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United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology Laboratory, Rm. 304, B-011A, BARC-W, Beltsville, Maryland 20705
Walter Gams
Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 TC Utrecht, The Netherlands
Lisa A. Castlebury
United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology Laboratory, Rm. 304, B-011A, BARC-W, Beltsville, Maryland 20705
Orlando Petrini
Tèra d'Sott 5, CH-6949 Comano, TI, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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Trichoderma aggressivum sp. nov. and T. aggressivum f. europaeum f. nov. are described. These forms cause the green mold epidemic in commercially grown Agaricus bisporus in North America and Europe, respectively. In the literature they have been reported as T. harzianum biotypes Th 4 and Th 2, respectively. They are strongly separated from their closest relative, T. harzianum, in sequences of the ITS-1 region of nuclear rDNA and an approximately 689 bp fragment of the protein coding translation elongation factor gene (EF-1
). They are distinguished from the morphologically similar T. harzianum and T. atroviride (the latter also known as biotype Th 3) most readily by rate of growth. Of these, only T. harzianum grows well and sporulates at 35 C, while T. atroviride is the slowest growing. Trichoderma aggressivum f. aggressivum and f. europaeum are effectively indistinguishable morphologically although they have subtly different growth rates at 25 C on SNA and statistically significant micromorphological differences. Based on findings of this study, descriptions of T. harzianum and T. atroviride are expanded. A key to Trichoderma species commonly found associated with commercially grown A. bisporus is provided.
Key words: green mold disease, Hypocrea, Hypocreales, ITS, systematics, translation elongation factor 1 alpha gene (EF-1), Trichoderma aggressivum, Trichoderma atroviride, Trichoderma harzianum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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) and most recently in Spain (Hermosa et al 1999
). In each case, the causative fungus colonizes compost and results in crop loss. The seriousness of the disease is indicated by losses of 30–100% experienced in 1995 in Chester County, Pennsylvania (Seaby 1998
).
Four genetically distinct "biotypes," all initially identified as Trichoderma harzianum Rifai, have been associated with the disease (Muthumeenakshi et al 1994
, Castle et al 1998
, Gams and Meyer 1998
, Ospina-Giraldo et al 1998, 1999
, Chen et al 1999 a, b
, Hermosa et al 1999, 2000
). However, of these only those designated as Th 2, from Europe, and Th 4, from North America, are associated with appreciable loss. Biotype Th 1 is now recognized as T. harzianum s. str. as it includes the ex-neotype strain of T. harzianum (Gams and Meyer 1998
). It also includes biological control strains (Hermosa et al 2000
). None of the strains of this biotype cause losses in mushroom production despite their occurrence in mushroom growing houses. Biotype Th 3 has been reidentified as the common saprobe T. atroviride P. Karst. (Gams and Meyer 1998
, Castle et al 1998
, Dodd et al 2000
).
Trichoderma harzianum is commonly cited in diverse publications. It is frequently encountered in cultivated and natural soils (e.g., Roiger et al 1991
, Danielson and Davey 1973
). It is used in biological control of fungi that induce plant diseases (e.g., Papavizas 1985
, Chet and Inbar 1994
, Agosin and Aguilera 1998
, Hjeljord and Tronsmo 1998
, Hermosa et al 2000
) and it has growth-promoting capacities that may or may not be integral to biological control (e.g., Bailey and Lumsden 1998
, Harman and Björkman 1998
, Jinantana and Sariah 1998
). Degradation of organochlorine pesticides is attributed to it (Katayama and Matsumura 1993
). A new trichothecene toxin, harzianum A[1], is produced by T. harzianum (Corley et al 1994
), and T. harzianum was found to prevent synthesis of aflatoxin B1 by Aspergillus flavus in liquid culture medium (Shantha 1999
).
Given the strong biological divergences between the aggressive biotypes Th 2 and Th 4 and the innocuous T. harzianum s. str., there is reason to suspect that the former are not T. harzianum. Although several studies have suggested phenotypic or phylogenetic distinction between the aggressive biotypes and the non-aggressive Th 1 (T. harzianum) and Th 3 (T. atroviride), no formal taxonomy has been proposed.
