Control of growth of wood decay basidiomycetes by Trichoderma spp. and other potentially antagonistic fungi

Isolates of Trichoderma, Penicillium, and Aspergillus were tested against Trametes versicolor and Neolentinus lepideus by agar interaction tests and by measuring the toxicity of culture filtrates. Some isolates were able to overgrow and kill both of the decay fungi in agar culture but others were totally ineffective. Although the filtrate from most isolates produced some growth inhibition of the two target fungi, the extent of the control varied widely. Three fungi that produced the greatest inhibitory effects (two Trichoderma and one Aspergillus isolate) were subsequently tested against a range of brownand white-rot Basidiomycetes. The filtrates of all three isolates produced varying degrees of inhibition against each of the Basidiomycetes. However, the Aspergillus filtrate generally produced greater inhibition and was more effective against the white-rot Basidiomycetes. The implications of the results on the testing and use of nondecay antagonistic fungi for the biological control of wood decay are discussed. At present, broad-spectrum poisons are the only practical method of ensuring that most wooden materials will remain free from fungal decay. While certain physical methods will protect wood from decay (e.g., excluding moisture), these are often not practical for many wooden commodities which, by the nature of their use, are subjected to fluctuating environmental conditions. Increased concern over the environmental effects of chemical biocides and associated legislative constraints on the use of some chemical treatments has meant that radical alternatives are now being sought to replace these preservatives. One possible alternative approach for protecting wood is the use of biological control systems, where microorganisms could be used to inhibit colonization and decay by woodrotting fungi. The basic concept is to employ the natural ecological antagonisms of selected organisms (most often micro fungi) against target wood decay fungi. The idea of using biological control as a means of protecting wood from decay is not new. Early studies on the use of biological control in wood have examined decay control in tree stumps (33), felled timbers (21,22), and electrical distribution poles (32). In recent years, the need to find alternative wood preservation strategies has resulted in increased interest in biological control for a variety of uses in the wood preservation industry. This is mirrored by the large number of publications on this topic Much of the research work has concentrated on the use of Trichoderma spp. as possible control agents for wood decay fungi (5,6,8,10,11,20,24,25, 27,28,34). Trichoderma has received so much attention because of the encouraging results with this genus in earlier studies (21,22,32) and the fact that Trichoderma spp. have been widely studied as potential control agents for a wide range of plant pathogens in agricultural systems (29,37). Other microorganisms have also been studied for application as biological control agents to protect wood from decay including bacteria (2-4,30) and other microfungi (19,23,26,34). Biological control systems in agriculture have traditionally been used against single pests or disease agents and their success is due in part, to this specificity of interaction. In wood, however, a control agent would require a broader specificity because most wood structures can be decayed by a range of fungi. The target specificity of control agents is also likely to be peculiar to individual isolates because most micro fungi, and Trichoderma spp. in particular, exhibit huge interspecies and interstrain The authors are respectively, Lecturer in Microbiology, Dundee Inst. of Technology, Bell St., Dundee, Scotland DD1 1HG; and Project Leader, Biodeterioration and Preservation of Wood, USDA Forest Serv., Forest Prod. Lab., Madison, WI 53705. Bruce would like to thank the USDA Forest Serv. and Oregon State Univ. for funding in support of this work, which was undertaken during his visit to the Forest Prod. Lab. The authors would also like to thank E.E. Nelson, USDA Forest Serv., Pacific Northwest Res. Sta., Corvallis, OR, for supplying many of the Trichoderma cultures used in this study. Thispaper was recieved for publication in November 1989. Forest Products Research Society 1991. Forest Prod. J. 41(2):63-67. FOREST PRODUCTS JOURNAL Vol. 41, No. 2 63 variability in metabolize production (36). This paper describes work undertaken to examine the interactive effects of potential biological control strains against a range of wood decay Basidiomycetes with particular attention to the production of soluble metabolizes by the antagonists. Materials and methods Fifteen Trichoderma, 2 Penicillium, and 1 Aspergillus isolates were initially tested to find their reaction during interaction tests on agar plates against Neolentinus (=Lentinus) lepideus (Fr.:Fr.) Redhead and Ginns FPRL 7f) and Trametes (=Coriolus) versicolor