Canadian Forest Service, 506 West Burnside Road, Victoria, British Columbia V8Z 1M5, Canada

Background and objectives
The development of biological control agents to suppress competing forest vegetation in timber-production areas, as an alternative to herbicide use and manual silvicultural control, is important to forest managers in Canada. Invasive Rubus spp., namely R. strigosus (syn. R. ideaus var. strigosus), R. parviflorus, and R. spectabilis, are among the top 20 forest weeds in Canada and effectively compete with newly planted or naturally regenerated conifers in reforestation sites, reducing conifer growth and survival [1]. This study continues work into the development of an indigenous pathogen, Fusarium avenaceum (FA), as a potential biological control agent to infect and suppress growth of these Rubus spp. when applied in inundative doses.

Materials and methods
The fungal pathogen was isolated and purified from a diseased R. strigosus stem and selected through screening tests in which sufficient virulence was detected after inoculation of detached leaves and whole plants. Several agar, liquid and grain growth media were evaluated to obtain maximum mycelial and spore production, and temperature conditions for optimum growth and germination were determined. To enhance fungal virulence through formulation, amendments to FA inoculum assayed included nutrients (sucrose, neopeptone, malt, sodium alginate), humectants (starch, psyllium hydrophilic muciloid), dispersants (Tween 80, wetting agents), stickers/surfactants (Silwet®, Intac®, soybean oil), and formulation into an invert emulsion. Increasing host susceptibility through wounding with low doses of the glyphosate was attempted by herbicide applications followed by FA inoculum. The presence of phytotoxins produced by FA grown in a rice medium was also investigated.

Results and conclusions
FA, when grown on rice grains [2], and subsequent inoculum combined with an organosilicone surfactant, 0.4% Silwet®, induced greater foliar necrosis than other formulations. Extensive foliar damage occurred with this formulation within 24-48 h on R. strigosus and R. parviflorus, resulting in large areas of necrotic leaf tissue, leaf curl and death. R. strigosus was the most susceptible to the FA+Silwet® treatment, with 89% of treated plants demonstrating >51% foliar injury within 7 days of treatment. For treated R. parviflorus and R. spectabilis, 44 and 6.25% of plants, respectively, showed >51% foliar injury with this treatment. All treated plants flushed new leaves by 3 weeks, and the new foliage and stems were free of necrotic symptoms. Among the other formulations assayed, either a low rate of necrosis was obtained or the amendment alone produced necrosis similar to the formulated inoculum. The incorporation of low doses of glyphosate was not further pursued, as the combined effect of FA+glyphosate did not exceed that of glyphosate alone. Extraction and analysis of FA-infested rice filtrates for metabolite production showed that a single toxin, moniliformin, was present at levels of 3300 p.p.m. No butenolide or enniatin B was detected. The active phytotoxin(s) or their analogues may be useful under biorational strategies to control target plants by applying fungal metabolites. An initial host-range test demonstrated that several economically important conifer seedlings did not show disease symptoms when inoculated with the FA+Silwet® formulation.

Based on this research, FA inoculum produced on rice appears to have several suitable attributes for investigation as a candidate biological control agent. Further research will expand trials using inundative applications of FA inoculum on invasive Rubus spp. in forest sites to determine optimum formulation, application and evaluation techniques, timing of applications with regard to environmental conditions and target plant phenology, and further host-range testing. In vitro screening of FA phytotoxins by tissue culture techniques is under way.

1. Oleskevich C, Shamoun SF, Punja ZK, 1996. Canadian Journal of Plant Science 76, 187-201.
2. Abbas HK, Boyette CD, Hoagland RE, 1995. Phytoprotection 76, 17-25.