Analyses based on molecular sequence data suggest that Th 2 and Th 4 are phylogenetically distinct from Th 1 (Muthumeenakshi et al 1994
, Castle et al 1998
, Gams and Meyer 1998
, Ospina-Giraldo et al 1998, 1999
, Chen et al 1999a, b
, Hermosa et al 2000
). Most phylogenetic analyses of the ITS region of rDNA have included aggressive biotype Th 2, but not Th 4. In a consensus tree clustering of Th 1 and Th 2 was only weakly supported (54% bootstrap) but Th 1 isolates were distinguished from Th 2 isolates with 75% bootstrap support (Gams and Meyer 1998
). Hermosa et al (2000)
found modest support (71% bootstrap in a NJ tree) for a group that included one strain of Th 4 and two of Th 2. Ospina-Giraldo et al (1999)
are the only authors to have included a large number of strains of all three T. harzianum biotypes in an ITS analysis. Their data, however, do not support their main conclusion that the aggressive biotypes are genetically distinct from biocontrol strains (Th 1). Chen (1998)
and Romaine et al (1999)
included several each of Th 2 and Th 4 in analyses of ß-tubulin sequences, and the aggressive biotypes were resolved independent of each other in a single clade that received 100% bootstrap support in NJ analysis. Also RAPD-PCR analysis reliably distinguished the aggressive from the non-aggressive biotypes (Chen et al 1999a, b
).
If there is a solid phenotypic and genotypic basis for it, there should be a taxonomic distinction between a fungus that is capable of commercially effective control of fungus-induced plant disease and other beneficial or neutral activities and a fungus that causes major losses in a crop of considerable economic importance. In this paper, we identify significant distinguishing phenotypic and genotypic character differences, and propose a species- and form-level taxonomy to reflect them.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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) and TEF1 rev (5'-GCC ATC CTT GGA GAT ACC AGC-3') (Christian Kubicek pers comm). PCR reactions were performed with an initial 2 min at 94 C followed by 30 cycles of 30 s at 94 C, 30 s at 55 C and 1 min at 72 C, followed by a final 10 min at 72 C. Template DNA for sequencing was prepared directly from PCR products with the QIAquick PCR purification kit (Qiagen, Valencia, California). DNA sequences were determined using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California). Sequence reactions (20 µL total) were performed according to the manufacturer's instructions and the products were analyzed directly on an ABI PrismTM 377 DNA Sequencer (Applied Biosystems). Both strands were sequenced for each isolate using the primers EF1–728F (Carbone and Kohn 1999
) and TEF1 rev.
Molecular analyses –
Published ITS-1 sequences were retrieved from GenBank for 36 isolates representing the T. harzianum morphological complex, Trichoderma sect. Trichoderma and sect. Longibrachiatum (Table I
, Fig. 1
). ITS-1 sequences were determined for five isolates of the T. harzianum complex from Cameroon (Table I
) using the methods described. In addition, two strains that had been identified by Ospina-Giraldo et al (1998)
as Th 4 (C.P.R. 170 = G.J.S. 99–30, AF05764; C.P.R. 178 = G.J.S. 99–29, AF057644) were resequenced because in preliminary trees using the deposited sequences neither clustered with Th 2 nor Th 4.
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) was amplified, sequenced and compared for 21 isolates of T. harzianum and nine representative isolates of sect. Trichoderma. Within the T. harzianum morphological complex isolates of all three recognized ITS sequence types (Th 1, Th 2 and Th 4) were represented. Representative strains of the former Th 3 biotype, now known to be T. atroviride, were also included (Table I
).
Raw DNA sequences were aligned and edited using SEQUENCHER 3.1 (Gene Codes, Ann Arbor, Michigan) and ClustalX 1.81 (Thompson et al 1997
), respectively. Alignments were manually adjusted using the program Genedoc 2.5.000 (Nicholas et al 1997
). Within PAUP 4.0b3a (Sinauer Associates, Sunderland, Massachusetts), unrooted trees were produced with neighbor-joining (NJ) analysis for ITS-1 sequence data and both NJ and maximum parsimony (MP) analysis for EF-1. The Kimura-2 parameter distance calculation was employed for NJ analysis and 1000 bootstrap replications were performed to assess the relative stability of the branches. For parsimony analysis, the heuristic search option with 1000 random addition sequences, MULPARS on and TBR branch-swapping options were employed. Relative stability of clades was assessed with 500 parsimony bootstrap replications. To root the ITS-1 and EF-1 trees, sequences from T. sects. Longibrachiatum and Trichoderma were added as the outgroup because these sections have previously been shown to be outside of the group that include T. harzianum (Kindermann et al 1998
). For the EF-1 trees, sequences from T. sect. Trichoderma were used as the outgroup.
Phenotype characterization –
The ninety-nine strains of T. harzianum biotypes Th 1, Th 2, Th 4 and T. atroviride (formerly Th 3) examined for this study are listed in Table I
. Most of the strains identified as biotype Th 4 had previously been identified by RAPD analysis (Chen et al 1999a
), Th 2–4 specific PCR (Chen et al 1999b
), and/or ß-tubulin sequences (Romaine et al 1999
, P. Romaine pers comm). The Th 2 strains had been previously reported in the literature by Muthumeenakshi et al (1994)
. Several of the Th 1 strains were identified on the basis of their morphological similarity to strains for which there were either published or newly generated DNA sequences.
