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Previous Article | Next Article 
Applied and Environmental Microbiology, July 2003, p. 4192-4199, Vol. 69, No. 7
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.7.4192-4199.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Peter R. Mills,2 and Richard M. Cooper1*
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY,1 Department of Microbial Biotechnology, Horticultural Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom2
Received 1 November 2002/ Accepted 16 April 2003
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ABSTRACT |
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INTRODUCTION |
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Muthumeenakshi et al. (35, 36) and Castle et al. (12) described the molecular classification of T. harzianum groups associated with mushroom compost. Groups Th2 and Th4 represent aggressive strains indigenous to Europe and North America, respectively, and nonaggressive European strains were designated Th1 and Th3. Aggressiveness is a term usually used to describe the degree of pathogenicity of plant pathogens, but here it refers to the reduction in A. bisporus yield resulting from unknown interactions with T. harzianum. For example, in controlled inoculations Th2 reduced quality and yield by up to 80% (46). The forms responsible for green mold have been recently separated from T. harzianum in growth rate and in sequences of the ITS1 region of nuclear rRNA and a fragment of the protein-encoding translation elongation factor gene (EF-1
) (42). On this basis, Samuels et al. (42) renamed Th4 and Th2 as Trichoderma aggressivum sp. nov. and T. aggressivum f. sp. europaeum f. sp. nov., respectively.
Mycoparasitism by T. harzianum can take the form of directed growth towards a potential host followed by attachment, coiling, formation of hooked branches, and sometimes appressorium-like structures and penetration (4, 15). Initial recognition of host fungi may involve a fucose-binding lectin (2). Penetration of the host cell wall could have both enzymatic and mechanical components (21). Chitinolytic degradation was localized to penetration sites under constricted hyphae in the invasion of Sclerotium rolfsii sclerotia (5). Mycoparasitic structures have been induced to form on nylon fibers. Mycoparasitic signal transduction may involve G protein(s) and is mediated by cyclic AMP (37).
The role of depolymerases dominates reports of T. harzianum mycoparasitism to plant pathogens (11, 20, 25, 26, 27, 41). A serine protease, PRB1, was produced in the presence of cell walls or mycelium of several pathogenic fungi (32), and Flores et al. (23) obtained improved biocontrol of Rhizoctonia solani by Trichoderma harzianum transformants that overexpressed PRB1. An endochitinase (ECH42) was implicated in the mycoparasitism of R. solani based on improved disease control by multiple-copy transformants (11). Differential expression of T. harzianum chitinases during mycoparasitism of R. solani and S. rolfsii was host dependent (26). Expression of ech42 from T. atroviride occurred before mycoparasitic contact following molecular exchange with the host R. solani (29), but in contrast, chit 33 was expressed during, but not before, overgrowth of R. solani by T. harzianum (18). Also three to seven extracellular ß-1,3-glucanases were produced by isolates of T. harzianum exposed to fungal cell walls, autoclaved mycelia, or laminarin, and some of these enzymes were linked to mycoparasitism (20, 40).
T. harzianum is a highly competitive fungus in the soil (52). The rapid growth rate of Trichoderma spp. and the abundant asexual production of numerous propagules could classify them as ruderals (38), or opportunists. However, their versatile complement of lytic enzymes suggests a more combative strategy (53). The ability to switch strategies based on environmental conditions and community structure might explain their ubiquity in soils. This hypothesis of versatility is consistent with investigations of Trichoderma spp. for intraspecific competition (54) and interspecific competition under field conditions (52). The outcomes of these interactions are dependent on many factors, such as resources, temperature (15, 28, 49), and tolerance to inhibitory bacteria from soil or compost (43). The mechanism(s) by which aggressive isolates of T. harzianum inhibit growth of A. bisporus is unknown.
Our objective was to identify traits linked with aggressiveness and, in particular, to test the hypotheses that saprotrophic growth of T. harzianum is a crucial factor and that extracellular depolymerases are required for aggressiveness. The role of depolymerases in the saprotrophic lifestyle of T. harzianum has not been widely studied. We used both aggressive and nonaggressive strains of T. harzianum to determine (i) the presence of mycoparasitic structures in interaction zones with A. bisporus, (ii) growth rates in compost in the presence and absence of A. bisporus, (iii) the presence of depolymerases induced by cell walls isolated from wheat straw (a major component of compost) and from A. bisporus (the putative host), and (iv) the presence of depolymerases produced in colonized compost.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fungal inoculation of compost.
