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Actinomycetes as antagonists of litter decomposer fungi
applied soil ecology 38 (2008) 109–118
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Actinomycetes as antagonists of litter decomposer fungi B.A.T. Dinishi Jayasinghe *, Dennis Parkinson Department of Biological Sciences, University of Calgary, 2500 University Drive, NW, Calgary, Alberta, Canada T2N1N4
article info
abstract
Article history:
The actinomycete communities of North American forests and impacts of their activities on
Accepted 21 September 2007
decomposer fungi have not been thoroughly investigated. In the current study, the community size and species richness of the forest floor actinomycete communities in cool
Keywords:
temperate aspen poplar and lodgepole pine forests of Alberta, Canada, was investigated.
Actinomycetes
Based on the isolations conducted on chitin agar, both aspen and lodgepole forests had
Decomposer fungi
relatively large actinomycete communities (104–105 CFU g1 soil). One hundred and fifty-six
Forest floor
different actinomycete strains were isolated and the genus Streptomyces predominated in
Antagonism
both forests. The community size as well as the number of different actinomycete strains
Colonization
was higher in aspen forests than in pine forests, but the exact reasons for these differences
Organic material
are not clear at this stage. It may be that favorable conditions created by endogeic earthworms (e.g. burrowing and mixing of organic material, dispersal of actinomycetes) increase the density and diversity of actinomycetes in aspen litter. It is also possible that pine litter anti-microbial compounds, waxy material and phytotoxic products of pine litter decomposition may have negative affects on actinomycetes in pine forests. Both in vitro and microcosm studies showed that the frequently isolated actinomycetes could act as antagonists for some common leaf litter and wood decomposer fungi. Of the litter decomposer fungi, fast growing genera (e.g. Mucor, Penicillium, Trichoderma) were more tolerant of actinomycete antagonism than slow and moderately slow-growing genera (e.g. Cladosporium, Mortierella). The colonization of organic substrates by some actinomycetes did reduce the degree of subsequent colonization by susceptible decomposer fungi. # 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Ecological studies of actinomycetes in soils of been conducted in various habitats including grasslands (Lee and Hwang, 2002), beach sands (Suzuki et al., 1994), underground caves (Groth et al., 1999), rice-paddies (Hayakawa et al., 1988), orchards (Lee and Hwang, 2002) and sub-glacial ice of Antarctica (Priscu et al., 1999). According to these studies, actinomycetes constitute a significant component of the microbial population in most soils and in some soils they are more numerous than all other bacteria. Some actinomycetes have been shown to play an important part in recycling of complex organic materials such as lignocelluloses (Craw-
ford, 1978). Therefore, actinomycetes are important members of forest floor decomposer community. However, there have been few studies of actinomycete communities in forest soils (a cool temperate pine forest in Lancashire (Davies and Williams, 1970), a tropical rain forest in Singapore (Wang et al., 1999), mountain forests of Japan (Hayakawa et al., 1988), a pine forest in north east India (Das and Mishra, 1989), a sclerophyllous forest in New South Wales of Australia (Gerrettson-Cornell and Kelly, 1981) and a forest formed on a worked-out peat bog in Russia (Zvyaginstev et al., 1996)). These studies showed that factors controlling the density and diversity of actinomycetes in the forest soil have been suggested to be the relative moisture content, pH, nature
* Corresponding author. Tel.: +1 403 220 5948; fax: +1 403 289 9311. E-mail address: [email protected] (B.A.T. Dinishi Jayasinghe). 0929-1393/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2007.09.005
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and abundance of the organic matter, abundance of mesofauna and above ground vegetation. In the present study, the nature of the forest floor actinomycete communities in relation to the abundance of organic matter, pH, moisture content was investigated in North American cool temperate aspen poplar and lodgepole pine forests. A number of actinomycetes, however, may be acting as antagonists of soil fungi. Therefore, potential effects of actinomycetes on the litter decomposer fungi were also investigated. According to the results, compared to the lodgepole pine forests, the density as well as the diversity of actinomycetes was highest in aspen forests but the Streptomycetes were predominant in both forests. Microcosms studies showed that the frequently isolated actinomycetes could act as the antagonists for the common litter and wood decomposer fungi. The antagonistic impacts of actinomycete species on pathogenic fungi is well known and a few species have been used as biological control agents (Yuan and Crawford, 1995; ElShanshoury et al., 1996; Jones and Samac, 1996; El-Tarablily et al., 1997; Berg et al., 2000; Getha and Vikineswary, 2002; Bressan, 2003). While there are examples of actinomycetes antagonistic to VAm mycorrhizal fungi (Krishna et al., 1982) and ectomycorrhizal fungi (Keast and Tonkin, 1983; Fridman et al., 1989; Richter et al., 1989), there are very few data on such impacts of actinomycetes on decomposer fungi (e.g. lysis of cell walls of Mucor ramammianus by Streptomyces spp. (Jones et al., 1968) and inhibition of southern pine sap-wood decomposers (Lenzites saepiaria, Polyporus versicolor and Lentinus lepideus) by an isolate of Streptomyces sp. (De Groot, 1971)). Since saprophytic fungi are important, members of the forest decomposer community in temperate forests ecosystems, any antagonistic impact by actinomycete species could have marked effects on organic matter decomposition rates and fungal community structure in the forest floor. As the distributions of actinomycetes in North American forests remain largely undescribed, the first step of the present study was to determine the nature of two forest floor actinomycete communities, which would allow selection of frequently isolated actinomycete strains for further in vitro and in vivo studies of their potential antifungal antagonism. The objectives of the current study were: (1) To determine the community size and, composition of actinomycetes at different depths of organic layers in lodgepole pine and aspen poplar forests. (2) To determine the ability of frequently isolated actinomycetes to inhibit the growth of common decomposer fungi. (3) To determine the impact of actinomycetes on colonization of organic matter by decomposer fungi.
2.
Materials and methods
2.1.
Study site
continental with long winters and hot summers. Even though the soil usually remains frozen in the winter (from November to March) Chinook winds may frequently thaw the upper soil layers (Coxson and Parkinson, 1987). The mean annual maximum and minimum temperatures for the duration of the present study (2001 July–June to 2003 June–July) were 10.2 8C and 2.5 8C, respectively, and the mean annual precipitation was 607.7 mm. These forests were reported free of earthworms until the late 1980s (Dymond et al., 1997) when the epigeic earthworm, Dendrobaena octaedra was found. More recently (about 5 years ago), several species of deep- to middepth burrowing earthworms, including Lumbricus terrestris, Aporrectodea caliginosa, and Octolasion tyrtaeum were also found colonizing aspen forests in this area (Migge, 2001). The organic layers of pine forests can be distinguished into L (litter), F (fermentation) and H (humus) materials. However, the F and H materials could not be separated easily, presumably as a result of the feeding and mixing actions of D. octaedra, and binding of organic particles together by their casts. In most parts of the aspen poplar forests, the organic layers and the upper mineral layer were completely mixed by burrowing earthworms (L. terrestris).
2.2.
In summer 2003, five soil cores (5.5 cm diameter, 19.5 cm depth) were taken at random from each of five discrete stands (30 m 30 m) of both pine and aspen forests. Each soil core was separated into three layers (sub-samples) according to the depth (0–4 cm, 4–8 cm, 8–12 cm). Soil sub-samples were sieved (2 mm mesh) and during sieving, woody debris, live plants and earthworms were removed. Actinomycetes were isolated from samples (three replicates) using dilution plate method (diluted to 105) with spread plates using chitin agar supplemented with 50 mm mL1 nystatin (Wellington and Toth, 1994). The plates were incubated at room temperature (22–24 8C) for 14–21 days, and actinomycete colonies were recognized on the basis of morphological characteristics. Total actinomycete colony counts, presence/absence of each morphological form, and the 10 most common strains from each forest were recorded. Actinomycete colonies with different morphologies were transferred to Czapek-Dox agar slants (Sigma1) and were stored at 4–6 8C for further studies. Actinomycetes were identified to genus levels according to the methods suggested in Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994) and Bergey’s Manual of Systematic Bacteriology—volume 4 (Williams et al., 1989). Genus identification results of some of the 20 most frequently isolated actinomycete strains were confirmed by the similarity index in the Sherlock Microbial Identification system, which is based on the fatty acid composition of actinomycetes (Microbial ID Inc., Newark, DE).
