Copyright © Copyright 2006 Centraalbureau voor Schimmelcultures, P.O.
Box 85167, 3508 AD Utrecht, The Netherlands. Hypocrea rufa/Trichoderma viride: a reassessment, and
description of five closely related species with and without warted
conidia *Correspondence: Gary J. Samuels,
Gary/at/nt.ars-grin.gov You are free to share–to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights. | ||||
Abstract The type species of the genus Hypocrea (Hypocreaceae,
Hypocreales, Ascomycota, Fungi), H. rufa, is re-defined and
epitypified using a combination of phenotype (morphology of teleomorphs and
anamorphs, and characteristics in culture) and phylogenetic analyses of the
translation-elongation factor 1α gene. Its anamorph, T. viride,
the type species of Trichoderma, is re-described and epitypified.
Eidamia viridescens is combined as Trichoderma viridescens
and is recognised as one of the most morphologically and phylogenetically
similar relatives of T. viride. Its teleomorph is newly described as
Hypocrea viridescens. Contrary to frequent citations of H.
rufa and T. viride in the literature, this species is relatively
rare. Although both T. viride and T. viridescens have a wide
geographic distribution, their greatest genetic diversity appears to be in
Europe and North America. Hypocrea vinosa is characterised and its
anamorph, T. vinosum sp. nov., is described. Conidia of T.
vinosum are subglobose and warted. The new species T. gamsii is
proposed. It shares eidamia-like morphology of conidiophores with T.
viridescens, but it has smooth, ellipsoidal conidia that have the longest
L/W ratio that we have seen in Trichoderma. Trichoderma scalesiae, an
endophyte of trunks of Scalesia pedunculata in the Galapagos Islands,
is described as new. It only produces conidia on a low-nutrient agar to which
filter paper has been added. Additional phylogenetically distinct clades are
recognised and provisionally delimited from the species here described.
Trichoderma neokoningii, a T. koningii-like species, is
described from a collection made in Peru on a fruit of Theobroma
cacao infected with Moniliophthora roreri. Keywords: Bayesian phylogeny, biogeography, biological control, cacao, endophytes, Hypocrea, Hypocreales, Hypocreaceae, molecular identification, morphological key, nomenclature, species identification, systematics, translation elongation factor 1-alpha | ||||
INTRODUCTION Trichoderma viride Pers. (Hypocreales, Hypocreaceae) is one of the most commonly reported species of fungi. In only the two years 2004 and 2005 T. viride appeared in nearly 200 articles that were abstracted by CAB. The species is encountered in widely diverse contexts; a few examples of activities include organochlorine degradation as a soil fungus (Smith 1995), biological control in fungus-induced plant disease (Brown & Bruce 1999; Brown et al. 1999), and as the cause of disease in button mushrooms in India (Mishra & Singh 2005). It is said to effect seed germination of flowering plants (Celar & Valic 2005), and enhance phosphorus uptake by plants (Rudresh et al. 2005). It produces enzymes (Nobe et al. 2004), degrades cellulosic agricultural waste to alcohol (Baig et al. 2004), colonises leaf litter (Osono 2005) and is a normal inhabitant of soils (Roiger et al. 1991, Hagn et al. 2003). Do all these citations refer to only one species, T. viride? Kullnig et al. (2001) detected a shockingly high level of misidentification of strains that were reported in the literature as T. harzianum. If this experience is representative of the genus, as it is likely, then not all of these reports actually refer to T. viride. One example that is representative of the degree of inaccuracy in identification is that of a biocontrol fungus reported in the literature as T. viride (Bastos 1988, 1996 a, b) that was ultimately described as the new species T. stromaticum Samuels & Pardo-Schultheiss (Samuels et al. 2000); these two species are distantly related and morphologically and biologically highly dissimilar. Obviously, it is important to clarify the identity of T. viride, otherwise the literature is meaningless. Bisby in 1939 stated that essentially there was only one species of Trichoderma, T. viride. In spite of some discordant indications, that view held sway until 1969 when Rifai (1969) monographed the genus and characterised T. viride as the only species having globose, warted conidia. This immediately raised suspicion about all reports of activity by Trichoderma species prior to 1969. Even with the description of T. saturnisporum and T. ghanense, both having warted conidia and both being members of T. sect. Longibrachiatum Bissett (Samuels et al. 1998), T. viride stood out because its conidia were globose as compared to ellipsoidal in the other species. Scanning electron microscopy (Meyer & Plaskowitz 1989) revealed the existence of two distinct patterns of conidial ornamentation within strains identified as T. viride, viz. more and less strongly warted. Strains having the less strongly warted conidia were segregated as T. asperellum Samuels et al. (Lieckfeldt et al. 1999; Samuels et al. 1999). In a study of variation within the morphological species T. viride, in addition to recognising T. asperellum and T. viride s. str., Lieckfeldt et al. (1999) noted the existence of two additional ITS-defined groups that had warted conidia, which they referred to as Vd and Ve. The group Vd was very closely related to Vb in its ITS1 and 2 sequences and its morphology. The group Ve was more distantly related and was phenotypically diverse, some of the few included strains having smooth conidia and others having warted conidia. They (Samuels et al. 1999) determined that the group Vb was “true” T. viride by comparison with the over two-hundred-year-old type specimen of the species that is preserved in Leiden. Despite differences in ITS sequences, Samuels et al. (1999) could not see consistent phenotypic differences between Vb and Vd that would support recognition of Vd as a separate taxon. Bissett (1991a) proposed to include H. rufa/T. viride and its relatives in Trichoderma sect. Trichoderma, including also T. koningii Oudem. and T. atroviride P. Karst. The monophyly of this group either as Trichoderma sect. Trichoderma (e.g. Kullnig-Gradinger et al. 2002) or more recently simply as “the viride clade” (Samuels 2006), has been affirmed by DNA sequence analysis. Since the work of Lieckfeldt et al. (1999) we have obtained many additional specimens and cultures referable to the viride clade and are able to propose a revised taxonomy for this clade. In the present work we re-evaluate T. viride groups Vb and Vd and recognise group Vd as a distinct species. Since the middle of the 19th century (Tulasne & Tulasne 1865), T. viride has been recognised as the anamorph of Hypocrea rufa (Pers.: Fr.) Fr., the type species of