Studies on production and ecophysiology of fungal antagonists for improved biological control of Fusarium oxysporum f.sp. Iycopersici and Monilinia laxa
This work investigated the production of the biocontrol agents: Penicillium oxalicum to control Fusarium oxysporum f.sp. Iycopersici in the rhizosphere; and Epicoccum nigrum and Penicillium frequentans to control Monilinia laxa in the phyllosphere. Ecophysiological studies were carried out to categorise different inoculum types, and to test their biocontrol efficacy. A method was developed for the induction of submerged conidiation of P.oxalicum for the first time. This was achieved by transferring 24 h cultures to a nitrogen free medium, and further stimulated by a high C:N ratio with 20 mM calcium. Optimum submerged conidial concentrations obtained were 35x10 6 spores mr1. The hydrophobicity of submerged and aerial conidia of P.oxalicum was similar. However, different results were obtained depending on the method used. Appearance of both spore types under the SEM was similar in size and shape. However, submerged spores were covered by a filamentous matrix, absent in aerial ones. Viability of aerial and submerged P.oxalicum spores was higher than 80% after 27 weeks, when stored fresh at either 4 or 25°C, but aerial spores survived slightly better. Freeze-drying severely affected viability, especially of submerged spores. Aerial spores effectively colonised sterile soil in tomato seedbeds with water potentials in the range 1-7 (-MPa), and this was further favoured by the addition of nutrients. Aerial conidia of P.oxalicum applied to seedbeds were able to significantly (P<0.05) reduce Fusarium wilt of tomato at concentrations as low as 6x10 4 spores mr 1 substratum, indicating that the amount of the antagonist needed is not a limiting factor for the practical application of this antagoniSt. Aerial spores were slightly more effective than submerged ones in the control of the disease. However, mycelium was ineffective. Coating of tomato seeds with formulations of aerial spores of P.oxalicum in alginate or methyl cellulose significantly (P<0.05) enhanced the growth promotion effect of the antagonist in vitro, which may be related to the ability of the fungus to control the disease. E.nigrum spores were produced by solid fermentation on wheat grains at different water activities (aw). Maximum levels of sporulation (7-11x10 6 spores g-1 grain) were obtained at high aw (0.996) or reduced aw (0.98) adjusted with a mixture glycerol/water. E.nigrum and P.frequentans were both produced in culture medium at reduced aw, to improve their ecological competence in the phyllosphere and therefore their biocontrol ability. E.nigrum produced at reduced aw showed improved germ tube extension and in some cases colony growth rate when placed on medium at reduced aw, showing water stress tolerance of such modified inocula. Furthermore, such inocula showed an enhanced ability to compete with the pathogen, M.laxa, at reduced aw, shown by a higher Niche Overlap Index (the proportion of the carbon compounds utilised by M.laxa that were also utilised by E.nigrum). E.nigrum spores produced at reduced aw had improved survival when stored fresh at 4 or 25°C. Freeze-drying severely affected the viability of both spore types (produced at high or reduced aw). E.nigrum and P.frequentans produced at reduced aw accumulated low molecular weight polyols as compatible solutes. Improvement of biocontrol of peach twig blight was obtained in the case of E.nigrum, which is more sensitive to conditions of low water availability than the xerotolerant P.frequentans. However, both fungi accumulated glycerol as the main compatible solute, indicating that different accumulation mechanisms may be responsible for the different tolerance to low water availability. Glycerol was also the main compatible solute in E.nigrum spores produced by solid fermentation at reduced aw. E.nigrum produced at high or reduced aw was also able to control brown rot of cherries, under optimum conditions for the development of the disease. The results presented in this work show that the conditions during the production of biocontrol agents are critical in determining their efficacy. Therefore, when developing mass-production systems it is necessary to aim not only for high propagule numbers but also for inoculum quality, defined by parameters such as ecological competence or survival during storage.