2.3. The study was conducted in aspen (Populus tremuloides L.) and lodgepole pine (Pinus contorta Loud.) forests in the lower subalpine zone (Williams, 1990) of the Canadian Rockies of Southwest Alberta (518010 N/1158010 W). All the study sites were located on well-drained eastern mountain slopes of the Kananaskis Valley at about 1350 m elevation. The soil is classified as Orthic Eutric Brunisol and the climate is
Isolation and identification of actinomycetes
In vitro antagonism tests
The 20 most commonly isolated actinomycete strains were tested in vitro using the dual culture method (modified procedure of Fuhrmann, 1994) for antagonism toward 16 litter-decomposing fungi isolated from the same study area. It was assumed that the most commonly isolated actinomycetes and frequently occurring decomposer fungal species from the
applied soil ecology 38 (2008) 109–118
same forest soils may frequently interact with each other in the soil environment. Therefore, impacts of dominant actinomycete isolates on decomposer fungi will be greater than the impacts of rare or less frequently isolated actinomycete isolates. The test fungi are common inhabitants of upper layers of both aspen and pine forests (Widden and Parkinson, 1973; Visser and Parkinson, 1975). Litter decomposer fungi: Chrysosporium pannorum, Cladosporium cladosporioides, Cladosporium herbarum, Mortierella alpina, Mortierella ramanniana var. angulispora, Mortierella ramanniana var. ramanniana, Mortierella vinacea, Mucor hiemalis var. 1, Mucor hiemalis var. 2, Mucor racemosus, Paecilomyces carneus, Penicillium chrysogenum, Penicillium montanense, Trichoderma polysporum, Trichoderma sp. 2, Zygorrhynchus moelleri (from a previously prepared culture collection by Dr. Beryl Zaitlin and identification of these fungi was confirmed using Compendium of Soil Fungi (Domsch et al., 1980)) and The genus Penicillium (Pitt, 1979). Wood decomposer fungi: Cystoderma sp., Phyllotopsis nidulans and Pholiota squarrosa (from a previously prepared and identified culture collection by Dr. S. Visser). Test media for the dual culture method were selected based on the growth performances of both test actinomycetes and fungi. Media for litter decomposer fungi were V8 agar (V8A), potato dextrose agar (PDA), malt extract agar (MEA), and modified medium formulated by mixing (Czapek-Dox) CZA and PDA (1:1). Media for wood decomposer fungi were oatmeal agar (OA) and PDA. Size of the culture plates was determined based on the growth rates of fungi on each test medium (90 mm diameter plates for fast growing and 45 mm for slow growing). Plates were inoculated with actinomycetes 24 h or 48 h prior to the fungal inoculation. Spores of 4 of the 20 actinomycetes were uniformly spotted equidistantly near the periphery of the each plate and when these strains were visibly growing, 5-mm diameter agar plugs taken from a growing edge of a 5-day-old test fungal colony was transferred to the centre of the test agar plate surface (3 replicates). Cultures were incubated at room temperature (22–24 8C) and when growing edges of control fungi (without any actinomycete inoculum) were at the edge of the plates, the diameters of the test fungal colonies toward each actinomycete were measured. Actinomycete antagonism against the fungi was evaluated using percentage inhibition values (I) on each medium. I¼
radius of control fungus radius of test fungus 100 radius of control fungus
In the presence of a very highly antagonistic actinomycete strain, growth of the test fungal colony was completely or highly inhibited regardless of the direction of the actinomycete growth. As this restricted the determination of antagonistic activity of the other three actinomycete strains, in such cases the experiment was repeated with the plates with a single individual actinomycete colony of the highly antagonistic strains.
2.4. Studies of nature of the interactions between fungi and actinomycetes The morphology of fungal mycelia adjacent to the actinomycete colonies was examined under both a dissecting micro-
111
scope (10) and a compound microscope (400) for abnormal growth patterns and to ascertain whether or not there were direct contacts between actinomycete and fungal colonies. The response of spores of five susceptible fungi (M. ramanniana var. angulispora, M. ramanniana var. ramanniana, M. vinacea, P. montanense, Trichoderma sp. 2) and five resistant fungi (M. alpina, M. hiemalis var. 1, M. hiemalis var. 2, M. racemosus, P. chrysogenum) (based on the in vitro tests) to the actinomycete antifungal compounds was evaluated as the ability of their fresh spores to form viable colonies on agar blocks (5 mm 5 mm) cut from clear zones formed in dual culture plates with highly antagonistic actinomycetes (Str108-52 and Str-330-9) after 5 days of incubation. To determine whether diffusible antifungal compounds are produced even in the absence of susceptible fungi, the same procedure was repeated using agar blocks cut from area surrounding the actinomycete colonies in pure culture plates. The response of both susceptible and resistant fungal hyphae to highly antagonistic actinomycetes was evaluated as ability of their mycelial plugs cut from colonies adjacent to actinomycete colonies to produce viable colonies on the fresh agar plates with the same medium.
2.5. Ability of fungi to colonize substrates with highly and weakly antagonistic actinomycetes Soil plates were prepared using 30 g of sieved (<4 mm) field moist soil taken from the 0–4 cm depth of the aspen forests in the Kananaskis valley. Two highly antagonistic actinomycetes (Str-108-52 and Str-330-9) and two weakly antagonistic actinomycetes (Str-266-90 and Str-73-44) strains were selected as the test actinomycetes on the basis of the in vitro tests. For each actinomycete strain, two 9-cm Petri dishes with 25 g of three times autoclaved bran (bran:distilled water (w/v) = 5:2) were inoculated with 1 mL of actinomycete spore suspension and mixed well with a sterile spatula. The covered Petri dishes were kept at room temperature (22–24 8C) and were shaken occasionally to ensure the uniformity of colonization. Two, 5, 10 and 14 days after the inoculation of bran with actinomycetes, 25 bran flakes with each different actinomycetes and sterile control bran were added to previously prepared Petri dishes with natural soil (three replicates). Plates were incubated at room temperature for 7 days. Then five bran flakes were removed carefully from each soil plate, washed three times (sterile distilled water) and plated on fresh MEA. Plates were incubated for 7 days and fungi growing were identified. Percentage of bran particles with each different fungi was calculated.
2.6. Impacts of colonization of organic soil by antagonistic actinomycetes on subsequent colonization by the decomposer fungi Five milliliters of a spore suspension (density 106– 108 spores mL1) of one of the most antagonistic actinomycete strain Str-108-52 (based on in vitro tests) was added to each of ten 25 g samples of sterile soil and mixed with a sterile spatula. Each soil–spore mixture was spread into each of 10 sterile glass Petri dishes and incubated at room temperature (22–24 8C) for 2 weeks or until the appearance of whitish actinomycete
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colonies. Sterile distilled water (0.5 mL) was added to the soil every 4 days and mixed. The final actinomycete population number was determined for each of the soil replicates using the dilution plate and the spread plate method. Moisture contents of sterile soil and soil colonized with actinomycetes were adjusted to the moisture level of natural soil. An empty 9.5-cm diameter sterile Petri dish lid was placed in the middle of a 15-cm diameter sterile glass Petri dish and the uncovered area was filled with 20 g of natural soil. The 9.5cm diameter lid was carefully removed and the uncovered area was filled with 10 g soil colonized with the actinomycetes. The boundary between natural soil and soil colonized with actinomycetes was marked with a 9.5-cm diameter sterile metal ring. Control plates were prepared using sterile soil in centre instead of soil colonized with the actinomycetes. To determine the fungal growth into sterile soil and soil colonized with actinomycetes, 18 plates were prepared and on each sampling day (day 0, day 5, day 10, day15, day 20 and day 25 after set up) 3 replicates of 1 g of soil and 10 soil particles (mainly organic particles) were taken from three predetermined distances from the boundary between 2 soil types (between boundary and 5 mm away from the boundary, 7 mm and 15 mm away from the boundary). The types of different fungal species at each sampling point were determined by incubating 10 soil particles taken from each sampling point on the surface of MEA plates for 7 days. The actinomycete counts of soil plates were determined using the dilution plate and the spread plate method.
2.7.
Data analyses
The data were analyzed with Statistical Product and Service Solution for Windows, Rel. 12.0 (SPSS Inc., Chicago, IL). Statistical significance was determined at probability level, p 0.05. Data were tested for normality and homogeneity of variance and if necessary appropriate transformations were done. Analysis of variance with a General Linear Model was used to determine the significant differences among the actinomycete numbers in different layers of aspen and pine organic profiles. Significance differences between means were determined by Tukey’s HSD test. Student’s t-tests for independent samples were performed to compare the actinomycete numbers in same depths of aspen and pine organic profiles. The most frequently isolated actinomycetes were based on percentage counts of isolation plates. For in vitro antagonism, the percentage inhibition values were analyzed by a General Linear Model analysis of variance with Tukey’s HSD test post hoc.
Fig. 1 – Actinomycete densities in soil depth profiles of aspen and pine forests (mean W standard deviation). (Within one type of forest bars sharing the same letter indicates no significance at p = 0.05.)
significantly higher ( p < 0.05) than those of pine forests. But there was no significance difference in actinomycete counts between 0 cm and 4 cm layers of the two forests ( p > 0.05). Ninety-four different actinomycete strains were found in the aspen forests and 80 were found in the pine forests. Of these, 18 strains were common to both forest types. Both forest types had higher numbers of Streptomycete strains than other actinomycete strains (Table 1). Sixteen of the 20 most frequently isolated actinomycete strains belonged to the genus Streptomyces and 9 of these were common to both forests (Table 2).
