- Email: [email protected]
In vitro activity of 6-pentyl-α-pyrone, a metabolite of Trichoderma harzianum, in the inhibition of Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici
Mycol. Res. 98 (10): 1207-1209 (1994)
1207
Printed in Great Britain
In vifro activity of 6-pentyl-U-pyrone, a metabolite of Trichoderma harzianum, in the inhibition of Rhizocfonia solani and Fusarium oxysporum f. sp. lycopersici
R. SCARSELLETTI A N D J. L. FAULL Department of Biology, Birkbeck College, London W C I E 7HX, U.K.
The compound 6-pentyl-a-pyrone [6-p-p]produced by Trichoderma harzianum IMI 288012 was used in plate tests against Rhiwctonia solani and Fwarium oxysporum f. sp. lycopersici. The addition of 0.3 mg ml-' 6-p-p to agar medium caused a 69.6% reduction of growth of Rhizocfonia sohni and 31.7% reducfion of growth of Fwarium oxyspomm f. sp. lycopersici after 2 d. When used in spore germination tests 0 4 5 mg ml-l 6-p-p was found to inhibit completely the germination of Fusarium spores. A strong relationship was found between the production of the pyrone by T. harzianum and the antagonistic ability of this h g u s in vitro.
A possible role for secondary metabolites produced by Trichodema spp. has emerged over the years from the study of the interactions between these fungi and other saprotrophs or plant pathogens (Dennis & Webster, 1971a, b, c; Moss, Jackson & Rogers, 1975). Numerous metabolites have been identified from cultures of Trichodsrma spp. and have been shown to have adverse effects on the growth of different species of fungi in vifro (Godtfredsen & Vangedal, 1965; Ooka ef al., 1966; Meyer & Ruesser, 1967; Dennis & Webster, 1971a; Hou, Ciegler & Hesseltine, 1972; Fuji ef al., 1978).The compound 6-pentyl-a-pyrone [6-p-p] was first isolated from cultures of T. viride. It was identified as one of the major components of the coconut aroma characteristic of many strains of Trichoderma (Collins & Halim, 1972). In vifro 6-p-p inhibited various soil fungi and reduced the incidence of R. solani damping-off of lettuce in pot trials (Claydon ef al., 1987). The objective of this study was to provide evidence of a causal link between 6-p-p production by T. harzianum and its in vifro antagonism to target plant pathogens.
MATERIALS A N D M E T H O D S Fungal strains Trichodema harzianum Rifai (THI) (IMI 288012) was used in this study. Target fungi included one isolate of Fusarium oxysporum f. sp. lycopersici W. C. Snyder & H. N. Hansen (IMI 141140) and one isolate of R. solani J. G. Kuhn (RSPW23) (University of Hull, U.K.). Trichodema and Fusarium strains were maintained on silica gel in glass vials at 4 OC while Rhiwctonia was maintained on 2% malt agar (MA) slopes at 20'. Each fungus was subcultured on 2% MA and grown at 25' in the dark.
Exfraction and purification of 6-pentyl-a-pyrone from cultures of Trichoderma harzianum Liquid cultures of THl were prepared by inoculating 300 ml of sterilized (0.103 x lo6 N m-2 for 15 min) Potato Dextrose Broth (PDB) (Difco)contained in 1 1 conical flasks with 4 ml of a spore suspension containing approximately I x lo7 spores ml-'. The flasks were incubated at 20° on a reciprocal shaker in the dark for 7 d. Each liquid culture was filtered through filter paper (Whatman no. I) in a Buchner funnel and the culture filtrate was extracted by steam distillation. The distillate (300 ml) was extracted by liquid partition with an equal volume of ethyl acetate (AnaIar). The solvent was dried over anhydrous sodium sulphate and then evaporated under vacuum in a rotary evaporator at 35O. The dried residue was resuspended in 1ml of ethyl acetate and transferred dropwise into the still-pot of a microdistillation apparatus containing two or three glass anti-bumping chips. The still pot was kept in a water bath at 95O which allowed the solvent to evaporate. The residue was distilled under reduced pressure in a paraffin oil bath at 120°. The 6-p-p was collected on a cold finger in a bath of dry CO, and methylene chloride. 6-p-p purity was determined by Nuclear Magnetic Resonance spectrometry, Gas Chromatography and Mass Spectroscopy. Concentrations of 6-p-p were determined from the absorbance at 302 nm using an ultraviolet spectrophotometer (Unicam SP800).
