Clonal Species Trichoderma parareesei sp. nov. Likely Resembles the Ancestor of the Cellulase Producer Hypocrea jecorina/T.

reesei
Lea Atanasova, Walter M. Jaklitsch, Monika Komon-Zelazowska, Christian P. Kubicek and Irina S. Druzhinina Appl. Environ. Microbiol. 2010, 76(21):7259. DOI: 10.1128/AEM.01184-10. Published Ahead of Print 3 September 2010. Downloaded from http://aem.asm.org/ on February 11, 2012 by guest

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We showed that the extremely efficient cellulase-producing strains are present in all these phylogenetic species and that in general their carbon metabolisms are very similar. prov. 17). parareesei in conidiation intensity. Druzhinina1* Research Area of Gene Technology and Applied Biochemistry. where it destroyed canvas and other cellulose-containing materials of the U. Institute of Chemical Engineering. Getreidemarkt 9/1665. It is unique among industrial fungi. jecorina/T. Here we provide the formal taxonomic description of Trichoderma parareesei on the basis of macro.at. Thus. A-1030 Vienna. and the South Pacific region (1. reesei and form at least two new phylogenetic species.01184-10 Copyright © 2010. jecorina/T. Trichoderma reesei was originally collected on the Solomon Islands during World War II. jecorina/T.1 and Faculty Centre of Biodiversity. In that work we also provided some details on the ecophysiological characteristics of H.and micromorphologies.org/. 76. All Rights Reserved.APPLIED AND ENVIRONMENTAL MICROBIOLOGY. jecorina (4).S. Other putative anamorphs of H.asm. reesei was indistinguishable from mycelial cultures of the pantropical ascomycete Hypocrea jecorina. parareesei nom. reesei indicate that divergence probably occurred due to the impaired functionality of the mating system in the hypothetical ancestor of both species. p. 7259–7267 0099-2240/10/$12. reesei and T. 2010. Kuhls et al. TUCIM num- .00 doi:10. parareesei has previously been found to be essentially altered compared to the sequence of H. jecorina/T. Mailing address: Research Area of Gene Technology and Applied Biochemistry. Rennweg 14. No details on carbon utilization profiles or temperature-dependent growth rates have been presented. jecorina have more recently been identified as frequent inhab* Corresponding author. sympatric sister species devoid of sexual reproduction and which is constituted by the majority of anamorphic strains previously attributed to H. as T. their origin. have recently shown that the majority of anamorphic strains are genetically isolated from H. and all genetically improved mutant strains used in biotechnology today have been derived from it.zserv. The more frequent one (T. 7259 Downloaded from http://aem. Austria2 Received 18 May 2010/Accepted 25 August 2010 We have previously reported that the prominent industrial enzyme producer Trichoderma reesei (teleomorph Hypocrea jecorina. (4). such as coding fragments of the RNA polymerase B subunit II (rpb2) and GH18 chitinase (chi18-5). reesei. In this paper we present the formal taxonomic description of this new species. Druzhinina et al. army (18). 7. reesei is essentially evolutionarily derived. A phylogenetic analysis of relatively conserved loci. (14) found that T. [4]) was shown to be closely related to and also sympatric with H. We show that this species likely resembles the ancestor of H. although the clonal species are more versatile and efficient in the utilization of their preferred substrata. Institute of Chemical Engineering. which is still valid. Ascomycota.2‡ Monika Komon ´-Zelazowska.ac. Phenotype microarray analyses performed at seven temperature regimens support our previous speculations that T. Austria. reeseiᰔ† Lea Atanasova. Fax: 4315880117299. Nov. nov. Getreidemarkt 9/1665. 12). This and the fact that at least one mating type gene of T. showed that T. and mycoparasitism have been found. † Supplemental material for this article may be found at http://aem . and phylogenetic analysis. photosensitivity. American Society for Microbiology.asm. ᰔ Published ahead of print on 3 September 2010. the anamorph of the pantropical saprotrophic ascomycete Hypocrea jecorina.1‡ Walter M. and the NCBI GenBank sequence accession numbers are listed in Table 1. Phone: 4315880117202. we show that the sexually reproducing and correspondingly more polymorphic H. jecorina/T.tuwien. Likely Resembles the Ancestor of the Cellulase Producer Hypocrea jecorina/T. reesei