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1 United States Department of Agriculture, Agricultural Research Service,
Systematic Botany and Mycology Laboratory, Rm 304, B-011A, Beltsville,
Maryland 20705, U.S.A.
2 The Pennsylvania State University, Department of Plant Pathology, Buckhout
Laboratory, University Park, Pennsylvania 16802, U.S.A. Current address:
New Zialand Institute of Crop and Food Research Ltd., Private Bag 4704,
Christchurch, New Zealand
3 The Pennsylvania State University, Department of Plant Pathology, Buckhout
Laboratory, University Park, Pennsylvania 16802, U.S.A. Current address:
Agronomy College, Department of Plant Protection, Zhongkai Agrotechnical
College, Guangzhou 510225, China
4 Tèra d'Sott 5, CH-6949 Comano, Ticino,
Switzerland
5 Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box
85167, 3508 TC Utrecht, The Netherlands. Current address: Agricultural
Institute of Slovenia, Hacquetova 17, 1001 Ljubljana, Slovenia
6 Technische Universität Wien, Abteilung für Mikrobielle
Biochemie, Institut für Biochemische Technologie und Mikrobiologie,
Getreidemarkt 9/172, A-1060 Wien, Austria.
*
Correspondence: Gary J. Samuels,
Gary{at}nt.ars-grin.gov
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO SPECIES OF...DISCUSSION AND TAXONOMIC...THE SPECIES OF THE...References |
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Trichoderma koningii s. str. is an uncommon species restricted to Europe and eastern North America; T. caribbaeum var. aequatoriale, T. koningiopsis, and T. ovalisporum were isolated as endophytes of trunks of Theobroma species in tropical America, and T. ovalisporum from the woody liana Banisteropsis caapi in Ecuador; T. koningiopsis is common in tropical America but was isolated also from natural substrata in East Africa, Europe and Canada, and from ascospores in eastern North America, and as an endophyte in Theobroma species; T. stilbohypoxyli, originally described as a parasite of Stilbohypoxylon species in Puerto Rico, is found to be more common in the tropics, besides an endophytic isolate from Fagus in U.K. The additional new species are known almost exclusively from their teleomorphs. Isolates of T. ovalisporum and T. koningiopsis may have biological control potential. A morphophenetic key and a set of tools for molecular species identification were developed.
Taxonomic novelties: Trichoderma austrokoningii/Hypocrea austrokoningii Samuels & Druzhinina sp.nov., T. caribbaeum var caribbaeum/H. caribbaea Samuels & Schroers sp.nov., T. caribbaeum var. aequatoriale Samuels & H.C. Evans var.nov., T. dingleyae/H. dingleyae Samuels & Dodd sp.nov., T. dorotheae/H. dorotheae Samuels & Dodd sp.nov., T. intricatum/H. intricata Samuels & Dodd sp.nov., T. koningiopsis/H. koningiopsis Samuels, C. Suarez & H.C. Evans sp.nov., T. petersenii/H. petersenii Samuels, Dodd & Schroers sp.nov., T. rogersonii/H. rogersonii Samuels sp.nov., T. stilbohypoxyli Samuels & Schroers sp.nov., T. taiwanense/H. taiwanensis Samuels & M.L. Wu sp.nov.
Keywords Actin / barcode / Bayesian phylogeny / local BLAST / biogeography / biological control / cacao / calmodulin / endophytes / GCPSR / Hypocrea / Hypocreales / Hypocreaceae / ISTH / ITS1 and 2 / molecular identification / morphological key / nomenclature / rDNA / RNA polymerase / sequence similarity search / species identification / systematics / translation elongation factor 1-alpha
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO SPECIES OF...DISCUSSION AND TAXONOMIC...THE SPECIES OF THE...References |
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What is T. koningii? Despite the fact that the genus Trichoderma Pers. was proposed late in the 18th century, prior to 1984 only 35 species were included in the genus, and before 1969 very few of these were reported in the literature subsequent to their original description. Trichoderma koningii, described in 1902 (Oudemans & Koning 1902), was included by Rifai (1969) as one of the nine "aggregate" or "morphological" species that he recognized. Bissett (1991a) included it in Trichoderma sect. Trichoderma, which includes the type of the genus, T. viride Pers., on the basis of the morphology of the conidiophore. Lieckfeldt et al. (1998) confirmed membership of T. koningii in sect. Trichoderma using ITS1 and 2 sequences of the rDNA gene cluster, and PCR fingerprinting, a result that has been affirmed in additional publications with other genes (e.g. Kullnig-Gradinger et al. 2002). Lübeck et al. (2004) showed that infra-species variation was greater than inter-species in ITS in the T. koningii aggregate species. Essentially, in that study ITS1 and 2 were not helpful in separating closely related species of sect. Trichoderma, but the authors found that UP-PCR fingerprinting could distinguish T. koningii from T. viride and other members of Trichoderma sect. Trichoderma. The first version of an oligonucleotide barcode based on ITS1 and 2 implemented in TrichOKEY program (Druzhinina et al. 2005) is able to identify the T. koningii/T. ovalisporum/H. muroiana species triplet and attribute it to the "Pachybasium A" clade after Kullnig-Gradinger et al. (2002).
