Production, Survival and Evaluation of Liquid Culture-produced Inocula of Coniothyrium minitans Against Sclerotinia sclerotiorum

Growth of Coniothyrium minitans on potato dextrose broth was compared with that on an inexpensive molasses-yeast liquid medium at 18-22°C in static culture. Biomass and conidial production were, in general, similar, although the rate of biomass production was quicker and conidial production was slightly greater per unit volume of medium in the molasses-yeast medium. Air-dried biomass from molasses-yeast liquid culture containing mycelia, pycnidia and conidia of C. minitans was mixed (12%, w/w) with kaolin to give a kaolin-biomass dust. The ability of C. minitans to survive and subsequently infect and reduce the viability of sclerotia of Sclerotinia sclerotiorum from this kaolin-biomass dust was found to be little affected by storage for 48 weeks between 4 and 15°C but was decreased by higher storage temperatures. The kaolin-biomass dust preparation did not differ from a standard maizemeal-perlite inoculum of C. minitans in its ability to infect sclerotia of S. sclerotiorum or reduce their viability or carpogenic germination in glasshouse and field pot bioassays. Further, when either inoculum was applied once to glasshouse soil naturally infested with S. sclerotiorum prior to planting three successive crops of lettuce, the pattern of disease control, reduction of sclerotial numbers/ plot, infection of sclerotia, reduction of sclerotial viability and survival in soil were similar for both inocula. The potential for the commercial development of liquid-culture-produced inocula of C. minitans is discussed.     

Some Nutritional Factors Affecting Production of Biomass and Antifungal Metabolites of Coniothyrium minitans

The ability of the mycoparasite Coniothyrium minitans to utilize a range of C and N sources and vitamins for growth, pycnidial formation and antifungal metabolite production was examined using a defined liquid medium. Coniothyrium minitans was able to use all the C sources tested, with the exception of D -xylose, and all the N sources tested, although growth was generally better on organic N sources rather than NO 3 -N. Increasing C:N ratios from 9:1-202:1 with N constant (2.0 g L -l L -alanine) resulted in steadily increasing yields, whereas increasing C:N ratios with C constant (40.0 g L -l D -glucose) gradually decreased yield. Addition of thiamine to the glucose-alanine basal medium resulted in the greatest increase in growth but biomass was still less than that achieved using an undefined molasses-yeast medium. Pycnidial production was generally low or failed to occur in the basal medium + C + N sources in the absence of vitamins, but addition of thiamine consistently led to abundant pycnidial formation. Molasses-yeast static culture provided greater biomass and conidial yields than molasses-yeast shaken culture. Incorporation of C. minitans culture medium into potato dextrose broth (10% v/v) resulted in consistent reduction in growth of Sclerotinia sclerotiorum irrespective of C, N or vitamin content of the basal medium or whether molasses-yeast medium was used. This is the first report of consistent production of antifungal metabolites by C. minitans .     

Risk Analysis of Sclerotinia sclerotiorum for Biological Control of Cirsium arvense in Pasture: Ascospore Dispersal

Natural levels of Sclerotinia sclerotiorum ascsospores in the Canterbury region were determined over 3 years by trapping depositing ascospores in dishes containing a selective agar. Mean levels in 'horticulture', 'biocontrol-pasture', 'mixed cropping' and 'pasture' strata were 115, 56, 10 and 3 ascospores m -2 day -1 , respectively. Ascospore deposition downwind of small experimental biocontrol sites was measured on 2 days in 1994 and 9 days in 1997 in late spring. Exponential depletion models scaled up to represent a 1 ha biocontrol site, revealed that dispersing ascospores declined to natural levels at downwind distances of 2.5-7.9 m. These results imply that biological weed control in pasture using S. sclerotiorum creates no greater risk of crop disease than does horticulture, and that under the conditions of our experiments, an isolation distance of 8 m would have sufficed. However, such a safety zone may be inadequate under certain meteorological conditions not encountered in the experiments when ascospores may disperse in larger numbers over longer distances. To complete the information required to build a mechanistic model of spore dispersal (beyond the scope of this paper) which would cope with a variety of meteorological conditions, two studies were conducted on the dynamics of apothecium formation and ascospore release. In a two-year study, apothecium formation was confined to the spring (September-November), and population size peaked in mid October. In a 5-day study, ascospore release occurred during the daytime, reaching a maximum late morning on frost-free days and a lower maximum mid afternoon on days with morning frost.     

Compatibility of Phloxine B, an Insecticidal Photoactive Dye, with Selected Biocontrol Fungi

The incorporation of the photoactive red food dye, phloxine B, into integrated pest management strategies has been suggested for the control of various insect pests. Inclusion of such a chemical pest control agent may interfere with a microbial pest control agent. Thus, representative fungi with potential for biocontrol were tested for their responses to phloxine B (0.01%) at different temperatures. Growth rates at selected temperatures were inhibited by dye in the presence of light for Coniothyrium minitans and Verticillium lecanii. Trichoderma virens growth was inhibited in light and dark. At optimal growth temperatures, Beauveria bassiana growth was inhibited only by the photoactivated dye species, while growth of Stilbella erythrocephala was not affected by the dye even in the presence of light.     

Importance of Pollen and Senescent Petals in the Suppression of Alfalfa Blossom Blight (Sclerotinia sclerotiorum) by Coniothyrium minitans

The effect of pollen and senescent petals on the suppression of alfalfa (Medicago sativa L.) blossom blight (Sclerotinia sclerotiorum) by the mycoparasite Coniothyrium minitans was investigated. When incubated at 20°C for 39 h, germination of conidia of C. minitans and ascospores of S. sclerotiorum was 99.9 and 98.6%, respectively, in the presence of alfalfa pollen (9×10⁴ pollen grains mL^?1), whereas spore germination of both organisms was <0.5% in the absence of pollen (in water). In the presence of a commercial pollen product, Swiss? pollen granules (mainly bee pollen), germination was 99.6% for C. minitans and 98.3% for S. sclerotiorum when the pollen concentration was 1.0% (w/v). When the pollen concentration was reduced to 0.1% (w/v), germination was reduced to 13.0% for C. minitans and 10.8% for S. sclerotiorum. Tests on detached alfalfa florets showed that the colonization of alfalfa florets by S. sclerotiorum was significantly suppressed by C. minitans in the presence of pollen (1.0% Swiss? pollen granules), especially when C. minitans was inoculated 1-day before S. sclerotiorum. In vivo inoculation tests revealed that the efficacy of C. minitans in the protection of alfalfa pods from the infection by S. sclerotiorum was affected by the time at which C. minitans was applied. When C. minitans was applied on young blossoms of alfalfa at the anthesis stage, pod infection was 96.6% for the treatment of C. minitans+S. sclerotiorum and 99.6% for the treatment of S. sclerotiorum alone. However, when C. minitans was applied on senescent petals of alfalfa at the pod development stage, pod infection was 8.0% for the treatment of C. minitans+S. sclerotiorum compared to 90.8% for the treatment of S. sclerotiorum alone. These results suggest that timing of the application of C. minitans is critical for the mycoparasite to compete with S. sclerotiorum for the source of nutrients from pollen and senescent petals, and for its control of alfalfa blossom blight caused by S. sclerotiorum.