was known only from the single isolate QM 6a for 50 years. which probably ensured the survival of this species in ancient and sustainable environment such as tropical forests. parareesei. jecorina. parareesei is genetically invariable and likely resembles the ancestor which gave raise to H. all suggesting that the latter species occupies a separate ecological niche and has much stronger opportunistic potential than H.1 1 Christian P. The isolates are stored at Ϫ80°C in 50% glycerol in the Collection of Industrial Microorganisms of Vienna University of Technology (TUCIM). strong interest in this fungus has also recently reemerged in attempts to produce second-generation biofuels to reduce carbon dioxide emissions and the dependence on fossil fuels (oil) (15. jecorina/T. is used in the biotechnological industry for the production of cellulolytic and hemicellulolytic enzymes and recombinant proteins (13. Vol. on the basis of sequences of the internal transcribed spacer of the rRNA gene cluster and randomly amplified polymorphic DNA (RAPD) fingerprinting analysis. Accordingly. University of Vienna. A-1060 Vienna. reesei. parareesei possesses a relatively high opportunistic potential. ‡ These two authors contributed equally to the manuscript and are listed in alphabetical order. carbon utilization profiles at different temperatures. jecorina but exclusively asexual (clonal or agamospecies). T. Jaklitsch. Kubicek. Trichoderma reesei. Hypocreales. Dikarya) has a genetically isolated. A-1060 Vienna. complemented by multivariate phenotype profiling and molecular evolutionary examination. The strains used in this work. however. Vienna University of Technology.org/ on February 11. No. they established the anamorph-teleomorph connection. Vienna University of Technology. Striking differences between H. Austria. jecorina and its sister species. 21). In contrast. 2012 by guest itants of soils in tropical forests of Southeast Asia. 21 Clonal Species Trichoderma parareesei sp. South America. For convenience.1128/AEM. E-mail: druzhini@mail. and Irina S. MATERIALS AND METHODS Material studied.

The micromorphology of conidiation structures.). Growth rates on different carbon sources were analyzed using a phenotype microarray system for filamentous fungi (Biolog Inc.25% Phytagel... Phytotoxicity assays were carried out using seeds of garden cress (Lepidium sativum) from Austrosaat AG (Vienna.S.S. collection of George Szakacs. Growth rates at 15. previously ech42) and rpb2 (RNA polymerase subunit II ß) was performed as described previously (9). jecorina/T.P. PCR fragments were purified (PCR purification kit. Conidial inocula were prepared by rolling a sterile. GenBank accession no. schweinitzii T. 25. The assay was designed on the basis of a 24-well plate.J.S.org/ on February 11.J. 0. Mycelia were harvested after 2 to 4 days of growth on 3% malt extract agar (MEA. 2012 by guest Other species from Trichoderma section Longibrachiatum Trichoderma sp. reesei as well as other species from Trichoderma section Longibrachiatum used in this studya Taxon C. parareesei and T.J. 42.78.J.J.P. 24. 3419 of H. and the comparative morphology of cultures were determined and morphological terms were applied as described by Jaklitsch (10). Indonesia Celebes. ATCC 18903 CBS 121275 G. Germany) DNeasy plant minikit following the manufacturer’s protocol.6). Phenotype microarrays. TABLE 1. 20. 86-401 G.S. which would prohibit the use of each locus for phylogenetic analysis. TUB F-728 TUB F-733 G. CBS. APPL. implemented in the same software.J.3) (19).P. CA). ATCC. 1837 T. wetted cotton swab over sporulating areas of the plates. 524 Trichoderma sp. QM 6a G. 48. 30. parareesei and H. Surface sterilization of the seeds was performed in 96% ethanol for 15 min. Briefly.J. Hungary. Europe HM183006 HM183007 HM183011 DQ087242b HM183009 HM183008 HM183010 HM182981 HM182982 HM182986 EU401511b HM182984 HM182983 HM182985 a Strains of T.P. parareesei sp. numbers of the original isolators’ collections are listed in Table 1. 1282. Summaries of the model parameters and nucleotide characteristics of the loci used are given in the Table 2. Amplification of fragments of chi18-5 (GH18 chitinase CHI18-5.K. Statistical analyses were performed using the Statistica software package (version 6.S.K. nov.J. saturnisporum H. H. 40. Merck.. Metropolis-coupled Markov chain Monte Carlo (MCMC) sampling was performed using the MrBayes program (version 3. G. ENVIRON. 