AbstractAll living organisms produce volatile compounds that can have a significant effect on the ecological balance within their community. In wood, volatile compounds can build up and become toxic to other resident microorganisms. The major aim of this study was to evaluate the production of volatile compounds by Trichoderma species and evaluate their importance as a mechanism of control of Serpula lacrymans. Volatile interactions between a range of Trichoderma isolates grown on 3 different media (malt extract, minimal media and sawdust) and 4 S. lacrymans isolates demonstrated that levels of inhibition were influenced by Trichoderma isolate, media type and sensitivity of the S. lacrymans strain. The growth inhibition of S. lacrymans consistently resulted in the production of yellow pigmentation in the Serpula mycelium. The greatest levels of inhibition were seen when the Trichoderma were grown on the malt extract media. However, significant levels of inhibition were still recorded when the Trichoderma spp. were grown on a minimal medium (with a carbonmitrogen ratio similar to that found in Scots pine sawdust) and sawdust. The growth inhibition of S. lacrymans on the minimal media and sawdust is likely to provide a much more realistic guide to levels of inhibition that might be expected in the field. Gas chromatograph-mass spectrometry and associated principal component analysis of the Trichoderma VOCs resulted in the identification of 8 volatile compounds, mainly ketones, which were implicated as the active VOCs involved in the inhibition of S. lacrymans. The range and quantity of volatiles produced was dependent on the Trichoderma isolate and the media on which it was growing. If Trichoderma. is to be successful as a remedial treatment of dry rot in buildings, the volatiles identified as active inhibitory compounds must also be produced by the potential biocontrol agent in wood. Therefore, compounds of particular interest were 3-octanone, 2-nonanone and 2-heptanone, as they were produced by the most inhibitory treatment (T. aureoviride when grown on malt extract) but also produced on the sawdust.
After exposure of the S. lacrymans strains to the Trichoderma VOCs, the Serpula cultures were assayed for production of 3 enzymes, cellulase, tyrosinase and peroxidase. The results demonstrated that the Trichoderma VOCs did not affect cellulase production. Therefore, while there would be a decrease in cellulase production, due to the reduction in S. lacrymans biomass, caused by the inhibition of growth by the Trichoderma VOCs, S. lacrymans would still be capable of degrading cellulose. Tyrosinase production was not detected in any of the cultures of S. lacrymans. Since yellow pigmentation was detected in plates of S. lacrymans inhibited by the Trichoderma VOCs it is unlikely that tyrosinase plays a role in this pigmentation in Serpula. Increased peroxidase production was detected in all plates of S. lacrymans displaying yellow pigmentation, suggesting that increased peroxidase production is linked to increased pigmentation associated with a stress reaction in S. lacrymans.
SDS-PAGE analysis of protein production indicated changes in the protein profiles of S. lacrymans isolates when exposed to inhibitory VOCs from Trichoderma. One protein band in particular, at molecular weight 22.4 kDa, appeared to be closely linked to levels of growth inhibition with production of this protein completely inhibited by the VOCs from T. aureoviride, which gave the greatest levels of inhibition of the S. lacrymans isolates. The protein was unaffected by VOCs from T. pseudokoningii, which gave very little or no inhibition of growth of S. lacrymans, while VOCs from T. viride (which gave on average between 35-50% inhibition of growth) caused a marked reduction in synthesis of this protein. Analysis of this protein by electrospray ionisation did not, however, result in the identification of this protein. Removal of the antagonistic VOC stress demonstrated that the inhibition of S. lacrymans growth was transient, with not only growth resuming but production of all proteins previously inhibited also resuming. This work has shown that VOC production by Trichoderma spp. can have a fundamental role in microbial ecology of ecosystems dominated by fungi and for the first time has shown both up and down regulation of protein production in S. lacrymans after exposure and subsequent removal of inhibitory Trichoderma VOCs.
|Date of Award||Feb 2003|