Growth rates were determined on PDA and SNA, a defined, low-sugar medium (Nirenberg 1976
). Vigorously growing colonies were established on cornmeal dextrose agar (CMD, Difco cornmeal agar +2% (w/v) dextrose) at 20 C. After a few days when the colonies were visibly growing, but before conidial production, a 5 mm-diam plug was taken from the actively growing edge of the colony and inoculated onto freshly prepared medium. Vented 9 cm-diam plastic Petri dishes contained 20 mL of freshly made medium. The inoculum plug was placed mycelium-side-down approximately 1.5 cm from the edge of the Petri dish. The Petri dishes were incubated in darkness (except for the brief exposure to cool white fluorescent light when they were measured) at 15, 20, 25, 30 and 35 C. They were examined at 24-hourly intervals when the colony radius, measured from the edge of the inoculum plug, was recorded as was colony appearance, time of first appearance of green conidia, and any yellow pigmentation in the medium or conidia. Each growth trial consisted of a single Petri dish for each strain at each temperature. The growth trials were repeated three times at roughly weekly intervals, and the average radius was taken from the three independent measurements.
Colony characters were observed from PDA and CMD. All micromorphological data were taken within 1 wk from colonies grown on CMD containing the antibiotics streptomycin and neomycin at 20–21 C under conditions of 12 h darkness/12 h cool white fluorescent light. All phenotypic characters, including morphology and colony characters, are given in Table II . The slope is the linear growth of the colony per hour in mm. Descriptive statistics were used to evaluate the phialides. Phialide range (the difference between the longest and shortest phialide observed), standard deviation, and variance were used as measures of spread.
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Phenotype analysis –
Exploratory data analysis was undertaken using Systat 8.0 (Wilkinson 1996
). Strains were grouped according to their molecular type into putative species, viz. "T. harzianum s. str.," Th 2, Th 4 and T. atroviride. Thirty-seven characters were derived from the phenotype (Table II
). The mean for each micromorphological character was determined for each putative species. A new worksheet that included the means for each species was prepared, containing the matrix to be used for ordination analysis. Discrete or binary characters taken from colony appearance and also data derived from growth trials were inserted into the matrix. The measurements reported in Table II
are minimum, maximum, mean ± standard deviation and upper and lower 95% confidence intervals. These data are based on the mean measurement of each object in each collection.
Principal coordinates analysis (PCO, Kovach 1999
) was used as a multivariate ordination analysis to detect consistency of phenotype characters within the DNA-defined groups. PCO is a generalized form of principal components analysis used to study the correlations among a large number of variables. The chi-squared distance measure was used.
Discriminant analysis was performed on a reduced set of characters using Systat 8.0 to verify the goodness of fit of the outcome of the ordination analysis, but the results are not presented in detail.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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Alignment and comparison of the 689 bp fragment of the EF1- for the 21 representatives of the T. harzianum group and 9 representatives of T. sect. Trichoderma revealed 378 constant, 70 variable and 241 parsimony-informative characters (Fig. 2
, Treebase number S634). Although NJ and MP analyses of the ITS-1 and EF1- alignments produced different tree topologies (compare Figs. 1, 2
), there were no apparent conflicts between the two. Two main groups were revealed within the T. harzianum complex: one (Th 1) containing the ex-type cultures of T. harzianum and T. inhamatum, biocontrol strains and other non-mushroom-aggressive strains, and one containing only the two mushroom-aggressive types, Th 2 and Th 4. The Th 1 group was not well supported by the bootstrap test (56%) but within this group there were two strongly supported groups. One of these groups contained the ex-neotype culture of T. harzianum (CBS 226.95). The T. inhamatum ex-type culture (CBS 273.78) also grouped in the Th 1 group, forming a weakly supported group (64% bootstrap) with one other strain. In contrast to Th 1, the mushroom-aggressive group was well supported and within it the Th 2 strains formed a group of their own, which also had good bootstrap support (99%). The fourth biotype, T. atroviride, consistently grouped among the outgroup T. sect. Trichoderma strains.
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Most of the taxonomically useful characters were derived from growth rate, colony appearance (Fig. 5 ) and odor. Growth rates on PDA (Fig. 4A ) and SNA (Fig. 4B ) were the most easily observed and consistent characters. Although repeating each experiment independently three times helped to minimize inconsistencies in conditions of the experiment, there were often differences among the three radius measurements for most of the strains. The median difference between the minimum and the maximum measurements for individual strains grown on PDA for 64 h at 30 C was 8.1 mm. For twenty-five strains the differences had a range of 12 mm or more and seven strains showed spreads of 20 mm or more. In general T. harzianum was the fastest growing and grew best at higher temperatures whereas T. atroviride grew slowest at all temperatures.