Bissett (1991a) divided Trichoderma species among several sections. Among them was sect. Trichoderma, which included T. viride. Chaverri & Samuels (2004) proposed a move towards the classification based on phylogenetic clades rather than dividing the genus into sections. They referred to the "Rufa Clade," named for Hypocrea rufa, the type species of the genus, which included members of sections Trichoderma and species from the "Pachybasium A" Clade. The latter group includes T. hamatum (Bonord.) Bainier, the type species of Pachybasium Sacc., and other species. It was refered to as the "Hamatum clade" by Jaklitsch et al. (2006a). In the present work we refer to the combined "Rufa Clade" and the "Pachybasium A" Clade as the "Viride Clade." Trichoderma koningii and the species discussed in the current paper belong to that clade.
Lieckfeldt et al.
(1998) narrowly defined the
morphology of T. koningii and linked it to a teleomorph, Hypocrea
koningii Lieckfeldt et al. Lieckfeldt et al.
(1998) and Lübeck et
al. (2004) demonstrated
genetic diversity within the T. koningii aggregate species.
Lieckfeldt et al.
(1998) noted four additional,
morphologically similar and phylogenetically closely related species that they
identified as H. cf. muroiana or Hypocrea sp. One
of the strains identified by Lieckfeldt et al.
(1998) as H. cf.
muroiana has since been described as H. stilbohypoxyli B.S.
Lu & Samuels (Lu & Samuels
2003). Later, in a revision of T. viride, Lieckfeldt
et al. (1999) found
nine ITS haplotypes among isolates that conformed to the broadly defined
morphospecies T. koningii, of which one was true T. koningii
in the narrow sense of Lieckfeldt et al.
(1998). Holmes et al.
(2004) distinguished T.
ovalisporum Samuels & Schroers from T. koningii s. str. and
other members of the T. koningii morphological aggregate on the basis
of sequences of the protein-encoding gene translation-elongation factor
1-
(tef1) and conidium morphology. In addition to these T.
koningii-like species, Holmes et al.
(2004) designated four clades
of Trichoderma collections that have the T. koningii
morphology as "Tkon 20," "Tkon 21," "Tkon
22," and "Tkon 3."
Since the study of Lieckfeldt et al. (1999) we have received many additional collections from geographically and biologically diverse sources that can be assigned generally to sect. Trichoderma and specifically to the T. koningii aggregate species. In the present work we examine the phenotypic and phylogenetic diversity found within the T. koningii aggregate species, and develop a taxonomy for those fungi by combining results of morphological, cultural, and molecular-phylogenetic analyses.
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MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO SPECIES OF...DISCUSSION AND TAXONOMIC...THE SPECIES OF THE...References |
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Cultures used for study of anamorph micromorphology were grown on CMD or, less frequently SNA (without filter paper, Nirenberg 1976), at 20 or 25 °C for 7–10 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. Approximately 20 mL of agar was poured into Petri dishes.
We did not observe any difference in anamorph morphology between CMD and SNA but there was a tendency for more reliable conidial production on SNA than on CMD. Conidial pustules of Trichoderma isolates grown on these two media appeared to be more similar to how they appear in nature than conidia formed on other commonly-used media, including potato-dextrose agar, malt agar and oatmeal agar (Gams et al. 1998).
Morphological analysis of microscopic characters was undertaken from material that was first hydrated in the case of herbarium material, or wetted in the case of living cultures, in 3 % KOH. The KOH was subsequently replaced by distilled water. Measurements were made from KOH or water; we did not observe any differences between the two reagents. Where possible, 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 will be found. The parameters used for analysis are listed in Table 3. Chlamydospores were measured by inverting a 7–10 d old CMD culture on the stage of a compound microscope and observing with a 40 x objective. Data were gathered using a Nikon DXM1200 digital camera and Nikon ACT 1 software and measured using Scion Image (release Beta 4.0.2; Scioncorp, Frederick, MD).
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Five types of 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 8.00) was used for FL.
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 about 15 µm on a Microtome-Cryostat (International Equipment Co., Needham Heights, MA). The sections were first floated in water and then placed on slides to make semi-permanent preparations following Volkmann-Kohlmeyer & Kohlmeyer (1996). Slides are deposited with the specimens.
Growth rate trials were performed in darkness on potato-dextrose agar (PDA, Difco or Sigma) 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 96–120 h. Each growth-rate trial was repeated three times and the results of the three were averaged.
The slope of the growth curve, which reflects rate of growth per hour, is determined by linear regression. Regression is used to characterize the manner in which the colony radius changes (x's) with the time (y's) when measurements of colony radius are made. By revealing how the mean of the y measures changes as the various x measures change, the regression line is understood to describe the regression of y (colony radius) over x (time of measurement). This regression line is the slope of the growth curve; it is the predicted value of each colony radius for each time of measurement and essentially reports growth per hour (see http://www.animatedsoftware.com/statglos/sgregres.htm).