L. ex Fr.) Pilate (MAD 697) as representative brownand white-rot fungi, respectively. The Trichoderma isolates used in this study included five strains of T. pseudokoningii Rifai, two T. polysporum (Link. ex Pers.) Rifai (IMI 206040)), and three unidentified Trichoderma spp. isolated from various cellulosic materials. The two Penicillium and one Aspergillus strain were isolated from the interior of a creosotetreated distribution pole. The method used to study interactions between the micro fungi and decay fungi was similar to that used by Rayner and Todd (31). Mycelial plugs removed from the growing margins of cultures of either N. lepideus or T. versicolor were placed at one side of a petri dish containing malt extract agar (2% malt extract, 1.5% agar) and incubated at 27°C and 70 percent relative humidity for 4 days. After this time, cores removed from the margins of actively growing cultures of the microfungi were placed at the opposite sides of the dishes and the plates were incubated under the same conditions for up to 6 weeks. Because of the failure of many of the Trichoderma and the two Penicillium species to grow at 27°C, combinations containing these organisms were reincubated at 23°C while the remainder were reincubated at 27°C. Plates were examined daily to determine the outcome of interactions between the organisms and were assessed on the bases of whether 1) either organism was overgrown by its competitor and the rate at which overgrowth occurred; 2) contact and overgrowth was accompanied by browning and lysis of the Basidiomycete mycelium; and 3) the Basidiomycete was completely killed by the microfungus. Lysis of the Basidiomycete mycelium was confirmed by microscopic examination of samples from the zone of contact between the two colonies where the Basidiomycete mycelium had released a brown pigment into the agar medium. In plates where the Basidiomycete had been overgrown by the antagonist, cores were plated onto malt extract agar containing 4 ppm benomyl to test viability of the decay fungus. Lack of growth from these cores after 3 weeks’ incubation at 27°C indicated that the Basidiomycete had been killed by the antagonist. In addition to interaction studies, the same 15 microfungi were tested to determine whether they produced any soluble products that were inhibitory to N. lepideus or T. versicolor This was accomplished using a method described by Bruce et al. (9). Each of the microfungi was grown in 100 ml of 3 percent malt extract broth for 7 days at either 23° or 27°C, and any mycelium was removed by filtration and the culture filtrate was sterilized by passing through a 0.2 μm millipore membrane. Ten ml of sterile filtrate was then added to an equal volume of strengthened agar (2% malt extract, 3% agar) held at 50°C and poured into petri dishes to produce a solid medium for inoculation with the Basidiomycetes. Cores (7 mm diameter) removed from the margins of actively growing cultures of N. lepideus or T. versicolor were inoculated in the centers of the plates that were then incubated in the dark at 27°C. Controls were prepared by adding uninoculated malt extract broth (3%) to the strengthened agar. Three replicate plates were set up for each test. Inhibition of growth of the Basidiomycetes was recorded as the difference in mean radial growth of the Basidiomycetes in the presence or absence of the fungal filtrates after 6 days for the T. versicolor or 8 days for the N. lepideus. These values were then used to calculate the inhibition of hyphal extension as a percentage of hyphal extension in the absence of the filtrate. After the initial screening of the culture filtrates, the three organisms that produced the highest levels of inhibition against N. lepideus and T. versicolor were selected for further testing against a wide range of wood decay Basidiomycetes. The T harzianum (T25), T. hamatum (T150), and Aspergillus isolates were incubated in 1000 ml of malt extract broth (3%) for 7 days and filter-sterilized as previously described. A 500-ml portion of the filtrate from each micro fungus was heat treated in an oven at 90°C for 2 hours to determine the effect this might have on its inhibitory activity. Solid agar plates were then made as before by adding strengthened agar to either heat-treated or nonheat-treated filtrate. Replicate plates and controls were then inoculated as previously described with cores of the following fungi: brown-rot fungi — Coniophora puteana (Schum.:Fr.) (MAD 515); Fibroporia vaillantii (DC:Fr.) Parm (FP 90877R); Fomoptsis meliae (Underw.) Gilbn. (FPI0002R); Gloeophyllum trabeum (Pers.:Fr.) Murr. (MAD 617R); Neolentinus lepideus (Fr.:Fr.) Redhead and Ginns (MAD 534); Antrodia (=Poria) carbonica (Over.) Ryvet Gilbn. (MAD 141); and Postia (=Poria) placenta (Fr.) M. Larset Lomb. (MAD 697); white-rot fungi – Bjerkandera adusta (Willd.:Fr.) Karst. (L 1539sp.); Trametes versicolor (L. ex Fr.) Pilate (MAD 697); Ganoderma applanatum (Pers.) Pat. (MAD 7823s); Heterobasidion annosum (Fr.) Bref. (=Fomes annosus (Fr.) Kars