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Growth of all strains was slower on SNA than on PDA. However, a somewhat greater differentiation among the groups was seen in growth rates on SNA (Fig. 4B ) than on PDA. At 25 C on PDA the various biotypes did not exhibit different growth rates. On SNA T. atroviride grew considerably more slowly than any of the other groups. At 25 C on SNA, Th 4 grew significantly faster than all other groups. At 30 C on SNA Th 2 was somewhat slower than T. harzianum and Th 4. At 35 C on both PDA and SNA T. harzianum grew well and sporulated whereas the other biotypes were barely able to grow; this difference was especially marked on PDA.
A coconut odor was constantly associated with colonies of T. atroviride grown on CMD and PDA (less pronounced on SNA) but no distinctive odor was detected in the other biotypes.
Mature conidia of the Trichoderma strains included in this study are green, but the first sign of conidial production is white tufts. Green conidia of T. harzianum Th 1 and T. atroviride were first observed on average within 44–50 h in cultures grown on PDA in darkness at 25 C. At 30 C green conidia were seen in cultures of T. harzianum within, on average, 44 h and as early as 24 h. Green conidia were not seen in cultures of T. atroviride, Th 2 or Th 4 until 15–20 h later. Overall, conidia were slower to form on SNA than on PDA, and were slowest to form in Th 2. This was especially evident at 20 C on PDA when green conidia in Th 2 were not observed, on average, before 75 h, which is approximately 10 hours later than in T. harzianum, T. atroviride and Th 4.
On PDA after 96 h at 30 C in darkness (Fig. 5 ) conidial production of all three groups is diffused rather than being pustulate. The entire culture of T. harzianum is conidial whereas conidial production in the other groups is restricted to concentric rings and/or the center of the colony. At 30 C conidial production in T. atroviride is also restricted to concentric rings.
On CMD after 96 h at 30 C in darkness (Fig. 5 ) conidia of T. harzianum are uniformly dispersed throughout the colony, although one or more concentric rings of conidial production may be visible in addition to the dispersed conidiation. Conidia of T. atroviride are more or less restricted to pronounced concentric rings with the advancing edge of the colony still sterile. In the aggressive biotypes, conidia are formed in pustules. The pustules may be large, up to 5 mm diam, and discrete, or much smaller and then coalescent, often forming concentric rings or a dense broad band at the edge of the colony. The aspect of disposition of conidia in the culture of the aggressive biotypes can vary from one growth trial to the next, but in contrast to T. harzianum and T. atroviride, the tendency to form pustules is usually strong. The characters of colonies of the various groups grown for one week are less marked than at 96 h, especially on PDA, but differences are still evident (Fig. 5 ).
There is a tendency for cultures of T. harzianum to produce a diffusing yellow pigment (28 of 35 cultures). One culture of T. atroviride produced a yellow pigment (G.J.S. 97–26). Distinct yellow pigmentation was observed in young conidia of 5 of 31 strains of Th 4, but never a diffusing pigment. Yellow pigment was not observed in any of the 5 cultures of Th 2 that we studied.
On the basis of their micromorphology, all of the strains that we studied could be assigned, respectively, to T. harzianum in the sense of Gams and Meyer (1998)
(Figs. 6–16
) or T. atroviride (Bissett 1992
) (Figs. 31–37
). All have green conidia that are approximately 3 µm diam, smooth and subglobose to slightly ovoidal, with a mean L/W of 1.1–1.2. Conidia of T. atroviride are significantly longer and wider than are those of the other groups, and conidia of T. harzianum are significantly shorter and narrower than all the others. Conidial sizes of the two aggressive biotypes are not statistically different from each other. Conidia of all are dark green, but the green of conidia of all biotypes except T. atroviride tends slightly toward yellow whereas conidia of T. atroviride lack yellow. This is especially noticeable for T. atroviride in colonies grown on CMD at 25 C (Fig. 5
).
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Most Trichoderma species eventually produce chlamydospores in the hyphae. These are remarkably uniform in appearance (Figs. 16, 37 ), being unicellular, subglobose to clavate, and smooth. Under the specific conditions of this research, where the presence or absence of chlamydospores was evaluated within one week in cultures grown on CMD at 20 C with alternating cool-white fluorescent light and darkness, all 22 strains of T. atroviride produced chlamydospores. In contrast, chlamydospores were found in 9 of 33 strains of T. harzianum and one each of Th 2 and Th 4. In the collection of Th 4 that produced chlamydospores, only 11 were found. The chlamydospores found in T. atroviride and Th 4 were the same size and significantly larger than those produced by T. harzianum and Th 2, which were indistinguishable in size.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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provides much greater resolution of the representatives of the T. harzianum complex than did ITS-1 sequences, a result that parallels what O'Donnell (2000)
found in Fusarium. Chen (1998)
observed good resolution of biotypes Th 1, Th 2, and Th 4 using ß-tubulin.