Principal Components Analysis (PCA), a multivariate analysis (Multivariate Statistical Package, version 1.131; Kovach Computing Services, U.K.), was utilized to determine patterns of variation of phenotype within phylogenetically defined groups. The eigenanalysis is shown in Table 2 and graphical output is shown in Fig. 4. The standardized data used in PCA, and other data analyses, were obtained using Systat version 10 (SPSS Inc., Chicago, IL, U.S.A.).
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Dry cultures of Trichoderma species were prepared by placing all or part of a culture growing in 9-cm-diam Petri dish in a cardboard two-slide micro-slide holder (e.g. VWR Scientific, West Chester, PA, U.S.A.) and drying them for ca. 2 h over low heat of a fruit dryer. Dry cultures were prepared so as to preserve essential characters of conidiophore branching and phialides.
DNA EXTRACTION, AMPLIFICATION AND SEQUENCING
The extraction of genomic DNA was performed as reported previously
(Dodd et al.
2002).
The PCR for amplification of the internal transcribed spacers 1 and 2 of the rDNA gene cluster (ITS1 and 2 including the 5.8S RNA gene) was performed in a 50 µL reaction volume using 5 µL of 10 x PCR buffer (Applied Biosystems), 200 µM dNTPs, 25 pmole of each primer (ITS1 and ITS4), 1.25 units AmpliTaq Gold (Applied Biosystems), and about 10–50 ng of template DNA. The reaction mixture was placed in a 0.2 mL PCR tube. The PCR was carried out on a PT-200 PCR system (MJ Research, Waltham, MA, U.S.A.) according to the following protocol: initial activation of AmpliTaq Gold at 95 °C for 10 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min; and a final extension period at 72 °C for 10 min. Five µL of the PCR product was analyzed on 1 % agarose gel in TAE buffer. The positive PCR reactions were purified using the Qiagen QIAquick PCR purification kit (Qiagen, California, U.S.A.) following the manufacturer's instructions. The concentration of the PCR products in ng/µL was determined on 1 % agarose gel electrophoresis in TAE buffer with Lambda Hind III DNA as a marker.
Similarly, 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), which resulted in a PCR product of approximately 600 bp, and was sequenced in both directions. The primers for amplification of the calmodulin-encoding gene (cal) were CAL-228F and CAL-737R (Carbone & Kohn 1999). Initially a fragment of actin gene (act) was amplified using the primers Fung.ACT.F1 and Fung.ACT.R1 and the conditions described by Wirsel et al. (2002). Based on the sequences obtained, two Trichoderma-specific act primers were designed Tact1 (5'-TGGCACCACACCTTCTACAATGA) and Tact2 (5'-TCTCCTTCTGCATACGGTCGGA). These two primers were used for amplification of act for all the isolates in this study. Additionally two sequencing primers for act were designed called Tact500F (5'-ATTCCGTGCTCCTGAG) and Tact511R (5'-CTCAGGAGCACGGAAT) and were used for sequencing reactions.
The portion of the RNA polymerase subunit B 2 (rpb2) gene was amplified and sequenced as described by Chaverri & Samuels (2004) using fRPB2-5F and fRPB2-7cR (Liu et al. 1999) as forward and reverse primers, respectively.
DNA sequences were obtained using the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, California). Products were analyzed directly on a 3100 DNA sequencer (Applied Biosystems). Both strands were sequenced for each gene.
ANALYSIS OF SEQUENCE DATA
Sequences were edited and assembled using Sequencher 4.1 (Gene Codes, WI).
Clustal X (Thompson et al.
1997) was used to align the sequences; the alignment of each locus
was manually edited using MacClade and GeneDoc 2.6
(Nicholas & Nicholas
1997). The sequences were deposited in GenBank
(Table 1) and alignments were
deposited in TreeBase
(http://herbaria.harvard.edu/treebase/),
submission number SN 1008). The multiple sequence alignment file for the
tef1 locus is also available at
http://www.isth.info/phylogeny/koningii.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. The modelblock3. nex which is compatible with the current version of PAUP* v. 4.0b10 was downloaded from http://workshop.molecularevolution.org/software/modeltest/files/modelblock3. Both hierarchical LRT and AIC output strategies were considered, although the preference was given to the last one. The unconstrained GTR + I + G substitution model was selected for all tree loci.
Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed
with four incrementally heated chains with the default heating coefficient
= 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 more than 200
sequences. Alignments of the other two loci (cal and act),
neither of which exceeded 100 sequences, were analysed using 3 million
generations. To check for potentially poor mixing of MCMCMC, each 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 first 2000 trees for tef1 and the first 500 for
cal and act using the "burn" command. According
to the protocol of Leache & Reeder
(2002), PP values lower then
0.95 were not considered significant, while values below 0.9 are not shown on
phylograms and radial trees. Model parameter summaries after MCMC run and
burning first samples were collected. For tef1 mean substitution
values were estimated as G
T =1, CT = 3.33, CG = 1.14,
AT = 1.32, AG = 5.98, AC = 1.43; nucleotide frequencies were
estimated as 0.19(A), 0.28(C), 0.17(G), 0.36(T); alpha parameter of gamma
distribution shape was 0.23. For cal mean substitution values were
estimated as GT =1, CT = 4.43, CG = 0.83, AT = 1.15,
AG = 3.55, AC = 1; nucleotide frequencies were estimated as
0.26(A), 0.26(C), 0.24(G), 0.24(T); alpha parameter of gamma distribution
shape was 0.1. For act mean substitution values were estimated with a
high affinity to pyrimidine transitions (CT = 81.9); other transitions
were GT =1, CG = 0.3, AT = 0.85, AG = 0.83, AC =
0.61; nucleotide frequencies were estimated as 0.2(A), 0.3(C), 0.24(G),
0.26(T); alpha parameter of gamma distribution shape was 0.09. The genetic
distance was computed in PAUP* v. 4.0b10 under the GTR + I model.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSKEY TO SPECIES OF...DISCUSSION AND TAXONOMIC...THE SPECIES OF THE...References |
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PHYLOGENETIC ANALYSES OF SEQUENCE DATA
The position of T. koningii-like species on the
Hypocrea/Trichoderma genus phylogeny is shown on the radial tree
obtained after the analysis of partial rpb2 sequences
(Fig. 1). This complex species
takes the terminal position on the sect. Trichoderma branch, which
consists of "Pachybasium A" and "Viride Clades"
("Rufa Clade" in Chaverri &
Samuels 2004, and Druzhinina
et al. 2005). It is interesting to note the relatively
short genetic distances within clades and species on this branch. The
neighbouring H. voglmayrii, which was recently described from the
Austrian Alps (Jaklitsch et al.
2006a), or species from "Hypocreanum" and "Lutea
Clades" are separated by longer evolutionary distances.
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The high degree of similarity of teleomorphs and anamorphs within the T. koningii species complex led us to anticipate the higher level of sequence similarity of protein-encoding DNA sequences. Therefore we chose phylogenetic markers with relatively big introns such as (i) the partial sequence of the translation elongation factor 1-alpha (tef1) covering the fourth (large) and fifth (short) introns (Kopchinskiy et al. 2005), (ii) the partial actin (act) and (iii) the partial calmodulin (cal) genes with two introns each. Since tef1 is the most variable locus (>50 % of variable sites) it was selected as a reference phylogenetic marker and, consequently, sequenced for all investigated strains. The cal and act genes were used to apply the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept of Taylor et al. (2000) to representatives of the main groups detected by the phylogenetic analysis of tef1.
In order to examine the phylogeny of T. koningii-like strains with respect to their position to the "Viride Clade" we aligned a portion of the tef1 gene for a large number of isolates. First, we attempted to analyse T. koningii-like strains against a background of few representatives of the nearest clades such as T. viride VD and T. viride VB (data not shown). However, the log probability plotted against the number of up to 5 million generations did not reach a stationarity. This indicated a low reliability of the resulting tree. Moreover, trees obtained in different runs with equal priors showed inconsistent topologies and were poorly resolved. Our strategy to solve this obstacle was based on the inclusion of the maximum known variability within the "Viride Clade" in the multiple sequence alignment, including T. viride VB, T. viride VD, H. stilbohypoxyli, T. erinaceus, T. atroviride and several potentially new taxa. The repeated consecutive use of intermediate phylogenetic analyses, rearrangements of sequences in MSA and realignments, particularly of the highly variable forth (large) intron of tef1, made it possible to produce the most correct final MSA file (available at www.isth.info/phylogeny/koningii). In this file, sequences of the T. koningii and T. viride complexes were aligned to representatives of T. asperellum and T. hamatum as members of the next neighbouring phylogenetic clade. As expected, likelihood estimations reached stationarity over generations, indicating reproducibility of the MCMC analyses. Fig. 2 represents a radial Bayesian phylogenetic tree obtained after 5 million chain generations.
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Analyses of cal and act sequences did not produce problems during repeated MCMC runs; results are shown in Fig. 3.
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In the tef1 tree, the most basal position of the LKB is occupied by the highly supported multifurcating clade of T. koningiopsis. Trichoderma koningiopsis presents a genetically variable species because only few internal nodes within that species are well-supported and are of numerous long intraspecific genetic distances. Although strains of T. koningiopsis have the same position on the LKB on the trees of Fig. 3, its identity as a distinct monophyletic clade is not statistically supported in analyses of the cal and act genes. These findings could indicate the presence of a relatively intensive recombination process due to sexual reproduction, despite the fact that the vast majority of the several strains of T. koningiopsis studied were derived directly from substrata with no known teleomorph.
