Mierob Ecol (1990) 19:303-309

MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1990

Hyphal and Mycelial Interactions Between Agaricus bisporus and Scytalidium thermophilum on Agar Media Huub J. M. Op den Camp, ~* Claudius K. Stumm, ~ Gerben St raat sm a) Piet J. L. Derikx,1 and Leo J. L. D. van Griensven 2 tDepartment of Microbiology,Faculty of Science, University of Nijmegen, Toernooiveld, NL6525 ED Nijmegen, The Netherlands; and 2Mushroom ExperimentalStation, Postbus 6042, NL5960 AA Horst, The Netherlands

The interaction between Agaricus bisporus and Scytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy. A. bisporus combatively replaces S. thermophilum in culture on agar media. The antagonistic effect o f A. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration o f S. thermophilum protoplasm, resulting in a total

Abstract.

loss o f viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 m m . d -~ following contact with S. thermophilum mycelinm.

Introduction The edible white button mushroom, Agaricus bisporus (Lange) Imbach, is cultivated on a composted mixture o f horse manure, wheat straw, chicken manure, and gypsum [22]. The composting is necessary to prepare a selective substrate on which the growth o f A. bisporus is pr om ot e d to the practical exclusion o f other microorganisms [9, 19]. During composting a succession o f microorganisms takes place. Towards the end o f the (composting) process thermophilic fungi become abundant, notably Scytalidium thermophilum (Cooney & Emerson) Austwick, Humicola grisea Traanen var. thermoidea Cooney & Emerson, and Humicola insolens Traanen [2, 6, 9, 16, 20], which were considered to be synonymous [20]. Fr om a recent study on the population dynamics o f S. thermophilurn [20] the following could be concluded: (1) S. thermophilum promotes growth o f A. bisporus; (2) the presence o f S. thermophilum is important for successful colonization o f the substrate by A. bisporus mycelium; and (3) precolonization o f the compost by S. thermophilum results in a selective medium. When the compost is fully colonized by A. bisporus, S. thermophilum can no longer be recovered [16, 20], even though S. thermophilum survives and * Corresponding author.

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grows at t h e o p t i m u m g r o w t h t e m p e r a t u r e o f A . bisporus (24~ It is g e n e r a l l y a c c e p t e d t h a t t h e m i c r o b i a l b i o m a s s f o r m e d d u r i n g c o m p o s t i n g h a s a role i n the n u t r i t i o n o f A. bisporus [8, 24]. G r o w t h o f A. bisporus o n h e a t - k i l l e d m y c e l i u m f r o m S. thermophilum ( s y n o n y m o u s t o Humicola grisea var. thermoidea) a n d o t h e r f u n g i as sole C, N , a n d P s o u r c e [5, 7] a n d t h e p r o d u c t i o n o f the a p p r o p r i a t e e x t r a c e l l u l a r d e p o l y m e r i z i n g e n z y m e s h a v e b e e n r e p o r t e d [7, 14]. S i n c e u n t i l n o w o n l y d e a d b i o m a s s h a s b e e n tested, t h e a i m o f t h e p r e s e n t s t u d y was to e x a m i n e t h e i n t e r a c t i o n b e t w e e n l i v i n g A. bisporus a n d S. thermophilum o n agar w h e r e t h e h y p h a e c o u l d r e a d i l y b e e x a m i n e d m i c r o s c o p i c a l l y .

Methods

Organisms and Media Agaricus bisporus strain Horst | U1 and Scytalidium thermophilum CBS 671.88, isolated from compost [20], were used throughout. The fungi were cultivated on yeast/glucose agar [20], malt/ peptone agar [30 g malt extract, 5 g mycological peptone, and 12 g agar per liter demineralized water], or on a compost extract/glucose agar (CEA) [21], at 24"C in the dark. When necessary, S. thermophilum was pregrown at 45~