Hypocrea Fr. Like T. viride, H. rufa is possibly the most common name used in the identification of Hypocrea specimens. Hundreds of specimens in herbaria throughout the world are labelled “Hypocrea rufa”. However, even a quick glance at specimens shows that a plethora of species has been lumped under this name. For example, species such as H. minutispora B.S. Lu et al./T. minutisporum Bissett and H. pachybasioides Yoshim. Doi/T. polysporum (Link: Fr.) Rifai have both been incorrectly identified as the only distantly related H. rufa. Webster (1964) provided the first modern description of H. rufa. It is a species that has a stroma that starts out semieffused and whitish to tan to reddish brown and pruinose and with age becomes darker and cushion-shaped; the ascospores are hyaline. In our continuing work with the viride clade we have found that especially the young stroma of most members of the clade is distinctive of a number of often sympatric species that are best distinguished by their Trichoderma anamorphs (Samuels et al. 2006a). We have found indistinguishable teleomorphs for both T. viride groups, Vb and Vd. This calls for a redefinition and redescription of H. rufa. In the present work we refine the description of H. rufa and provide an epitype for the species, we describe as new a teleomorph for T. viride group Vd, redescribe Hypocrea vinosa with its new anamorph T. vinosum, and describe the new species T. gamsii, T. neokoningii and T scalesiae. | ||||
MATERIALS AND METHODS Isolates including NCBI GenBank accession numbers of gene sequences investigated in this study are listed in Table 1. The locations in European countries are indicated with coordinates and map sheets (MTB = Messtischblatt). Collections and analysis of phenotype The isolates originated from three natural sources: isolations from
ascospores of Hypocrea specimens, direct isolations by a variety of
means from soil or dead herbaceous tissue, and isolations as endophytes from
sapwood of living stems of Theobroma and related tree species, and
from Fagus sylvatica. Isolation of the stem endophytes was done as
reported by Evans et al.
(2003). A smaller number of
isolates was obtained from the American Type Culture Collection (ATCC),
Biologische Bundesanstalt (Berlin), the Centraalbureau voor Schimmelcultures
(CBS), and from individual colleagues. Cultures derived from single
part-ascospores that were germinated on cornmeal agar with 2 % dextrose (CMD,
Difco cornmeal agar + 2 % dextrose w/v) and isolated by means of a
micromanipulator; usually two or more single-spore cultures were combined in a
single stock culture, and such polyspore cultures were used in all subsequent
analyses. The working set of cultures is maintained on cornmeal agar slants at
ca. 8 °C, in 20 % glycerine at -80 °C, or in liquid
nitrogen.Representative isolates are deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS) and the American Type Culture Collection, Manassas, VA (ATCC). Isolates listed as C.P.K. are those maintained in the collection of Christian P. Kubicek, Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Vienna. Kornerup & Wanscher (1978) was used as the colour standard. The name of the most commonly cited collectors, G.J. Samuels and W.M. Jaklitsch, are abbreviated as G.J.S. and W.J. Cultures used for study of anamorph micromorphology were grown on CMD, PDA or SNA (Nirenberg 1976), at 20 or 25 °C for 5–11 d under alternating 12 h cool white fluorescent light and 12 h darkness; in the descriptions that follow, these alternating light conditions are referred to when the word “light” is used. Morphological analyses of microscopic characters were undertaken from material that was first hydrated in the case of herbarium material, or wetted in the case of living cultures, in 3 % KOH. Autolytic activity, which is here defined as usually circular excretions at the tips of hyphae, was assessed under direct microscopic observation using a 10 × objective. Coilings, defined as circularly oriented and coiled intercalary or terminal parts of hyphae, were detected in the same way as autolytic excretions. Measurements were made from KOH or water mounts and we did not observe any differences when the respective reagents were used. Where possible, at least 30 units of each parameter were measured for each collection. Ninety-five percent confidence intervals of the means (CI) are provided; this figure represents the interval within which 95 % of the individuals of the parameter was found in the analysed isolates. The parameters used for analysis are listed in Table 2. Chlamydospores were measured by inverting a 7–15 d old CMD culture on the stage of a compound microscope and observing with a 40 × objective. Data were gathered using a Nikon DXM1200 or a Nikon Coolpix 4500 digital camera and Nikon ACT 1 software and measured either directly on the microscope or by using Scion Image (release Beta 4.0.2; Scioncorp, Frederick, MD). Five types of light microscopy were used, viz. stereo microscopy (stereo), bright field (BF), phase contrast (PC), Nomarski differential interference contrast (DIC), and epifluorescence (FL). The fluorescent brightener calcofluor (Sigma Fluorescent Brightener 28 C.I. 40622 Calcofluor white M2R in 2 molar phosphate buffer at pH 8.0) was used for FL. Scanning electron microscopy (SEM) was done by one of two methods. Material for SEM studies was obtained from cultures that were grown on PDA for up to 2 weeks at 20–25 °C. Agar blocks with abundant conidia were prepared for SEM. For Figs 8 a–h all SEM procedures followed the protocols of Meyer & Plaskowitz (1989), and for Fig. 10h those of Carta et al. (2003) and Erbe et al. (2003). Sections of Hypocrea stromata were prepared by rehydrating small blocks of substratum supporting stromata in 3 % KOH. The blocks were supported by Tissue Tek O.C.T. embedding medium 4583 (Miles, Inc., Elkhart, IN) and sectioned at 12–15 μm on a Microtome-Cryostat (International Equipment Co., Needham Heights, MA, or Leitz Kryostat 1720, Leica Microsystems, Vienna). Growth rate trials were performed in darkness on potato-dextrose agar (PDA, Difco, Biolab, or Merck) and SNA following the procedure described by Samuels et al. (2002) with the addition that cultures were also grown at 25 °C under 12 h darkness/12 h cool white fluorescent light for 5–7 d. Each growth-rate trial was repeated three times and the results of the three were averaged. The slope of the growth curve was determined using the mean of the colony radius (see Samuels et al., 2006a). DNA extraction and sequencing methods The extraction of genomic DNA was performed as reported previously
(Dodd et al. 2002). A
portion of translation elongation factor 1 alpha (tef1) was amplified
using the primers EF1-728F (Carbone &
Kohn 1999) and TEF1 rev
(Samuels et al. 2002)
or TEF1LLErev (Jaklitsch et al.