Plate culture test using 6-pentyl-a-pyrone Five ml filter sterilized (Millipore type GS 0.22 ym) aqueous solutions of 6-p-p were added to 10 ml of 2 % MA in 9 cm Petri dishes to obtain two sets of concentrations, 0.3 and 0-2 mg of 6-p-p ml-' of agar. Distilled water was used in
1208
Activity of 6-pentyl-a-pyrone in uifro control plates in place of 6-p-p solution. Each plate was inoculated with a 5 mm plug of R. solani or F. oxysporum f. sp. lycopersici. The plates were incubated at 25' in the dark and examined after 2, 5, 7 and 9 d. The diam. of the colonies was measured and the percentage inhibition of growth was calculated from mean values. Antibiotic assay disc test using 6-pentyl-a-pyrone
A few drops of an ethyl acetate solution of 6-p-p was applied to each antibiotic assay disc allowing complete evaporation of the solvent between each application until the disc was loaded with 100 ~1 of the solution (1.1mg 6-p-p). Similarly, clean ethyl acetate was applied to each control disc. When completely dry, the discs were placed onto 2% MA plates which were then inoculated in the centre with an 8 mm plug of R. solani with the distance between the edge of the inoculum plug and that of the disc being 1.5 cm. The control disc was placed at the same distance on the opposite side of the inoculum. The plates were incubated at 25' in the dark and were examined after 48 h and again after 72 h. The radial growth of the fungus on the side facing the control disc was compared with that opposing the 6-p-p disc and the percentage inhibition was calculated on mean values as follows: [(rl-r,/rl)] x 100, where r, is the radius of the colony towards the control disc and r, the radius towards the 6-p-p disc. Spore germination test
Six solutions containing 1.14, 0.91, 0.68, 0.45, 0.22 and 0.11 mg ml-' of 6-p-p in ethyl acetate were prepared. One ml of each solution was dried in Eppendorf tubes using a Hetovac. The dry residue was resuspended with 1ml of PDB containing 1x lo6 spores of F. oxysporum f. sp. lycopersici. Control tubes contained the spore suspension only. The tubes were incubated at 25' in the dark for 18 h, when the number of germinated spores or swollen spores was recorded by observing and counting under a microscope. At the end of the spore germination test 0.1 ml of the spore preparation were spread onto 2 % M A plates. The remaining spore preparation was centrifuged at high speed (8160g) using a Micro Centaur centrifuge for 3 min. The supernatant was discarded and the pellet resuspended in 1ml of sterile PDB, shaken and centrifuged again. The whole procedure was repeated five times. After the last rinse 0.1 ml of the spore suspension was spread onto 2% M A plates. Spores from the control tubes were treated in the same way. All plates were incubated at 25' in the dark and examined for spore germination over a number of days up to I wk.