3.2.
Intensity of antagonistic activity (in vitro tests)
All actinomycetes were antagonistic to some degree to a minimum of five decomposer fungi on one or more test media. However, 15 actinomycete strains were more antagonistic to test fungi than others because they could inhibit at least half of the test decomposer fungi by more than 40% on one or more test media (Fig. 2). Based on the maximum number of decomposer fungi inhibited (Fig. 2) and the intensity of the
Table 1 – Actinomycete genera and strains isolated from aspen and pine forests Actinomycete genera
3.
Results
3.1.
Actinomycete community size and species richness
In pine forest (Fig. 1) actinomycete counts decreased significantly ( p < 0.05) with soil depth. In aspen forests the 8–12 cm layer had the lowest actinomycete numbers but there was no significant difference ( p > 0.05) between the actinomycete counts of 0–4 cm and 4–8 cm layers. The actinomycete counts in the 4–8 cm and 8–12 cm layers of aspen forests were
Forest Pine
Streptomyces spp. Actinomadura spp. Nocardia spp. Pseudonocardia spp. Unidentified species
51 4 2 0 23
Total
80
(63.8) (5) (2.5) (0) (28.8)
Aspen 60 6 5 1 22
(63.8) (6.4) (5.3) (1.1) (23.4)
94
Percentages of total are given in parenthesis. Simple coefficient of similarity for aspen and pine forests = 20.6%.
applied soil ecology 38 (2008) 109–118
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Table 2 – Commonly isolated actinomycete strains from aspen and pine forests Actinomycete strain
Genus
Percent of plates with isolate Aspen
Str-73-44 Str-64-39 Str-330-9 Str-108-52 Str-90-47 Str-210-56 Str-266-90 Str-21-14 Str-62-38 Str-18-6 Str-89-49 Str-275-28 Str-40-25 Str-31-23 Str-265-91 Str-201-59 NonStr-5-3 NonStr-6-3 NonStr-309-30 NonStr-312-5
Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Non-Streptomyces Non-Streptomyces Non-Streptomyces Non-Streptomyces
100 100 100 100 100 100 62 80 86 94 97 97 97 97 0 0 92 86 0 97
Pine 0 0 100 100 94 96 94 0 0 0 0 100 96 0 94 94 97 0 100 100
Fig. 2 – Numbers of litter and wood decomposer fungal species inhibited more than 40% by each actinomycete isolate at least on one agar medium.
inhibition (Fig. 3), Str-108-52 and Str-330-9 were the most antagonistic actinomycete strains and they showed highest inhibition against eight and five test fungi, respectively. Mucor spp., P. chrysogenum and Z. moelleri were the most resistant fungi to actinomycete antagonism and these fungi never showed more than 55% growth reduction against any actinomycete strain (Fig. 4). Mortierella species and P. montanense were the most susceptible fungi and except for M. alpina, they were inhibited by at least by 12 actinomycetes on all test media (Fig. 4). Wood decomposer fungi were more sensitive to actinomycete antagonism than leaf litter decomposers and Pholoita squarrosa and P. nidulans were completely inhibited by some actinomycetes (Fig. 4).
3.3. fungi
Nature of the interactions between actinomycetes and
Visual and microscopic observations indicated that there were no direct contacts between the actinomycete colonies and the inhibited fungal colonies indicating production of diffusible antifungal compounds. However, in some fungal–actinomycete interactions (e.g. Str-108-52 vs. Trichoderma sp. 2), there were no clear zones between actinomycete and fungal colonies (I < 5%), but actinomycetes were capable of lysing the test fungal hyphae using hyperparasitism. Germinating spores of highly susceptible fungi as well as resistant fungi showed aberrant and aborted hyphae on dual culture-agar blocks cut from the area surrounding colonies of highly antagonistic actinomycetes and they did not form viable colonies, presumably as a result of the diffusible antifungal compounds in agar blocks. The same results were observed when spores of susceptible fungi (except T. polysporum) were placed on pure culture-agar blocks. Similar to the fresh spores, all the mycelial plugs of susceptible fungi except
Fig. 3 – Maximum inhibition levels reported by different fungi against actinomycetes on different test media (mean W standard deviation). The actinomycete strain which showed the highest inhibition and test media are given in parenthesis. Fungi: Cp—Chrysosporium pannorum, Cc—Cladosporium cladosporioides, Ch—Cladosporium herbarum, Ma—Mortierella alpina, Mrr—Mortierella ramanniana var. angulispora, Mra—Mortierella ramanniana var. ramanniana, Mv—Mortierella vinacea, Mh1—Mucor hiemalis var. 1, Mh2—Mucor hiemalis var. 2, Mr—Mucor racemosus, Pca—Paecilomyces carneus, Pch—Penicillium chrysogenum, Pm—Penicillium montanense, Tp— Trichoderma polysporum, T2—Trichoderma sp. 2, Zm— Zygorrhynchus moelleri, Cy—Cystoderma sp., Pn— Phyllotopsis nidulans, Ps—Pholiota squarrosa; media: PDA— potato dextrose agar, CZPDA—Czapek-Dox potato dextrose agar, V8A—V8 agar, OA—oatmeal agar.
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fungal threads bound soil particles to each other. In soil plates with soil colonized with actinomycetes (1010 CFU g1), there was no visible growth of fungi even at the end of the test period indicating the suppression of fungal invasion by actinomycetes. Twenty-one fungi were isolated from natural soil surrounding the soil already colonized with actinomycetes (Mucor heimalis, M. racemosus, T. polysporum, two unidentified Trichoderma spp., C. cladosporioides, C. herbarum, M. alpina, M. ramanniana var. angulispora, M. ramanniana var. ramanniana, M. vinacea, one unidentified Mortierella sp. and seven Penicillium spp.). Only P. chrysogenum was able to colonize soil with actinomycetes located between 1 mm and 5 mm away from boundary within 5 days. After 5 days the fungus rapidly spread to the rest of the soil and at the end of the 10th day P. chrysogenum was isolated from all the sampling points. However, the actinomycete population size did not show any significant change from the population sizes of control plates due to the presence of P. chrysogenum (Fig. 5).
Fig. 4 – Ability of different actinomycete strains to inhibit growth of different fungi (I > 40%) on different test media. Abbreviations are same as in Fig. 3.
P. montanense, adjacent to actinomycete colonies could no longer produce new colonies on fresh agar medium. However, the peripheral hyphae of dual cultures of resistant fungi maintained their viability even after the after the direct contact with test actinomycetes. Therefore, spores appear to be more susceptible to antifungal compounds than hyphae but the intensity of this susceptibility depends on the species of actinomycetes.
3.4. Fungal colonization ability against highly and weakly antagonistic actinomycetes Results indicated that the colonization of bran by fungi in natural soil depended on three factors; stage of actinomycete colonization (early or late stages of colonization, e.g. number of days after inoculation) actinomycete type (antagonistic/ non-antagonistic) and intensity of colonization (complete or partial colonization of bran surfaces-based on visual surface area covered by actinomycetes). Bran particles cultured in soil at the early stage of actinomycete colonization (2 and 5 days after the inoculation) were completely colonized by several fungal genera. Even in the later stage of actinomycete colonization (after 14 days), fungi belonging to genus Penicillium and genus Mucor were able to colonize the bran particles with weakly antagonistic actinomycetes (Table 3). Nevertheless bran particles colonized with Str-330-9 and Str-108-52 at the same stage were free of any fungal inoculum or sparsely colonized with P. chrysogenum.
3.5.
Ability of fungi to colonize soil with actinomycetes
Control plates with sterile central soil were completely covered by Mucor species within 2–3 days and a network of
4.
Discussion
The actinomycete counts (104 CFU g1) of pine forests were comparable with those reported from a cool temperate pine forest with a mor type litter layer in United Kingdom (Davies and Williams, 1970). Actinomycetes grow extensively in soils rich in organic matter and previous studies have shown that the number of actinomycetes in soil is positively correlated to the level of organic matter and moisture content (Henis, 1986; Hayakawa et al., 1988). In the current study, the decrease in actinomycete community size with transition from the 0–4 cm layer to the 4–8 cm and 8–12 cm layers of both forests corresponded with the decrease in organic matter and moisture content down the profiles but there was no ( p < 0.05) or very little (R2 < 0.3) linear correlation between any of the tested soil properties and actinomycete numbers. Therefore, other than the soil moisture and organic matter content, there may be a combination of other environmental and biological factors, which control the actinomycete distribution in the litter layers of current study sites. Presence of anti-microbial compounds and waxy material on the pine needle surface, and negative impacts of phytotoxic products of pine litter decomposition on actinomycete community (Perry et al., 1987) may be two reasons for the low abundance and species richness of actinomycetes in pine litter than the aspen litter. According to a study done in a pine forest in North-east India (Das and Mishra, 1989), the soil actinomycete community was negatively influenced by phytotoxic products of pine litter decomposition, such as dihydroxybenzoic acid, vanillic acid and quinic acid. Unlike the pine needles comparatively resistant to microbial decomposition, texture of aspen leaves may also support colonization of actinomycetes. The favorable conditions (e.g. burrowing and mixing of organic material, dispersal of actinomycetes) created by endogeic earthworm activity, which was high in aspen forest also may responsible for the higher abundance and species richness of actinomycetes in aspen forests. As the exact reasons for these differences are not clear at this stage further studies should be done under fields and laboratory conditions.