2005). The PCR product of approximately 600 bp covers the large
4th and the short 5th introns of the gene. A fragment
covering the internal transcribed spacers 1 and 2 (ITS1 and 2) of the rRNA
gene cluster was amplified using ITS1 and ITS4 as the forward and reverse
primers, respectively (White et
al. 1990). DNA sequences were obtained using the BigDye
Terminator cycle sequencing kit (Applied Biosystems, Foster City, California).
Products were analysed directly on a 3100 DNA sequencer (Applied Biosystems).
Both strands were sequenced for each locus.Molecular phylogenetic analysis Sequences were edited and assembled using Sequencher 4.1 (Gene Codes,
Wisconsin). Clustal X 1.81 (Thompson
et al. 1997) was used to align the sequences; the
alignment of each locus was manually edited using MacClade or GeneDoc 2.6
(Nicholas & Nicholas
1997). The sequences were deposited in GenBank
(Table 1). The MSA file for the
tef1 locus is also available at
http://www.isth.info/phylogeny/rufa.php.The interleaved NEXUS file was formatted using PAUP* v. 4.0b10 (Sinauer Associates, Sunderland, MA) and manually formatted for the MrBayes v3.0B4 program. The Bayesian approach to phylogenetic reconstructions (Rannala & Yang 2005) was implemented using MrBayes 3.0B4 (Huelsenbeck & Ronquist 2001). The MODELTEST3-06 package (http://bioag.byu.edu/zoology/crandall_lab/modeltest.htm) was used to compare the likelihood of different nested models of DNA substitution and select the best-fit model for the investigated data set. Both hierarchical LRT and AIC output strategies were considered, although the preference was given to the latter. The unconstrained GTR + I + G substitution model was selected for the tef1 locus. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed with four incrementally heated chains (with the default heating coefficient λ = 0.2, heats for cold chains 1 and heated chains 2, 3 and 4 are 1, 0.83, 0.71 and 0.63, respectively) that were simultaneously run for 5 million generations for the tef1 alignment, which comprised 238 sequences. To check for potentially poor mixing of MCMCMC, the analysis was repeated at least three times. The convergence of MCMCMC was monitored by examining the value of the marginal likelihood through generations. Convergence of substitution rate and rate heterogeneity model parameters were also checked. Bayesian posterior probabilities (PP) were obtained from the 50 % majority rule consensus of trees sampled every 100 generations after removing the 2000 first trees using the “burn” command. According to the protocol of Leache & Reeder (2002), PP values lower than 0.95 were not considered significant while values below 0.9 are not shown on the resulting phylogram. Model parameter summaries after MCMC run and burning first samples were collected. For tef1 mean substitution values were estimated as G↔T = 1, C↔T = 3.55, C↔G = 1.28, A↔T = 1, A↔G = 4.68, A↔C = 1.5; nucleotide frequencies were estimated as 0.19 (A), 0.27 (C), 0.2 (G), 0.34 (T); alpha parameter of gamma-distribution shape was 0.29. Genetic distance was computed in PAUP* v. 4.0b10 under the GTR + I model. | ||||
RESULTS Phylogeny The majority of members of Trichoderma section
Trichoderma share the same or very similar alleles of internal
transcribed spacers 1 and 2 (ITS1 and 2), rendering this locus inappropriate
for recognition of some species within the section. Therefore, to infer
genetic diversity of the H. rufa/T. viride group we used intron
sequences of the translation elongation factor 1-alpha (tef1), the
most powerful phylogenetic marker as yet established in the genus. The
resulting Bayesian phylogram (Fig.
1), which was obtained from 238 sequences, corresponds well to the
previous analysis of related species with T. koningii-like morphology
(Samuels et al.
2006a). Considering the analysis of phenotypes, it is obvious that
there are two diverged groups named “Large Viride” and
“Large Viridescens” clades, both of them with significant
statistical support. Isolates of H. rufa form a compact clade
composed of mainly European but also North American, Asian and Pacific strains
showing its cosmopolitan nature. The “Large Viride” clade includes
additional unresolved lineages that apparently represent unnamed species. The
description of these taxa requires further sampling and therefore will be
discussed in subsequent publications. In this study we have focused on the
single endophytic strain from the Galapagos Islands, T. scalesiae sp.
nov., which belongs to the “Large Viride” clade but at the same
time occupies the most distant position from H. rufa. The largest
group on the tree, the “Large Viridescens” clade, splits into two
independent evolutionary lineages. The terminal position of the larger one
represents a compact and well defined subclade with significant statistical
support that contains isolates of the former Vd group
(Lieckfeldt et al.