Interactions between S. t h e r m o p h i l u m and A. b i s p o r u s The fungi were inoculated opposite to each other, 5 or 10 cm apart, on solid media in Petri dishes with diameters of 8.5 and 14 cm, respectively. Agar blocks (3 • 3 x 3 mm) taken from the mycelial growth front of the fungi pregrown on the same solid media were used as inocula. The bigger dishes were used to follow growth during a longer period of time. Growth was measured as mm radial extension. For microscopical examination, fiat blocks of agar were cut from the region of the mycelial growth front and examined by means of Leitz differential interference contrast (DIC) and phase contrast. In order to preserve both the original position of the hyphae and the hyphal content, care was taken to prevent squashing of the agar. Experiments on all media were performed at least in triplicate and repeated three times. In another experiment, S. thermophilum was pregrown at 45"C on yeast/glucose agar, while A. bisporus was cultivated at 24~ on a cellophane sheet lying on malt/peptone agar. A. bisporuswas not able to penetrate the cellophane. When the mycelia had reached a diameter of about 5 cm, the cellophane with A. bisporuswas peeled offthe agar and layered onto the S. thermophilummycelium. Incubation was continued at 24"C. In a control experiment the mycelium of S. thermophilum was covered with an autoclaved cellophane sheet without A. bisporus.After one day the S. thermophilurn hyphae were checked microscopically and viability was tested by transferring agar blocks from the growth front into liquid agar medium at 45"C which was then allowed to solidify. The latter procedure allowed outgrowth ofhyphae after incubation to be detected easily, since light refraction at the edges of the blocks was annihilated. The cellophane experiments were repeated in triplicate.

Results G r o w t h o f A . bisporus a n d S. thermophilum o p p o s e d o n c o m p o s t / g l u c o s e agar is s h o w n i n Fig. 1. P r i o r to c o n t a c t , t h e l i n e a r g r o w t h rate o f A. bisporus a n d S. thermophilum was 2.7 + 0.4 a n d 3.2 + 0.1 m m . d -1, r e s p e c t i v e l y (n = 5).

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Fig. 1. Growth ofA. bisporus (e) and S. thermophilum (ll) during an interaction experiment in a large Petri dish with compost/glucose agar. The arrow indicates the point where mycelia o f both fungi make contact. Growth o f A. bisporus in the absence o f S. thermophilum is plotted as a control (0).

Time (d)

When the growth fronts reached each other, extension of S. thermophilum stopped, while that of A. bisporus continued with the same rate, resulting in overgrowth of the S. thermophilum mycelium (Fig. 2). About 5 days after contact an acceleration of the growth rate ofA. bisporus along the radius line between the two inocula points was observed (Fig. 2), resulting in a linear growth rate (after 25 d) of 5.3 ___ 0.1 m m . d -t. In a control experiment A. bisporus grew at a rate of 2.7 + 0.5 m m . d - ' for up to 30 d and thereafter decreased slightly. On the other agar media tested no growth stimulation was observed, although S. thermophilum was inhibited and covered by the A. bisporus mycelium. At their optimal temperatures (24~ and 45~ for A. bisporus and S. thermophilum, respectively), the hyphal morphology of both fungi grown singly was similar (Figs. 3a and 3b). When S. thermophilum was grown at 240C septation frequently increased and intercalary spore formation of the distal regions of the hyphae was observed (Fig. 3c). The growth rate was lower than at 45~ but remained constant (Fig. 1). When the fungi were opposed to each other, growth of S. thermophilum stopped when the distance between the growth fronts had decreased to about 1 ram. Microscopical examination showed that the content of the S. thermophilure hyphae was coagulated and shrunk (Fig. 3d). After prolonged incubation (about 4 days) the A. bisporus hyphae penetrated into the disintegrated S. thermophilum mycelium (Figs. 3e and 3f). In the cellophane overlay experiment, after one day of incubation, hyphae from control mycelium contained dense and rather homogeneous cytoplasm (Fig. 3h), while those covered with the A. bisporus overlay had a highly vacuolated, foam-like content (Fig. 3g). In phase contrast the cytoplasm appeared to be brighter compared to the control hyphae, probably due to loss of solutes and/or uptake of water, also indicated by the slight swelling of the affected hyphae. In viability tests of agar blocks from the growth front of S. thermophilum rnycelium covered by cellophane sheet for one day, the blocks from the control had a fur-like covering of growing hyphae after 2 days, whereas only single tiny rnycelia developed from blocks cut from under A. bisporus. This indicated that

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Fig. 2. A. bisporus (white mycelium) and S. thermophilum growing together on compost/glucose agar. The photograph was taken after 15 d of growth. A mycelial sector ofA. bisporus penetrates and overlayers the S. thermophilum mycelium. t h e m a j o r i t y o f t h e S. thermophilum h y p h a e h a d b e e n k i l l e d . A f t e r 7 d a y s n o v i a b l e S. thermophilurn c o u l d b e s u b c u l t u r e d i n d i c a t i n g c o m p l e t e k i l l i n g o f S. thermophilum. S i m i l a r a n t a g o n i s t i c effects o f A . bisporus w e r e o b s e r v e d a g a i n s t s o m e o t h e r t h e r m o p h i l i c fungi i s o l a t e d f r o m c o m p o s t , e.g., Thermomyces lanuginosus T s i k l i n s k y , Rhizomucor miehei ( C o o n e y & E m e r s o n ) S c h i p p e r , a n d Talaromyces thermophilus S t o l k ( d a t a n o t s h o w n ) .