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Table 3 – Frequency of fungi from bran particles at different stages of actinomycetes colonization Actinomycete strain
Fungi
Percentages of bran particles with fungi Day 2
Day 5
Day 7
Day 14
Control
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
100.0 66.7 40.0 0.0 0.0 53.3 53.3 0.0 13.3
93.3 40.0 46.7 20.0 26.7 40.0 13.3 26.7 0.0
93.3 13.3 60.0 40.0 40.0 40.0 20.0 0.0 0.0
100.0 26.7 40.0 13.3 13.3 53.3 0.0 13.3 40.0
Str-108-52
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
66.7 26.7 40.0 40.0 6.7 20.0 0.0 0.0 0.0
66.7 13.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0
93.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
60.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Str-330-9
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
80.0 0.0 0.0 0.0 0.0 20.0 0.0 0.0 0.0
53.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
53.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
26.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Str-266-90
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
100.0 60.0 26.7 40.0 0.0 53.3 40.3 26.7 0.0
93.3 40.0 26.7 40.0 0.0 26.7 40.0 53.3 0.0
80.0 6.7 0.0 0.0 40.0 0.0 26.7 53.3 20.0
93.3 60.0 0.0 0.0 0.0 13.3 20.0 13.3 6.7
Str-73-44
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
100.0 60.0 73.3 0.0 53.3 53.3 6.7 0.0
100.0 60.0 60.0 20.0 80.0 13.3 26.7 0.0
100.0 93.3 0.0 40.0 13.3 20.0 0.0 0.0
100.0 40.0 0.0 13.3 33.3 0.0 40.0 0.0
With regard to the different actinomycetes encountered in the litter layers of aspen and pine forests, the genus Streptomyces was dominant and exhibited the highest relative abundance throughout the organic profiles, accounting for 63.8% of all actinomycetes isolated. This predominance supports the findings of Lee and Hwang (2002) where 70–80% of isolates actinomycetes in Korean Mountain forest soils were those belonging to genus Streptomyces. However, in these mountain forests Streptomyces was especially high (more than 80%) in soils with neutral pH and moderate moisture contents (9.1–13.1%). But in the current study sites Streptomyces was predominant in all soils regardless of moisture content, pH and organic matter content. Davies and Williams (1970) showed that the
63 of 67 pine litter actinomycetes were Streptomyces species (Hayakawa et al., 1988). According to the in vitro tests a large proportion of most commonly isolated actinomycetes, mostly Streptomyces strains, were antagonists against most of the test decomposer fungi. Some actinomycete strains had broad spectra of antifungal activity (e.g. Str-108-52, Str-330-9, Str-201-59) while others inhibited a limited number of fungal species (e.g. Str64-39, Str-5-3) that could not be inhibited by even actinomycetes with broad spectrum of antifungal activity. For example, Str-21-14, which inhibited only five fungal species, was one of the few actinomycetes, which could inhibit the most resistant test fungal species P. chrysogenum. This suggests that a single actinomycete strain will be unlikely to provide broad-
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Fig. 5 – Number of fungi isolated from soil previously colonized with Str-108-52 surrounded by natural soil (mean W standard deviation).
spectrum control of diverse decomposer fungi. Therefore, the ultimate impact of actinomycetes on decomposer fungi may be determined by the antifungal activities of all soil actinomycetes combined. The results of the in vitro tests indicated that actinomycetes could inhibit the growth of decomposer fungi using antifungal compounds and hyperparasitism. There are many reports related to antibiotic substances which induced malfunctions such as stunting, distortion, swelling, hyphal protuberances or highly branched appearance of fungal germ tubes (Getha and Vikineswary, 2002; Gunji et al., 1983). Examination under the light microscope showed fungal colony margins adjacent to the actinomycete colonies did not show any of these changes except the lysing of hyphae and aborted hyphae of germinating spores. But in some affected fungal colonies, e.g. all the Mortierella spp. except M. alpina, colorless, sterile hyphae were observed in colony margins adjacent to the antagonistic actinomycete colonies. Of the litter decomposer fungi, fast growing genera (e.g. Mucor, Penicillium, Trichoderma) were more tolerant to actinomycete antagonism than slow and moderately slow-growing genera (e.g. Cladosporium, Mortierella) and wood decomposer fungi. Actinomycetes are generally slow growing and fast growing fungi may out grow the action of inhibition. P. chrysogenum is the source for penicillin while Trichoderma species are capable of producing several antibiotics including viridin, gliotoxin (Harman et al., 2004), trichodermin, tricho-
virins II (Jaworski et al., 1999). Therefore, apart from the fast growing nature of these fungi, the developed high resistance levels to their own antibiotics (in order to avoid autolysis) may be the major reason for their high resistance against antagonistic actinomycetes. The inhibition of fungi by actinomycetes was greater for most actinomycete–fungal combinations (high percentages inhibition values) on V8A and PDA used in in vitro antagonistic tests, reflecting possible antifungal compound production and diffusion on these media. In the current study some actinomycete strains which seem to be strong fungal inhibitors (Str-108-52 and Str-275) showed hyperparasitism instead of antibiosis against some highly resistant decomposer fungal species (e.g. Trichoderma spp.) suggesting that even though antifungal compounds are not effective against some fungi, actinomycetes have other mechanisms to inhibit decomposer fungi. Results of the experiment done with bran colonized with actinomycetes revealed that most of the decomposer fungi, except P. chrysogenum, could not colonize the organic substrates well colonized by antagonistic actinomycetes. The results were consistent with the in vitro tests, which showed that P. chrysogenum is one of the most resistant fungi. However, the limited growth of P. chrysogenum in plates inoculated with bran with established antagonistic actinomycetes indicated that the highly antagonistic actinomycetes may control the growth of fungi regardless of the resistance level of fungi. Most of decomposer fungi could not colonize organic material well colonized by antagonistic actinomycetes. Even though P. chrysogenum could colonize the organic material well colonized by antagonistic actinomycetes, it could not preclude actinomycete population density. In sterile soil it was not possible to maintain a population of actinomycetes less than 108 CFU g1. Therefore, quantitative determination of impact of actinomycete population density on colonization of organic matter by decomposer fungi was not possible. In both experiments, only a few fast growing fungal species (e.g. Mucor species, Penicillium species, Trichoderma species) were detected in the sterile substrates and substrates with actinomycetes, even though the natural soil contains many decomposer fungi. Slow growing fungal species may colonize these substrates later but the current experimental period was not long enough to detect them. According to the both experiments, the colonization of organic substrates by actinomycetes does reduce the degree of subsequent colonization by susceptible decomposer fungi.
5.
Conclusion
The forest floors of both aspen and lodgepole forests in the Kananaskis valley had relatively large actinomycete communities (104–105 CFU g1 soil) with 156 different actinomycetes strains. The genus Streptomyces was predominant in both forest types. Aspen litter layers contained the highest actinomycete densities as well as the highest species richness of actinomycetes. According to in vitro tests, most of the common temperate forest floor actinomycetes were antagonists of decomposer fungi. Decomposer fungi varied significantly in their sensitivity to different actinomycete strains. Of the litter decom-
applied soil ecology 38 (2008) 109–118
poser fungi, fast growing genera (e.g. Mucor, Penicillium, Trichoderma,) were more tolerant to actinomycete antagonism than slow and moderately slow-growing genera (e.g. Cladosporium, Mortierella). Despite antifungal metabolite production and the hyperparasitism demonstrated in in vitro tests viability tests, no hard evidence indicated the exact mechanism(s) responsible for suppression of fungal growth by actinomycetes under microcosm conditions. The initial colonization of organic substrates by some actinomycetes did reduce the degree of subsequent colonization by susceptible decomposer fungi. Even though the actinomycete-resistant decomposer fungi could share the same substrate with antagonistic actinomycetes, the presence of actinomycetes could reduce the growth of actinomyceteresistant fungi. Suppression of decomposer fungi could be the result of antifungal metabolite production and/or hyper parasitism.
references
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Actinomycetes as antagonists of litter decomposer fungi B.A.T. Dinishi Jayasinghe *, Dennis Parkinson Department of Biological Sciences, University of Calgary, 2500 University Drive, NW, Calgary, Alberta, Canada T2N1N4
article info
abstract
Article history:
The actinomycete communities of North American forests and impacts of their activities on
Accepted 21 September 2007
decomposer fungi have not been thoroughly investigated. In the current study, the community size and species richness of the forest floor actinomycete communities in cool
Keywords:
temperate aspen poplar and lodgepole pine forests of Alberta, Canada, was investigated.