1999), described as H. viridescens below. Similar to
H. rufa, this species has mainly European origin, also nearly all
primary European nodes include North American, Central American, Asian and
Pacific isolates, suggesting the absence of recent allopatric speciation in
this group of isolates. Another well-supported clade in the vicinity of H.
viridescens is composed of isolates of H. vinosa. As in the
“Large Viride” clade this branch contains representatives of
several well-supported speciation nodes composed of strains that are closely
related to H. viridescens and H. vinosa and undoubtedly
represent yet undescribed species of Hypocrea/Trichoderma. This
diversity will be discussed in subsequent publications following further
investigations and sampling. The material summarised in this study is
sufficient to prove the existence of another phylogenetic species with
eidamia-like morphology that occupies the second independent lineage within
the “Large Viridescens” clade. The new species T. gamsii
forms a homogeneous clade mainly represented by isolates from undisturbed
soils in Sardinia and Central Russia. As in the case of H.
viridescens and H. rufa, T. gamsii did not evolve as a result of
any geographic isolation since we also sampled isolates from North America and
Australia. We describe the most distant member of the “Large
Viridescens” Clade, once again a single strain, as T.
neokoningii. The detailed analysis of the highly variable intron
sequences of the tef1 gene has clearly shown that, despite their
close relationship, H. rufa, H. viridescens, H. vinosa, and a large
group of isolates that we describe here as T. gamsii represent
distinct sympatric phylogenetic species.Most of the Trichoderma species that have warted conidia fall within one of these two large clades. Exceptions include T. saturnisporum and T. ghanense, both of which are members of the distantly related T. sect. Longibrachiatum (Samuels et al. 1998) and clade Ve (Fig. 1). Clade Ve will be discussed in a future publication. All members of the “Large Viridescens” clade are characterised by the formation of peculiar, percurrently proliferating phialides that are diagnostic of Eidamia viridescens, the ex-type of which (CBS 433.34) falls in T. viridescens. Phenotype: anamorph and cultures DNA sequences referred eighty-seven strains to the “Large
Viridescens” clade and thirty-four to the “Large Viride”
clade. All of these fungi are typical of Trichoderma in producing
copious amounts of green conidia in pustules or in extensive
“lawns” on CMD, PDA and SNA. There was a tendency for conidia to
form more quickly on SNA than on CMD or PDA and often conidia did not form on
either of the latter media while they did form on SNA. Of the three media, SNA
is overall better for the study of fungi in the viride clade in terms of more
reliable production of conidia. The endophytic T. scalesiae only
produced conidia on SNA to which a 1 cm2 piece of sterile filter
paper had been added; the conidia only formed at the interface of the paper
and the agar and on the paper itself.There was a tendency for yellow pigment to develop in conidia in colonies of the “Large Viridescens” clade grown on PDA and SNA at 25 °C for two weeks, and a yellow pigment often diffused through CMD. No pigment was noted on SNA. Diffusing yellow pigmentation was not noted in colonies belonging to the “Large Viride” clade. A more or less strong coconut odour was detected in PDA and CMD cultures of most members of the “Large Viridescens” clade. Conidia tended to form in pulvinate to hemispherical pustules < 1–3 mm diam. Distinct pustules measuring 1–5 mm were formed in T. viride/H. rufa on CMD. While pustules formed in T. viridescens/H. viridescens reached 3 mm diam, most often they measured less than 1 mm and often no pustules were formed, the conidiophores arising in more or less continuous cottony lawns. Often conidiophores formed apart from the larger pustules in the aerial hyphae and in minute tufts. Pustules in both groups tended to be cottony, and individual fertile branches could be seen; often conidiophores protruded beyond the surface of a pustule, producing a single phialide or a few fertile branches near the tip, the rest of the conidiophore remaining sterile or nearly so. The pustules of T. viridescens were usually less compact than in T. viride, and transparent under a 10 × objective. In pustules of T. viride produced on CMD, conidia often appeared to form at the surface of the pustule. In all cases, after one week at 20–25 °C, conidia were deep green to dark green 27–28D–F6–8, although lighter green conidia were observed in younger cultures. In some cultures of T. viridescens grown at 25 °C under alternating light on CMD and SNA, conidial masses were yellow. Conidia of T. neokoningii on PDA often were yellow at first. Often conidia of members of the “Large Viridescens” clade became greenish yellow when mounted in 3 % KOH. Most of the fungi discussed in this work produce colonies that are recognizable as typical of Trichoderma in producing green conidia in abundance on most media. The exception is T. scalesiae, which only produced conidia sparingly on SNA to which a 1 cm2 piece of sterile filter paper had been added. Conidiophores in this species were irregularly branched, similar to what was described for T. paucisporum Samuels et al. (Samuels et al. 2006b) and for synanamorphs of pustulate species of Trichoderma (Chaverri et al. 2004). Conidia were held in drops of clear liquid, which appeared yellow to pale green because of the conidia, at the tips of the phialides. The following results pertain to the remaining species discussed in this work. It is difficult or impossible to define a conidiophore in Trichoderma. Conidiophores are mostly formed in pustules. As was noted above, pustules tend to be composed of intertwined hyphae that terminate in fertile branching systems. For the purposes of the present discussion, the conidiophore is referred to as the terminal branching system of intertwined hyphae that form the pustule. Various types of conidiophores were encountered in this study, and