After 9 d of incubation R. solani had completely covered the control plates and plates containing both concentrations of 6-p-p. Completely developed sclerotia were produced in control plates. Sclerotial initials were visible in the plates containing 0.2 mg ml-' of 6-p-p, while no sclerotia were in the plates containing 0.3 mg ml-' of 6-p-p. After a month of incubation sclerotia had developed normally in the 0,2 mg ml-' plates while they were just starting to appear in the 0.3 mg ml-' plates. For Fusarium, the addition of 6-p-p to the agar medium caused a loss of pigmentation and a reduction in mycelial growth. The degree of these alterations was more severe in the plates with the higher concentration of 6-p-p. When 1.1 mg of 6-p-p were applied on to antibiotic assay discs the radial growth of R. solani was reduced with respect to the control by 66% at 2 d and 57% at 3 d after inoculation. Inhibition started at a distance of 8 mm between the disc and the colony edge. Ejfecf of 6-pentyz-a-pyrone on the germination of Fusarium spores
Spores of F. oxysporum f. sp. lycopersici did not germinate when incubated in aqueous solutions containing 0.45 mg ml-' of 6-p-p or greater (Table 2). At concentrations of 6-p-p higher than 0.45 mg ml-' all spores appeared unimbibed. At concentrations of 0.45 mg ml-' a small percentage of spores appeared swollen as in the control (Table 2). The percentage of normally swollen spores and of germinated spores increased progressively at lower concentrations of 6-p-p. Spores from the 6-p-p preparation did not germinate when inoculated onto fresh agar medium but when repeatedly rinsed with PDB these spores achieved 100% germination under the same conditions. Table I. Inhibition of growth (%) of target fungi on 2% malt agar containing different concentrations of 6-pentyl-a-pryone Days of incubation 6-pentyl(entries show % a-pyrone inhibition) concentration (mg ml-') 2 5 7 9
Fwarium oxysporum f. sp. lycopersici Rhizoctonia solani
0.2 0.3 0.2 0.3
14.2 11.5 7 3 1.2 31.7 30.0 28.9 19.7 55.5 26.7 0.0 0.0 6 9 6 51.1 22.1 0.0
Table 2. Spore germination (%) and proportion of swollen spores of Fwarium orysporum 6. sp. lycopersici incubated in different concentrations of 6-pentyl-a-pyrone 6-pentyl-a-pyrone concentration % germinated % swollen % germinated spores spores washed spores (mg mlkl)
RESULTS Activity of 6-penfyl-a-pyrone on the growth of iargef fungi
Aqueous solutions of 6-p-p added to the agar medium caused a reduction of the growth rate of R. solani and of Fusarium (Table I).
Control 1.14 0.91 068 045 022 0.11
15.4 0.0 0.0 0.0 0.0 10.2 15.2
100.0 0.0 0.0 0.0 1.25 43.25 94.5
100.0 100.0 100.0 100.0 100.0 100.0 100.0
1209
R. Scarselletti and J. L. Faull DISCUSSION Previous work using the dual culture technique of Dennis & Webster ( 1 9 7 3 ~ )has shown that of three isolates of T. harzianum tested only one significantly reduced hyphal growth of Rhizoctonia solani before contact (Scarselletti, 1992). Furthermore this isolate prevented sclerotium production by R. solani and could overgrow F. oxysporum f. sp. lycopersici. The effect on growth rate could be observed when the two paired fungi were as far as 7 mm apart. This isolate alone was shown to produce 6-pentyl-a-pyrone. Results from the present experiments show that when 6-p-p was applied onto antibiotic assay discs the growth rate of R. solani was similarly reduced and that inhibition started at a distance of 8 mm between the colony margin of the target fungus and the disc. Incorporation of 6-p-p into the agar medium also caused inhibition of sclerotia formation by R. sobni and a lack of pigmentation in Fusarium. Aqueous solutions of 6-p-p also inhibited the germination of Fusarium spores. The inhibition of germination was persistent unless the compound was removed. Results from the spore viability tests suggest that the compound was strongly bound to or into the spores and that, regardless of its volatile nature, its effect could not be eliminated by simply dispersing the spores over a wider, more aerated environment. Although ultrastructural analysis of spores and hyphal tips is necessary, the hydrophobic nature of 6-p-p (log KOct,H20= 2.793) (Scarselletti, 1992) could substantiate the hypothesis of a mechanical impediment to water absorption by the fungal cells due to the formation of a hydrorepellent film on the cell wall. However, phenomena like the inhibition of pigmentation in Fusarium and the inhibition of sclerotia production by Rhizoctonia seem to indicate that 6-p-p interferes with more complex metabolic activities of fungi. The similarities between the activity of T H I and of 6-p-p together with the lack of any of the aforementioned effects by (Accepted 16 March 1994)
isolates of T. harzianum which do not produce 6-p-p, strongly supports a causal relation between 6-p-p and antagonistic ability of T. harzianum in vifro. The Authors would like to thank Dr E. Hedgley for his assistance in the isolation and characterization of 6-pentyl-apyrone.
REFERENCES Claydon, N., Allan, M., Hanson, 1. R. & Avent, A. G. (1987).Antifungal alkyl pyrones of Trichoderma harzianum. Transactions of the British Mycological Society 88, 503-513.