Actinomycetes
Based on the isolations conducted on chitin agar, both aspen and lodgepole forests had
Decomposer fungi
relatively large actinomycete communities (104–105 CFU g1 soil). One hundred and fifty-six
Forest floor
different actinomycete strains were isolated and the genus Streptomyces predominated in
Antagonism
both forests. The community size as well as the number of different actinomycete strains
Colonization
was higher in aspen forests than in pine forests, but the exact reasons for these differences
Organic material
are not clear at this stage. It may be that favorable conditions created by endogeic earthworms (e.g. burrowing and mixing of organic material, dispersal of actinomycetes) increase the density and diversity of actinomycetes in aspen litter. It is also possible that pine litter anti-microbial compounds, waxy material and phytotoxic products of pine litter decomposition may have negative affects on actinomycetes in pine forests. Both in vitro and microcosm studies showed that the frequently isolated actinomycetes could act as antagonists for some common leaf litter and wood decomposer fungi. Of the litter decomposer fungi, fast growing genera (e.g. Mucor, Penicillium, Trichoderma) were more tolerant of actinomycete antagonism than slow and moderately slow-growing genera (e.g. Cladosporium, Mortierella). The colonization of organic substrates by some actinomycetes did reduce the degree of subsequent colonization by susceptible decomposer fungi. # 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Ecological studies of actinomycetes in soils of been conducted in various habitats including grasslands (Lee and Hwang, 2002), beach sands (Suzuki et al., 1994), underground caves (Groth et al., 1999), rice-paddies (Hayakawa et al., 1988), orchards (Lee and Hwang, 2002) and sub-glacial ice of Antarctica (Priscu et al., 1999). According to these studies, actinomycetes constitute a significant component of the microbial population in most soils and in some soils they are more numerous than all other bacteria. Some actinomycetes have been shown to play an important part in recycling of complex organic materials such as lignocelluloses (Craw-
ford, 1978). Therefore, actinomycetes are important members of forest floor decomposer community. However, there have been few studies of actinomycete communities in forest soils (a cool temperate pine forest in Lancashire (Davies and Williams, 1970), a tropical rain forest in Singapore (Wang et al., 1999), mountain forests of Japan (Hayakawa et al., 1988), a pine forest in north east India (Das and Mishra, 1989), a sclerophyllous forest in New South Wales of Australia (Gerrettson-Cornell and Kelly, 1981) and a forest formed on a worked-out peat bog in Russia (Zvyaginstev et al., 1996)). These studies showed that factors controlling the density and diversity of actinomycetes in the forest soil have been suggested to be the relative moisture content, pH, nature
* Corresponding author. Tel.: +1 403 220 5948; fax: +1 403 289 9311. E-mail address: [email protected] (B.A.T. Dinishi Jayasinghe). 0929-1393/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2007.09.005
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applied soil ecology 38 (2008) 109–118
and abundance of the organic matter, abundance of mesofauna and above ground vegetation. In the present study, the nature of the forest floor actinomycete communities in relation to the abundance of organic matter, pH, moisture content was investigated in North American cool temperate aspen poplar and lodgepole pine forests. A number of actinomycetes, however, may be acting as antagonists of soil fungi. Therefore, potential effects of actinomycetes on the litter decomposer fungi were also investigated. According to the results, compared to the lodgepole pine forests, the density as well as the diversity of actinomycetes was highest in aspen forests but the Streptomycetes were predominant in both forests. Microcosms studies showed that the frequently isolated actinomycetes could act as the antagonists for the common litter and wood decomposer fungi. The antagonistic impacts of actinomycete species on pathogenic fungi is well known and a few species have been used as biological control agents (Yuan and Crawford, 1995; ElShanshoury et al., 1996; Jones and Samac, 1996; El-Tarablily et al., 1997; Berg et al., 2000; Getha and Vikineswary, 2002; Bressan, 2003). While there are examples of actinomycetes antagonistic to VAm mycorrhizal fungi (Krishna et al., 1982) and ectomycorrhizal fungi (Keast and Tonkin, 1983; Fridman et al., 1989; Richter et al., 1989), there are very few data on such impacts of actinomycetes on decomposer fungi (e.g. lysis of cell walls of Mucor ramammianus by Streptomyces spp. (Jones et al., 1968) and inhibition of southern pine sap-wood decomposers (Lenzites saepiaria, Polyporus versicolor and Lentinus lepideus) by an isolate of Streptomyces sp. (De Groot, 1971)). Since saprophytic fungi are important, members of the forest decomposer community in temperate forests ecosystems, any antagonistic impact by actinomycete species could have marked effects on organic matter decomposition rates and fungal community structure in the forest floor. As the distributions of actinomycetes in North American forests remain largely undescribed, the first step of the present study was to determine the nature of two forest floor actinomycete communities, which would allow selection of frequently isolated actinomycete strains for further in vitro and in vivo studies of their potential antifungal antagonism. The objectives of the current study were: (1) To determine the community size and, composition of actinomycetes at different depths of organic layers in lodgepole pine and aspen poplar forests. (2) To determine the ability of frequently isolated actinomycetes to inhibit the growth of common decomposer fungi. (3) To determine the impact of actinomycetes on colonization of organic matter by decomposer fungi.
2.
Materials and methods
2.1.
Study site
continental with long winters and hot summers. Even though the soil usually remains frozen in the winter (from November to March) Chinook winds may frequently thaw the upper soil layers (Coxson and Parkinson, 1987). The mean annual maximum and minimum temperatures for the duration of the present study (2001 July–June to 2003 June–July) were 10.2 8C and 2.5 8C, respectively, and the mean annual precipitation was 607.7 mm. These forests were reported free of earthworms until the late 1980s (Dymond et al., 1997) when the epigeic earthworm, Dendrobaena octaedra was found. More recently (about 5 years ago), several species of deep- to middepth burrowing earthworms, including Lumbricus terrestris, Aporrectodea caliginosa, and Octolasion tyrtaeum were also found colonizing aspen forests in this area (Migge, 2001). The organic layers of pine forests can be distinguished into L (litter), F (fermentation) and H (humus) materials. However, the F and H materials could not be separated easily, presumably as a result of the feeding and mixing actions of D. octaedra, and binding of organic particles together by their casts. In most parts of the aspen poplar forests, the organic layers and the upper mineral layer were completely mixed by burrowing earthworms (L. terrestris).
2.2.
In summer 2003, five soil cores (5.5 cm diameter, 19.5 cm depth) were taken at random from each of five discrete stands (30 m 30 m) of both pine and aspen forests. Each soil core was separated into three layers (sub-samples) according to the depth (0–4 cm, 4–8 cm, 8–12 cm). Soil sub-samples were sieved (2 mm mesh) and during sieving, woody debris, live plants and earthworms were removed. Actinomycetes were isolated from samples (three replicates) using dilution plate method (diluted to 105) with spread plates using chitin agar supplemented with 50 mm mL1 nystatin (Wellington and Toth, 1994). The plates were incubated at room temperature (22–24 8C) for 14–21 days, and actinomycete colonies were recognized on the basis of morphological characteristics. Total actinomycete colony counts, presence/absence of each morphological form, and the 10 most common strains from each forest were recorded. Actinomycete colonies with different morphologies were transferred to Czapek-Dox agar slants (Sigma1) and were stored at 4–6 8C for further studies. Actinomycetes were identified to genus levels according to the methods suggested in Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994) and Bergey’s Manual of Systematic Bacteriology—volume 4 (Williams et al., 1989). Genus identification results of some of the 20 most frequently isolated actinomycete strains were confirmed by the similarity index in the Sherlock Microbial Identification system, which is based on the fatty acid composition of actinomycetes (Microbial ID Inc., Newark, DE).