these were largely related to the medium and to the clade. In Type 1 (Fig. 3d, e, i), a well-developed main axis was not readily visible, or it was short and sometimes sinuous. Branching was highly irregular; branches were not paired and phialides tended to arise singly from the main axis. Phialides were often hooked or sinuous (Fig. 3d, e, j, k), cylindrical or somewhat swollen at or below the middle. This type of conidiophore was only found in the “Large Viride” clade, especially in T. viride. The Type 2 conidiophore (e.g. Figs 6, 11, 13, 14) was formed by all clades. In the Type 2 conidiophore there was a more or less readily discernable, well-developed main axis, from which lateral branches arose at or near 90°; the lateral branches were longer with distance from the tip and secondary branches were shorter with distance from the point of departure of the branch from the main axis. Branches often arose in pairs and produced secondary branches in pairs. Phialides tended to terminate branches in cruciate whorls of 3–4. The phialides were straight, cylindrical or somewhat swollen at or below the middle. In Type 3, which was common in the “Large Viridescens” clade, including T. vinosum, T. gamsii and T. neokoningii, the most distinctive characteristic is the production of percurrently proliferating phialides (Figs 5f, g, i, k; 10d–g; 12c, e, f, g; 14h–j), the branching system itself is highly variable in extent and form. At its simplest, a single phialide percurrently produces a second phialide (Fig. 14 h). What appears to be continuing percurrent proliferation of phialides results in a submoniliform chain of five or more cells (Figs 5g, 10 e, 12e, f), each cell of the chain being often abruptly swollen in the middle and separated by the cell above and below by a conspicuous septum. A main axis was discernable or not and was often reduced to a few, short, verticillately disposed branches or a reticulum of branches (e.g. Figs 5i–k; 10d, 12f–h, 14i–j). The most extreme form of the third branching type was observed in old pustules on CMD and PDA, where chains of percurrently proliferating phialides having subglobose bases and extremely long, cylindrical beaks arose from swollen, subglobose cells (Figs 5k, 10f, 12f). Percurrently proliferating phialides having this morphology were also seen occasionally on more typically branched conidiophores (Figs 5d; 12e), on conidiophores that produced typical, non-proliferating, phialides. Proliferating phialides were rarely seen on SNA. Conidiophores of DIS 328g (Vb 1) arose within well-developed pustules; they formed a reticulum with short fertile branches. The branches tended to be sinuous or curved and to be broader than is found in other clades that are studied here. The conidiophores produced often unicellular lateral branches, each of which terminated in 2–4 phialides. The phialides in DIS 328g are shorter than in any strain included in this study and have a smaller L/W ratio; they were often hooked or sinuous. The “Large Viridescens” clade includes CBS 433.34, which is the ex-type culture of Eidamia viridescens A.S. Horne & H.S. Williamson. This species was described based on conidiophores produced on PDA; the original illustration is highly suggestive of what we have seen in the “Large Viridescens” clade, especially the extreme form described above as Type 3 and illustrated in T. viridescens (Fig. 5k) and T. vinosum (Fig. 10f). Conidiophores produced by this culture on SNA (Fig. 6f, g) were typical of Trichoderma, with a more or less uniformly branched conidiophore and typical phialides. The culture remained sterile on PDA but produced a coconut odour and a diffusing yellow pigment. On CMD mononematous conidiophores bearing green conidia in appressed phialides developed, but no pustules and no proliferating phialides were seen. These conidiophores were suggestive of the synanamorph conidiophores described by Chaverri et al. (2004) for species of Hypocrea/Trichoderma having conidiophore elongations. Intercalary phialides were seen in some isolates but were neither common nor restricted to any particular clade (e.g. Figs 3k, 12d, 13g, 14k). Various conidial types were observed in this study. These, like conidiophore types, were largely typical of clades. Most of the strains produced warted conidia. Conidia of T. gamsii (Fig. 12i), T. neokoningii (Fig. 14l) and T. scalesiae (Fig. 15h) are smooth. Trichoderma viride (Figs 3m, n; 8a–e), DIS 328g (Vb 1), G.J.S. 04-40 (Vb 2), and T. vinosum (Fig. 10h–j) have nearly globose conidia that have a length/width ratio 1.0–1.2. Conidia in G.J.S. 03-151/G.J.S. 02-87 (Vd 1) are ellipsoidal, length/width of 1.2–1.4. Conidia of individual collections of T. viridescens vary from subglobose to ellipsoidal (Figs 6i–m, 8f–h); although the mean L/W of all collections in this clade varies from 1.1–1.3, there is considerable overlap between this clade and DIS 328g. Conidia of T. gamsii and T. neokoningii are unusual in being ellipsoidal. Both of these species produce T. koningii-like conidiophores and conidia. Conidia of T. viride are much more coarsely warted than any of the other clades considered here. Warted conidia are also produced by members of clade Ve. Conidia in this clade are subglobose to ellipsoidal. Ornamented conidia were observed for most members of this clade. Conidial warts, while often large, are widely spaced and thus are not as conspicuous as in members of the “Large Viride” and “Large Viridescens” clades that are the focus of the present work. Chlamydospores were inconsistently produced in most clades. Chlamydospores formed in abundance in T. gamsii (Fig. 13i) and T. neokoningii (Fig. 14m). Chamydospores were especially abundant in T. scalesiae (Fig. 15i). Chlamydospores were typical of Trichoderma in being globose to subglobose and terminal at the ends of hyphae or intercalary within hyphal cells. Optimal temperature for growth on PDA for all clades except T. scalesiae and Vd 1 was 25 °C. The optimum for T. scalesiae was 30 °C and the two isolates in Vd 1 exhibited considerable variation at 30 °C (35–70 mm radius after 72 h). Trichoderma