Sequences of the EF-1 not only clearly separated the aggressive biotypes from Th 1 but also distinguished them from each other, with Th 4 appearing basal to Th 2. These results correlate well with those of principal coordinate and discriminant analyses of phenotype. These independent but complementary results lead us to conclude that the aggressive biotypes are taxonomically separable from T. harzianum and we propose below the new species, T. aggressivum, for the aggressive biotypes Th 2 and Th 4. In addition to its specialization for growth on mushroom compost, T. aggressivum is recognized by its slow growth at 35 C on PDA and SNA, late in vitro sporulation, and by the tendency for its conidiophores to be less uniformly branched than in T. harzianum.
The biotypes Th 2 and Th 4 are also taxonomically distinct from each other. Hermosa et al (2000)
distinguished them on the basis of their ITS-1 sequences. Using RAPD analysis, Chen et al (1999b)
found high genetic similarity between Th 2 and Th 4 but concluded that they were distinct clones that arose independently of each other. Sequences of EF-1 also confirm that Th 2 and Th 4 are sister groups. Given the colony differences noted by Seaby (1996)
and here, and the geographic isolation of the respective biotypes, we regard the European and American biotypes as biological forms of T. aggressivum. Our results do not support a derivation of the aggressive biotypes from T. harzianum. Rather they suggest that T. harzianum and the aggressive biotypes have had independent origins.
The origins of the aggressive biotypes and their closest relatives are still completely unknown. ITS-1 sequences of an isolate of a species of Hypocrea (CBS 978.70) that has green ascospores and that occurs on decorticated wood in the Netherlands are identical to those of T. aggressivum f. europaeum (Gams and Meyer 1998
). However we doubt that T. aggressivum is the anamorph of that Hypocrea because of morphological differences. Furthermore, conidia of CBS 978.70 are much larger, 4–6 x 2.5–4.0 µm, than in T. aggressivum and are ellipsoidal (Samuels unpubl).
Gams and Meyer (1998)
did not search for phenotypic differences between the European biotype, Th 2, and T. harzianum, despite the differences in ITS-1 sequences. Castle et al (1998)
only commented that the two aggressive biotypes differed from the type of T. harzianum in having a less regular branching system, again despite finding differences in ITS-1 sequences and RAPD patterns. Biotypes Th 2 and Th 4 are not as regularly branched as is typical T. harzianum, but this difference can be difficult to observe for those not familiar with typical T. harzianum. The illustrations presented here (Figs. 6–14, 17–21, 23–29 ) show the variations in branching.
Seaby (1996)
distinguished between the aggressive mushroom competitors and Th 1 using colony characters and conidium size. Colony morphology does tend to be constant within a species but variation must be allowed for and it is difficult to interpret colony morphology of an uknown without reference to an identified culture. Figure 5
shows variation in colony morphology on different media after two different time intervals. Conidia of T. harzianum consistently form much earlier than in any of the other groups and T. harzianum grows faster than any of the other groups. In general, on CMD and SNA, conidia of Th 2 and Th 4 tend to be formed in more or less distinct pustules, although the pustules may be confluent and thus obscure, while conidiophores of T. harzianum and T. atroviride are more diffuse. Because our sample of Th 2 strains was too small to provide a good picture of taxon variability we do not emphasize colony morphology in our taxonomy. We did not find any significant differences in conidium size between Th 2 and Th 4. Both have slightly larger conidia than T. harzianum, at least when measured on CMD.
The correlation between the DNA-defined groups, T. harzianum s. str., Th 2, Th 4 and T. atroviride and their respective phenotypes was almost perfect. Despite its diverse phenotype, T. harzianum was clearly separated from the other groups. The analysis depends upon study of a large number of geographically separated specimens and cultures in order to accurately account for intra-taxon variation. Multivariate analysis of quantifiable phenotype characters was an effort to document both the phylogenetic proximity and to highlight diagnostic differences in phenotype. In this regard, the power of using independently derived data—in this case DNA sequence data—as a grouping factor cannot be overstated.
It is now widely acknowledged that Th 3 is T. atroviride in sect. Trichoderma (Gams and Meyer 1998
, Hermosa et al 2000
) despite the difficulty in distinguishing between T. atroviride and T. harzianum on the basis of morphology alone. Our results augment Bissett's (1992)
redescription of the species by showing that T. atroviride is slow-growing relative to T. harzianum and is unable to grow, or grows poorly, at 35 C on PDA and SNA. In addition, chlamydospores form in abundance on CMD in cultures of T. atroviride within one week, unlike T. harzianum where chlamydospores are less common and are also smaller than in T. atroviride. Finally, T. atroviride is characterized by a strong odor of coconut in PDA cultures. With these additional characters T. atroviride can be easily identified.