To simulate green mold disease, T. harzianum and A. bisporus were inoculated into tubes (3 by 5 cm, polypropylene) containing mushroom compost (formula 8; supplied by Mushroom Unit, HRI). A. bisporus spawn (infested rye grain) was rolled in profusely sporulating cultures of T. harzianum and coated with conidia (ca. 1 x 106 conidia per grain) to produce the T. harzianum inoculum. Three grains of this inoculum were placed at the bottom of each tube with three uninoculated spawn grains and covered with ca. 30 g of fresh mushroom compost, which was lightly compressed before the tube was sealed with a lid. There were no significant differences between T. harzianum strains in CFU load per grain (see below). To determine growth by T. harzianum in the absence of A. bisporus, conidia were coated onto rye grain as described above. Tubes were placed in a 25°C incubator with high relative humidity in the absence of light for 3 weeks, and lids were left loose to allow gaseous exchange.
Fungal growth was initially determined visually by using two scales. A. bisporus growth was measured on a scale of 0 (no visible growth) to 3 (full colonization of the tube). The growth of T. harzianum was determined by the distance (in millimeters) at which characteristic green sporulation was detected from the inoculum source. Subsequently, a T. harzianum-selective medium (THSM) was developed to quantify T. harzianum in compost (55).
Quantification of T. harzianum growth in mushroom compost.
The growth of T. harzianum in compost was determined on THSM by counting CFU. Two or three strains from each T. harzianum group were tested in three different batches of commercial compost. Compost was removed from the tube, and a top layer (1 cm thick) containing the inoculum source was discarded. A sample of 10 g was taken from the remaining compost and added to 100 ml of 100 mM sodium tetrapyrophosphate (Sigma, Poole, United Kingdom) in distilled water (13). Samples were steeped for 60 min at room temperature followed by 2 min of continuous agitation in a Stomacher Lab-Blender 400 (Seward Laboratories, London, United Kingdom).
Colony counts were log10 transformed, treated as geometrical with a normal distribution, and subjected to one-way analysis of variance (ANOVA) with Minitab for Windows. The results were compared with analysis of the original counts, which had a Poisson distribution and were analyzed by using Genstat. The two methods gave comparable results, and therefore, ANOVA was chosen for convenience.
The grain inocula for all T. harzianum strains were tested for differences in numbers of conidia. A sample of 10 rye grains of each T. harzianum inoculum was subjected to spore retrieval in 100 mM sodium tetrapyrophophate (2-min agitation in the stomacher), and log dilutions were incubated on five replica plates of THSM. The resulting CFU per grain were analyzed by ANOVA, and no significant differences were found between T. harzianum strains and the numbers of conidia on the grain introduced to the compost. Therefore, direct comparison of the CFU retrieved from compost for each strain was made with no significant biased effects from different quantities of inoculum.
Dual cultures of A. bisporus and T. harzianum.
Dual cultures were produced on agar media, in compost, and on spawn grain. Mycelial disks (5-mm diameter) of A. bisporus and T. harzianum were cut from the advancing edge of colonies and placed 3 cm apart on a cellophane disk on various agar media. Media included distilled water agar (DWA) (2% agar in distilled water), MEA (2% agar and 2% Oxoid malt extract), compost filtrate agar (CFA) (5% blended oven-dried compost was infused in 1 liter of distilled water for 3 h and filtered through four layers of muslin and 2% agar was added, adapted from reference 39), blended compost agar (1 g of blended, autoclaved compost per petri dish covered with 20 ml of DWA). In compost, A. bisporus and T. harzianum inocula were added at opposite ends of a cylinder containing hand-compressed compost. Interactions on spawn grains were followed after adding grains to profusely sporulating cultures of T. harzianum and incubating on 2% MEA. All combinations were incubated at 25°C in the absence of light.
SEM.