Discussion T h e r e s u l t s p r e s e n t e d i n t h i s r e p o r t c o r r e s p o n d well w i t h t h o s e o b t a i n e d s t u d y ing t h e i n t e r a c t i o n o f A. bisporus a n d S. thermophilum o n c o m p o s t s u b s t r a t e [20]. O n a g a r m e d i u m A. bisporus m y c e l i u m e n c r o a c h e d i n t o t h e S. therrnophilure m y c e l i u m , t o t a l l y t a k i n g o v e r its d o m a i n . A f t e r t h i s " r e p l a c e m e n t " , v i a b i l i t y o f S. thermophilum w a s s t r o n g l y r e d u c e d .

Fig. 3. Microscopical observations on the interaction of S. thermophilum and A. bisporus. Hyphae from the growth front of S. thermophilurn (a) and A. bisporus (b), singly grown at 45"C and 24"C, respectively. Differential interference contrast (DIC), bar = 25 t~m. (c) Hyphae from the growth front of S. thermophilum, singly grown at 24"C. Note septation and intercalary spore formation. DIC, bar = 25 gin. (d) S. thermophilum hyphae from a mycelium about 1 mm distant from an A. bisporusgrowth front. The cytoplasm is shrunk and coagulated. DIC, bar = 25 ~m. (e) A. bisporus hyphae (growing from the right to the left) penetrating S. thermophilum mycelium; not only the hyphal tips but also distal regions of the latter fungus are altered; (f) stronger magnification showing the difference between the light refractive, living hyphae ofA. bisporusand the coagulated cell content ofS. thermophilum. DIC, bar = 50 and 25 #m, respectively. (g, h) S. thermophilurn hyphae from an overlay experiment. Whereas in the control hyphae (h) the thread-like mitochondria are visible, in the affected hyphae the cytoplasm is highly disintegrated (g). Phase contrast, bar = 10 #m.

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Some classical literature [10, 25] already describes the b e h a v i o r o f a s c o m y c e t e and basidiomycete fungi in m i x e d cultures. M o r e recent studies have focused on interactions during successions o f fungi i n v o l v e d in degradation o f dung [ 13, 23] and w o o d [4, 11 ]. In an overview, R a y n e r and W e b b e r [ 17] proposed a scheme to describe fungal interactions. Competitive, neutralistic and mutualistic interactions were distinguished. O n basis o f the definitions o f these authors A. bisporus should be classified as a c o m b a t i v e c o m p e t i t o r o f S. thermophilum leading to replacement o f the latter (secondary resource capture). The antagonistic effect can be mediated at a distance. H y p h a l interference [ 11, 12] and mycoparasitism [1 ] were not observed. Although some preliminary results exist on the nature (diffusible a n d / o r volatile) o f the c o m p o u n d s i n v o l v e d in the antagonistic effects o f A. bisporus [15], detailed studies were not performed. Previously [3] we reported on the presence o f an alternative respiratory pathway i n A. bisporus, while S. thermophilum lacked this pathway. T h e p r o d u c t i o n o f CO, an inhibitor o f the n o r m a l respiratory pathway, by A. bisporus, however, c a n n o t account for the killing p h e n o m e n o n observed in this study since it is a reversible inhibition. The growth p r o m o t i n g effect o f S. thermophilum on A. bisporus was demonstrated only on compost/glucose agar. On other agar media S. thermophilum was inhibited by A. bisporus but no growth stimulation o f the latter was found. Renard and Cailleux [18] also did not find growth stimulation on malt agar. Use o f compost/glucose agar could be helpful in elucidating the m e c h a n i s m o f growth p r o m o t i o n . Other thermophilic fungi were also reported to cause enhanced growth o f A. bisporus, and S. thermophilum enhanced growth o f different species o f basidiomycete m u s h r o o m s [20] in compost. However, interactions were not studied in detail. On a potato agar plate, growth o f Pleurotus sapidus was accelerated as soon as Aspergillus glaucus or Aspergillus sydowi colonies were reached [25]. There might be a correlation between m e d i u m c o m p o s i t i o n and product form a t i o n by, or c o m p o s i t i o n of, growth p r o m o t i n g fungi. In the final stage o f composting, t h e r m o p h i l i c fungi colonize the substrate to a high density resulting in a selective substrate. A. bisporus, the next organism in the succession, has an ecological advantage in being c o m p e t i t i v e / c o m b a t i v e , and f u r t h e r m o r e this fungus is able to use the dead biomass as nutrient source [5, 7, 24].

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