Conidium "fitness" in Trichoderma : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy, Lincoln University

A major constraint for use of biocontrol agents (BCAs) is inconsistent performance under changeable environmental conditions. This study aimed to develop new knowledge of effects of growth conditions, to increase persistence and efficiency of the key agent Trichoderma atroviride LU132, and to understand the factors which may influence conidium “fitness” for biocontrol formulations of conidia, that are robust (long-surviving) and active against target plant pathogens. Effects of culture conditions, (incubation period, temperature, nutrients, water activity and pH) productivity, germinability and bioactivity of T. atroviride LU132 conidia were assessed, in assays against the soil-borne plant pathogen, Rhizoctonia solani. Conidium fitness was assessed after storage and in glasshouse pot experiments. Biochemical and ultrastructural characteristics of conidia produced in different culture conditions were also examined, in relation to conidium fitness. The influence of incubation temperatures (20, 25, or 30°C) on the production of conidia was assessed under constant light over a 25 d period. Two measures of quality of the resulting conidia were also determined; - germination and subsequent bioactivity against Rhizoctonia solani. Maximum conidium production occurred at 25°C after 20 d but was less at 25 d. Conidia produced at 30°C germinated more rapidly and gave the greatest bioactivity against R. solani in comparison with incubation at 20 or 25°C. An incubation period of 25 d gave the greatest bioactivity compared with shorter incubation periods. To examine the effects of extending incubation time on conidium production, germination and bioactivity, the experiment was extended at 25°C for up to 50 d, which resulted in a second peak of conidium production at 45-50 d. These conidia had optimum germination after 20 and 25 d incubation, and optimum bioactivity was achieved with conidia harvested after 15 d. Therefore, temperatures near 25°C and incubation period of 15 d were shown to be optimum for production of T. atroviride LU132. Formulations of T. atroviride based on optimised production of conidia may not result in optimal bioactivity. This is the first report indicating that the temperature at which conidia of T. atroviride are produced affects germination and bioactivity. Conidium production of this biocontrol strain was shown to be a continuous process, and a scheduled dark/light regime increased conidium production. Furthermore, conidium production is likely to be on 20 d base cycle, which is probably dependent on colony age rather than abiotic factors. This is also the first report of bimodal conidium production in a Trichoderma biological control agent. Identification of the production and storage factors that affect conidium fitness can assist the success of biological control agents. Conidia from the culturing regimes which resulted in greatest and least bioactivity against R. solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Conidia were examined after storage at 30˚C and at 0 or 50% relative humidity (RH) over six months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH. The greatest number of conidia and greatest germination resulted from C to N ratios of 5:1 or 160:1, amended with sucrose at 25°C, but greatest bioactivity resulted from conidia produced at 30˚C. However, fewer conidia were produced at 30˚C, and the least germination and bioactivity resulted from conidia produced at 20°C, both amended with dextrose. The conidia adapted to high temperature of 30°C (amended with dextrose) or nourished at C to N ratio of 5:1 (amended with sucrose) showed the greatest conidium fitness. Further experiments assessed effects of temperature and hydrocarbon type. Interactions of temperatures (20 or 30°C) vs sugars (dextrose, 4.2 g/L or sucrose, 4.2 g/L in constant C:N ratios of 5:1) were examined for bioactivity and colonisation potential in pot experiments with ryegrass in the presence of R. solani. Conidia produced at 20°C with dextrose (4.2 g/L in constant C:N ratio of 5:1) gave the greatest bioactivity, where rhizosphere and bulk soil assessments were carried out. The bimodal population cycle in T. atroviride LU132 recurred in pot experiments (recorded as colony forming unit (CFU)) in a manner similar to that observed in agar plates, but in an approx. 15 d cycle, indicating that simulated natural conditions shortened the Trichoderma life cycle. Biochemical and ultrastructural studies were carried out to determine relationships between quality variations and cellular characteristics for conidia produced in different culturing conditions. The effect of culture conditions on trehalose accumulation was most marked, while differences in arabitol and mannitol were much less. The least trehalose accumulation was detected in conidia produced at 20°C (13 mg/g dry conidia). This could justify the least conidium survival and bioactivity during storage. Fatty acids detected in conidia by gas chromathography were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1 c9) and linoleic acid (18:2 c9, 12). Linoleic acid was the most abundant (overall mean of 26%), and stearic acid was the least abundant (8%). The conidium production treatment at 20°C gave the greatest amount of fatty acids (66 µg/g of dry conidia), giving conidia deep dormancy or other deterioration effects, while the C:N 5:1 treatment which gave high bioactivity after storage gave the least conidium fatty acid content (12 µg/g dry conidia). Ultrastructural differences of conidia were linked to differences in conidium survival and successful biocontrol establishment. Low electron density of conidium contents and accumulation of lipid droplets were associated with less integrity and viability. Conidia produced at 20°C showed significant disorganisation of cellular structures. This research has provided new insights which can form the basis of efficient production of Trichoderma-based biocontrol agents. Additional insights into the basis of conidium fitness in T. atroviride LU132 have also been provided.