Samples of cellophane were removed from the interaction zone, where the two fungal colonies made contact on agar media. Straw samples were taken from interaction zones in compost cylinders, and entire spawn grains were also examined. Samples were attached to a cryoscanning electron microscopy (SEM) stub and then flash frozen in liquid nitrogen. The sample stub was introduced to the initial chamber where ice was removed by sublimation. Subsequently, the sample was sputter coated with gold and viewed in a JEOL (Tokyo, Japan) JSM6310 SEM. Crystals on the A. bisporus mycelial surface were analyzed by energy dispersive X-ray microanalysis (Oxford Instruments AN10000 X-ray analyzer; Gatan, Osney Mead, United Kingdom). Three samples from the interaction zones of two replicates of aggressive and nonaggressive isolates were removed and examined for each treatment.
Induction of T. harzianum depolymerases in vitro.
T. harzianum strains were cultured in 100 ml of basal medium (16) in 250-ml conical flasks and incubated at 25°C in the absence of light for 3 days. The basal medium was buffered to pH 6.0 with 50 mM morpholineethanesulfonic acid (MES) and supplemented with 1% (wt/vol) glucose to produce an extensive mycelium (ca. 500 mg). The resulting mycelium was washed in 50 ml of sterile, basal medium and transferred to an additional 100 ml of basal medium and starved overnight (ca. 16 h). Finally, the mycelium was transferred to 100 ml of basal medium supplemented either with blended (ca. 1-cm lengths) untreated wheat straw (1% wt/vol) or with extracted A. bisporus cell walls (1% wt/vol) (method adapted from reference 51) as the sole source of carbon. Samples of culture fluids were removed after 12, 24, 48, and 72 h and clarified by centrifugation at 3,000 x g.
Extraction of T. harzianum depolymerases from sterile, commercial mushroom compost.
Commercial mushroom compost was autoclaved three times for 1 h at 121°C with intervals of 12 h at room temperature to allow germination of heat-activated thermophiles. Tubes containing compost were then inoculated with T. harzianum and incubated as described above. Samples were collected after 1, 2, and 3 weeks. Enzymes were extracted with a buffer designed to prevent denaturation of the enzymes and to allow maximum desorption from the compost substrate; the buffer comprised 5 mM dithiothreitiol, 200 mM KCl, and 5% polyvinyl pyrrolidone in 50 mM sodium phosphate (pH 6.0) (Sigma) (16). Cold extraction buffer was added to the compost in a ratio of 5 ml:1 g (vol/wt [fresh weight]). Samples were agitated for 1 min in a Stomacher Lab-Blender 400, steeped on ice for 15 min, and agitated again for 1 min in the blender. Solid matter was removed by filtration through two layers of muslin, and the filtrate was clarified by centrifugation at 4,000 x g for 15 min then 23,000 x g for 15 min. The supernatant was dialyzed exhaustively against 25 mM MES (pH 6) at 4°C overnight, and then extracts were concentrated ca. fivefold against 30,000-Mr polyethylene glycol (Sigma) at 4°C. Before isoelectric focusing on gels, samples were desalted with PD-10 columns (Amersham Biosciences, Rainham, United Kingdom) prepacked with G-25 Sephadex.
Enzyme assays.
All enzyme activities were first characterized with respect to optimal pH, temperature, and time for assays (for linear kinetics). Chitinase, laminarinase, xylanase, and cellulase activities were assayed by following the release of reducing sugars from the appropriate polymeric substrates (16). The substrates, 1-mg/ml laminarin, 25-mg/ml colloidal chitin (crab shell chitin), birch wood xylan (all three from Sigma), and cellulose (comminuted, insoluble Whatman no. 1 filter paper) were prepared in 25 mM MES (pH 5.5). The temperatures and incubation times used were 37°C for laminarinase (20 min) and chitinase (60 min) and 50°C for cellulase (20 min) and xylanase (20 min). The general protease activity was determined with azocasein 3% (wt/vol) in 50 mM MES (pH 5.5). To 50-µl test samples, 100 µl of azocasein substrate (both preincubated at 37°C) was added, and the reaction mixtures were incubated for 180 min at 37°C (9). Chymoelastase-like and trypsin-like protease activities were measured against the nitroanilide substrates (Sigma) suc-(Ala)2-Pro-Phe-pNA and benzoyl-Phe-Val-Arg-pNA, respectively (8, 47, 48). Reaction mixtures at 25°C contained 40 µl of test sample, 160 µl of Tris-HCl buffered to pH 8.0 (0.1 M), and 50 µl of substrate (2 mM) in dimethyl sulfoxide. The release of nitroanilide was monitored for 10 min.
