Alan Bruce

Effect of substrate composition on production of volatile organic compounds from Trichoderma spp. Inhibitory to wood decay fungi

Abstract The production of volatile organic compounds (VOCs) by two Trichoderma isolates grown on sand infused with either malt extract broth or a minimal medium with the carbon:nitrogen levels similar to that found in Scots pine wood was examined. The VOCs were trapped in tubes packed with chromatographic adsorbant, then analysed by Integrated Automated Thermal Desorption-GC-MS. A total of 45 individual VOCs were recorded. Principle Component Analysis was used to establish that the production of individual VOCs was dependent on both the Trichoderma isolated and the growth media type. The inhibitory effect of the VOCs from the two Trichoderma isolates was also tested on four common wood decay fungi. Although VOCs from both Trichoderma species grown on a malt agar medium consistently inhibited the growth of all four target fungi, effects were negligible when the isolates were grown on a minimal agar medium. The importance of the production of individual VOCs on the inhibition of the wood decay fungi and the implications for the biological control of such fungi are discussed.

Production of Volatile Organic Compounds by Trichoderma in Media Containing Different Amino Acids and Their Effect on Selected Wood Decay Fungi

Summary The paper describes an experiment to evaluate the effect of subtle changes to the amino acid composition of the growth medium on the production of volatile organic compounds (VOCs) by a Trichoderma aureoviride isolate and the consequent inhibition of wood decay fungi. A synthetic medium was produced to represent the C:N ratio and major amino acid content of Scots pine sapwood. Changes in the amino acid content of the medium produced very significant differences in the VOC output produced by the Trichoderma isolate and in the levels of inhibition of the target wood decay fungi. Principal component analysis of the VOC profiles indicated that aldehyde and ketone components were associated with the greatest inhibition of the target fungi. The implications of the results for the use of Trichoderma isolates as biological control agents of decay fungi in wood are discussed.

Effect of volatiles from bacteria and yeast on the growth and pigmentation of sapstain fungi

Sapstain fungi affect the appearance of wood due to colonisation by pigmented hyphae but without producing significant strength losses. This is due to the production of melanin in the fungal cell walls of the staining fungi. Any biological control strategy targeted against this type of deterioration would therefore be considered successful if it inhibited either fungal growth or pigment production. Previous work has established that specific bacterial and yeast isolates selected on the basis of agar screening studies could significantly reduce levels of staining in wood block tests. This paper presents the results of a study to examine the role of volatile organic compounds (VOCs) produced by four bacterial and three yeast isolates on the growth and pigment production by a range of five sapstain fungi on three media types. VOCs from three of the four bacterial strains tested completely inhibited growth of the five target sapstain fungi but only when the antagonists were grown on tryptone soya media. When antagonists were grown on either malt agar or a low nutrient medium levels of inhibition were either significantly reduced or non-existent. Yeast antagonists generally produced lower levels of growth inhibition than the bacteria but a Williopsis mrakii isolate gave 100% inhibition of three of the five sapstain fungi. Production of inhibitory VOCs was highly dependent on the specific antagonist as well as its growth substrate and all five sapstain fungi showed varying sensitivities to the VOCs produced. Not all fungi were inhibited, growth of O. piliferum and A. pullulans being stimulated by the VOCs from antagonists but only when grown under low nutrient conditions. In some instances, where growth was only slightly reduced, the level of pigmentation of the sapstain colony was significantly reduced compared with corresponding controls. The implications of this work for the biological control of sapstain fungi are discussed.

Chitinase and laminarinase production in liquid culture by Trichoderma spp. and their role in biocontrol of wood decay fungi

Abstract This paper describes macro- and microassay methods to assess the production of the lytic enzymes laminarinase and chitinase, by a range of Trichoderma isolates, and investigates the effect of nutrient composition, glucose amendment and the addition of basidiomycete cell wall material on the production of these lytic enzymes. Large interstrain and interspecies differences exist in the levels of production of both enzymes. Total activities of the enzymes are greater when isolates are cultured in malt medium, but specific chitinase and laminarinase activities are higher in low nutrient conditions. Glucose repressed the production of both laminarinase and chitinase, although this effect was not common to all Trichoderma isolates for the latter enzyme. Cell walls of both N. lepideus and T. versicolor induced increased chitinase activity but this effect was Trichoderma species specific. Mycoparasitism involving lytic enzymes has been described as the mechanism of action of Trichoderma species in the biological control of commercially important plant pathogens. However, little information is available on the significance of this mechanism for the biological control of wood decay fungi. The importance of mycoparasitism as a mechanism in the biological control of wood decay fungi is briefly discussed.

Identification of volatile organic compounds (VOCs) from bacteria and yeast causing growth inhibition of sapstain fungi

Abstract This paper describes an experiment to identify volatile organic compounds (VOCs) from a range of three bacteria and one yeast strain that had previously been shown to be inhibitory to selected sapstain fungi. The bacteria and yeast were cultured on two media, malt extract (ME) and tryptone soya (TS) and the VOCs trapped on chromatographic adsorbant before being analysed by Integrated Thermal Desorption—GC-MS. Since sapstain fungi were only inhibited by VOCs produced on the TS media, it was possible to use Principle Component Analysis to highlight the individual VOCs that are most likely to be responsible for the inhibition. A number of ketones together with dimethyl disulphide and dimethyl trisulphide were highlighted. The importance of VOC production by organisms during the biological control of sapstain is discussed.