2.3. The study was conducted in aspen (Populus tremuloides L.) and lodgepole pine (Pinus contorta Loud.) forests in the lower subalpine zone (Williams, 1990) of the Canadian Rockies of Southwest Alberta (518010 N/1158010 W). All the study sites were located on well-drained eastern mountain slopes of the Kananaskis Valley at about 1350 m elevation. The soil is classified as Orthic Eutric Brunisol and the climate is
Isolation and identification of actinomycetes
In vitro antagonism tests
The 20 most commonly isolated actinomycete strains were tested in vitro using the dual culture method (modified procedure of Fuhrmann, 1994) for antagonism toward 16 litter-decomposing fungi isolated from the same study area. It was assumed that the most commonly isolated actinomycetes and frequently occurring decomposer fungal species from the
applied soil ecology 38 (2008) 109–118
same forest soils may frequently interact with each other in the soil environment. Therefore, impacts of dominant actinomycete isolates on decomposer fungi will be greater than the impacts of rare or less frequently isolated actinomycete isolates. The test fungi are common inhabitants of upper layers of both aspen and pine forests (Widden and Parkinson, 1973; Visser and Parkinson, 1975). Litter decomposer fungi: Chrysosporium pannorum, Cladosporium cladosporioides, Cladosporium herbarum, Mortierella alpina, Mortierella ramanniana var. angulispora, Mortierella ramanniana var. ramanniana, Mortierella vinacea, Mucor hiemalis var. 1, Mucor hiemalis var. 2, Mucor racemosus, Paecilomyces carneus, Penicillium chrysogenum, Penicillium montanense, Trichoderma polysporum, Trichoderma sp. 2, Zygorrhynchus moelleri (from a previously prepared culture collection by Dr. Beryl Zaitlin and identification of these fungi was confirmed using Compendium of Soil Fungi (Domsch et al., 1980)) and The genus Penicillium (Pitt, 1979). Wood decomposer fungi: Cystoderma sp., Phyllotopsis nidulans and Pholiota squarrosa (from a previously prepared and identified culture collection by Dr. S. Visser). Test media for the dual culture method were selected based on the growth performances of both test actinomycetes and fungi. Media for litter decomposer fungi were V8 agar (V8A), potato dextrose agar (PDA), malt extract agar (MEA), and modified medium formulated by mixing (Czapek-Dox) CZA and PDA (1:1). Media for wood decomposer fungi were oatmeal agar (OA) and PDA. Size of the culture plates was determined based on the growth rates of fungi on each test medium (90 mm diameter plates for fast growing and 45 mm for slow growing). Plates were inoculated with actinomycetes 24 h or 48 h prior to the fungal inoculation. Spores of 4 of the 20 actinomycetes were uniformly spotted equidistantly near the periphery of the each plate and when these strains were visibly growing, 5-mm diameter agar plugs taken from a growing edge of a 5-day-old test fungal colony was transferred to the centre of the test agar plate surface (3 replicates). Cultures were incubated at room temperature (22–24 8C) and when growing edges of control fungi (without any actinomycete inoculum) were at the edge of the plates, the diameters of the test fungal colonies toward each actinomycete were measured. Actinomycete antagonism against the fungi was evaluated using percentage inhibition values (I) on each medium. I¼
radius of control fungus radius of test fungus 100 radius of control fungus
In the presence of a very highly antagonistic actinomycete strain, growth of the test fungal colony was completely or highly inhibited regardless of the direction of the actinomycete growth. As this restricted the determination of antagonistic activity of the other three actinomycete strains, in such cases the experiment was repeated with the plates with a single individual actinomycete colony of the highly antagonistic strains.
2.4. Studies of nature of the interactions between fungi and actinomycetes The morphology of fungal mycelia adjacent to the actinomycete colonies was examined under both a dissecting micro-
111
scope (10) and a compound microscope (400) for abnormal growth patterns and to ascertain whether or not there were direct contacts between actinomycete and fungal colonies. The response of spores of five susceptible fungi (M. ramanniana var. angulispora, M. ramanniana var. ramanniana, M. vinacea, P. montanense, Trichoderma sp. 2) and five resistant fungi (M. alpina, M. hiemalis var. 1, M. hiemalis var. 2, M. racemosus, P. chrysogenum) (based on the in vitro tests) to the actinomycete antifungal compounds was evaluated as the ability of their fresh spores to form viable colonies on agar blocks (5 mm 5 mm) cut from clear zones formed in dual culture plates with highly antagonistic actinomycetes (Str108-52 and Str-330-9) after 5 days of incubation. To determine whether diffusible antifungal compounds are produced even in the absence of susceptible fungi, the same procedure was repeated using agar blocks cut from area surrounding the actinomycete colonies in pure culture plates. The response of both susceptible and resistant fungal hyphae to highly antagonistic actinomycetes was evaluated as ability of their mycelial plugs cut from colonies adjacent to actinomycete colonies to produce viable colonies on the fresh agar plates with the same medium.
2.5. Ability of fungi to colonize substrates with highly and weakly antagonistic actinomycetes Soil plates were prepared using 30 g of sieved (<4 mm) field moist soil taken from the 0–4 cm depth of the aspen forests in the Kananaskis valley. Two highly antagonistic actinomycetes (Str-108-52 and Str-330-9) and two weakly antagonistic actinomycetes (Str-266-90 and Str-73-44) strains were selected as the test actinomycetes on the basis of the in vitro tests. For each actinomycete strain, two 9-cm Petri dishes with 25 g of three times autoclaved bran (bran:distilled water (w/v) = 5:2) were inoculated with 1 mL of actinomycete spore suspension and mixed well with a sterile spatula. The covered Petri dishes were kept at room temperature (22–24 8C) and were shaken occasionally to ensure the uniformity of colonization. Two, 5, 10 and 14 days after the inoculation of bran with actinomycetes, 25 bran flakes with each different actinomycetes and sterile control bran were added to previously prepared Petri dishes with natural soil (three replicates). Plates were incubated at room temperature for 7 days. Then five bran flakes were removed carefully from each soil plate, washed three times (sterile distilled water) and plated on fresh MEA. Plates were incubated for 7 days and fungi growing were identified. Percentage of bran particles with each different fungi was calculated.
2.6. Impacts of colonization of organic soil by antagonistic actinomycetes on subsequent colonization by the decomposer fungi Five milliliters of a spore suspension (density 106– 108 spores mL1) of one of the most antagonistic actinomycete strain Str-108-52 (based on in vitro tests) was added to each of ten 25 g samples of sterile soil and mixed with a sterile spatula. Each soil–spore mixture was spread into each of 10 sterile glass Petri dishes and incubated at room temperature (22–24 8C) for 2 weeks or until the appearance of whitish actinomycete
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colonies. Sterile distilled water (0.5 mL) was added to the soil every 4 days and mixed. The final actinomycete population number was determined for each of the soil replicates using the dilution plate and the spread plate method. Moisture contents of sterile soil and soil colonized with actinomycetes were adjusted to the moisture level of natural soil. An empty 9.5-cm diameter sterile Petri dish lid was placed in the middle of a 15-cm diameter sterile glass Petri dish and the uncovered area was filled with 20 g of natural soil. The 9.5cm diameter lid was carefully removed and the uncovered area was filled with 10 g soil colonized with the actinomycetes. The boundary between natural soil and soil colonized with actinomycetes was marked with a 9.5-cm diameter sterile metal ring. Control plates were prepared using sterile soil in centre instead of soil colonized with the actinomycetes. To determine the fungal growth into sterile soil and soil colonized with actinomycetes, 18 plates were prepared and on each sampling day (day 0, day 5, day 10, day15, day 20 and day 25 after set up) 3 replicates of 1 g of soil and 10 soil particles (mainly organic particles) were taken from three predetermined distances from the boundary between 2 soil types (between boundary and 5 mm away from the boundary, 7 mm and 15 mm away from the boundary). The types of different fungal species at each sampling point were determined by incubating 10 soil particles taken from each sampling point on the surface of MEA plates for 7 days. The actinomycete counts of soil plates were determined using the dilution plate and the spread plate method.
2.7.
Data analyses
The data were analyzed with Statistical Product and Service Solution for Windows, Rel. 12.0 (SPSS Inc., Chicago, IL). Statistical significance was determined at probability level, p 0.05. Data were tested for normality and homogeneity of variance and if necessary appropriate transformations were done. Analysis of variance with a General Linear Model was used to determine the significant differences among the actinomycete numbers in different layers of aspen and pine organic profiles. Significance differences between means were determined by Tukey’s HSD test. Student’s t-tests for independent samples were performed to compare the actinomycete numbers in same depths of aspen and pine organic profiles. The most frequently isolated actinomycetes were based on percentage counts of isolation plates. For in vitro antagonism, the percentage inhibition values were analyzed by a General Linear Model analysis of variance with Tukey’s HSD test post hoc.
Fig. 1 – Actinomycete densities in soil depth profiles of aspen and pine forests (mean W standard deviation). (Within one type of forest bars sharing the same letter indicates no significance at p = 0.05.)
significantly higher ( p < 0.05) than those of pine forests. But there was no significance difference in actinomycete counts between 0 cm and 4 cm layers of the two forests ( p > 0.05). Ninety-four different actinomycete strains were found in the aspen forests and 80 were found in the pine forests. Of these, 18 strains were common to both forest types. Both forest types had higher numbers of Streptomycete strains than other actinomycete strains (Table 1). Sixteen of the 20 most frequently isolated actinomycete strains belonged to the genus Streptomyces and 9 of these were common to both forests (Table 2).
