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Applied and Environmental Microbiology, December 1998, p. 5053-5056, Vol. 64, No. 12
Department of Applied Plant Science,
Received 19 February 1998/Accepted 3 September 1998
Trichoderma harzianum biotypes Th1, Th2, and Th3
produced volatile metabolites in vitro which had similar fungistatic
effects on the growth of Agaricus bisporus. Metabolites
present in agar colonized by these strains also inhibited mycelial
growth of A. bisporus, although the reduction in growth was
less in the presence of metabolites produced by biotype Th2 than
that in the presence of metabolites produced by Th1 or Th3.
A. bisporus produced metabolites in liquid culture
that inhibited the growth of Th1 and Th3 but stimulated the growth of
Th2. A compound(s) responsible for the inhibition and stimulation was
extracted from A. bisporus culture filtrate and from
compost-grown fruit bodies with n-butanol, but the identity
of the compound(s) was not determined. We suggest that the stimulation
of Th2 by metabolites produced by A. bisporus and the
relatively low level of inhibition of A. bisporus by
Th2 facilitate colonization of compost by both fungi. However,
as compost colonization reaches a maximum, a change in the
competitive balance in favor of Th2 results in the inhibition of
fruit body production by A. bisporus and the
devastating green mold epidemics affecting mushroom production.
One of three biological forms of
Trichoderma harzianum Rifai, group 2 described by
Muthumeenakshi et al. (12) and designated biotype Th2 in
this paper, is the main agent responsible for green mold epidemics
affecting the commercial mushroom (Agaricus bisporus) in the
British Isles (13, 17). Two other forms of T. harzianum, biotypes Th1 and Th3, also have been identified but are
excluded during compost pasteurization (13). A fourth
T. harzianum biotype, which is similar to but
genetically distinct from biotype Th2 and was designated group 4 by
Muthumeenakshi et al. (11), has caused serious compost
infestation in North America (15).
Of the three T. harzianum biotypes found in the British
Isles, biotype Th2, although rarely isolated from compost raw
materials, once introduced, often on infected spawn, colonizes compost
effectively (13, 14). During severe green mold epidemics,
when no mushrooms are produced, T. harzianum Th2
predominates in affected compost. In contrast, biotypes Th1 and Th3,
which are commonly found in compost raw materials but are infrequently
found in pasteurized or colonized compost, rarely cause problems during
mushroom production. If introduced, however, Th1 and Th3 will colonize
a restricted area of compost from which A. bisporus is
excluded (13). The relative abilities of the three
biotypes of T. harzianum to colonize compost in
competition with A. bisporus and their influence on A. bisporus growth may be associated with secondary
metabolite production. The present study was made to determine, in
vitro, the biological activity of compound(s) produced by T. harzianum and A. bisporus, separately, and to
determine their possible relationship to green mold disease.
Fungal cultures.
Three strains of A. bisporus
(commercial spawn strains 130, 229, and 280), all U3 types, and three
isolates each of T. harzianum biotypes Th1, Th2, and
Th3 were used in the study. One isolate each of Th1 (IMI 359823), Th2
(IMI 359824), and Th3 (IMI 359825) were deposited in the International
Mycological Institute (IMI) culture collection; the other two isolates
of each biotype were identical at the molecular level (10)
to those deposited. Subsequent to the completion of our study, Th3 was
confirmed to be Trichoderma atroviride (11).
These Trichoderma isolates have been tested for their
aggressiveness in compost trials (16), and the Th2 isolates
continue to cause green mold disease.
Data analysis.