Several recent publications indicate that even the restricted morphological concept of T. harzianum (Bissett 1991
, Gams and Meyer 1998
) is genetically and phenotypically diverse. In the present work T. harzianum was shown to be highly variable in most of its phenotype characteristics. Grondona et al (1997)
also found variation in physiological and biochemical characters for the species. Furthermore, there is substantial variation in the chromosome arrangements of different strains of the same Trichoderma species, including Trichoderma harzianum (Gomez et al 1997
, Harman et al 1998
). The apparent large number of ITS (e.g., Fujimori and Okuda 1994
, Muthumeenakshi et al 1994
, Gams and Meyer 1998
, Ospina-Giraldo et al 1998
, Dodd et al 2000
, Hermosa et al 2000
, Kullnig et al 2000
) and TEF-1 genotypes indicates significant allelic variation in these genes.
At least some of the genetic and phenotypic diversity could be accounted for by sexual or parasexual outcrossing, as has been suggested for Aspergillus flavus by Geiser et al (1998)
. Although T. harzianum has not been linked to a teleomorph yet we have found a likely teleomorph in a Hypocrea that is very similar to T. harzianum in its ITS-1, EF-1 sequences and anamorph phenotype (E. Lieckfeldt pers comm, P. Chaverri pers comm). In addition to sexual lineages in the species, the existence of diversity in chromosomal arrangement argues for the existence of genetically distinct and possibly clonal lines within T. harzianum.
This diversity leads to the possibility that T. harzianum is a species complex. Whether it can be further subdivided is beyond the scope of the present research. Publications on T. harzianum such as those of Grondona et al (1997)
, Hermosa et al (2000)
, Kullnig et al (2000)
and the present work beg a more complete, polygenic and polyphasic study of the species that includes many more strains than any of the individual studies have included. Additional phenotype characters, such as isozymes and physiological characters, and additional protein-coding genes should also be evaluated. Models for such a study include elements of the work of Lieckfeldt et al (1999)
with Trichoderma, O'Donnell et al (2000)
with Fusarium, and Harrington et al (2001)
with Ophiostoma.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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1. Trichoderma harzianum Rifai, Mycol. Pap. 116:38. 1969. Figs. 6–16
Optimum temperature for linear growth on PDA 30 C, on SNA 25 C. After 96 h at 30 C in darkness on PDA conidia filling most Petri dishes; conidia formed densely over the center and in undulating concentric rings toward the edge; no pustules formed; in many colonies conidia first yellow, becoming yellow-green; often with yellow pigment diffusing in medium. After 96 h at 30 C in darkness on CMD, Petri dish filled with conidia that are dark green, uniformly dispersed except for the center of the colony which remains sterile, little tendency to form pustules although ill-formed cottony pustules are formed to a greater or lesser extent in individual colonies. On PDA at 30 C in darkness, green conidia first seen in most strains at 40–48 h with the earliest seen after 24 h (G.J.S. 95–69).
Uniformly branched conidiophores common and often forming over 150 µm of the length of terminal branches. Within these systems branches tend to be paired with the longest branches forming near the base of the system and nearest the main axis. Branches toward the tip and secondary branches tending to be held at 90° with respect to the axis from which they arise; further from the tip of the branching system the angle of branching tends to less than 90° with respect to the axis above. Cells supporting the phialides equivalent in width to, or at most only slightly wider than, the base of phialides arising from them. Phialides typically held in whorls of 2–4 and held at 90° with respect to the hyphae from which they arise, or solitary; those in whorls typically flask-shaped, enlarged in the middle, sharply constricted below the tip to form a narrow neck and slightly constricted at the base. Terminal phialides in a whorl or solitary, typically cylindrical or at least not conspicuously swollen in the middle and longer than the subterminal phialides. Intercalary phialides not observed. Conidia subglobose to ovoidal, lacking a visible basal abscission scar, smooth. Chlamydospores not observed in most cultures, globose to subglobose, terminal or intercalary in hyphae.
2. Trichoderma aggressivum Samuels & W. Gams, sp. nov. forma aggressivum Figs. 17–22
Trichodermati harziano Rifai simile sed conidiophora magis irregulariter ramosa; phialides (4.0–)5.7–7.8(–21.0) x (1.3–)2.7–3.5(–4.3) µm (in parte latissima); chlamydosporae raro formate, 5.3–8.7 µm diam; coloniae lentius crescentes in agaro PDA temperatura 35 C. In America boreali.
HOLOTYPUS: ex culto sicco in agaro DAOM 222156 (BPI 748201).
Optimum temperature on PDA 25–30 C, on SNA 30 C. After 96 h in darkness at 30 C conidia forming in a restricted central 3–4 cm-diam area as well as in a raised marginal band of green adjacent to the plastic edge of the Petri dish; on CMD the amount of conidial production variable depending upon the culture; typically no yellow pigment diffusing into the agar (CBS 450.95). After 96 h darkness at 30 C on CMD, conidia beginning to form in a marginal ring of white pustules. Green conidia typically appearing within 50 h on PDA at 30 C in darkness, with conidia first forming as early as within 40 h in many cultures.