vinosum was unusual in having a temperature optimum of 20–25 °C and in reaching no more than 5 mm colony radius after 4 days at 30 °C. On SNA most isolates reached a radius of no more than 40 mm, and usually less, after 72 h at 25–30 °C. On SNA only DIS 328g (Vb 1), T. viride and T. viridescens demonstrated a clear optimum at 25 °C. On SNA, the optimum for T. scalesiae was 25–30 °C, as it was for T. vinosum. The two isolates of Vd 1 were too variable to show a temperature optimum on SNA. G.J.S. 04-40 (Vb 2) was the fastest growing strain on SNA, reaching 65 mm after 72 h at 30 °C. This temperature differential was not observed on PDA. At 25 °C on PDA DIS 328g (Vb 1), G.J.S. 04-40 (Vb 2), T. gamsii, Vd 1 and Vd 2 reached or exceeded a radius of 45 mm after 72 h. Despite their phylogenetic complexity, both T. viride and T. viridescens showed very little variation in growth rate among their many isolates, both reaching a radius of 30–40 mm after 72 h at 25 °C. Significantly, growth of isolates in both of these clades, as well as in T. vinosum and DIS 328g (Vb 1), was more than 20 mm slower at 30 °C than at 20 °C. Trichoderma scalesiae was the slowest growing, reaching only 10 mm on SNA after 72 h at 25–30 °C and 18 mm on PDA at 30 °C. The fastest growing isolate at 30 °C was G.J.S. 04-40 (Vb 2) reaching 45 mm, although G.J.S. 03-151 (Vd 1) reached a radius of 70 mm after 72 h at 30 °C. Clade Vd 3, which is a sister to T. viridescens, comprises two distinct groups of isolates. The North American isolates (G.J.S. 00-67, G.J.S. 97-243) cannot be distinguished from T. viridescens in any of their morphs and aspects. The Taiwanese isolates (G.J.S. 94-9 – G.J.S. 94-11) grow significantly more slowly than T. viridescens. Phenotype: teleomorph The stromata of the species included in this study are morphologically and
anatomically so similar that they often cannot be distinguished. The youngest
stage, when it could be observed, was semieffused, velutinous to conspicuously
hairy and light tan in colour (Figs 4a,
d; 2a, c). As
perithecial development continued, the stroma became pulvinate to tuberculate
or turbinate, and assumed a brown to rufous colour. Occasionally
“albino” stromata, off-white to pale yellow, were observed in
H. rufa (Fig. 2f) and
in H. vinosa (Fig.
16i), in the latter only in an immature state. Often a velvety
scurf was also present on mature stromata, the result of short hyphal hairs
protruding from the stroma surface (Figs
2k, l;
4 l;
9b, e). Ostiolar openings were
usually not visible macroscopically, or were barely visible as lighter areas
on the stroma surface, sometimes with darker margins. The stroma surface, when
observed in the compound microscope, was composed of small
pseudoparenchymatous cells. Typically brown pigment was unevenly deposited in
the walls of these cells giving a mottled appearance to the rehydrated stroma
(Figs 4j,
9a). The stromata typically
have a pigmented cortical layer underlain by a region of loosely arranged
hyphae. Asci were cylindrical and had a thin ring in the apex; they typically
contained 8 uniseriate ascospores. Ascospores were hyaline, spinulose and
disarticulated early to form two halves, or part-ascospores. The
part-ascospores were dimorphic, the distal part was subglobose to broadly
conical and the proximal part was ellipsoidal or oblong to narrowly
wedge-shaped. Ascospore sizes were clade-specific. G.J.S. 02-87 (Vd 1), a
teleomorphic member of the “Large Viridescens” clade from Sri
Lanka, had the smallest ascospores. Ascospores of H. vinosa were
longer in the distal part than in all other species and the width of its
proximal and distal parts was greater than in all others. Ascospores of H.
rufa and H. viridescens are nearly identical in size. Vb 3
includes two Hypocrea collections from, respectively, Virginia and
North Carolina. While these two collections are sympatric with, but
phylogenetically distinct from H. rufa, we did not observe any aspect
of their teleomorph, anamorph or cultural phenotypes that would serve to
distinguish them from that species.Biogeography Most of the clades that included more than one strain did not show strong
biogeographic bias. Hypocrea vinosa was originally described from New
Zealand and, in this work, it is restricted to New Zealand and Australia. The
“Large Viride” and “Viridescens” clades are widely
distributed but are more common in North America and Europe. These are not
tropical fungi. Trichoderma viridescens has been found in Peru at
high elevation. We have seen only one isolate of T. viride from a
tropical region, i.e. G.J.S. 92-15, from Brazil. However, two members of the
“Large Viride” clade, DIS 328g (Ecuador) and G.J.S. 04-40
(Brazil), originated in South America. These two endophytic isolates
apparently represent two distinct species. Trichoderma neokoningii
was isolated in a tropical region in Peru. On the basis of our collecting,
T. viridescens is far more common and possibly more widespread than
T. viride. Trichoderma viride and T. viridescens are common
in Europe as anamorphs, but uncommon as teleomorphs if compared to common
species like H. minutispora. There is a tendency for isolates
originating in a geographic area (e.g. Taiwan or Europe) to cluster together
but there was an equally strong tendency for clades to comprise strains of
mixed origin (e.g. Japan, United Kingdom and U.S.A.). Trichoderma
gamsii includes strains from widely separated locations, viz. the
Tyrrhenian island of Sardinia (Italy), U.S.A. (Texas), Russia and Australia.
The clade Vd 3 comprises two biogeographically distinct sister clades.
Isolates G.J.S. 00-67 and G.J.S. 97-243 are from eastern U.S.A. Isolates
G.J.S. 94-9 – G.J.S. 94-11 were collected in Taiwan.The isolates G.J.S. 04-40 and DIS 328g were isolated as endophytes from trunks of Theobroma cacao and Th. gileri, respectively, and T. scalesiae was isolated as an endophyte from woody, above-ground tissue of Scalesia pedunculata. Definition of species Fig. 1, with T.
asperellum as outgroup, demonstrates the considerable known and yet to be
described taxonomic diversity in a large part of T. sect.