Data were subjected to analysis of variance;
arc sine transformation was employed for percentage data. Least
significant difference (LSD) values are calculated from the standard
errors of the means (SEM) × Volatile T. harzianum metabolites are toxic to
A. bisporus.
Trichoderma spp. produce both
volatile and nonvolatile metabolites that adversely affect growth of
different fungi (1, 3, 5, 6, 9). To determine the effects of
volatile metabolites released by the three T. harzianum
biotypes on the growth of A. bisporus, discs (4-mm
diameter) excised (with a no. 2 cork borer) from the leading edge of a
12-day-old colony of each of the three strains of A. bisporus were placed at the center of 2% malt extract agar (MEA)
in petri dishes (90-mm diameter) and incubated for 10 days prior to the
interaction study. Discs from three isolates each of T. harzianum biotypes Th1, Th2, and Th3, excised from the leading
edge of cultures grown on potato dextrose agar (Oxoid, Basingstoke,
United Kingdom), were placed on 2% MEA immediately before the
interaction took place. The petri dish lids were removed, and a plate
containing A. bisporus was placed over a plate
containing T. harzianum. The two plates were held
together with adhesive tape. For each strain of A. bisporus, three replicate plates for each T. harzianum isolate and two sets of controls were used. One control
set comprised A. bisporus cultures paired with plates
of uninoculated MEA, and the second set comprised A. bisporus paired with Rhizoctonia solani which was
inoculated and grown on 2% MEA in the same manner as T. harzianum. R. solani was included as a control
since Hutchinson and Cowan (7) found that growth of
Aspergillus niger, Pestalotia rhododendri, and several bacteria in the headspace gases from cultures of a strain of
T. harzianum could be accounted for solely by the
presence of CO2 and ethanol produced by the
T. harzianum cultures. R. solani has a growth rate similar to T. harzianum and
presumably releases similar levels of CO2. All treatment
and control plates were randomized and incubated at 25°C in the dark.
Diffusible T. harzianum metabolites are toxic to
A. bisporus.
To assess the toxic effects on
A. bisporus of substances released by the T. harzianum strains in the agar, we overlaid 2% MEA with a
sterilized cellophane membrane (Courtauld Films; 50-µm thick).
The plates were left overnight to allow excess water to evaporate. A
disc (4-mm diameter) excised (with a no. 2 cork borer) from the leading
edge of an actively growing T. harzianum culture was
placed on the cellophane at the center of each agar plate and incubated
at 25°C in darkness for 2 days. The cellophane and adhering fungus
were removed, and a disc (4-mm diameter) of A. bisporus, excised from the leading edge of a 12-day-old culture, was placed at the center of the plate. The plates were randomized and
incubated at 25°C for 12 days, and the diameters of the A. bisporus cultures were measured. Three replicate plates were
established for each treatment, and the experiment was repeated twice.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Metabolites Produced by Trichoderma
harzianum Biotypes and Agaricus bisporus on Their
Respective Growth Radii in Culture
ABSTRACT
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TABLE 1.
Growth of A. bisporus strains in the
presence of volatiles released by T. harzianum biotypes
Th1, Th2, and Th3
TABLE 2.
Growth of A. bisporus strains 12 days
after inoculation onto agar containing diffusible substances produced
by T. harzianum biotypes Th1, Th2 and Th3
Metabolites produced by A. bisporus. The ability of the slow-growing mycelium of A. bisporus to colonize and dominate a nonsterile mushroom compost has prompted speculation that it may produce antibiotic(s). Certain metabolites of A. bisporus have been isolated, and antibiotic potential has been attributed to their derivative or analogues (2). We assessed the effect of a metabolite(s) produced by A. bisporus in fruit bodies and in liquid culture on the growth of T. harzianum.
A. bisporus mycelia from 7-day-old stationary liquid cultures (each, 25 ml) in yeast extract-glucose medium (YEG medium) (containing 0.1 g of yeast extract, 10 g of glucose, 0.14 g of sodium glutamate, 0.2 g of potassium chloride, 0.2 g of magnesium sulfate, and 0.2 g of calcium chloride per 1 liter of distilled water) were transferred to 100 ml of fresh YEG medium in 250-ml conical flasks and incubated in an orbital incubator (40 rpm, 25°C). Culture filtrate (100 ml) was removed from different flasks each day (on days 9, 12, 15, 18, and 21) and filter sterilized (0.2-µm-pore-size filters), and 20 ml from each of three replicate flasks was stored at
20°C until used. The effect of the culture
filtrates on the growth of T. harzianum was assessed by
cutting a well (6-mm diameter) with a no. 3 cork borer at the center of
2% MEA (25 ml) in petri dishes (90-mm diameter). A. bisporus culture filtrate (150 µl) was pipetted into each
well and immediately covered by a disc (8-mm diameter, mycelium-side
down) cut with a no. 4 cork borer from the leading edge of an actively
growing T. harzianum colony. Three replicate plates
were established for each treatment. The plates were then placed in an
incubator at 25°C. Filter-sterilized, uninoculated medium (150 µl)
was used in control plates. Growth radii were measured twice at right
angles, 24 h after inoculation. The experiment was repeated twice,
and all treated plates were compared with the controls to calculate the
percentage stimulation or inhibition of growth of T. harzianum.