Branches typically arising from main axis singly, not paired, main axis often sparingly branched with long ‘internodes' between branches (20–50 µm). Branches arising at 90° or less with respect to the axis above the branch. ‘Paired’ branching systems not conspicuous. Phialides in whorls typically flask-shaped, enlarged in the middle, sharply constricted below the tip to form a narrow neck and slightly constricted at the base, often held in compact heads at less than 90° with respect to the terminal phialide. Terminal phialides of a whorl or solitary phialides typically cylindrical or at least not conspicuously swollen in the middle and often longer than phialides formed below the tip. Solitary phialides common. Intercalary phialides not seen. Conidia subglobose to ovoidal, smooth. Chlamydospores rare, typically not observed on CMD after 7 d (only observed in one culture, G.J.S. 94–5).
3. Trichoderma aggressivum Samuels & W. Gams forma europaeum Samuels & W. Gams, forma nov. Figs. 23–30
Trichodermati aggressivo Samuels et W. Gams f. aggressivo simile sed coloniae lentius crescentes, in agaro SNA temperatura 25 C post 72 h (49.7–)50.5–60.2(–62.7) mm radio; phialides (4.0–)4.3–5.9(–6.1) x (2.3–)2.9–3.7(–5.0) µm (parte latissima). Chlamydosporae raro formatae, (5.7–)7.7–11.3(–14.3) µm diam. In Europa.
HOLOTYPUS: ex culto sicco in agaro CBS 100526 (BPI 748204).
Optimum temperature on PDA 25–30 C, on SNA 25 C. Colonies diverse in appearance; aerial mycelium cottony, barraging; conidia forming either in the middle or in a raised ring around the margin; no tendency to form pustules; no yellow pigment. After 96 h at 30 C in darkness on CMD colonies sterile, nearly invisible. On PDA at 30 C in darkness, green conidia are first seen in most strains at 66 h with the earliest seen after 24 h.
Conidiophore branching typically unilateral, main axis often sparingly branched with long ‘internodes' between branches (20–50 µm). Branches arising at 90° or less with respect to the axis above the branch. ‘Paired’ branching systems not conspicuous. Cells supporting the phialides at most only slightly wider than the phialides. Phialides in whorls typically flask-shaped, enlarged in the middle, sharply constricted below the tip to form a narrow neck and slightly constricted at the base, often held in compact heads at less than 90° with respect to the terminal phialide. Terminal phialides of a whorl, or solitary phialides typically cylindrical or at least not conspicuously swollen in the middle and longer than phialides formed below the tip. Solitary phialides common. Intercalary phialides infrequently observed. Conidia subglobose to ovoidal, lacking a visible basal abscission scar, smooth. Chlamydospores abundant in one of six strains on CMD (CBS 433.95), otherwise absent.
Commentary.
The only differences in micromorphology between T. aggressivum f. aggressivum and f. europaeum are the statistically longer phialides in f. europaeum (95% confidence intervals 7.5–7.8 µm vs 6.5–7.0 µm, respectively). The two forms can be distinguished by the faster growth of f. aggressivum at 25 C (radius of 60 mm after 72 h vs 50 mm). Seaby (1996)
documented differences in colony morphology. We noted in f. aggressivum after 96 h darkness at 30 C on CMD the highly consistent formation of conidia in a marginal ring of white pustules that had not yet turned green.
Intercalary phialides, a short spur-like phialide that projects immediately below a terminal phialide (Samuels et al 1998
), were infrequently seen in f. europaeum but not in f. aggressivum, T. harzianum or T. atroviride.
4. Trichoderma atroviride P. Karsten, Finl. Mögelsvamp. p. 21. 1892. Figs. 31–37
Optimum temperature on PDA 25–30 C, on SNA 30 C. After 96 h at 30 C darkness on CMD, conidia forming in the middle of the colony in an area ca 4 cm diam, uniformly dispersed and not pustulate or in confluent, dense pustules that have a radial arrangement. On PDA after 96 h at 30 C, colony sharply delimited and with a more or less dense central disk within which most conidia form. No pustules observed. Coconut odor typically noticed on CMD and PDA.
Branching of conidiophores typically unilateral although paired branches are common. Branches typically arising at 90° or less with respect to the branch above the point of branching. Phialides straight or sinuous, sometimes hooked; in whorls of 2–4, often solitary; the terminal phialide of a whorl and solitary phialides often cylindrical and constricted only below the tip to form a narrow neck; phialides formed below the terminus typically flask-shaped and enlarged in the middle, constricted to the tip and slightly at the base; cells supporting the phialides at most only slightly wider than the phialide base. Intercalary phialides not observed. Conidia subglobose to ovoidal, lacking a visible basal abscission scar, smooth. Chlamydospores abundant on CMD within 7 d, globose to subglobose, terminal or intercalary, (5.2–)8.5–12.0(–16.3) µm diam.