07-26 CBS 125862. 06-140 G. 30.K.03% Tween 40).. strain C. CBS 125862. Beltsville. Phytotoxicity assays. OK).J. in addition.P.S. and C. 9). Germany) at 25°C. Color was determined as described by Kornerup and Wanscher (11). reesei.S.S. and Trichoderma sp. The neutral evolution was tested by Tajima’s test. (5). USDA. USA Germany. nov. and 96 h using a microplate reader (Biolog Inc. The optical density (OD) at 750 nm (for detection of mycelial growth [2. American Type Culture Collection. 06-138 G. PCR amplification. Origin chi18-5 rpb2 Reesei subclade Trichoderma parareesei sp. Germany). C. 524 were published earlier (1. Indonesia New Caledonia New Caledonia Solomon Islands Cameroon Cameroon Brazil HM182987 HM182991 HM182990 HM182992 HM182989 HM182993 HM182987 HM183002 HM182999 HM183005 HM182998 HM183003 HM183004 HM183000 HM183001 HM182994 HM182997 HM182996 HM182995 HM182963 HM182966 HM182965 HM182967 HM182964 HM182968 HM182962 HM182977 HM182974 HM182980 HM182973 HM182978 HM182979 HM182975 HM182976 HM182969 HM182972 HM182971 HM182970 Downloaded from http://aem. strains were cultivated on 3% MEA for 5 days. Hypocrea jecorina 717 type 3426 661 665 3420 634 3692 160 282 1392 1282 158 159 1127 1337 917 3418 3419 155 CBS 125925. TUB. which were incubated at 15. 66. Samples without Trichoderma were used as controls. DNA extraction. Tulsa. and sequencing. 85-249 G. 97-177 G. Strains of T.95 were not considered significant.0b10) (22). 25. 86-404 Mexico Ghana Argentina Argentina Brazil Sri Lanka Ethiopia Celebes. Samuels.asm. The conidia were then suspended in sterile Biolog FF inoculating fluid (0. Phylogenetic analysis. gently mixed.K. and adjusted to a transmission of 75% at 590 nm (using a Biolog standard turbidimeter calibrated to the Biolog standard for filamentous fungi). (2) and Friedl et al. Austria) and strains CBS 125925.K.S. followed by washing of the seeds with sterile double-distilled water.J. strain no. 93-22 CBS 383. jecorina. 04-41 TUB F-730 G. Centralbureau voor Schimmelcultures. and genomic DNA was isolated using a Qiagen (Hilden. Indonesia French Guiana Puerto Rico Celebes.S. as described by Druzhinina et al.S.). collection of Gary J. 4. strain C. MICROBIOL. The possibility of intragenic recombination.7260 ATANASOVA ET AL.1.J. DNA sequences were aligned by use of the Clustal X program (version 1.P. 665 of T. MD. images of cultures grown on 3% MEA for 5 days at 25°C under alternating 12 h of light/12 h of dark were taken. jecorina. 85-236 G. 35. Other strain no.J. 85-230 G. Bayesian posterior probabilities (PPs) were obtained from the 50% majority rule consensus of trees sampled every 100 generations after removal of the first trees using the “burnin” command. As the sample size is relatively small (1.50.0B4) with two simultaneous runs of four incrementally heated chains for 1 million generations. with this step . sativum. 5]) was measured after 18.K.J. TUB F-1066 G. parareesei and strains C. Hayward. The number of discarded generations was determined for each run on the basis of visual analysis of the plot showing the generation versus the log probability of observing the data. A total of 90 ␮l of the conidial suspension was dispensed into each of the wells of the Biolog FF microplates (Biolog Inc.P. Qiagen) and sequenced at Eurofins MWG Operon (Ebersberg. PP values lower than 0. In addition. 85-229 G. 81-300 Taiwan Taiwan Ethiopia Colombia Georgia.J. 917 (QM 6a). Growth rate determination on agar plates and morphological observations. was tested by linkage disequilibrium-based statistics implemented in DnaSP software (version 4.S. the unconstrained GTRϩIϩG substitution model was applied to all sequence fragments (Table 2). StatSoft Inc. strain C.S. The interleaved NEXUS file was formatted using the PAUP* program (version 44. Indonesia Celebes.K.670 characters per 26 sequences for the biggest data set). b Previously published by Jaklitsch et al. Briefly.81) and visually checked in GeneDoc software (version 2. 12).K. pseudokoningii 523 524 1837 1254 1266 2002 1277 TUB F-1034 TUB F-1038 CBS 48978 CBS 33070. and 45°C in darkness. and C. where each well was inoculated with one seed of L. growth rates.J. 72. TU Budapest.S. Phylogenetic analysis was done essentially as described previously (4.P.S. 93-23 G. longibrachiatum T. (8). bers (CPK) are used for the strains throughout the article. and 37°C were recorded in a single experiment using 2 strains each of T.

