Natural Resources Canada, Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste. Marie, Ontario,Canada, P6A 5M7

Background and objectives
The successful regeneration of our northern Ontario forest, primarily spruce species, is severely affected by A. ostoyae and thinning of our southern Ontario stands promotes the infection rate of H.annosum. There are no control options available for A. ostoyae and the use of borax as a stump protectant against H.annosum is no longer an approved method. The current mandate is to reduce the use of chemical pesticides in forestry and develop biological control methods for the control of the pests affecting our forests. The endophytes Ascocoryne sarcoides and Tympanis hypopodia have been reported to be commonly associated with and antagonistic to decay fungi [1,2]. The objective of this study was to identify the mode of action of the most inhibitory strains of these endophytes and to develop methods to introduce them as part of the mycoflora of seedlings prior to outplanting.

Materials and methods
Strains of A. sarcoides and T. hypopodia were isolated from black spruce and jack pine respectively at the interfaces between decayed and healthy wood and screened for their inhibitory capabilities on 3% malt agar. The most inhibitory strain was used in further studies. To confirm their avirulence callus tissue of black spruce and jack pine were inoculated with spore suspensions and sampled biweekly, for up to 20 weeks, by isolating the fungus on malt agar and transferring the calli cells on half strength B5 medium to confirm their viability. Surface sterilized seeds of jack pine and black spruce were soaked in suspensions of 2% Pelgel of A. sarcoides and T. hypopodia containing 1x108 spores/mL for 10 minutes and air dried in a laminar flow chamber. Glass test tubes (4x25 cm) each containing 75 g of sterile peat moss-vermiculite 2:1 v/v at 100% water holding capacity and stoppered with a cotton plug were sterilized at 121C for 45 minutes. Each tube was inoculated with 2 treated seeds of the same tree species and the endophyte and incubated at 25C with a 16 hour photoperiod. Control seeds contained only the sticker. Isolations were made every 10 days to determine the infection rates and the rate of spread within the seedlings. The production of antifungal compound(s) was determined using the dialysis membrane overlay method and by culturing the endophytes in 3% malt extract and subsequently testing of the culture filtrates at dilution factors of 75, 50 and 25%.

Results and conclusions
A. sarcoides was common in spruce and found more often in trees infected with A.ostoyae than other root pathogens whereas the frequency of isolations of T.hypopodia was very low in jack pine. Bioassays with the isolated strains demonstrated a great variation amongst the strains of A. sarcoides for their abilities to inhibit the linear growth of A.ostoyae and H. annosum whereas the T.hypopodia strains isolated were more homogenous in their inhibitory capabilities. The seeds treated with the endophytes germinated marginally faster than the controls but there were no significant differences in the growth after 1 month. The endophytes survived for up to 21 days on the seed coat once it fell from the cotyledon. A. sarcoides was quicker to penetrate the tissues of the plants but both organisms inhabited the stem and roots of seedlings 14 days following germination. Control plants did not contain these microbes. It appears that the mode of action of these 2 endophytes is through the release of toxic metabolites and that T. hypopodia produces the more inhibitory compound(s). Greenhouse production with A.sarcoides treated seeds for outplanting in the field is underway to determine the biocontrol potential of this method.

1. Roll-Hansen F, and Roll-Hansen H, 1979. Eur. J. For. Pathol. 9, 275-280.
2. Funk A, 1967. Can. J. Bot. 45, 309-317.