Collins, R. P. & Halim, A. F. (1972). Characterization of the major aroma constituents of the fungus Trichoderma viride Pers. Journal of Agricultural and Food Chemistry 20, 437-438. Dennis, C. & Webster, J. ( 1 9 7 1 ~ )Antagonistic . properties of species-groups of Trichoderma. I. Production of nonvolatile antibiotics. Transactions of the British Mycological Society 57, 26-39.
Dennis, C. & Webster, J. (1971b). Antagonistic properties of species-groups of Trichoderma. 11. Production of volatile antibiotics. Transactions of the British Mycological Sociefy 57, 41-48.
Dennis, C. & Webster, J. (1971c). Antagonistic properties of species-groups of Trichoderma. 111. Hyphal interactions. Transactionsoffhe British Mycological society 5 7, 363-369.
Fuji, K., Fujita, E., Takaishi, Y., Fujita, T., Arita, i., Komatsu, M. & Hiratsuka, N. (1978).New antibiotics, trichopolyns A and B: isolation and biological activity. Experientia 34, 237-239. Godtfredsen, W. 0.& Vagendal, S. (1965).Trichodermin, a new sesquitelpene antibiotic. Acta Chemica Scandinavica 19, 1088-1103. Hou, C. T., Ciegler, A. & Hesseltine, C. W. (1972). New mycotoxin, trichotoxin A, from Trichoderma viride isolated from southern leaf blightinfected corn. Applied Microbiology 23, 183-185. Meyer, C.E. & Reusser, F. (1967).A polypeptide antibacterial agent isolated from Trichoderma viride. Experientia 23, 85-86. Moss, M. O., Jackson, R. M. & Rogers, D. (1975).The characterization of 6(pent-I-enyl)pyrone from Trichoderma viride. Phytochemisty 14,270&2708. Ooka, T., Shimojima, Y., Akinrnoto, T., Takodam, I., Senoh, S. & Abe, J. (1966).A new antibacterial peptide 'suzucacillin'. Agricultural and Biological Chemistry 30, 700-702. Scarselletti, R. (1992).A study on the production of an antifungal compound by Trichoderma harzianum and its effects on Fwarium wilt of tomato. Ph.D. Thesis, University of London.
1207
Printed in Great Britain
In vifro activity of 6-pentyl-U-pyrone, a metabolite of Trichoderma harzianum, in the inhibition of Rhizocfonia solani and Fusarium oxysporum f. sp. lycopersici
R. SCARSELLETTI A N D J. L. FAULL Department of Biology, Birkbeck College, London W C I E 7HX, U.K.
The compound 6-pentyl-a-pyrone [6-p-p]produced by Trichoderma harzianum IMI 288012 was used in plate tests against Rhiwctonia solani and Fwarium oxysporum f. sp. lycopersici. The addition of 0.3 mg ml-' 6-p-p to agar medium caused a 69.6% reduction of growth of Rhizocfonia sohni and 31.7% reducfion of growth of Fwarium oxyspomm f. sp. lycopersici after 2 d. When used in spore germination tests 0 4 5 mg ml-l 6-p-p was found to inhibit completely the germination of Fusarium spores. A strong relationship was found between the production of the pyrone by T. harzianum and the antagonistic ability of this h g u s in vitro.
A possible role for secondary metabolites produced by Trichodema spp. has emerged over the years from the study of the interactions between these fungi and other saprotrophs or plant pathogens (Dennis & Webster, 1971a, b, c; Moss, Jackson & Rogers, 1975). Numerous metabolites have been identified from cultures of Trichodsrma spp. and have been shown to have adverse effects on the growth of different species of fungi in vifro (Godtfredsen & Vangedal, 1965; Ooka ef al., 1966; Meyer & Ruesser, 1967; Dennis & Webster, 1971a; Hou, Ciegler & Hesseltine, 1972; Fuji ef al., 1978).The compound 6-pentyl-a-pyrone [6-p-p] was first isolated from cultures of T. viride. It was identified as one of the major components of the coconut aroma characteristic of many strains of Trichoderma (Collins & Halim, 1972). In vifro 6-p-p inhibited various soil fungi and reduced the incidence of R. solani damping-off of lettuce in pot trials (Claydon ef al., 1987). The objective of this study was to provide evidence of a causal link between 6-p-p production by T. harzianum and its in vifro antagonism to target plant pathogens.