3.2.
Intensity of antagonistic activity (in vitro tests)
All actinomycetes were antagonistic to some degree to a minimum of five decomposer fungi on one or more test media. However, 15 actinomycete strains were more antagonistic to test fungi than others because they could inhibit at least half of the test decomposer fungi by more than 40% on one or more test media (Fig. 2). Based on the maximum number of decomposer fungi inhibited (Fig. 2) and the intensity of the
Table 1 – Actinomycete genera and strains isolated from aspen and pine forests Actinomycete genera
3.
Results
3.1.
Actinomycete community size and species richness
In pine forest (Fig. 1) actinomycete counts decreased significantly ( p < 0.05) with soil depth. In aspen forests the 8–12 cm layer had the lowest actinomycete numbers but there was no significant difference ( p > 0.05) between the actinomycete counts of 0–4 cm and 4–8 cm layers. The actinomycete counts in the 4–8 cm and 8–12 cm layers of aspen forests were
Forest Pine
Streptomyces spp. Actinomadura spp. Nocardia spp. Pseudonocardia spp. Unidentified species
51 4 2 0 23
Total
80
(63.8) (5) (2.5) (0) (28.8)
Aspen 60 6 5 1 22
(63.8) (6.4) (5.3) (1.1) (23.4)
94
Percentages of total are given in parenthesis. Simple coefficient of similarity for aspen and pine forests = 20.6%.
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Table 2 – Commonly isolated actinomycete strains from aspen and pine forests Actinomycete strain
Genus
Percent of plates with isolate Aspen
Str-73-44 Str-64-39 Str-330-9 Str-108-52 Str-90-47 Str-210-56 Str-266-90 Str-21-14 Str-62-38 Str-18-6 Str-89-49 Str-275-28 Str-40-25 Str-31-23 Str-265-91 Str-201-59 NonStr-5-3 NonStr-6-3 NonStr-309-30 NonStr-312-5
Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Non-Streptomyces Non-Streptomyces Non-Streptomyces Non-Streptomyces
100 100 100 100 100 100 62 80 86 94 97 97 97 97 0 0 92 86 0 97
Pine 0 0 100 100 94 96 94 0 0 0 0 100 96 0 94 94 97 0 100 100
Fig. 2 – Numbers of litter and wood decomposer fungal species inhibited more than 40% by each actinomycete isolate at least on one agar medium.
inhibition (Fig. 3), Str-108-52 and Str-330-9 were the most antagonistic actinomycete strains and they showed highest inhibition against eight and five test fungi, respectively. Mucor spp., P. chrysogenum and Z. moelleri were the most resistant fungi to actinomycete antagonism and these fungi never showed more than 55% growth reduction against any actinomycete strain (Fig. 4). Mortierella species and P. montanense were the most susceptible fungi and except for M. alpina, they were inhibited by at least by 12 actinomycetes on all test media (Fig. 4). Wood decomposer fungi were more sensitive to actinomycete antagonism than leaf litter decomposers and Pholoita squarrosa and P. nidulans were completely inhibited by some actinomycetes (Fig. 4).
3.3. fungi
Nature of the interactions between actinomycetes and
Visual and microscopic observations indicated that there were no direct contacts between the actinomycete colonies and the inhibited fungal colonies indicating production of diffusible antifungal compounds. However, in some fungal–actinomycete interactions (e.g. Str-108-52 vs. Trichoderma sp. 2), there were no clear zones between actinomycete and fungal colonies (I < 5%), but actinomycetes were capable of lysing the test fungal hyphae using hyperparasitism. Germinating spores of highly susceptible fungi as well as resistant fungi showed aberrant and aborted hyphae on dual culture-agar blocks cut from the area surrounding colonies of highly antagonistic actinomycetes and they did not form viable colonies, presumably as a result of the diffusible antifungal compounds in agar blocks. The same results were observed when spores of susceptible fungi (except T. polysporum) were placed on pure culture-agar blocks. Similar to the fresh spores, all the mycelial plugs of susceptible fungi except
Fig. 3 – Maximum inhibition levels reported by different fungi against actinomycetes on different test media (mean W standard deviation). The actinomycete strain which showed the highest inhibition and test media are given in parenthesis. Fungi: Cp—Chrysosporium pannorum, Cc—Cladosporium cladosporioides, Ch—Cladosporium herbarum, Ma—Mortierella alpina, Mrr—Mortierella ramanniana var. angulispora, Mra—Mortierella ramanniana var. ramanniana, Mv—Mortierella vinacea, Mh1—Mucor hiemalis var. 1, Mh2—Mucor hiemalis var. 2, Mr—Mucor racemosus, Pca—Paecilomyces carneus, Pch—Penicillium chrysogenum, Pm—Penicillium montanense, Tp— Trichoderma polysporum, T2—Trichoderma sp. 2, Zm— Zygorrhynchus moelleri, Cy—Cystoderma sp., Pn— Phyllotopsis nidulans, Ps—Pholiota squarrosa; media: PDA— potato dextrose agar, CZPDA—Czapek-Dox potato dextrose agar, V8A—V8 agar, OA—oatmeal agar.
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fungal threads bound soil particles to each other. In soil plates with soil colonized with actinomycetes (1010 CFU g1), there was no visible growth of fungi even at the end of the test period indicating the suppression of fungal invasion by actinomycetes. Twenty-one fungi were isolated from natural soil surrounding the soil already colonized with actinomycetes (Mucor heimalis, M. racemosus, T. polysporum, two unidentified Trichoderma spp., C. cladosporioides, C. herbarum, M. alpina, M. ramanniana var. angulispora, M. ramanniana var. ramanniana, M. vinacea, one unidentified Mortierella sp. and seven Penicillium spp.). Only P. chrysogenum was able to colonize soil with actinomycetes located between 1 mm and 5 mm away from boundary within 5 days. After 5 days the fungus rapidly spread to the rest of the soil and at the end of the 10th day P. chrysogenum was isolated from all the sampling points. However, the actinomycete population size did not show any significant change from the population sizes of control plates due to the presence of P. chrysogenum (Fig. 5).
Fig. 4 – Ability of different actinomycete strains to inhibit growth of different fungi (I > 40%) on different test media. Abbreviations are same as in Fig. 3.
P. montanense, adjacent to actinomycete colonies could no longer produce new colonies on fresh agar medium. However, the peripheral hyphae of dual cultures of resistant fungi maintained their viability even after the after the direct contact with test actinomycetes. Therefore, spores appear to be more susceptible to antifungal compounds than hyphae but the intensity of this susceptibility depends on the species of actinomycetes.
3.4. Fungal colonization ability against highly and weakly antagonistic actinomycetes Results indicated that the colonization of bran by fungi in natural soil depended on three factors; stage of actinomycete colonization (early or late stages of colonization, e.g. number of days after inoculation) actinomycete type (antagonistic/ non-antagonistic) and intensity of colonization (complete or partial colonization of bran surfaces-based on visual surface area covered by actinomycetes). Bran particles cultured in soil at the early stage of actinomycete colonization (2 and 5 days after the inoculation) were completely colonized by several fungal genera. Even in the later stage of actinomycete colonization (after 14 days), fungi belonging to genus Penicillium and genus Mucor were able to colonize the bran particles with weakly antagonistic actinomycetes (Table 3). Nevertheless bran particles colonized with Str-330-9 and Str-108-52 at the same stage were free of any fungal inoculum or sparsely colonized with P. chrysogenum.
3.5.
Ability of fungi to colonize soil with actinomycetes
Control plates with sterile central soil were completely covered by Mucor species within 2–3 days and a network of
4.
Discussion
The actinomycete counts (104 CFU g1) of pine forests were comparable with those reported from a cool temperate pine forest with a mor type litter layer in United Kingdom (Davies and Williams, 1970). Actinomycetes grow extensively in soils rich in organic matter and previous studies have shown that the number of actinomycetes in soil is positively correlated to the level of organic matter and moisture content (Henis, 1986; Hayakawa et al., 1988). In the current study, the decrease in actinomycete community size with transition from the 0–4 cm layer to the 4–8 cm and 8–12 cm layers of both forests corresponded with the decrease in organic matter and moisture content down the profiles but there was no ( p < 0.05) or very little (R2 < 0.3) linear correlation between any of the tested soil properties and actinomycete numbers. Therefore, other than the soil moisture and organic matter content, there may be a combination of other environmental and biological factors, which control the actinomycete distribution in the litter layers of current study sites. Presence of anti-microbial compounds and waxy material on the pine needle surface, and negative impacts of phytotoxic products of pine litter decomposition on actinomycete community (Perry et al., 1987) may be two reasons for the low abundance and species richness of actinomycetes in pine litter than the aspen litter. According to a study done in a pine forest in North-east India (Das and Mishra, 1989), the soil actinomycete community was negatively influenced by phytotoxic products of pine litter decomposition, such as dihydroxybenzoic acid, vanillic acid and quinic acid. Unlike the pine needles comparatively resistant to microbial decomposition, texture of aspen leaves may also support colonization of actinomycetes. The favorable conditions (e.g. burrowing and mixing of organic material, dispersal of actinomycetes) created by endogeic earthworm activity, which was high in aspen forest also may responsible for the higher abundance and species richness of actinomycetes in aspen forests. As the exact reasons for these differences are not clear at this stage further studies should be done under fields and laboratory conditions.