Commentary. Phialides of T. atroviride and T. aggressivum f. aggressivum are indistinguishable but are significantly longer than those of T. harzianum and T. aggressivum f. europaeum. Solitary phialides are formed by all Trichoderma species, but the tendency for phialides to be held in whorls of three or more is strongly developed in T. harzianum and less so in either of the forms of T. aggressivum, where solitary phialides are common and conspicuous.
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TRICHODERMA SPECIES ASSOCIATED WITH COMMERCIALLY GROWN AGARICUS BISPORUS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
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provided a dichotomous key for 10 "taxa" of Trichoderma that are commonly associated with commercially grown Agaricus bisporus. This key was based largely on colony morphology, which can be difficult to intrepret as a taxonomic character. Nonetheless, most of the taxa included by Seaby can easily be distinguished on their combined microscopical characters and growth rates.
One species that Seaby included was T. pseudokoningii Rifai. Kuhls et al (1997)
and Samuels et al (1998)
found that T. pseudokoningii is rare. Most strains identified as this species were either T. longibrachiatum Rifai or T. citrinoviride Bissett. Seaby also noted two types of T. longibrachiatum, distinguishing them by their growth rates. We have included both T. longibrachiatum and T. citrinoviride in the key that follows but cannot comment on the different forms of T. longibrachiatum noted by Seaby.
KEY TO THE SPECIES OF TRICHODERMA COMMONLY ASSOCIATED WITH COMMERCIALLY GROWN MUSHROOMS
1. Conidia globose, L/W 1.0–1.2 . . . . . 2
1. Conidia oblong to ellipsoidal, L/W > 1.4 . . . . . 6
2. Conidia warted . . . . . T. viride (Samuels et al 1999
)
2. Conidia smooth . . . . . 3
3. Colonies growing well at 35 C, colonies at lower temperatures sporulating within 48 h in darkness; conidia 2.7–3.5 x 2.5–3.0 µm . . . . . T. harzianum
3. Colonies at best growing slowly at 35 C, colony radius never more than 15 mm on PDA after 72 h . . . . . 4
4. Conidia 3.0–3.8 x 2.8–3.5 µm, rapidly becoming dark green; colonies with a strong coconut odor; chlamydospores typically formed; colony radius on PDA after 72 h at 30 C 30–35 mm . . . . . T. atroviride
4. Conidia smaller, 3.0–3.5 x 2.8–3.0 µm, remaining pale green or with yellow hues; odor not sweet or coconut-like; chlamydospores typically not or tardily formed; colony radius on PDA after 72 h at 30 C 50–62 mm . . . . . 5
5. Colony radius on SNA at 25 C after 72 h 38–60 mm (mean = 50 mm); conidia typically not forming on PDA at 25 C before 58(46–70) h; known only from Europe . . . . . T. aggressivum f. europaeum
5. Colony radius on SNA at 25 C after 72 h 59–62 mm (mean = 60 mm); conidia typically forming within 51(47–54) h on PDA at 25 C; known only from North America . . . . . T. aggressivum f. aggressivum
6. Phialides short and wide, 5.5–7.5 µm long, 2.7–4.0 µm wide (L/W = 1.9), clustered along and at the tips of short lateral branches at the base of sterile elongations . . . . . T. hamatum (based on study of the ex-neotype culture, DAOM 167057, see also Bissett 1991
)
6. Phialides longer and narrower, 6.0–9.0 µm long, 2.0–3.0 µm wide (L/W 2.0–3.5), phialides often solitary, sterile elongations of conidiophores not formed . . . . . 7
7. Conidia 3.7–4.0 x 2.4–2.7 µm; solitary phialides frequent and conspicuous but whorls of phialides relatively uncommon; phialides 6.5–9 x 2.7–3.0 µm at the widest, L/W of phialides 2.3–3.5; intercalary phialides common; colony radius after 64 h on PDA at 40 C 44 mm . . . . . T. longibrachiatum (Bissett 1984
, Samuels et al 1998
)
7. Conidia 3.2–3.5 x 2.2–2.4 µm; solitary phialides less common than in T. longibrachiatum and whorls of phialides more common; phialides 6.0–6.7 x 2.8–3.0 µm at the widest, L/W of phialides 2.0–2.3; intercalary phialides less common than in T. longibrachiatum; colony radius after 64 h on PDA at 40 C 26 mm . . . . . T. citrinoviride (Bissett 1984
, Samuels et al 1998
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication March 1, 2001.
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LITERATURE CITED |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYTRICHODERMA SPECIES ASSOCIATED...LITERATURE CITED |
|---|
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= T. harzianum, 
= T. aggressivum f. aggressivum, 
= T. aggressivum f. europaeum, 
= T. atroviride
