3.5.6S

A MOLECULAR APPROACH FOR STUDYING AUTECOLOGY OF FUNGAL BCAs


D FUNCK JENSEN AND H GREEN

The Royal Veterinary and Agricultural University, Department of Plant Biology, Thorvaidsensvej 40, DK1871 Frederiksberg C, Copenhagen, Denmark.

Background and objectives
More than 30 biocontrol agents (BCAs) have been commercialized for the control of plant diseases. The BCAs must be present and active at the right place at the right time, namely at the specific microsites where the pathogen is found, and where the antagonist consequently has to work. Therefore, there is a need for autecology studies of the antagonists in experiments simulating the complex environment in which they are to be used. This is important both for optimizing production, formulation and delivery systems, and for obtaining the best performance of the BCAs in commercial operations. It is also important for understanding how the antagonist can be favoured by cultural practices. The BCAs are often difficult to distinguish from other related organisms and methods for specific measurement of the activity of antagonists are few [1]. One approach to overcome this and other problems is to use genetically marked BCAs and in the present work, a GUS transformed strain of Trichoderma harzianum was used. The objectives were to study the ecology of T. harzianum in the rhizosphere of cucumber plants, in relation to biocontrol of damping-off and root rot caused by Pythium ultimum.

Materials and methods
A GUS transformant of T. harzianum was constructed and tested for its phenotypically resemblance to the wild type according to [2]. Cucumber plants, Cucumis sativus L., were used, and the plant growing medium consisted of fine, 0 to 20 mm, light coloured sphagnum peat (either pasteurized or raw) mixed with vermiculite at a ratio of 3:2 (wt/wt). The medium was watered with a balanced nutrient solution to a water content of -0.98 kPA and pH was 5.5. The rhizosphere was separated from the bulk growing medium by nylon mesh. Two sets, A and B, of pot experiments were carried out. (A): The wild type, the transformant or a 1:1 combination of the wild type and the transformant, was incorporated in the growing medium as a peat bran formulation (0.001g/g dry matter). The antagonists were then monitored after application as described in [3]. In brief, the methods were as follows: Dilution plating on selective substrate combined with GUS staining for revealing colonies of the transformant. Measurement of activity of the transformant by extraction and spectrophotometrically quantification of GUS from rhizosphere samples. Results were further supported by studies of spore germination in rhizosphere samples by use of FDA staining and microscopy. Direct observation of the transformant on the rhizoplane by microscopy following incubation of roots with GUS substrate (X-gluc). Longitudinal root sections of regions with localized colonization by the transformant were studied in more detail.
In (B), oospores of P. ultimum (5x104 per g dry matter) were incorporated in the rhizosphere and the effect of the antagonist on the primary infections was scored using a disease indexing system.The antagonistic effect on secondary spread and infection was determined by planting healthy seedlings at defined distances from diseased plants in growing medium with or without the antagonist. Healthy and diseased roots were incubated with X-gluc to reveal GUS-activity of the antagonist.

Resuits and conciusions
The GUS-transformant can be used for studying spore germination, hyphal growth, formation of conidiophores and sporulation in the rhizosphere. In pasteurized peat the transformant was active in the rhizosphere only for a few days following application after which conidia or chlamydospores were formed and it remained as inactive conidia also on the surface of healthy roots. The antagonist, however, was very active in and around wounds on the roots and it actively colonized dead and dying roots infected by P. ultimum both in raw and pasteurized peat. The primary and secondary infections and the secondary spread of the pathogen by hyphal growth were restricted by incorporating the antagonist in pasteurized growing medium. The results shows that it is important to have information about the autecology of the antagonists in natural systems for improving the efficiency of BCAs used in plant production.

References
1. Jensen DF, Woiffhechel H, 1995. in: Hokkanen H, Lynch JM, Biocontrol Agents: Benefits and Risks, Cambridge Univ. Press, Cambridge, 177-189.
2. Thrane C, LObeck M, Green H, Degefu Y, Allerup S, Thrane U, Jensen DF, 1995. Phytopathology 85, 1428-1435.
3. Green H, Jensen DF, 1995. Phytopathology 85, 1436-1440.