Trichoderma. Despite the existence of several clades that no doubt
merit taxonomic recognizion, in the current work we emphasize the “Large
Viride” and “Large Viridescens” clades.Each of these large clades includes several well-supported internal clades, making it difficult to delimit species. In most cases, more or less distinct phenotypic apomorphies lead to our decision as to where to draw species boundaries. The greatest phylogenetic diversity is found in the “Large Viridescens” clade. At the most distant point of this clade, T. gamsii and T. neokoningii can be distinguished because they both have smooth, ellipsoidal conidia. Trichoderma gamsii is a common species in Europe and North America. Trichoderma neokoningii is only known from a single culture that was collected in Peru as a hyperparasite on a destructive pathogen of Theobroma cacao. Clade Vd 2 includes European and middle-eastern (Iran) isolates that also have smooth, ellipsoidal conidia. Clade Vd 1 includes isolates from Sri Lanka and Ghana that have ellipsoidal, warted conidia. One of these, G.J.S. 02-87 (Sri Lanka), produces a H. rufa-like stroma but it has smaller ascospores than either H. rufa or H. viridescens. We did not observe an eidamia-like morphology in Vd 1 or Vd 2. Hypocrea vinosa is distinguished from H. viridescens primarily on the basis of its faster rate of growth and on its larger ascospores. It has a conspicuous eidamia-morphology when grown on CMD. Clade Vd 3 is phenotypically and biogeographically diverse. We had originally included all of these isolates within T. viridescens. As was noted above, the North American isolates (G.J.S. 00-67, G.J.S. 97-243) cannot be distinguished from H. viridescens, whereas the remaining isolates, all from Taiwan, have a noticeably slower rate of growth than T. viridescens. Their relationship to T. viridescens is indicated by the dotted line in Fig. 1. Hypocrea/Trichoderma viridescens is a widely distributed species that is common in Europe. It is phenotypically, phylogenetically and geographically diverse, but the phenotypic diversity overlapped to such an extent that we were not able to subdivide the species. Hypocrea/T. viridescens is characterised by north- and south-temperate distribution, relatively slow growth, conidiophores that tend to produce paired branches on SNA, subglobose to nearly ellipsoidal, warted conidia, a coconut odour on PDA and CMD, and the conspicuous eidamia-morphology found on PDA and CMD. The most distant point of the “Large Viride” clade is T. scalesiae. This unusual species was isolated as an endophyte from the trunk of an endemic daisy tree in the Galapagos Islands. It only produced few conidia on conidiophores that are atypical in Trichoderma. Even in the absence of conidial development, it is recognizable as a Trichoderma by its strong odour of coconut and also by the production of abundant chlamydospores that are typical of Trichoderma. A single clade that is sister to H. rufa/T. viride includes Vb 1, Vb 2 and Vb 3. The two isolates of Vb3 were isolated in the mid Atlantic states of the U.S.A. and they cannot be distinguished from T. viride (with which they are sympatric) morphologically. Apart from the phylogenetic difference indicated by sequences of tef1, we cannot observe any way to taxonomically separate them from H. rufa/T. viride. The single strains that comprise Vb 1 and Vb 2 were isolated as endophytes from trunks of, respectively, Theobroma gileri and Th. cacao in Ecuador and Brazil. Both of them have a faster growth rate than H. rufa/T. viride, a difference that is especially marked on SNA, and Vb 2 grows faster than any of the clades included in the present study. Both of these, but especially Vb 2, have somewhat smaller conidia than T. viride. Conidiophores of Vb 2 are Type 1 described on page 144 and typical of T. viride. The unusual conidiophores of DIS 328g (Vb 1) and the short broad phialides distinguish this clade from its closest relatives, Vb 2, Vb 3 or T. viride. The data suggest that these two endophytic strains represent distinct species; their taxonomy will be discussed in a future publication. As was the case with H./T. viridescens, H. rufa/T. viride is phylogenetically and phenotypically diverse but we did not find any hiatus in the characters that would enable us to recognise more than a single species. The hallmark of T. viride is its remarkably consistent, rather slow rate of growth, strongly warted, globose to subglobose conidia and this is consistent with the type specimen of T. viride (Fig. 8a, b and Samuels et al. 1999). Moreover, the conidiophores found in T. viride, with often solitary, hooked phialides, are consistent with what Tulasne & Tulasne (1865) illustrated for their concept of H. rufa and T. viride. What we have called T. viridescens could have perhaps been selected as being typical of T. viride, given the overlap in phenotype characters of the anamorph, but conidia in this group are not so strongly warted and the tendency is for ellipsoidal conidia rather than globose. The ex-type culture of Eidamia viridescens (CBS 433.34) was included in our analysis. Thus we name this clade T. viridescens, with Hypocrea viridescens sp. nov. as its teleomorph. | ||||
KEY TO TAXONOMIC AND PHYLOGENETIC SPECIES OF TRICHODERMA
SECT. TRICHODERMA DISCUSSED IN THIS PAPER
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DESCRIPTIONS OF THE SPECIES Continuous characters not provided in the descriptions are given in Table 2.
