Efficacy of Trichoderma asperellum oil formulations on the control of cocoa black pod disease (Phytophthora megakarya).

The objective of this study was therefore to develop a formulation of conidia of T. asperellum with the aim of improving its efficacy. The formulations developed were oily dispersions. It was a combination of solvents consisting of groundnut oil or palm oil with structural agents and emulsifying-dispersing agents. Emulsification tests were carried out and the stability of the emulsions evaluated. The evaluation of the effect of co-formulants on the growth of conidia of T. asperellum was done by reading the optical densities of the formulated samples on multi-plates using a plate reader. The test on detached cocoa pods was done by treating the cocoa pods with selected formulations at 1.10(7) conidia/ml and inoculation of the treated cocoa pods was done 24 hours later with zoospores of P. megakarya at 1.10(5) zoospores/ml. The growth of necrosis on the fruits was measured daily. The screening of co-formulants and emulsification tests ended up with the selection of two formulations. The first composed of conidia of T. asperellum, groundnut oil, Tensiofix NTM and Tensiofix 869. The second differed from the first by utilisation of palm oil as the solvent. These formulations proved stable when diluted in water with 1% and 0.5% of sedimentation respectively after 24 hours. The viability test of the conidia indicated that the different formulations selected did not have a fungitoxic effect. The test on detached cocoa pods showed an improved efficacy of T. asperellum to control the disease. The growth rates of necrosis were 6.29 mm/day, 7.25 mm/day and 31.6 mm/day for treatment with formulation 1, pure conidia and control treated with water respectively.

Compatibility between Clonostachys isolates with a view to mixed inocula for biocontrol.

To aid the development of compatible biocontrol inocula, a prescreening method for the prediction of compatibility of fungal antagonists was developed. Compatibility between 18 Clonostachys isolates with known antagonistic capabilities against Phytophthora palmivora was tested using intra- or interisolate pairings (dual cultures) on water agar plates, a hyphal interaction experiment and a modified double host-range experiment. Almost all inter- or intraisolate pairings of Clonostachys isolates showed growth inhibition zones and did not show free hyphal intermingling. A hyphal interaction experiment on water agar demonstrated that the aggressiveness of a Clonostachys isolate and its susceptibility to mycoparasitism were unrelated phenomena. However the level of aggressiveness and/or susceptibility of an isolate were largely dependant on the isolate with which it was challenged. The degree of growth-inhibition caused by an isolate was unrelated to the hyphal damaged it caused or received. In the double host-range experiment all possible pairs from four Clonostachys isolates were inoculated in different ratios (10 000-fold range) on plates precolonized with one of two P. palmivora isolates. The results showed that antagonistic capabilities of certain combinations were affected by the Clonostachys isolates. The primary host, P. palmivora, did not affect antagonistic capabilities; whereas inoculum ratio did. Of note, it was not possible to predict the outcome of the double host range on the basis of the results of the hyphal interaction experiment. In conclusion the competitive abilities of Clonostachys isolates depend on the partner with which they are applied and less on resource availability. The double host-range test as developed here might provide the most representative tool to date to test compatibility of fungal antagonists to be used in biocontrol inocula. However the link between the results of the double host-range test and field efficacy of biocontrol inocula remains to be investigated.

Preservation methodology for Rosellinia species.

Many small (temporary) collections of fungi maintained by plant pathologists during their research receive inadequate attention to ensure stability. Maintaining collections of fungi in pure and viable conditions, minimising physiological and morphological changes is, however, a necessity. The objective of this study was to find preservation techniques for three Rosellinia isolates used in our plant pathogenic research. Various inert and nutritious carriers, solid as well as liquid, were used to test their suitability for conserving these Rosellinia isolates. Different cryoprotectants, cooling rates and thawing rates were tested to optimise liquid nitrogen storage procedures. Survival and/or growth rate were assessed over time. Rosellinia bunodes was the most difficult to store with survival not exceeding six to nine months using traditional storage methods in mineral oil and silica gel. Storage of Rosellinia necatrix and Rosellinia pepo was successful for periods up to at least 16 months in several carriers and for up to two years for R. necatrix in silica gel. Storage in liquid nitrogen proved no problem for R. necatrix or R. pepo with a 100% survival in all cases, although radial growth rates after recuperation were affected by cryoprotectant and thawing rates. Storage of R. bunodes was more difficult and survival as well as growth rates were affected by cryoprotectant and thawing rates. Cooling rates did not affect radial growth in any of the isolates. The results showed that development of a generalised procedure for storage of our Rosellinia species was not possible and successful storage protocols had to be developed for individual isolates.