In the LKB of the tef1 tree, T. ovalisporum and T. koningii s. str. possess two equally supported clades. The insignificant support of nodes where both of these species diverge from the main stem suggests approximately simultaneous speciation even for both taxa. However, the divergence was clearly allopatric, because T. koningii is common in North America and Europe, while T. ovalisporum is an endophyte from South America. The phylogenetic position of these two species is not contradicted by cal or act trees, although these genes did not always provide significant support. Compared to other species such as T. koningiopsis, T. koningii s. str. appears to be a relatively homogeneous taxon represented by strains from North America and Europe. Trichoderma koningii strains have almost identical sequences in the hypervariable large intron of tef1. Only one strain (DAOM 167073 from Québec) appeared distinct from numerous other strains, which had very similar or identical sequences, irrespective of their broad geographic distribution.
The upper part of the LKB on all three trees has a stepped structure with well-supported internal nodes (except the cal tree). Based on the concordance between the three loci trees, it consists of at least six phylogenetic species. In general, their phylogeny may be attributed to allopatric speciation because T. taiwanense is Asian, both T. dorotheae and T. dingleyae are isolated, known only from Australia and New Zealand, while strains of T. caribbaeum var. aequatoriale have a South American origin. The terminal position is occupied by T. petersenii. It consists of strictly North American and European clades. Trichoderma intricatum, which is located basal to the species listed above, may be an exception because it is represented by one Asian and one Caribbean strain.
Results of phylogenetic analysis show that additional species having T. koningii-like morphology have evolved independently from the taxa of the LKB. The majority of these species appear on the "Small Koningii Branch" (SKB), which is segregated from the LKB by taxa of the "Viride Clade" (Figs 2, 3). The first species on the SKB is T. rogersonii, which is represented by mainly North American and a few European strains. The terminal part of the SKB (tef1 tree) consists of a number of long lineages that lead to geographically separated strains. The divergence among these strains may be explained by allopatric speciation. Three of these six strains originated from Australia and New Zealand, one from Europe, one from the U.S.A., and one from Taiwan. Based on both tef1 and cal loci, Taiwan and North American strains form the most terminal well-supported clade, although on the act tree this clade also includes a European strain. Thus, there is no concordance between topologies of the act tree and trees inferred from sequences of the other two loci. This finding makes it difficult to draw conclusions about phylogenetic species on the terminal part of the SKB.
The third lineage that is characterized by the koningii morphology is represented by the single species T. stilbohypoxyli.
PHENOTYPE: ANAMORPH
For summary of continuous characters see
Table 3.
A total of eighty-six strains were studied. Typical of Trichoderma, very little aerial mycelium forms on CMD or SNA, and mycelial production on PDA is typically lush. There is variation among the strains of individual clades as to whether conidiophores form in aerial mycelium or in complex cottony pustules on CMD as well as in relative amounts of conidial production. Conidial production on CMD and SNA tends to occur at the margin of the colony. Discrete conidial pustules sometimes form on CMD and SNA, but on PDA pustules are not formed, rather, conidia form in dense, effused areas. On CMD and SNA pustules are at most 1.5 mm diam and usually smaller, hemispherical, uniformly cottony. Entirely fertile, somewhat plumose conidiophores can often be seen within pustules (e.g. Figs 176, 197). Usually projecting sterile hairs or conidiophores that are only fertile at the apex are absent, but occasionally long, apically fertile conidiophores are seen in T. austrokoningii (Figs 55, 97), T. dingleyae (Fig. 138), and T. koningiopsis (Fig. 212). Conidiophores also form in the aerial mycelium. One isolate of Trichoderma dorotheae (G.J.S. 99-202) formed hemispherical pustules in addition to conidiophores in the aerial mycelium. Conidiophores in the pustules were not easily discerned (Figs 156, 161). In older cultures of this species phialides appeared to proliferate percurrently to form a second phialide (Fig. 158). The newly formed phialides were often abruptly swollen in the middle. This aspect is also seen in Eidamia viridescens A.S. Horne & H.S. Williamson (1923), which is T. viride VD of recent publications (e.g. Lieckfeldt et al. 1999, Dodd et al. 2003, Holmes et al. 2004) and distinct from "true"T. viride (VB in Figs 2, 3). Pustules generally are compact, formed of intertwined hyphae that tend to branch dichotomously near the surface and to produce short branches that sometimes act as phialides, or for the cells near the surface of the pustules to swell and produce two or more short cells. In addition, verticillium-like conidiophores arise from near the surface of the pustules. Conidia are dark green (27E–F7–8). There is variation in the time and temperature at which conidia appear. Most of the isolates of T. dingleyae and T. dorotheae lost their ability to produce conidia after storage on cornmeal agar slants at ca. 8 °C.
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Conidiophores of T. taiwanense were unusual in often being conspicuously enlarged and verrucose at the base (Figs 290–293).