of thick. Twenty-nine sequences in total were analyzed. parsimony informative No. loosely arranged. 76.09 0. parareesei follow.5 to 3. Merck. thin. MycoBank accession number. ends becoming fertile. no distinct odor produced.12 0. straight or distinctly sinuous side branches.2 100 300 0. more or less arranged in concentric zones. or terminally on a supporting cell often similar in shape (intercalary phialides) or on few-celled cylindrical branches.12 0.VOL. Optimum growth at 30 to 37°C on all media. paired. and with thickenings to 5.5 ad 11 ␮m longae. Afterwards. the plants were inspected under a stereomicroscope and the lengths of their stems were measured. Consecutively. Darmstadt. erect. Differt a Trichodermate reesei absentia teleomorphosis. consisting of a loose reticulum bearing several straight or sinuous main axes with highly variable. 2010 TRICHODERMA PARAREESEI ANCESTOR OF T.2 ␮m longa. MB 515503 (Fig. radially arranged primary hyphae and numerous delicate secondary hyphae forming a reticulum. Morphology and growth rates of T. 1B3 to 3B3.20 0.20 0. 1 g of KH2PO4 and KNO3. of discarded first generations Total tree length (substitutions/site) a b Partial exon 852 80 685 0. The morphology and growth rates of T. 2.08 0.3 ad 6.5 ␮m when old. at higher temperatures often narrower.00 4/0. first white with sterile ends or elongations. 2012 by guest Alpha (shape parameter of the gamma distribution) and potential scale reduction factor (PSRF) values were estimated after GTR MCMC sampling and burning. (sub)globose to pyriform.5 to 5 ␮m. Autolytic activity and coilings lacking or inconspicuous.5 ad 3. flexible main axis often sterile at the tip. glabra. length/width ratios of 0. after ca. constant Parameters of MCMC analysis Mean nucleotide frequencya (A/C/G/T) Substitution ratesa A7C A7G A7T C7G C7T G7T Alphaa No.23 2/2 1 4/0. parareesei.3 Not applicable 1. conidiophoribus sinuosis et conidibus parvioribus. uniformioribus in forma et magnitudine. Pustules loose. 0. Individual seeds were introduced into 20 of the 24 wells filled with synthetic low nutrition agar (SNA) medium (for 1 liter. becoming fertile. or radially emergent from the main axis.3 to 0.24/0. Nucleotide properties of phylogenetic markers and MCMC parametersb Phylogenetic marker Parameter rpb2 chi18-5 7261 Concatenated data set Fragment characterization No. Shrubs loosely disposed and firmly attached to surface hyphae. Druzhinina & Atanasova. less commonly intercalary in wider hyphae. holding solitary phialides in right angles along their length. of chains/temp (␭) Sampling frequency No. 3.9 to 2.to 4-␮m-wide hyphae.10 0. slightly increased in number relative to that at lower temperatures. has been deposited in MycoBank under accession no. 3. Conidia viridia.0 to 4.44 0.5 g MgSO4 ⅐ 7H2O and KCl. of MCMC generations (106)/no. mostly in thin. Aerial hyphae scant. parareesei. RESULTS Taxonomy. Germany). Phialides in agaro CMD lageniformes vel ampulliformes. thick walled. REESEI TABLE 2.25 0.58 Partial exon 818 88 680 0.670 168 1.5 ad 3. Colony on CMD at 25 to 37°C hyaline. concentric zones. ellipsoidea vel oblonga. 2. 1). The sequence of Trichoderma parareesei Jaklitsch. concentrated on the agar surface.23 0. 6 to 22 ␮m long by 4 to 16 ␮m wide. and several tree-like side branches.06 0. in right angles or inclined upwards.org/ on February 11.asm. and 20 g of agar-agar. terminally 2. 4. Conidiation starting after 1 day on simple conidiophores of a straight. unpaired. 0.23 0. 3 days. The plates were incubated at 25°C under a rhythmic illumination cycle (12 h of light/12 h of dark) for 8 days.23/0. short. mycelium dense.03 0.6 mm in diameter.8 ␮m latae.5 ␮m wide.18 1/2 1.2 100 300 0. 