Mark P. McQuilken

Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of Coniothyrium minitans

The effects of growth media, temperature, pH and light on the development of four isolates of Coniothyrium minitans (CONIO and CH8 (colony type 3), G4 (colony type 4) and G9 (colony type 5)) were examined. Conidial germination, pycnidial production and hyphal extension rate were initially studied on seven different agar-based growth media at 18–20 °C. Potato-dextrose agar (PDA) and malt extract agar (MEA) consistently gave the greatest conidial germination, pycnidial production and hyphal extension rate for all four isolates. Growth and development on molasses-yeast agar was equivalent to that on PDA and MEA except that hyphal extension rate was slower. Subsequently, the effects of temperature, pH and light on the development of C. minitans were investigated on PDA only. The temperature range of conidial germination and pycnidial production of the four isolates was between 10–25° with the optimum at approximately 20°. Hyphal extension occurred over a greater temperature range, between 4 and 25°, with a maximum extension rate of approximately 3·5 mm d − for all isolates occurring between 20–25°. Conidial germination, pycnidial production and hyphal extension occurred over a pH range between 3–8 with optimum values for all growth assessments occurring between pH 4·5 and 5·6. Increasing light period from continuous dark, to 12 h light 12 h dark or continuous light had no effect on conidial germination or extension growth, but significantly increased pycnidial production. Isolates G4 and G9, previously characterized by sparse production of pycnidia in comparison with CONIO and CH8, consistently exhibited a reduced production of pycnidia on all media, at all temperatures and pH ranges, and all light regimes tested. This demonstrates the stability of this character among these isolates of C. minitans . The significance of these results for improving production of inoculum of this biocontrol agent and in the identification and classification of isolates of C. minitans is discussed.

Production of macrosphelide A by the mycoparasite Coniothyrium minitans

Coniothyrium minitans, a mycoparasite of sclerotia of Sclerotinia sclerotiorum and Sclerotium cepivorum, produced four closely related metabolites inhibitory to fungal growth. The major metabolite, identified as macrosphelide A, had IG(50) values (the concentration of metabolite to inhibit growth by 50%) of 46.6 and 2.9 microgram ml(-1) against S. sclerotiorum and S. cepivorum, respectively. This is the first report of both antifungal activity due to macrosphelide A as well as isolation of macrosphelide A from C. minitans.

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 car...

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 .

Antimicrobial activity of Coniothyrium minitans and its macrolide antibiotic macrosphelide A

Aims:  Assessment of antimicrobial activity of the mycoparasite Coniothyrium minitans and its macrolide antibiotic macrosphelide A.

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and β-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor β-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas β-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, β-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.

Aspects of biocontrol of fungal plant pathogens

In recent years there have been considerable changes in attitude towards the use of chemicals in agriculture. Increasing public awareness concerning the quantities and types of chemicals used and their potential impact on the environment has led to more stringent regulations on their use and, in some cases, removal from the market. Consequently, interest has focused on alternatives to chemicals, particularly for pest and disease control. The use of biological disease control measures is one of the strategies available and much experimental work is being carried out to assess its commercial applicability.

Potential for biocontrol of sclerotinia rot of carrot with foliar sprays of Contans® WG (Coniothyrium minitans)

Abstract A glasshouse and field trial were conducted to evaluate foliar sprays of Contans® WG (Coniothyrium minitans) conidial suspensions for control of sclerotinia rot of carrot and infection of Sclerotinia sclerotiorum sclerotia by C. minitans. In the glasshouse trial, foliar sprays (1×104–108 conidia mL−1) decreased the viability of sclerotia recovered from diseased plants and increased infection by C. minitans. In the field trial, three successive foliar sprays applied at 14-day intervals failed to reduce foliage disease severity, but significantly reduced viability of sclerotia recovered from diseased plants/crop debris and increased infection by C. minitans. No significant differences in sclerotial viability or infection were observed between two conidial concentrations (2.4 and 4.8×106 conidia mL−1). Foliar sprays of Contans® WG have potential for reducing viability of sclerotia produced on diseased foliage.

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

Abstract The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.

Effect of Storage on the Survival and Biocontrol Activity of Pythium oligandrum in Pelleted Sugar Beet Seed

Seeds of sugar beet were pelleted with oospores of Pythium oligandrum and stored for 6 years at 8 20IC. Mycelium of P. oligandrum grew from pelleted seed when plated on cornmeal agar (CMA) within 48 h from 100% of seeds stored for 0, 2 and 4 years, and from 93% of seeds stored for 6 years. The germination of oospores removed from pelleted seed immediately after pelleting was 30% on CMA after 18 h of incubation, but storage gradually reduced germination to only 16% after 48 h of incubation for oospores removed from seed stored for 6 years. The biocontrol activity of P. oligandrum -pelleted seed was also tested after 6 years of storage in mixes of soil naturally infested with Pythium spp. and Aphanomyces cochlioides , and sand. P. oligandrum -pelleted seed had no effect in reducing damping-off due to the combined effect of Pythium spp. and A. cochlioides in 5 and 1% (v/v) soil-sand mixtures. However, in the 1% (v/v) soil-sand mixture, P. oligandrum significantly reduced Pythium spp.-induced damping-off from...