MATERIALS A N D M E T H O D S Fungal strains Trichodema harzianum Rifai (THI) (IMI 288012) was used in this study. Target fungi included one isolate of Fusarium oxysporum f. sp. lycopersici W. C. Snyder & H. N. Hansen (IMI 141140) and one isolate of R. solani J. G. Kuhn (RSPW23) (University of Hull, U.K.). Trichodema and Fusarium strains were maintained on silica gel in glass vials at 4 OC while Rhiwctonia was maintained on 2% malt agar (MA) slopes at 20'. Each fungus was subcultured on 2% MA and grown at 25' in the dark.
Exfraction and purification of 6-pentyl-a-pyrone from cultures of Trichoderma harzianum Liquid cultures of THl were prepared by inoculating 300 ml of sterilized (0.103 x lo6 N m-2 for 15 min) Potato Dextrose Broth (PDB) (Difco)contained in 1 1 conical flasks with 4 ml of a spore suspension containing approximately I x lo7 spores ml-'. The flasks were incubated at 20° on a reciprocal shaker in the dark for 7 d. Each liquid culture was filtered through filter paper (Whatman no. I) in a Buchner funnel and the culture filtrate was extracted by steam distillation. The distillate (300 ml) was extracted by liquid partition with an equal volume of ethyl acetate (AnaIar). The solvent was dried over anhydrous sodium sulphate and then evaporated under vacuum in a rotary evaporator at 35O. The dried residue was resuspended in 1ml of ethyl acetate and transferred dropwise into the still-pot of a microdistillation apparatus containing two or three glass anti-bumping chips. The still pot was kept in a water bath at 95O which allowed the solvent to evaporate. The residue was distilled under reduced pressure in a paraffin oil bath at 120°. The 6-p-p was collected on a cold finger in a bath of dry CO, and methylene chloride. 6-p-p purity was determined by Nuclear Magnetic Resonance spectrometry, Gas Chromatography and Mass Spectroscopy. Concentrations of 6-p-p were determined from the absorbance at 302 nm using an ultraviolet spectrophotometer (Unicam SP800).
Plate culture test using 6-pentyl-a-pyrone Five ml filter sterilized (Millipore type GS 0.22 ym) aqueous solutions of 6-p-p were added to 10 ml of 2 % MA in 9 cm Petri dishes to obtain two sets of concentrations, 0.3 and 0-2 mg of 6-p-p ml-' of agar. Distilled water was used in
1208
Activity of 6-pentyl-a-pyrone in uifro control plates in place of 6-p-p solution. Each plate was inoculated with a 5 mm plug of R. solani or F. oxysporum f. sp. lycopersici. The plates were incubated at 25' in the dark and examined after 2, 5, 7 and 9 d. The diam. of the colonies was measured and the percentage inhibition of growth was calculated from mean values. Antibiotic assay disc test using 6-pentyl-a-pyrone
A few drops of an ethyl acetate solution of 6-p-p was applied to each antibiotic assay disc allowing complete evaporation of the solvent between each application until the disc was loaded with 100 ~1 of the solution (1.1mg 6-p-p). Similarly, clean ethyl acetate was applied to each control disc. When completely dry, the discs were placed onto 2% MA plates which were then inoculated in the centre with an 8 mm plug of R. solani with the distance between the edge of the inoculum plug and that of the disc being 1.5 cm. The control disc was placed at the same distance on the opposite side of the inoculum. The plates were incubated at 25' in the dark and were examined after 48 h and again after 72 h. The radial growth of the fungus on the side facing the control disc was compared with that opposing the 6-p-p disc and the percentage inhibition was calculated on mean values as follows: [(rl-r,/rl)] x 100, where r, is the radius of the colony towards the control disc and r, the radius towards the 6-p-p disc. Spore germination test
Six solutions containing 1.14, 0.91, 0.68, 0.45, 0.22 and 0.11 mg ml-' of 6-p-p in ethyl acetate were prepared. One ml of each solution was dried in Eppendorf tubes using a Hetovac. The dry residue was resuspended with 1ml of PDB containing 1x lo6 spores of F. oxysporum f. sp. lycopersici. Control tubes contained the spore suspension only. The tubes were incubated at 25' in the dark for 18 h, when the number of germinated spores or swollen spores was recorded by observing and counting under a microscope. At the end of the spore germination test 0.1 ml of the spore preparation were spread onto 2 % M A plates. The remaining spore preparation was centrifuged at high speed (8160g) using a Micro Centaur centrifuge for 3 min. The supernatant was discarded and the pellet resuspended in 1ml of sterile PDB, shaken and centrifuged again. The whole procedure was repeated five times. After the last rinse 0.1 ml of the spore suspension was spread onto 2% M A plates. Spores from the control tubes were treated in the same way. All plates were incubated at 25' in the dark and examined for spore germination over a number of days up to I wk.