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Table 3 – Frequency of fungi from bran particles at different stages of actinomycetes colonization Actinomycete strain
Fungi
Percentages of bran particles with fungi Day 2
Day 5
Day 7
Day 14
Control
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
100.0 66.7 40.0 0.0 0.0 53.3 53.3 0.0 13.3
93.3 40.0 46.7 20.0 26.7 40.0 13.3 26.7 0.0
93.3 13.3 60.0 40.0 40.0 40.0 20.0 0.0 0.0
100.0 26.7 40.0 13.3 13.3 53.3 0.0 13.3 40.0
Str-108-52
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
66.7 26.7 40.0 40.0 6.7 20.0 0.0 0.0 0.0
66.7 13.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0
93.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
60.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Str-330-9
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
80.0 0.0 0.0 0.0 0.0 20.0 0.0 0.0 0.0
53.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
53.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
26.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Str-266-90
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
100.0 60.0 26.7 40.0 0.0 53.3 40.3 26.7 0.0
93.3 40.0 26.7 40.0 0.0 26.7 40.0 53.3 0.0
80.0 6.7 0.0 0.0 40.0 0.0 26.7 53.3 20.0
93.3 60.0 0.0 0.0 0.0 13.3 20.0 13.3 6.7
Str-73-44
P. chrysogenum Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Mucor hiemalis Mucor sp. 2 Trichoderma sp. Unidentified
100.0 60.0 73.3 0.0 53.3 53.3 6.7 0.0
100.0 60.0 60.0 20.0 80.0 13.3 26.7 0.0
100.0 93.3 0.0 40.0 13.3 20.0 0.0 0.0
100.0 40.0 0.0 13.3 33.3 0.0 40.0 0.0
With regard to the different actinomycetes encountered in the litter layers of aspen and pine forests, the genus Streptomyces was dominant and exhibited the highest relative abundance throughout the organic profiles, accounting for 63.8% of all actinomycetes isolated. This predominance supports the findings of Lee and Hwang (2002) where 70–80% of isolates actinomycetes in Korean Mountain forest soils were those belonging to genus Streptomyces. However, in these mountain forests Streptomyces was especially high (more than 80%) in soils with neutral pH and moderate moisture contents (9.1–13.1%). But in the current study sites Streptomyces was predominant in all soils regardless of moisture content, pH and organic matter content. Davies and Williams (1970) showed that the
63 of 67 pine litter actinomycetes were Streptomyces species (Hayakawa et al., 1988). According to the in vitro tests a large proportion of most commonly isolated actinomycetes, mostly Streptomyces strains, were antagonists against most of the test decomposer fungi. Some actinomycete strains had broad spectra of antifungal activity (e.g. Str-108-52, Str-330-9, Str-201-59) while others inhibited a limited number of fungal species (e.g. Str64-39, Str-5-3) that could not be inhibited by even actinomycetes with broad spectrum of antifungal activity. For example, Str-21-14, which inhibited only five fungal species, was one of the few actinomycetes, which could inhibit the most resistant test fungal species P. chrysogenum. This suggests that a single actinomycete strain will be unlikely to provide broad-
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Fig. 5 – Number of fungi isolated from soil previously colonized with Str-108-52 surrounded by natural soil (mean W standard deviation).
spectrum control of diverse decomposer fungi. Therefore, the ultimate impact of actinomycetes on decomposer fungi may be determined by the antifungal activities of all soil actinomycetes combined. The results of the in vitro tests indicated that actinomycetes could inhibit the growth of decomposer fungi using antifungal compounds and hyperparasitism. There are many reports related to antibiotic substances which induced malfunctions such as stunting, distortion, swelling, hyphal protuberances or highly branched appearance of fungal germ tubes (Getha and Vikineswary, 2002; Gunji et al., 1983). Examination under the light microscope showed fungal colony margins adjacent to the actinomycete colonies did not show any of these changes except the lysing of hyphae and aborted hyphae of germinating spores. But in some affected fungal colonies, e.g. all the Mortierella spp. except M. alpina, colorless, sterile hyphae were observed in colony margins adjacent to the antagonistic actinomycete colonies. Of the litter decomposer fungi, fast growing genera (e.g. Mucor, Penicillium, Trichoderma) were more tolerant to actinomycete antagonism than slow and moderately slow-growing genera (e.g. Cladosporium, Mortierella) and wood decomposer fungi. Actinomycetes are generally slow growing and fast growing fungi may out grow the action of inhibition. P. chrysogenum is the source for penicillin while Trichoderma species are capable of producing several antibiotics including viridin, gliotoxin (Harman et al., 2004), trichodermin, tricho-
virins II (Jaworski et al., 1999). Therefore, apart from the fast growing nature of these fungi, the developed high resistance levels to their own antibiotics (in order to avoid autolysis) may be the major reason for their high resistance against antagonistic actinomycetes. The inhibition of fungi by actinomycetes was greater for most actinomycete–fungal combinations (high percentages inhibition values) on V8A and PDA used in in vitro antagonistic tests, reflecting possible antifungal compound production and diffusion on these media. In the current study some actinomycete strains which seem to be strong fungal inhibitors (Str-108-52 and Str-275) showed hyperparasitism instead of antibiosis against some highly resistant decomposer fungal species (e.g. Trichoderma spp.) suggesting that even though antifungal compounds are not effective against some fungi, actinomycetes have other mechanisms to inhibit decomposer fungi. Results of the experiment done with bran colonized with actinomycetes revealed that most of the decomposer fungi, except P. chrysogenum, could not colonize the organic substrates well colonized by antagonistic actinomycetes. The results were consistent with the in vitro tests, which showed that P. chrysogenum is one of the most resistant fungi. However, the limited growth of P. chrysogenum in plates inoculated with bran with established antagonistic actinomycetes indicated that the highly antagonistic actinomycetes may control the growth of fungi regardless of the resistance level of fungi. Most of decomposer fungi could not colonize organic material well colonized by antagonistic actinomycetes. Even though P. chrysogenum could colonize the organic material well colonized by antagonistic actinomycetes, it could not preclude actinomycete population density. In sterile soil it was not possible to maintain a population of actinomycetes less than 108 CFU g1. Therefore, quantitative determination of impact of actinomycete population density on colonization of organic matter by decomposer fungi was not possible. In both experiments, only a few fast growing fungal species (e.g. Mucor species, Penicillium species, Trichoderma species) were detected in the sterile substrates and substrates with actinomycetes, even though the natural soil contains many decomposer fungi. Slow growing fungal species may colonize these substrates later but the current experimental period was not long enough to detect them. According to the both experiments, the colonization of organic substrates by actinomycetes does reduce the degree of subsequent colonization by susceptible decomposer fungi.
5.
Conclusion
The forest floors of both aspen and lodgepole forests in the Kananaskis valley had relatively large actinomycete communities (104–105 CFU g1 soil) with 156 different actinomycetes strains. The genus Streptomyces was predominant in both forest types. Aspen litter layers contained the highest actinomycete densities as well as the highest species richness of actinomycetes. According to in vitro tests, most of the common temperate forest floor actinomycetes were antagonists of decomposer fungi. Decomposer fungi varied significantly in their sensitivity to different actinomycete strains. Of the litter decom-
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poser fungi, fast growing genera (e.g. Mucor, Penicillium, Trichoderma,) were more tolerant to actinomycete antagonism than slow and moderately slow-growing genera (e.g. Cladosporium, Mortierella). Despite antifungal metabolite production and the hyperparasitism demonstrated in in vitro tests viability tests, no hard evidence indicated the exact mechanism(s) responsible for suppression of fungal growth by actinomycetes under microcosm conditions. The initial colonization of organic substrates by some actinomycetes did reduce the degree of subsequent colonization by susceptible decomposer fungi. Even though the actinomycete-resistant decomposer fungi could share the same substrate with antagonistic actinomycetes, the presence of actinomycetes could reduce the growth of actinomyceteresistant fungi. Suppression of decomposer fungi could be the result of antifungal metabolite production and/or hyper parasitism.
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