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7.5–8.5 µm. In two species, T. dingleyae and
T. caribbaeum var. aequatoriale, mean phialide length was
9.5–10 µm and in T. intricatum and T.
stilbohypoxyli mean phialide length was ca. 7.0 µm. Conidia of all collections included in this study were oblong to ellipsoidal or ovoidal and smooth; the mean length was 4.0 µm, the mean width was 2.7 µm, and the mean L/W was 1.4. The mean length of conidia in most collections ranged 3.8–4.0 µm; conidia in collections of T. dingleyae and T. koningii were longest, 4.1–4.3 µm, while the mean length of conidia of T. austrokoningii, T. ovalisporum, T. intricatum and T. stilbohypoxyli ranged 3.4–3.5 µm. The mean width of conidia of most collections ranged 2.7–3.0; conidia of T. dingleyae, T. dorotheae, T. intricatum and T. ovalisporum were somewhat wider, the mean ranging 3.0–3.2 µm, while conidia of collections of T. koningii, T. koningiopsis and T. stilbohypoxyli were somewhat narrower, ranging 2.6–2.7 µm. The mean length/width ratio of most collections ranged 1.4–1.5; the mean L/W ratio of conidia in collections of T. koningii was 1.6 and in T. ovalisporum and T. intricatum, which have broadly ellipsoidal to ovoidal conidia, the mean L/W was ca. 1.2.
Chlamydospores are produced sporadically by members of the various species and of the clades, only the two collections of T. intricatum failed to produce chlamydospores. Chlamydospores are typical of Trichoderma, being terminal or intercalary within hyphae, and globose to subglobose.
PHENOTYPE: TELEOMORPH
Most of the strains that we studied were derived from ascospores of
Hypocrea specimens. The most notable exception was T.
koningiopsis, a common tropical species that was most often encountered
as direct isolations from substrata, including as an endophyte from trunks of
trees of Theobroma cacao and Th. gileri, and only a few
isolates were derived from ascospores. Trichoderma koningii s. str.
was most often directly isolated from substrata but three cultures were
derived from ascospores of Hypocrea collections made in the United
States and one from the Netherlands. Trichoderma ovalisporum is known
only from four isolations, all from natural substrata. Trichoderma
caribbaeum and its variety aequatoriale (DIS 320c) are
represented by three strains; of these, two were isolated from ascospores of
Hypocrea specimens collected in, respectively, Guadeloupe (G.J.S.
97-3) and Puerto Rico (G.J.S. 98-43), where they were growing on
fructifications of black ascomycetes; the variety (DIS 320c) was isolated from
the trunk of a live tree of Theobroma gileri in Ecuador and may be an
endophyte. Despite strong phylogenetic similarity between DIS 320c, on one
hand, and G.J.S. 97-3/G.J.S. 98-43 on the other, DIS 320c is phenotypically
quite different from the other two and we regard it as a variety of T.
caribbaeum.
Stromata (when dry, Figs 24–35, 36–50) were typically 6C–D8, brownish orange to light brown, but in T. petersenii stromata are darker, 7–8E–F8, reddish brown; stromata were typically pulvinate and broadly attached or at most slightly free at the margins. Perithecial elevations, or mounds, were not visible; the stroma surface was plane or wrinkled. Ostiolar openings were not visible, at least in dry specimens, or were barely visible as viscid circular areolae or dots on the stroma surface. There was no reaction to KOH in any tissue. When young, stromata were semi-effused, light brown or tan and villose; the developing stroma retained the villose aspect, which eventually was lost. The villose aspect is the result of short hairs that arise from the cells of the surface of the stroma; these are 5–10 µm long, 2.5–3.5 µm wide, septate, unbranched, often spinulose. The stroma surface, seen in face view, appeared mottled with unevenly deposited brown pigment in the cell walls. The cells at the surface of the stroma, when seen in face view, were angular, 3–7 µm diam, with walls slightly thickened. The stroma comprised three anatomically distinct regions. The surface region was 15–25(–35) µm thick and pigmented, in section appearing yellow when mounted in lactic acid. Cells of the surface region were angular, 2.5–5 µm diam, with slightly thickened walls. The tissue immediately below the stroma surface consisted of compact to loosely disposed hyphae. The tissue below the perithecia was pseudoparenchymatous, the cells measured 5–10(–15) x 3–7(–10) µm; their walls were slightly thickened or not visibly thickened; cells were oriented perpedicular to the surface of the substratum. The stromata of T. taiwanense (G.J.S. 95-93) are atypical in the group because they are luteous, lack hairs and have conspicuous ostiola; however this specimen is old and possibly has lost the traits that are typical of this group.
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Asci were cylindrical, 60–70 x 4–5.7 µm, completely filled with ascospores; there was a thickening at the tip of each ascus. Ascospores were bicellular; they disarticulated at the septum into two part-ascospores early in development.
There was little variation in ascospore morphology or measurements among the 46 teleomorph collections that were studied. Differences are noted as follows: Part-ascospores were hyaline, spinulose, dimorphic; distal parts ranging (3.0–)3.5–4.0(–4.5) x (2.0–)3.1–3.8(–4.0) µm; proximal parts ranging (2.7–)3.7–4.5(–5.0) x (2.2–)2.7–3.2(–3.7) µm. The means of distal part-ascospores of most collections ranged 3.6–4.0 x 3.2–3.7 µm and of proximal part-ascospores 4.0–4.6 x 2.8–3.2 µm. The ascospores of H. intricata were somewhat smaller overall than in the other species. The distal part-ascospores of H. intricata were somewhat shorter and narrower than in the other species, ca. 3.3 x 3.1 µm. The proximal part-ascospores of H. intricata were also smaller, falling in the lower end of the range of spore dimensions overall. The distal part-ascospores of H. koningii were somewhat longer than most species (mean 4.1 µm) while those of H. petersenii were somewhat wider than in most species (mean = ca. 3.3 µm).