Filtrates of 9-day-old cultures of A. bisporus
suppressed the growth of T. harzianum biotypes Th1 and
Th3 by 6% and 5%, respectively, but stimulated the growth of biotype
Th2 by ca. 7%. These results varied little with the strain or age of
the culture (data not shown).
A water-soluble component(s) was extracted with n-butanol
from the culture filtrate and from A. bisporus fruit
bodies as follows. A. bisporus (strain 280) was grown
in YEG medium as described above for 12 days. Pooled culture filtrate
(200 ml) was clarified by vacuum filtration through Whatman no. 3 paper
and extracted three times with 200 ml of n-butanol. The
n-butanol extracts (600 ml) were pooled, dried over
anhydrous sodium sulfate, and evaporated in vacuo almost to dryness.
Three replicate sets of culture filtrates were extracted in this
manner. Mushroom caps (100 g), which had just begun to open, were
homogenized (Waring blender) in 200 ml of methanol. The methanol
extract was clarified by filtration through Whatman no. 3 paper, and
the methanol was vacuum evaporated. The aqueous residue was extracted
three times with 50 ml of n-butanol each time. The pooled
n-butanol extract was dried over anhydrous sodium sulfate
and then vacuum evaporated to dryness.
To determine the biological activity of the n-butanol
extracts, the residues were dissolved, each in 10 ml of sterile
distilled water, and from these solutions a 1:5 dilution series was
made to give 2 × 10
1, 4 × 102,
8 × 103, 1.6 × 103, 3.2 × 104, 6.4 × 105, and 1.3 × 105 dilutions of the extracts. Aqueous dilutions of
the n-butanol extracts were pipetted into wells cut in agar
plates and bioassayed with isolates of Th1, Th2, and Th3. Sterile
distilled water was used in control plates. Three replicate plates were
used for each treatment; the plates were placed in an incubator at
25°C in a randomized manner. The experiment was repeated three times,
and all treated plates were compared with the controls to calculate the
percentage of inhibition or stimulation of the T. harzianum biotypes.
The more-concentrated solutions of the extracts from both the culture
filtrates and the fruit bodies suppressed growth of all three
T. harzianum biotypes. As the extracts were diluted, the growth of Th1 and Th3 continued to be suppressed while that of Th2
was enhanced (Table 3). No investigations
have been made to determine the chemical composition of the
n-butanol extract or the properties of the components.
However, as n-butanol will dissolve a number of
water-soluble compounds from biological material, the extract is
unlikely to have been homogeneous. We do not know, therefore, if the
same compound(s) inhibited Th1 and Th3 as stimulated Th2. The fact that
the active compound(s) was extracted from the fruit bodies does,
however, suggest that it is a constituent component of the fungus.
Phenylhydrazines (4, 8) and phenolic compounds (18) with antibiotic activity have been isolated from fruit bodies of A. bisporus, but their extraction or
synthesis for bioassay against T. harzianum was beyond
the scope of this study.
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ACKNOWLEDGMENTS |
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We thank Sally Watson for statistical analysis.
A. Mumpuni thanks the British Council for financial support. This study was partially supported by the International Fund for Ireland through the Centre for Innovation in Biotechnology.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Applied Plant Science, The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, United Kingdom. Phone: 44 1232 255245. Fax: 44 1232 668375. E-mail: s.sharma{at}qub.ac.uk.
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Applied and Environmental Microbiology, December 1998, p. 5053-5056, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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