Studies on the application of biocontrol agents for the control of seedling diseases.
The controlled environment of greenhouses, the high value of crops, and the limited number of registered fungicides offer a unique niche for the biological control of plant diseases. During the past ten years, over 80 biocontrol products have been marketed worldwide. A large percentage of these that have been developed in greenhouses could predominate over chemical pesticides in the same way that biological control of greenhouse insects predominated in the United Kingdom. A review of the literature was undertaken to obtain information on biocontrol agents with specific reference to Trichoderma and Gliocladium spp. Literature on the application, types of formulations, limitations in formulation, registration and commercialization of these biocontrol agents were obtained. Trichoderma harzianum Strain KMD has been used successfully as a biological control agent against several soil-borne plant pathogens. Biological control agents should possess several desirable characteristics, including, ease of preparation and application, stability during transport and storage, abundant production of viable propagules and good shelf-life. A strain of Trichoderma harzianum KMD with potential biocontrol activity was used to determine the effect of culture conditions on spore shelf-life. The influence of four growing media were investigated on the spore ultrastructure and shelf-life, using a basal salts medium with C:N ratios of 3 and 14, and pH's of 4.0 and 7.0. Mycelial development and sporulation were positively affected by acidic conditions (pH 4.0). The effect of these culture parameters on viability and shelf-life were evaluated by counting colony forming units (c.f.u) before and after seven days of storage at 75% relative humidity (rH): The effect of carbon concentration on spore viability after seven days of storage was also determined by increasing concentrations of glucose while a constant C:N ratio of 3 or 14 at pH 4.0 was maintained at a 75% rH. Increasing carbon concentration and C:N ratios increased spore production times. Spore viability was greatest when harvested from a medium with a C:N of 14 at pH 4.0 even when storage time was increased to 45 days and rH was reduced to 12%. Ultrastructural studies showed that spores had two cell wall layers, with the outer being more electron-dense than the inner layer. This layer is the spore's first defense against adverse conditions. Spores obtained from this medium were larger, germinated better and had a longer shelf-life than spores from C:N 3 medium, possibly because the two cell wall layers acted as a thicker barrier against adverse conditions. Increasing carbon concentration, while maintaining a constant C:N ratio of 3 or 14 at pH 4.0 slowed down spore production. Viability of spores were similar when introduced on media with variable carbon concentrations but fixed C:N ratios. The ultrastructural differences and shelf-life studies, confirmed empirical results from liquid fermentation studies, that the pH and C:N ratio of the medium upon which spores of T harzianum KMD strain KMD were produced have critical effects on physical and chemical structure of the spores and viability. This, in turn, affects critical parameters for biocontrol agents spore germination and shelf-life. Ultrastructural studies of mycoparasitism of T harzianum KMD on a soil-borne pathogen, Rhizoctonia solani were investigated. The modes of antagonistic action by Trichoderma in biological control have not been fully elucidated. However several mechanisms have been described, such as mycoparasitism, antibiotics, production of inhibitors, which have been identified and shown to suppress soil-borne pathogens. Mycoparasitic activities of T harzianum KMD against R. solani were studied using in vitro bioassays and Scanning electron microscopy (SEM). The fungal growth in dual cultures revealed that T harzianum KMD made hyphal contact with the pathogen within four days of inoculation, leading to an inhibition of pathogen growth. SEM observations showed that T harzianum KMD bound firmly to R. solani hyphae by coiling around the hyphae. Penetration of the pathogens hyphae occurred by the formation of hooks, haustoria and appressoria-like structures by T harzianum KMD, followed by cell disruption. The pathogen's hyphae disintegrated and collapsed upon contact with T harzianum KMD. It is hypothesized that the outcome of the interaction of antagonist and pathogen was most likely determined by initial hyphal contact that triggered a series of events in pathogen destruction. An experimental trial was undertaken to evaluate various formulations if T harzianum KMD and Gliocladium virens Strain MM1 for growth stimulation and biocontrol of R. solani and Pythium sp. on a variety of crops under greenhouse conditions using three application techniques at various dosages. Preparations of isolates of biocontrol agents T harzianum KMD, G. virens MM1 and Bacillus subtilis Strain AW57 were evaluated for their efficacy in enhancing growth and preventing damping-off caused by Pythium sp. and R. solani on a variety of crops namely cabbage, cucumber, Namaqualand daisy and Eucalyptus. Percentage survival and plot weights were measured after 3-4 weeks of growth. The preparations that were used included chlamydospores of biocontrol fungi in milled oats, powders containing conidia in an experimental compound, an oil base, and a commercial