PHENOTYPE: COLONY MORPHOLOGY AND GROWTH RATE
Colony morphology is described from PDA at 25 and 30 °C in light or
darkness after 72–96 h. Colony morphology is more or less consistent
within a species. The cultures illustrated in Figs
6–14,
15–23 are representative
of the respective species. There is a tendency for conidia to form in
concentric rings that are more or less pronounced; this is especially clear in
T. petersenii. With the exception of T. dingleyae and T.
dorotheae, conidia tended to form in abundance and to be dark green;
conidial production in these two species is poor.
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No distinctive odour was detected in any cultures, or rarely a coconut odour in T. koningii.
BIOGEOGRAPHY
With few exceptions, a biogeographic bias was seen in the respective clades
(Fig. 2,
Table 3). The many reports in
the literature of wide distribution not withstanding, distribution of T.
koningii is limited to eastern North America and Europe. Trichoderma
koningiopsis is a common and cosmopolitan species, but it is more common
at tropical than at temperate latitudes.
Most of our isolates originated in the American tropics, but the species occurs in Canada (Ontario) and Germany, and its teleomorph has been found in the U.S.A. (Kentucky). It was also found in the rhizosphere of Coffea arabica from the main coffee-growing area in Ethiopia, where sampling was done from elevations of 1300–2000 m (T. Belayneh, pers. comm.). Trichoderma stilbohypoxyli was also revealed in this work to be a common tropical species, being widespread in tropical America and found in one location in Ghana, but it also occurs in the U.K.
Trichoderma petersenii and T. rogersonii are common and sympatric in eastern North America and central Europe; we have only seen T. rogersonii as isolations from ascospores but we have a single soil isolate of T. petersenii; stromata of T. petersenii have been collected also in Costa Rica. Trichoderma intricatum is known only from two ascospore-derived cultures that originate, respectively, in Puerto Rico and Thailand.
The most problematic clade from the point of biogeography is the clade comprising strains identified here as T. austrokoningii. This clade includes six isolates with unclear phylogenetic position because topologies of corresponding branches are nonconcordant among three loci. Most divergent are two strains, respectively, from Florida and Taiwan. The basal lineage on the tef1 tree comprises two strains (only one shown in Fig. 2) from the South Island of New Zealand. There is a single lineage/isolate from Russia and two closely related collections from tropical Australia. The sequence divergence in this clade suggests that additional sampling would resolve it into two or more species. Although collections in the two Australasian clades are physically relatively close to each other, their actual locations are climatically very different, tropical in the case of the two Australian cultures and south-temperate in the case of the collections from New Zealand.
We cannot say that any geographic region is more diverse than any other as regards the genetic diversity represented in Fig. 2. As was noted above, T. petersenii and T. rogersonii, T. koningii and T. koningiopsis are sympatric in eastern North America. Three species are found in Australia and New Zealand, viz. T. dingleyae, T. dorotheae and T. austrokoningii, although the latter species was found on the northern, tropical Queensland coast of Australia, whereas the other two were collected in south-temperate Nothofagus forests of Australia (Victoria) and New Zealand (S. Island).
SUBSTRATUM
The Hypocrea specimens from which most of the cultures were
derived were found either directly on ascomata of, often, members of the
Xylariaceae or on indeterminate black fungi on rotting decorticated
wood or bark of rotting trees. In only a few cases was a fungal substratum not
seen. The isolates taken directly from the substratum were taken from soil,
less frequently from fallen leaves and mushroom casing. Several isolates were
recovered as endophytes from the sapwood of stems of Theobroma
species or, in one case (T. ovalisporum, DIS 70a), a liana. The
Trichoderma endophytes of Theobroma gileri were reported by
Evans et al. (2003).
Five isolates of T. koningiopsis from Ecuador, represented in the
cladogram by G.J.S. 01-07, were isolated directly from pods of Theobroma
cacao infected with Moniliophthora roreri that had been placed
on cacao leaf litter in a search for parasites of the Moniliophthora.
Perhaps most interesting of the Trichoderma endophytes of woody
plants was T. stilbohypoxyli. We have found (Samuels, unpubl.) that
Trichoderma stem endophytes tend strongly to be specific to host
genus and to biogeography, but T. stilbohypoxyli was isolated as an
endophyte from trunks of ancient Fagus sylvatica in the United
Kingdom and Theobroma species in Ecuador and Brazil.