Yeast and bacteria as biological control agents against fungal discolouration of Pinus sylvestris blocks in laboratory-based tests and the role of antifungal volatiles.

Summary This paper reports on the use of yeast as biological control agents against mould and blue-stain fungi in laboratory-based Pinus sylvestris sapwood wood block incubation tests. All 5 yeast isolates tested significantly reduced discolouration of wood surfaces by moulds and most were also effective against staining fungi. The 2 most promising yeast isolates also significantly reduced radial growth of selected mould and stain fungi in vitro solely through the liberation of volatile compounds. Five bacterial isolates were also tested for antagonism toward mould and stain fungi and were effective biological control agents in wood block tests. Again, volatile production alone by the bacteria was shown to be sufficient to significantly reduce fungal growth in vitro. Most of the bacteria and yeasts significantly reduced wood discolouration by fungi when inoculated onto block surfaces 7 days before, or at the same time as the target fungi, and in some cases even when inoculated 7 days after the fungi. The possibility of using yeasts as biological control agents to prevent wood spoilage during kiln drying and short-term storage at sawmills is considered.

The effects of specific volatile organic compounds produced by Trichoderma spp. on the growth of wood decay basidiomycetes

Summary Previous work by Srinivasan et al. (1992) and Bruce et al. (1996) has shown that inhibition of wood decay fungi by volatile organic compounds produced by Trichoderma spp. is dependent on the type of growth media and age of the Trichoderma colony. Wheatley et al. (1997) identified five volatile organic compounds produced by Trichoderma spp. that may be inhibitory to wood decay fungi. The effects of four of these compounds (Acetone, 2-methyl-1-butanol, heptanal and octanal) were tested over a range of concentrations against four selected wood decay fungi. Fungi were incubated in malt extract broth under appropriate conditions and growth was estimated by biomass production and respiration rates. Growth of all four fungi was affected by at least one of the compounds, usually by inhibition but occasionally stimulation. All but two of the fungus/chemical combinations showed significant effects on biomass development (P < 0.05) and fifteen of the sixteen combinations produced a significant concentration effect on respiration rates (P < 0.05). The aldehydes, heptanal and octanal, were very effective against all four wood decay fungi. All four fungi were inhibited by more than 80% at 25 μg ml−1 by heptanal and three of the four fungi were totally inhibited at 250 μg ml−1. Acetone did not inhibit the four wood decay fungi and even stimulated fungal growth at some concentrations. 2-methyl-1-butanol was only effective at the highest concentration of 2500 μg ml−1. The implications of these results for the biological control of wood decay fungi and future studies are discussed.

Decay Resistance of Wood Removed from Poles Biologically Treated with Trichoderma

Soil block ter mapping of microbial inhabitants. The results indicate that material from pole interiors colonized by Trichoderma is able to resist decay by Lentinus lepideus and Antrodia carbonica. Any decay prevention Southern yellow pine Trichoderma was lost however when the wood was steam sterilized prior to exposure to the basidiomycetes. The implications of the results for the usc of biological control of internal decay in creosoted poles are discussed.

Assessment of the biocontrol potential of a Trichoderma viride isolate: Part II: Protection against soft rot and basidiomycete decay

A previous paper reported on the establishment of a field and fungal cellar trial set up to determine the biocontrol potential of a specific Trichoderma isolate against wood decay fungi. This paper reports on the analyses used to examine the protective effect of the selected isolate, and presents results indicating an initial protective effect against both basidiomycetes and soft rot fungi. The parameters assessed in the field and fungal cellar trials were soft rot decay, basidiomycete decay, Trichoderma colonisation, moisture content and nitrogen content. The results of these analyses show that the introduction of a biological control agent has had a significant effect on moisture content, decay and nitrogen content. A protective effect has been observed against soft rot and basidiomycete decay fungi in field samples.

Assessment of the biocontrol potential of a Trichoderma viride isolate : Part I: Establishment of field and fungal cellar trials

A field trial has been set up to assess the biological control potential of a Trichoderma viride isolate, T60. This isolate had been shown in previous laboratory tests to be particularly eAective against certain basidiomycete decay fungi. Wood was treated with T60 spores using vacuum pressure impregnation in a pilot preservation plant. Treated stakes were planted in the field site along with CCA-treated and untreated control samples. Replicate samples were also set up in an accelerated decay facility employed to give a comparison to the field trial results. This paper describes the setting up and monitoring of the field and fungal cellar trials, and presents results of moisture monitoring and sapstain assessment which indicate that Trichoderma viride isolate T60 has a marked eAect on the rate of sapstain development under certain conditions. The paper also discusses the eAcacy of pressure impregnation of spore suspensions for use as biological control agents # 2000 Elsevier Science Ltd. All rights reserved.