After 9 d of incubation R. solani had completely covered the control plates and plates containing both concentrations of 6-p-p. Completely developed sclerotia were produced in control plates. Sclerotial initials were visible in the plates containing 0.2 mg ml-' of 6-p-p, while no sclerotia were in the plates containing 0.3 mg ml-' of 6-p-p. After a month of incubation sclerotia had developed normally in the 0,2 mg ml-' plates while they were just starting to appear in the 0.3 mg ml-' plates. For Fusarium, the addition of 6-p-p to the agar medium caused a loss of pigmentation and a reduction in mycelial growth. The degree of these alterations was more severe in the plates with the higher concentration of 6-p-p. When 1.1 mg of 6-p-p were applied on to antibiotic assay discs the radial growth of R. solani was reduced with respect to the control by 66% at 2 d and 57% at 3 d after inoculation. Inhibition started at a distance of 8 mm between the disc and the colony edge. Ejfecf of 6-pentyz-a-pyrone on the germination of Fusarium spores
Spores of F. oxysporum f. sp. lycopersici did not germinate when incubated in aqueous solutions containing 0.45 mg ml-' of 6-p-p or greater (Table 2). At concentrations of 6-p-p higher than 0.45 mg ml-' all spores appeared unimbibed. At concentrations of 0.45 mg ml-' a small percentage of spores appeared swollen as in the control (Table 2). The percentage of normally swollen spores and of germinated spores increased progressively at lower concentrations of 6-p-p. Spores from the 6-p-p preparation did not germinate when inoculated onto fresh agar medium but when repeatedly rinsed with PDB these spores achieved 100% germination under the same conditions. Table I. Inhibition of growth (%) of target fungi on 2% malt agar containing different concentrations of 6-pentyl-a-pryone Days of incubation 6-pentyl(entries show % a-pyrone inhibition) concentration (mg ml-') 2 5 7 9
Fwarium oxysporum f. sp. lycopersici Rhizoctonia solani
0.2 0.3 0.2 0.3
14.2 11.5 7 3 1.2 31.7 30.0 28.9 19.7 55.5 26.7 0.0 0.0 6 9 6 51.1 22.1 0.0
Table 2. Spore germination (%) and proportion of swollen spores of Fwarium orysporum 6. sp. lycopersici incubated in different concentrations of 6-pentyl-a-pyrone 6-pentyl-a-pyrone concentration % germinated % swollen % germinated spores spores washed spores (mg mlkl)
RESULTS Activity of 6-penfyl-a-pyrone on the growth of iargef fungi
Aqueous solutions of 6-p-p added to the agar medium caused a reduction of the growth rate of R. solani and of Fusarium (Table I).