Protease inhibitors.
We selected commercially available inhibitors (Sigma) to five classes of protease: metallo-, cysteine, trypsin-like, aspartic, and serine proteases (6). Samples of T. harzianum culture fluids were incubated with EDTA (50 mM), iodoacetic acid (50 µM), leupeptin (50 µM), pepstatin (1 µM), and phenylmethylsulfonyl fluoride (1 mM) for 30 min before protease assays were conducted (47).
Isoelectric focusing of depolymerases.
Samples containing 100 µg of protein were flash frozen in liquid nitrogen, lyophilized, dissolved in 10 µl of distilled water, focused on precast polyacrylamide gels, and calibrated with pI markers of pH 3 to 9.5 (Amersham Biosciences). Chitinase, laminarinase, cellulase, xylanase, and protease activities were detected in replica gels of 1% (wt/vol) agarose in 50 mM MES (pH 6.0) containing the corresponding dye-complexed substrates (final concentration, 3.2 mg/ml): carboxy-methyl-chitin-Remazol brilliant violet, Remazol brilliant blue (RBB)-curdlan, ostazin brilliant red-hydroxyethylcellulose, RBB-xylan, and RBB-gelatin (Loewe GmbH, Berlin, Germany). Other cellulase components, cellobiohydrolase and ß-D-glucosidase were detected with 4-methylumbelliferyl ß-D-cellobioside (Sigma); a replicate gel was soaked in 10 mM gluconolactone in 50 mM MES (pH 5.5) for 10 min, to inhibit ß-glucosidase, before exposure to 4-methylumbelliferyl ß-D-cellobioside (17). Agarose overlays were usually removed after 30 min of incubation at temperatures predetermined as optimal for enzyme assays (usually 37°C, but 50°C for xylanase) and destained (50 mM acetate buffer [pH 5.4]:ethanol [96%], 1:2 [vol/vol]) to reveal activity as achromatic bands on a colored background (7). Distances migrated by the isozymes were recorded with transmitted light to maximize the contrast between the background and the achromatic zones on destained gels.
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RESULTS |
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Depolymerases. (i) Total enzyme activities on A. bisporus cell walls.
Isolates from all groups of T. harzianum secreted abundant extracellular chitinase, laminarinase, and protease when exposed to A. bisporus cell walls as the sole carbon source in liquid cultures. The concurrent appearances and maximum levels of protease (A405 of 0.37) and chitinase (2,033 nkat ml-1) after 24 h of growth by isolate T7 were followed by that of laminarinase (1,123 nkat ml-1) after 48 h. There was no significant difference between the groups in protease, chitinase, and laminarinase total activities.
The chymoelastase-like and trypsin-like activities of two representative aggressive isolates were significantly higher after 12 h of induction than those from nonaggressive groups. Typically, chymoelastase activity was 9.6- and 12.2-fold higher for Th2 and Th4, respectively, and trypsin-like activity was 4.9- and 6.3-fold higher for Th2 and Th4, respectively, than the activities of nonaggressive isolates. However, after 24 h, nonaggressive isolates had produced similar maximal levels of protease activity (data not shown). For all isolates (three from each group), chymoelastase-like activity was completely inhibited by phenylmethylsulfonyl fluoride, which is characteristic of serine proteases, and slightly inhibited by leupeptin (9.4%). The trypsin-like activity of all isolates (one per group) was significantly inhibited by leupeptin (89%), EDTA (58%), and pepstatin (37%).
(ii) Depolymerase isoforms on A. bisporus cell walls.
The main isoforms identified on isoelectric focusing gels are summarized in Table 2. The European groups, Th1, Th2, and Th3, all produced (after 12 and 24 h) a dominant laminarinase isoform (pI 4.83) while Th4 (North American) secreted an isoform of pI 6.25 and several minor acidic bands (Fig. 2a).
