Development of biocontrol methods for camellia flower blight caused by Ciborinia camelliae Kohn

Camellia flower blight is caused by the sclerotial-forming fungus Ciborinia camelliae Kohn, and is specific to flowers of most species of camellias. An investigation was conducted into the feasibility of a wide range of biological methods for control of the pathogen by attacking soil-borne sclerotia and thereby preventing apothecial production, and protecting camellia flowers against ascospore infection. Two methods were developed to assess the viability of C. camelliae sclerotia for subsequent use in sclerotial parasitism assays. One method involved washing and rinsing sclerotia twice in 13.5% NaOCl, followed by immersion in antibiotics, bisection of sclerotia fragments onto potato dextrose agar, and identification of C. camelliae mycelium after incubation. Another method used sclerotial softness as an indicator of viability. Softness was determined from the energy required to push a 2.76 diameter penetrometer with a force of 40 N into a 6 mm thick segment of healthy sclerotia (17 and 14 Nmm for medium and large sizes). This method was modified by recording as parasitised those sclerotia that were pierced under a 1-kg probe with a footprint of 6.2 mm², which delivered a pressure 1.6 MPa. The known mycoparasites Trichoderma virens, T. hamatum, T. stromaticum, Clonostachys rosea, Coniothyrium minitans and Sporidesmium sclerotivorum were not effective in reducing sclerotia viability. However, in screens of 400 candidate microorganisms isolated from sclerotial baits and decaying sclerotia, two isolates of Trichoderma and an isolate of Fusarium lateritium reduced the number of viable sclerotia by 38-50%. This indicated that moderately parasitic micro-antagonists were present in soil under camellia bushes. A range of soil treatments, which were postulated to stimulate micro-parasitic activity, was investigated in an attempt to reduce the viability and germination potential of the overwintering sclerotia. Urea, applied to soil at 50 kg N/ha, with Bio-Start soil conditioners in February and again in June reduced field populations of sclerotia from 294 to 105 sclerotia/m² by the following November. Covering soil under camellia bushes with 100 mm thick tree mulches for 9 months resulted in total suppression of apothecia and a 77% reduction in the population density of soil-borne sclerotia, compared to bare soil. However, tree mulches had no effect on the new generation of C. camellia sclerotia developing in fallen flowers. Mulches amended with lignin-degrading white-rot fungi offered potential for decay of newly formed sclerotia. In laboratory assays, pine (Pinus radiata) sawdust amended with Phanerochaete cordylines LU900 led to a 77-83% reduction in viability of sclerotia after 12 weeks, compared to a 1-7% reduction in sclerotia embedded in sawdust alone. Ligninolytic enzymes released by the isolate were shown to degrade the rinds of sclerotia, exposing them to microbial parasitism. The practical application of P. cordylines is dependent upon confirmation that it is non-pathogenic to camellia. Integration of the three soil treatments offers potential for longterm control of camellia blight. A single application of the fertiliser calcium cyanamide at 500-1000 kg/ha to soil under camellia bushes immediately before flowering (August), gave complete suppression of apothecial production, and can be used for short-term control of the disease. Potassium bicarbonate and ammonium bicarbonate at 300 kg/ha was less effective. Attempts to protect camellia flowers from infection by C. camelliae ascospores were only partially successful. Although isolates of Bacillus, Pseudomonas, Aureobasidium and Cladosporium spp. provided almost complete protection against camellia blight in petal assays, they did not prevent symptoms in whole flowers on camellia bushes, even after repeated applications with or without adjuvants. Weekly applications of the elicitor acibenzolar-S-methyl to camellia bushes, 3 weeks before and during flowing to induce systemic acquired resistance, were also not effective in controlling camellia blight. The genetic diversity of C. camelliae isolates was investigated using a universally primed polymerase chain reaction DNA fingerprinting technique. The 27 isolates from four regions in New Zealand were found to be distinctly different to those from United States, which comprised three isolates each from Georgia, Oregon and Virginia. However, a low level of genetic variation (<20%) existed between isolates, suggesting that biocontrol agents are likely to be effective for control of all isolates of C. camelliae. The successful control strategies developed in this study could be integrated into a programme for effective control of camellia blight. Urea applied to soil beneath camellia bushes followed by a 100 mm thick layer of tree mulch should result in total suppression of apothecia, thereby preventing infection in flowers, and resulting in a gradual decline in numbers of soil-borne sclerotia. Amendment of the mulch with lignin-degrading white-rot fungi may control newly formed sclerotia developing in fallen flowers that have been infected by air-borne inoculum from adjacent untreated areas. Alternatively, apothecial production can be totally suppressed with calcium cyanamide applied to soil under camellia bushes immediately before flowering each year.