2.5 ␮m lata. not zonate. transparent.. sporulatione multo magis abundante. turning pale to dark green.2 g glucose and sucrose.5 to 6. dark green pustules to nearly 2 mm in diameter formed.24/0.28/0.08 0. of characters No. maturing from inside.38 Downloaded from http://aem.6 (n ϭ 33). No diffusing pigment produced or agar turning slightly yellowish. directly.00 4/0. On CMD (Sigma corn meal agar plus 10% dextrose) mycelium covering the plate within 3 days at 25 to 37°C.30/026/0. becoming green and entirely fertile.11 0. Conidiophores (all branches) generally narrow. sp.20 0. first with straight to sinuous sterile elongations. concentrated in proximal areas and in one to several diffuse. less commonly fusoid or oblong. smooth.32 1/2 1. terminal.5 ␮m wide. sometimes in whorls of 3 on intercalary .365 0.37 0. nov. usually unpaired. Table 3 presents some characteristics of T.10 0.39 0.33/0. of runs PSRFa No. Chlamydospores at 30 to 37°C after 1 week uncommon. appearing as minute white shrubs 0. sometimes 2 celled.28/0.24/0. leaving 4 wells for the control of the fungal growth.06 21.09 0. Pregrown mycelium was introduced to 20 seedlings per strain. downwards 4. being repeated twice.2 100 200 0. Hyphal width decreasing with increasing temperature. flexuous.

asm. k. Downloaded from http://aem. o. (e and j to m) growth at 15°C. l. Scale bars: 15 mm (a to d). (d) culture on PDA after 4 days at 30°C (C. n. 917). SNA (b) and PDA (c). g. 717.P. p. (a to c. 15 ␮m (f. f.P. h. and n to t) strain C. SNA. p. MICROBIOL. m. (e to t) growth on CMD. 1.K. (e. g. i. (u) conidia (C. and r to t) growth at 25°C.K.7262 ATANASOVA ET AL. and 5 ␮m (o). and q) growth at 30°C.K. 2012 by guest FIG. APPL. (f) young conidiophore (3 days). 4 days).K. and j to m) strain C. 661. (i to n) conidiophores after 3 days (i and n) and after 9 days (j to m). (s and t) conidia (8 days). (e) conidiophore without cover glass (9 days).P. 10 ␮m (j. and q). (o and r) phialides at 2 days (o) and 8 days (r). and r to u). Trichoderma parareesei. . i. (a to c) Cultures after 4 days on CMD (a). (h) conidiophore of a shrub on growth plate (2 days).P. ENVIRON. 1127.org/ on February 11. 30 ␮m (e and h). n. (g) young conidiophore with sterile elongation (3 days). 25°C. (a to c. (p and q) chlamydospores (7 days). (d and u) Hypocrea jecorina (Trichoderma reesei). (f to i.

P.4–3. mycelium covering the plate within 3 to 4 days at 25 to 37°C. Additional isolates examined were CBS 125862 (C. REESEI TABLE 3. ellipsoidal or oblong with parallel sides 3. Autolytic excretions conspicuous at the colony margin. nov.5–3.5–11 (5–8) 2. Colony phenotype and anamorph characteristic of the species discussed 7263 Parameter T.5–3.3–3. Conidiation after 4 to 8 days at 25°C on CMD and SNA concentrated in a distal concentric zone. ellipsoidal to cylindrical at 37°C 3.3) 1. living cultures CBS 125925. on 4 September 1997. Conidiation on PDA and MEA conspicuously abundant. 2012 by guest Phialide Length (␮m) Width at widest point (␮m) Width at base (␮m) Length/width a b c 5. Habitat and distribution. thick walled. Diffusing pigment variable.5–4.VOL. reesei cultured in the same experiments is given here (Table 3). n ϭ 70. (7) noted that the fungus occurs in soil of the African oil palm (Elaeis guineensis) in South America. S1 in the supplemental material).2–4.7–3. denser. see Fig. mycelium covering the plate within 3 days at 25 to 37°C. parareesei sp.4) 1. smooth. Colony similar to CMD. Argentina.4) Data in parentheses represent the narrower ranges determined by the mean plus minus standard deviation. 661.0 (2.0–14.0) 2. phialides. eguttulate or with some minute guttules. uniformly ellipsoidal.8–4. ellipsoidal to cylindrical at 37°C. 76.5) 1.5)a 2. Phialides lageniform or ampulliform with often cylindrical neck. in numerous densely arranged and variably superposed bright yellow-green to dark green pustules (for cultures of T. Central America (Mexico).3–1. H.7–2.2 (1. 