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(iii) Total enzyme activities on wheat straw.
All groups of T. harzianum secreted cellulase, laminarinase, protease, and xylanase with wheat straw as the sole carbon source in liquid cultures. There were no significant differences between aggressive and nonaggressive strains with respect to the main activities, which were cellulase and xylanase (5 replicates per treatment). Cellulase levels were maximal at 48 h and ranged from 500 to 1,600 nkat/ml; xylanase was still increasing at 72 h when activities were 2,000 to 2,900 nkat/ml.
(iv) Depolymerase isoforms on wheat straw.
The protease and laminarinase isoforms were very similar, after 24 h of growth, to those produced on A. bisporus cell walls, although the pIs differed slightly. The main protease isoform for Th1 and Th3 had a pI of 6.06 (although after 12 h there was a doublet that included a pI 6.25 isoform) while Th2 produced a dominant isoform with a pI of 7.08 (Fig. 2d). Laminarinase again had a simple profile with a major band with a pI of 4.98 for Th1, Th2, and Th3 and a main form with a pI of 6.19 for Th4 (Fig. 2e). There were numerous endo-ß-1,4 glucanases (cellulases) for all groups after 24 h. The Th3 profile was distinct with ca. six acidic bands and one with a pI of 6.19. The Th4 profile also was clearly different except for a major alkaline band shared with Th1 and Th2. Th1 and Th2 isoforms were very similar with ≤4 main bands with pIs of 4.7 to 5.74 (Fig. 2f). Several ß-glucosidase isoforms were detected after 12 h, and cellobiohydrolases were detected after 24 h of incubation. The profiles were not linked with aggressiveness, but Th3 again appeared distinct (data not shown). Xylanase profiles of all isolates were similar, with several predominantly acidic forms and a doublet at pIs 9.24 and 9.37 (Fig. 2g). However, Th3 was again distinct with two additional acidic forms.
(v) Total enzyme activities in compost.
Significant activities of proteases and three main glycanases were readily detected from buffer-extracted compost colonized by T. harzianum. Total activities after 14 days of growth (data not shown) did not reveal consistent association with aggressiveness. Xylanase activity predominated at 600 to 1,000 nkat/ml and laminarinase activity ranged from 200 to 780 nkat/ml, whereas cellulase activity was only 40 to 150 nkat/ml. Group 3 was the only one that produced detectable chitinase activity (50 nkat/ml).
(vi) Depolymerase isoforms in compost.
Isoenzyme profiles obtained from extracts after 14 days of growth, when activities were highest, were quite different from those secreted in culture, although some isoforms were common to both situations.
Group 3 (TD7) produced abundant (ca. 15) cellulase isoforms with a broad pI range of 4.43 to 8.99. The numerous alkaline forms were generally absent from liquid cultures. All groups produced an isoform with a pI of 4.69. Isolate Th1(c) secreted two additional isoforms with pIs of 6.97 and 7.30 (Fig. 2 h). As with cellulase, nonaggressive group 3 produced substantially more xylanase isoforms than the other three groups. The group 3 profile primarily consists of alkaline isoforms not present in vitro and not produced by any other isolates. Another band unique to growth in compost with a pI of 7.36 was from Th1 and Th4, and an isoform with a pI of 8.92 was from Th1, Th3, and Th4 (Fig. 2i). Laminarinase isoforms were similar from all groups and far more complex than the single dominant form (pI 4.98 or 6.19) observed in vitro. The latter isoform from liquid culture (pI 6.19) may be analogous to that of pI 6.25 in compost. From compost, >5 forms were present, but they were not well resolved (Fig. 2j).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Depolymerases of T. harzianum are probably essential for both saprotrophic and parasitic growth. We detected these enzymes in T. harzianum-infested compost and found that, in some cases, the speed of enzyme production or the presence of unique isoenzymes (Table 2) distinguished the aggressive and nonaggressive isolates. Similar isoforms were generally present whether wheat straw or mushroom cell walls were used as the inducing substrate. Chitinases, laminarinases, and proteases produced on A. bisporus walls could function in the degradation of the host's cell wall (3, 33). The difference in protease production between T. harzianum groups, apparent after only 12 h of induction and the much greater production of a protease isoform (pI 6.22) by aggressive biotypes, could be linked with mycoparasitism. Proteases have been implicated previously in the degradation of fungal host cell walls by mycoparasites (23, 25). Aggressive isolates of T. harzianum also had unique chitinase