Applied Mycology Group, Biotechnology Centre, Cranfield University, Bedford MK43 OAL, UK

Background and objectives
Ulocladium atrum has been demonstrated to be a very effective biocontrol agent of Botrytis cinerea in the phyllosphere of a number of crops by pre-emptive exclusion of the pathogens and suppression of sporulation [1]. For biocontrol to be consistent in the field, environmental stress tolerance is critical for effective establishment. Tolerance of the biocontrol agent to water stress is thus particularly important both for effective control and for producing formulations of ecologically competent inoculum. Recent work by Magan and co-workers has demonstrated that physiological manipulation of biocontrol agents can improve the range of environmental conditions over which growth can occur, and sometimes results in improved control of both pre- and post-harvest diseases [2]. The present investigation was undertaken to (i) identify the water potential x temperature range for stress tolerance of U. atrum; (ii) improve the quality of U. atrum spores by physiological manipulation of endogenous reserves; and (iii) examine germinative capacity of such characterized spore inocula.

Materials and methods
Studies on growth of U. atrum: a 2% oatmeal agar medium was modified with ionic (NaCI) and non-ionic (glycerol, PEG200 solutes) and the effect of water stress determined over the range 0.28-13.6 MPa and 10-30C. Growth rates were determined and compared.
Analyses of endogenous reserves: the temporal accumulation of sugars and sugar alcohols in conidia of U. atrum were determined by destructive harvesting over 30 days. The conidia were freeze-dried, extracted in water, resuspended in acetonitrile:water (40:60), and analysed by HPLC. The glucose, trehalose, mannitol, arabitol, erythritol and glycerol were determined.
Germination and growth of characterized spores: characterized condia obtained from unstressed and stressed media were then compared for germination and germ tube extension by plating onto oat agar medium modified to different water stresses (-0.2, -2.1 and 7.0 MPa) with PEG2OO. Germination (%) and the length of the longest germ tube emerging from each spore were measured using image analyses and the number of germ tubes per spore treatment determined.

Results and discussion
Water stress tolerance of U. atrum: the biocontrol agent grew over a wide range of temperature, with optimum at 25-30C. Optimum growth was at sub-optimal water potentials in the range -1.25 to -3.0 MPa with growth rates of ca 2.25 mm/day. Growth at the lowest water potential tested (-13.6 MPa) was very slow (<0.5 mm/day).
Accumulation of endogenous reserves: there were marked temporal changes in sugar and polyol content of conidia over the 30-day incubation period. Generally, under non-stressed water availability conditions, glucose and trehalose were major components of the endogenous reserves of conidia. Under water stress, more of the low molecular-weight polyol glycerol, and the high molecular-weight polyols arabitol and mannitol, were present in conidia. The spores from the driest media were significantly larger than those from unstressed media.
Germination and germ tube growth: the temporal patterns of germination of spores from these treatments were compared for water stress tolerance on media where water was freely available and at water stress of -2.1 and -7.0 MPa. This showed that germination rates of spores from unstressed media were most rapid, regardless of stress treatment. However, germ tube length of spores from 44 and -7.0 MPa treatments were significantly longer than others in the driest conditions tested (-7.0 MPa) after 52 h. The numbers of germ tubes per conidium were also greater. This suggests that larger endogenous reserves, particularly of polyols, may enable better establishment and colonization of niches such as leaf surfaces under environmental stress. Thus physiological manipulation of fungal growth conditions could improve ecological competence of inocula as well as being a tool for improving quality of formulations for field applications.

1. Kohl J, Molhoek WML, van der Plas CH, Fokkema NJ, 1995. Phytopathology 85, 393-401.
2. Teixido N, Vinas I, Usall J et al., 1998. Mycological Research 102, in press.