RESULTS OF PRINCIPAL COMPONENTS ANALYSIS (PCA)
PCA was performed to determine the correlation between phenotype traits and
clades; the phylogenetic clades were used as the grouping factor. Only
characters of the anamorph and of colony morphology and growth rates and
geographic distribution were used in the analysis because they were common to
all isolates. Characters of the teleomorph were not utilized in PCA because
those characters were not common to all strains. Analysis of the teleomorph
characters did not resolve groups. In the PCA of the geographic and phenotype
characters listed in Table 3,
51 % of the variation is accounted for by the first three axes. While the
results of the Eigen analysis (Table
2) do not indicate a strong fit of the data to the model, a
scatter plot of the Eigenvalues reveals that isolates of the same
clade/species tend to group together (Fig.
4). The two geographically distinct isolates of T.
intricatum were separated because of the slow growth of G.J.S. 97-88.
Slow growth also pulled DIS 94c from the rest of the isolates of T.
koningiopsis, and CBS
979.70 from the rest of T. koningii. The four Puerto
Rican gatherings of T. stilbohypoxyli clustered together and distant
from the other 8 cultures; this may be because the Puerto Rican strains have,
on average, slightly longer and wider conidia than the others. The
phylogenetic diversity of T. koningiopsis and, especially, T.
austrokoningii is reflected in their wide dispersion on the scatter plot
of eigenvalues.
IDENTIFICATION OF SPECIES
Three methods of Hypocrea/Trichoderma species identification based
on the analysis of DNA sequences have been developed. Most recently, an
automated identification system using oligonucleotide DNA barcodes of ITS1 and
2 sequences was developed. If it is already available for the group under
investigation, a barcode is the easiest method to obtain an absolute result.
The second possibility is to perform a similarity search (BLAST) against a
pool of voucher sequences. This method is very useful because a search can be
made using multiple loci; however, results from this technique are unavoidably
uncertain, because the user must subjectively weigh every mismatch in the
resulting sequence alignment. Moreover, since gene evolution does not always
reflect the speciation process, it is highly recommended to obtain a
concordant result of several unlinked loci. The third method of molecular
species identification is the most reliable one but, at the same time, the
most laborious because it implies phylogenetic analyses and the application of
the Gene Concordance Phylogenetic Species Recognition (GCPSR) concept of
Taylor et al. (2000).
A detailed description of the application of each of these methods to the
T. koningii aggregate species is given below. Molecular
identification is available via a dedicated online "T. koningii
morphological species project", which is located at
www.isth.info/phylogeny/koningii.
Using ITS1 and 2 and the oligonucleotide barcode program TrichOKEY v. 1.1
The first version of DNA oligonucleotide barcode integrated in
TrichOKEY v. 1.0
(www.isth.info,
Druzhinina et al.
2005) is able to recognize Trichoderma sect.
Trichoderma and all species from the "Viride Clade" that
were known prior to this study. Thus, the barcode distinguished the T.
koningii aggregate species as a triplet of T. koningii/T.
ovalisporum/H. muroiana. We have investigated the inter- and
intraspecific variability of ITS1 and 2 sequences from the complex based on
the present larger sample size. Unique species-specific oligonucleotide
hallmarks for T. petersenii and T. rogersonii and T.
koningii s. str. have been discovered. Because all three species are
known from many specimens, all of which were considered in the development of
the barcode, the resulting identification is reliable ("standard"
in TrichOKEY v.1.1). In addition, a characteristic DNA signature that
is common to both isolates of T. intricatum is incorporated in the
program. Although, due to the low number of available isolates, the barcode
identification is of low reliability and needs to be confirmed by other
methods of sequence analysis. Other species from the T. koningii
aggregate species such as T. koningiopsis, T. caribbaeum, T. ovalisporum,
T. dingleyae, T. dorotheae, T. taiwanense and T. austrokoningii
are not distinguishable based on ITS1 and 2 sequences, at least based on the
observed diversity. Therefore, they will be identified as "T.
koningiopsis or 6 rare species with T. koningii
morphology" because T. koningiopsis is the most abundant and
cosmopolitan species, known from more isolates than the total number of
specimens from other species with ITS1 and 2 haplotype identical to it. Help
involving biogeography is provided for distinguishing these species. Thus, the
updated version of the ITS1 and 2 barcode (TrichOKEY v. 1.1) is able
to distinguish all sympatric species from the complex of the T.
koningii aggregate species.
Using tef1 and sequence similarity search program TrichoBLAST
The TrichoBLAST tool for Hypocrea/Trichoderma sequence
identification installed on
www.isth.info
(Kopchinskiy et al.
2005) determines the sequence from the database to which the query
sequence is most similar. The main TrichoBLAST database consists of
sequences of five phylogenetic markers (two introns and one exon of
tef1, partial exon of rpb2 and ITS1 and 2). With respect to
the present group, the first version of this database included only two
sequences of each tef1 intron from two strains of T. koningii s.
str. and one strain of T. ovalisporum. The remaining
biodiversity was not considered. In order to facilitate the identification of
species from the T. koningii morphological species, we have extracted
both most variable tef1 phylogenetic markers (forth large and fifth
short introns) from the type sequences of each <