isoforms with pIs of 7.23 and 7.36 after 12 h of induction. The concurrent synthesis of chitinase and protease may result from the induction of some proteases by chitin-containing substrates (25). Some T. harzianum endochitinases appear particularly potent in terms of antifungal activity, which might justify further study of chitinase isoforms specific to aggressive isolates (30). Geographic origins of isolates could be distinguished by an acidic laminarinase isoform (pI 4.83) in the European groups (Th1, Th2, and Th3) and a more alkaline isoform (pI 6.25) in the North American group (Th4). On straw in vitro and during axenic growth in compost, xylanase activity was higher than that of ß-1,3 glucanase and cellulase. Cellulose is the key structural domain, and xylan is the major matrix component of cereal cell walls (10), with most fungal pathogens of cereals using arabinoxylanases as their main cell wall-degrading enzymes (9, 16). Noncellulosic, mixed-linkage ß-D-glucans are a matrix component in wheat straw and may suffice to induce the glucanase (10).
This report is the first to characterize fungal depolymerase isoenzymes from axenic growth of T. harzianum in compost. Glycosidases from T. harzianum isolates have been detected in compost (43), but total activities were insufficient to distinguish isolates differing in aggressiveness, as were the total activities of glycanases produced on straw and in compost measured in this study. Some of the isoforms in compost, e.g., most of the detected xylanases, had pIs similar to those produced on straw in vitro, which suggests that straw degradation is a key factor in compost colonization. However, some isoforms, especially that of laminarinase, were only produced by T. harzianum in compost. This pattern may reflect the induction of new isoforms or be an experimental artifact resulting from complexing with phenolic components of compost.
In all three forms of culture, Th3 (European nonaggressive) isolates had isozyme profiles that differed from those of the other groups. Chitinase activity was recovered only from compost colonized by Th3 isolates. This group has been reclassified as T. atroviride (based on ITS1 sequence homology) (35) and has a slower growth rate (42). Th3 generally grows poorly in spawned compost and does not affect yield (46), although there have been occasional reports of Th3 isolates extensively colonizing mushroom compost (22).
In conclusion, the only consistent feature linked with the aggressiveness of Th2 and Th4 isolates was their ability to colonize mushroom compost rapidly and extensively, even in the absence of A. bisporus. Prolific production of depolymerases could contribute to both mycoparasitic or saprotrophic growth. Thus, ß-1,3 glucanases have substrates in A. bisporus and in wheat cell walls while chitinases and proteases may facilitate saprophytic growth on the rich fungal and bacterial compost microflora (some chitinases possess dual lysozyme activities) (50). Some depolymerases appeared to be associated with aggressiveness. Prima facie, these enzymes are candidates for analysis of their roles in aggressiveness by site-directed mutagenesis. However, this approach may be difficult because most fungi have multiple depolymerases and can compensate for the loss of any single enzyme with related enzyme(s) or by expressing a previously latent gene(s) (8). Our results strongly suggest that the antagonism of T. harzianum to A. bisporus is not primarily the result of mycoparasitism. Instead, the key factor seems to be best described as competitive saprophytic ability (24) and is consistent with the hypothesis of Deacon and Berry (19) that competition for nutrients is the most general mechanism in biocontrol. However, the ability of some Th3 isolates to colonize sterilized compost at levels similar to those of the aggressive groups and to produce equivalent levels and ranges of depolymerases indicates that these factors alone do not explain aggressiveness. Nonetheless, aggressiveness is dependent on extensive saprophytic colonization and, presumably, on associated competition, but competition and colonization could be a prerequisite to antagonism to A. bisporus, of which mycoparasitism could be but one of several components.
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ACKNOWLEDGMENTS |
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J.W. was supported by a BBSRC CASE studentship.
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FOOTNOTES |
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Present address: Molecular Sensing plc, Melksham, Wiltshire SN12 8LH, United Kingdom.
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REFERENCES |
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