product. Formulations of bacteria were prepared with and without Nutristart. The evaluation of three delivery methods were used namely, a seed coating using an adhesive, Pelgel®, capping (a preparation is capped on the surface and incorporated into planting media) and as a drench (preparation drenched on seed at planting). Various dosage levels 0.25, 0.5, 1, 5 and 10g/1 of each formulation was mixed with water and drenched on seed at planting. Growth promotion of seedlings varied for the different formulations of different biocontrol organism. Overall, plot weight was significantly increased on all crops tested. Plant growth of seedlings was consistently increased by all conidial formulations of T. harzianum KMD and G. virens MMI. The best application technique that effectively delivered the biocontrol agents to the target was seed treatment followed by drenching and capping. Most formulations significantly increased plot weight on all seedlings ranging from 2000-5000% when compared to controls and percentage survival was comparable to the controls. In most instances it was recorded that all biocontrol organisms effectively enhanced growth of seedlings equally well irrespective of other main effects. Most formulations of the different biocontrol organisms significantly reduced damping-off caused by Pythium sp. on eucalyptus and Namaqualand daisy. Formulations of T. harzianum KMD prepared with chlamydospores in milled oats and prepared with conidia effectively reduced damping-off on eucalyptus and Namaqualand daisy by 8-31% when compared to the controls. It was observed that biocontrol organisms T. harzianum KMD and G. virens MMI effectively reduced damping-off better than B. subtilis AW57. To effectively reduce damping-off caused by Pythium sp. seed treatment was the best application technique to deliver the biocontrol agent to the target. Biocontrol of damping-off caused by R. solani was achieved on all crops by all formulations of T. harzianum KMD, G. virens MMl and B. subtilis AW57. Disease was reduced by 1000 fold with the application of biocontrol organisms when compared to disease controls. Conidial formulations performed better in reducing disease than formulations prepared with chlamydospores applied as a drench or a seed treatment. In most instances the best dosage to apply formulations were doses that ranged from l-5g/1 for both growth stimulation and biocontrol of soil-borne pathogens. Severe stunting of seedlings occurred at high dosages of 109/I. The compatibility of the biocontrol agent T harzianum KMD with selected fungicides were determined on a variety of crops under greenhouse conditions. A commercial formulation of T harzianum KMD was used for this investigation. An in vitro assay was used to determine the sensitivity of T harzianum KMD to a range of rates of two fungicides, Benlate® and Previcur®. Trichoderma harzianum KMD was found least sensitive to both fungicides after 15 days of incubation at 25°C. The compatible mutants resulted in a lack of sporulation even when induced with UV light. Greenhouse trials were then carried out on cabbage, cucumber, Namaqualand daisy, eucalyptus and tomato. It was confirmed that T harzianum KMD achieved better growth and biocontrol activity against R. solani and Pythium sp. when applied without fungicides to infested and non-infested composted pine bark. Trichoderma harzianum KMD was only compatible to fungicides when applied as a seed treatment prior to planting. As a disease integrated management programme, seed treatment application of T harzianum KMD may be compatible with fungicides for control of damping-off of seedling diseases caused by R. solani and Pythium sp. The effect of environmental stress (oxidative injury, cold and drought) on the growth enhancement of a variety of greenhouse crops by a commercial formulation of T harzianum KMD was evaluated. In an absence of a disease colonization by T harzianum KMD on maize and cucumber roots in rhizotron studies increased root area by 3104 mm(2) and 1787, 48 mm(2) respectively. Oxidative stress was carried out by applying 0.05% NaOCl, to cabbage, cucumber and tomato seeds. This stress did not reduce vigor of seedlings and hence the effect of subsequent treatment with T harzianum KMD on stressed seeds was not determined. Treatments of imbibed but unemerged seeds of cucumber, tomato and white grain maize in cold temperatures (5-100C night/day) for varying periods reduced subsequent growth. Seeds treated with cold stress and T harzianum KMD did not display any growth enhancement. On cabbage, cucumber, tomato and white grain maize seeds sown in various media, which induced various levels of drought and water logging conditions, were not enhanced when seeds were coated with T harzianum KMD. Overall, T. harzianum KMD did not enhance growth under stressed conditions of oxidative injury, cold and drought. The results presented in this thesis shows that T harzianum KMD has potential against soilborne pathogens namely Pythium sp. and R. solani under greenhouse conditions. Applying conidial formulations of T harzianum KMD using seed treatment and applying it at the correct dosage may increase the turnover of seedling production in nurseries. Trichoderma harzianum KMD can replace toxic fungicides and. fumigants under greenhouse conditions. More trials and research are needed on a wider variety of crops and diseases if growth promotion and biological control of T harzianum KMD are to be fully exploited.