiments were conducted to determine the effects of urea and/or calcium hydroxide (lime) on pathogenicity. Histopathology studies showed that similar levels of infection structures were produced under both the water and urea treatments. Under the calcium hydroxide treatment, there were fewer surface hyphae and infection cushions produced at each assessment time while there was a 12 h delay in the production of infection hyphae. Separate detached leaf experiments showed that similar levels of disease were produced under the nitrogenous fertilisers to that of the water treatment. However, approximately 30% less infection was produced when calcium hydroxide was applied alone or in combination with urea at different application times compared to the water treatment. In whole plant experiments, significantly less disease was produced when calcium hydroxide was applied at inoculation or 24 h prior to inoculation with urea applied at the time of inoculation compared to the water treatment. Under these treatments, S. sclerotiorum isolate S36 produced disease severity scores of 2 and 4.2, compared to disease scores of 6.2 under the water control; while isolate LU459 produced disease severity scores of 1.5 and 0.83, compared to a disease score of 6.2 under the water control. As calcium hydroxide reduced the pathogenicity of both S. sclerotiorum isolates, biochemical and molecular studies were conducted to investigate its effect on oxalic acid production and expression of pg1-pg3 and acp1 genes. Results showed that maximum gene expression occurred when oxalic acid levels ranged between 3.8 and 4.5 µM. Under the high pH of the calcium hydroxide, oxalic acid production was delayed with a concomitant repression of the pg1-pg3 and acp1 genes. It is postulated that oxalic acid secreted by isolate S36 sequestered the readily available calcium (Ca²⁺) ions from the calcium hydroxide applied on the leaf surface. As a consequence, neutralisation of oxalic acid through chelation of Ca²⁺ ions to form calcium oxalate inactivated the oxalic acid, thereby preventing rapid decrease to the acid pH required for expression of pg 1-3 and acp1 genes. Observations of variable efficacy of the mycoherbicide in previous glasshouse and field trials could have been attributed to genetic variation of the host plant. In this study, Randomly Amplified Polymorphic (RAPD) DNA and Universally Primed Polymerase Chain Reaction (UP-PCR) were used to identify diverse R. acris individuals collected from Takaka, Taranaki, Salt Water Creek and Leithfield regions. Polymorphic band profiles produced by RAPD and UP-PCR primers indicated that R. acris individuals were genetically distinct from each other. Band profiles were used to construct an unrooted neighbour joining (NJ) dendrogram, from which 10 Takaka individuals were chosen for pathogenicity experiments. Results showed that all above-ground tissue of R. acris individuals was equally susceptible to isolate 536. However, some resistance of the below-ground crown tissue was observed 2 months after pathogen application. For example, R. acris individuals Tk 1.19, Tk 4.16 and Tk 6.8 had 1.01, 1.02 and 1.05 g fresh weight (FW) tissue, respectively, while R. acris individuals Tk 1.8 and Tk 1.18 had 0.22 and 0.15 g FW tissue, respectively, by the end of the experimental period. The effects of long term preservation methods on two S. sclerotiorum isolates were investigated. When stored at -80°C and dry stored at 4°C, sclerotial viability (100%) and growth were maintained and both isolates produced 100% infection in each R. acris leaf over the 30 month experimental period. When freeze dried, sclerotial viability for isolate 536 and LU459 was reduced by 94 and 50%, respectively, 30 months after storage. However, growth and virulence was not adversely affected and was similar to that of the -80°C and dry stored at 4°C methods. Mycelial inoculum stored at -80°C was not viable at the 6 or 12 month storage treatment times. The potential of using ascospore inoculum for mycoherbicide formulation preparation was investigated. Ascospores amended with inorganic phosphorous/glucose were 11% more viable and germ tubes were 29 µM longer than ascospores amended with water. Pathogenicity experiments showed that ascospores amended with water, inorganic phosphorous/glucose, starch, urea and KNO3 produced 100, 80, 80 and 78% infection in wounded leaves. Only 9% of wounded leaves became infected under calcium hydroxide amendment. Non-wounded leaves became completely infected when ascospores were combined with inorganic phosphorous/glucose while less than 5% infection was produced under all other treatments. An ascospore concentration of 2. 5x10⁵ ascospores mL⁻¹ produced 100% infection in R. acris leaves, which was an equivalent level of infection to that of the mycelial plug. Mycelial inoculum would be favoured for the mycoherbicide over ascospore inoculum because of the technical difficulties in producing high concentrations of ascospores by carpogenic germination. Moisture, fertilisers and R. acris itself (i.e., crown tissue) influenced the ability of S. sclerotiorum to infect plant tissue and may have contributed to mycoherbicide variability in previous trials. Timing of mycoherbicide application that is matched with host phenology, irrigation schedules and/or rainfall and fertiliser application should help enhance the efficacy of the mycoherbicide. Additional cultural practices, such as host wounding and/ or host defoliation combined with repeated mycoherbicide application, may promote greater infection of the crown biomass, thereby reducing the regenerative potential of R. acris.