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DISCUSSION This is the eighth article in our series dealing with members of the phylogenetically and taxonomically complex Trichoderma sect. Trichoderma (Dodd et al. 2002, 2003, Druzhinina et al. 2004, Holmes et al. 2004, Lieckfeldt et al. 1999, Lu & Samuels 2003, Samuels et al. 1999, 2006a, b). In the introduction to the current work we suggested that reports of T. viride refer to more than one species. We have shown that even the classical morphological concept of T. viride, i.e. a green-conidial species with globose, warted conidia, is paraphyletic. In the present paper we distinguish the two most common species of Trichoderma that have warted conidia and describe a new species that has warted conidia, T. vinosum. We augment the known diversity of morphological expression in Trichoderma to include the peculiar Eidamia morphology. During the course of the study, additional species having warted conidia or that were closely related to T. viride or T. viridescens were revealed to us. In addition we describe a second species, T. scalesiae, that but for DNA sequence analysis and careful examination of cultures on SNA, would be reported as a sterile unknown fungus. Although we present results from sequencing of only a single gene, tef1, the resulting clades conform to phenotypic apomorphies in defining species. Clearly, from Fig. 1 it can be seen that additional species remain to be characterized. These will be discussed in forthcoming publications. In the present work, as in the accompanying article in this issue that concerns T. koningii (Samuels et al. 2006a), the question that we have had to answer was how to delimit taxonomic species. As we continue to collect new specimens and submit them to DNA sequencing and phylogenetic analysis, we find within clades considerable homoplasy in the few morphological features that are available for analysis. Despite the formation of a teleomorph by several species, this morph is so highly conserved within the viride clade as to be virtually useless in species-level taxonomy while at the same time being diagnostic of the clade. Similarly, while the basic conidiophore morphology in the viride clade, defined here as “Type 2”, signals membership in the clade, it varies only little among the species. For this reason, one cannot fault earlier mycologists for having recognized few species of Trichoderma, or even later mycologists for having failed to recognize the differences between T. viride and T. viridescens. In both the T. viride and T. koningii works our strategy for recognition of taxonomic species was to closely integrate phenetic and phylogenetic characters. These two analyses were undertaken independently in two laboratories, and therefore were unbiased. Species were recognized when the concordance between both approaches were found. Although we clearly understand that the phylogeny of one single locus, in this case tef1, is useful to formulate a hypothesis concerning the phylogeny of the fungus, we also understand that use of this single gene region is not powerful enough to falsify a species hypothesis. Thus, we rely on the concordance between phenotype and genotype. It is also important to note that for some groups the high observed phylogenetic diversity enabled us to recognize a species (e.g. T. gamsii), while in other cases, when the diversity was low and both sets of characters were unclear, we refrained from proposing a new taxonomy (Vd 1–3). The formation of warted conidia in the viride clade seems to suggest that this character is derived within Trichoderma and that smooth conidia are the primitive state. However, we are not yet in a position to discuss evolution of most phenotypic characters in Trichoderma, and conidium ornamentation is not an exception. Tuberculate conidia are produced by completely unrelated species of Trichoderma, including T. saturnisporum and T. ghanense (both sect. Longibrachiatum, Samuels et al. 1994), and Bissett (1991b) illustrated warted conidia in T. virens (sect. Pachybasium) using scanning electron microscopy. On the other hand the eidamia-like morphology of conidiophores produced on rich media is apomorphic for the large viridescens clade defined here, and the coconut-like odour produced by several members of the entire viride clade. The level of knowledge of Trichoderma species is perhaps greater than for any other genus of fungi. We acknowledge the difficulty of identifying Trichoderma species on the basis of their phenotype, despite the fact that morphology-based keys to species, and illustrations of at least the most common species is available at www.nt.ars-grin.gov. However, every species is represented in GenBank by sequences of two or more genes and by a multiplicity of correctly identified strains (Samuels 2006). Moreover, all known species of Trichoderma may be identified using molecular markers at www.isth.info. The majority of species can be safely identified by the DNA barcoding based on ITS1 and 2 loci (Kopchinskiy et al. 2005). However, since species from Trichoderma section Trichoderma share the same or very similar alleles of ITS1 and 2, they should be identified by an integration of the barcode method (TrichOKEY) and Trichoderma similarity search TrichoBLAST (Kopchinskiy et al. 2005) as described in Druzhinina et al. (2006). If a researcher has access to a sequencing facility, there is no need to misidentify a species of Trichoderma. We strongly recommend that individuals check the identity of strains that are of interest and we urge editors of journals reporting properties of Trichoderma species to require that the identity of the strains be verified by members of the International Subcommittee on Taxonomy of Hypocrea (ISTH) which can be accessed at www.isth.info. | ||||
Acknowledgments We thank Hermann Voglmayr for the collection of Hypocrea teleomorphs, Svengunnar Ryman and Roland Moberg for support of excursions in Sweden. Drs Moberg and Hennig Knudsen were instrumental in identifying type material of Sphaeria rufa. Cultures were provided by Drs John Bissett (DAOM), Charles Howell (USDA), Toru Okuda (Nippon Roche), Walter Gams (CBS), Willies Soberanis (Tarpoto, Peru), Helgard Nirenberg (BBA, Berlin), and Doustmorad Zafari (Bu Ali SIna University, Hamadan, Iran). Dr. Adnan Ismaiel (BPI) and Mag. Monika Komon-Zelazovska obtained many sequences from Trichoderma cultures. Ms Ellen Bloch (NY) patiently located and expedited the loan specimens of Hypocrea. Mr James Plaskowitz (BPI) and Dr Eric Erbe (USDA, Beltsville), respectively, performed the electron microscopy. A.T. Gräfenhan kindly reviewed an earlier version of this paper. The financial support by the Austrian Science Fund (FWF Project P16465-B03) to W.M.J. is gratefully acknowledged. This study was supported in part by the United States National Science Foundation (PEET) grant 9712308, “Monographic Studies of Hypocrealean Fungi: Hypocrea and Hypomyces” to the Pennsylvania State University, Department of Plant Pathology. | ||||
Notes Taxonomic novelties: Hypocrea viridescens Jaklitsch &
Samuels sp.nov., Trichoderma viridescens (A.S. Horne &
H.S. Williamson) Jaklitsch & Samuels comb.nov., T.
gamsii Samuels & Druzhinina sp.nov., T. vinosum
Samuels sp.nov., T. neokoningii Samuels & Soberanis
sp.nov., T. scalesiae Samuels & H.C. Evans sp.nov. | ||||
References
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