Control 1.14 0.91 068 045 022 0.11
15.4 0.0 0.0 0.0 0.0 10.2 15.2
100.0 0.0 0.0 0.0 1.25 43.25 94.5
100.0 100.0 100.0 100.0 100.0 100.0 100.0
1209
R. Scarselletti and J. L. Faull DISCUSSION Previous work using the dual culture technique of Dennis & Webster ( 1 9 7 3 ~ )has shown that of three isolates of T. harzianum tested only one significantly reduced hyphal growth of Rhizoctonia solani before contact (Scarselletti, 1992). Furthermore this isolate prevented sclerotium production by R. solani and could overgrow F. oxysporum f. sp. lycopersici. The effect on growth rate could be observed when the two paired fungi were as far as 7 mm apart. This isolate alone was shown to produce 6-pentyl-a-pyrone. Results from the present experiments show that when 6-p-p was applied onto antibiotic assay discs the growth rate of R. solani was similarly reduced and that inhibition started at a distance of 8 mm between the colony margin of the target fungus and the disc. Incorporation of 6-p-p into the agar medium also caused inhibition of sclerotia formation by R. sobni and a lack of pigmentation in Fusarium. Aqueous solutions of 6-p-p also inhibited the germination of Fusarium spores. The inhibition of germination was persistent unless the compound was removed. Results from the spore viability tests suggest that the compound was strongly bound to or into the spores and that, regardless of its volatile nature, its effect could not be eliminated by simply dispersing the spores over a wider, more aerated environment. Although ultrastructural analysis of spores and hyphal tips is necessary, the hydrophobic nature of 6-p-p (log KOct,H20= 2.793) (Scarselletti, 1992) could substantiate the hypothesis of a mechanical impediment to water absorption by the fungal cells due to the formation of a hydrorepellent film on the cell wall. However, phenomena like the inhibition of pigmentation in Fusarium and the inhibition of sclerotia production by Rhizoctonia seem to indicate that 6-p-p interferes with more complex metabolic activities of fungi. The similarities between the activity of T H I and of 6-p-p together with the lack of any of the aforementioned effects by (Accepted 16 March 1994)
isolates of T. harzianum which do not produce 6-p-p, strongly supports a causal relation between 6-p-p and antagonistic ability of T. harzianum in vifro. The Authors would like to thank Dr E. Hedgley for his assistance in the isolation and characterization of 6-pentyl-apyrone.
REFERENCES Claydon, N., Allan, M., Hanson, 1. R. & Avent, A. G. (1987).Antifungal alkyl pyrones of Trichoderma harzianum. Transactions of the British Mycological Society 88, 503-513.
Collins, R. P. & Halim, A. F. (1972). Characterization of the major aroma constituents of the fungus Trichoderma viride Pers. Journal of Agricultural and Food Chemistry 20, 437-438. Dennis, C. & Webster, J. ( 1 9 7 1 ~ )Antagonistic . properties of species-groups of Trichoderma. I. Production of nonvolatile antibiotics. Transactions of the British Mycological Society 57, 26-39.
Dennis, C. & Webster, J. (1971b). Antagonistic properties of species-groups of Trichoderma. 11. Production of volatile antibiotics. Transactions of the British Mycological Sociefy 57, 41-48.
Dennis, C. & Webster, J. (1971c). Antagonistic properties of species-groups of Trichoderma. 111. Hyphal interactions. Transactionsoffhe British Mycological society 5 7, 363-369.
Fuji, K., Fujita, E., Takaishi, Y., Fujita, T., Arita, i., Komatsu, M. & Hiratsuka, N. (1978).New antibiotics, trichopolyns A and B: isolation and biological activity. Experientia 34, 237-239. Godtfredsen, W. 0.& Vagendal, S. (1965).Trichodermin, a new sesquitelpene antibiotic. Acta Chemica Scandinavica 19, 1088-1103. Hou, C. T., Ciegler, A. & Hesseltine, C. W. (1972). New mycotoxin, trichotoxin A, from Trichoderma viride isolated from southern leaf blightinfected corn. Applied Microbiology 23, 183-185. Meyer, C.E. & Reusser, F. (1967).A polypeptide antibacterial agent isolated from Trichoderma viride. Experientia 23, 85-86. Moss, M. O., Jackson, R. M. & Rogers, D. (1975).The characterization of 6(pent-I-enyl)pyrone from Trichoderma viride. Phytochemisty 14,270&2708. Ooka, T., Shimojima, Y., Akinrnoto, T., Takodam, I., Senoh, S. & Abe, J. (1966).A new antibacterial peptide 'suzucacillin'. Agricultural and Biological Chemistry 30, 700-702. Scarselletti, R. (1992).A study on the production of an antifungal compound by Trichoderma harzianum and its effects on Fwarium wilt of tomato. Ph.D. Thesis, University of London.