2010 TRICHODERMA PARAREESEI ANCESTOR OF T. near Iguazu Falls.5–3. only shrubs. but not originating at the same position.2–2. Argentina. and India (4). jecorina on 3% MEA.0 (2.2) 1. Conidiophores . TUB F-1066). Secondary hyphae forming a dense reticulum. and is deposited as a dry culture (WU 30015. n ϭ 75. reesei Colony type on CMD Colony radius (mm) at 48 h CMD 15°C 25°C 30°C 37°C PDA 15°C 25°C 30°C 37°C SNA 15°C 25°C 30°C 37°C Conidia Conidium Length (␮m) Width at widest point (␮m) Length/width Phialides Conidia formed abundantly in shrubs and welldefined pustules Only shrubs.7–2.K.5) 1. On potato dextrose agar (PDA).7) 8–19 34–44 44–55 37–58 9–16 36–49 45–59 42–59 9–15 32–40 40–60 32–64 Variable. Africa (Ghana and Ethiopia).6 (1.P. positively correlated with increasing temperature. 717. C.asm. mycelium covering the plate within 3 days at 25 to 37°C. often with a cylindrical neck. yellow if present.8–3. T.5) 1. conidiation more abundant.2–4. Anamorph morphology of Hypocrea jecorina/Trichoderma reesei.3–1. straight and symmetric or inequilateral and distinctly curved. The holotype was isolated from soil of a subtropical rain forest near Iguazu Falls. scar indistinct or pointed. To highlight differences in culture and anamorph morphologies. On CMD. farinose. Conidia formed in minute.2–2.8 (2.5 (2.2 (3. parareesei and H.3–3. no well-defined pustules formed. usually with distinct widening at or above the middle 4.6 (2–3.0b (1. Hojos-Carvajal et al. On SNA. and more regularly arranged in concentric zones.7c (1. jecorina/T.6–2. parareesei has been isolated from soils of subtropical and tropical areas in South America (Brazil.5–9. mostly dry heads 10 ␮m in diameter.0 (3. Argentina. jecorina/T.0 (1.3–6.9) Lageniform Downloaded from http://aem. no well-defined pustules formed 19–21 44–47 50–56 50–53 15–16 44–48 53–56 57–61 17–18 40–43 50–55 53–58 Uniformly ellipsoidal.5–6.K. more variable.5) Lageniform or ampulliform. with wide or constricted base. Colombia). Conidia green. usually with distinct widening at or above the middle. TUB F-728) from subtropical rain forest.5 (6–10) 2. a short description of H.org/ on February 11. Holotype. 4 September 1997. shape and size more variable. green.5–3.

jecorina. Figure 2A shows the temperature-dependent growth rates of both species calculated for the 16 best carbon sources (cluster I) for H.and micromorphological characters. We have previously reported that T. A detailed inspection of this subclade (Fig. The use of an integrated approach consisting of morphological observation and description. longibrachiatum and species related to it (H. parareesei and C. coarsely tubercular colony surface. Furthermore. The results show that both T. parareesei and H. Evolution of T. Development of the rootlets in the presence of Trichoderma was observed to be strain specific. parareesei is better adapted to growth in light and is more competitive with epigeal fungi than H.org/ on February 11. In contrast to T. strains of H. Thus.K. yet without statistical significance (ANOVA. Figure 2B shows examples of faster growth of T. DISCUSSION In this study we extend our earlier finding (4) of T. Moreover. jecorina (analysis of variance [ANOVA].K. strain C. (2). and H.P.K. The nucleotide properties of these loci and parameters of phylogenetic analyses are given in Table 2.P. parareesei and H. parareesei at 35°C on i-erythritol. where T. 667 of T. conidiation in T. jecorina) clearly inhibited maturation of Lepidium roots. 524 derived from the ancestral state.P. parareesei is evolutionarily older than H. 2012 by guest . Lepidium sativum. jecorina on the germination of seeds and on the growth of a model plant.K. In contrast. Macro. as estimated by Druzhinina et al.P. jecorina with respect to its ecophysiological adaptation (4). for H. jecorina. while four other strains form a statistically supported subclade derived from it. These data indicate that T. Here we performed phenotype microarrays (PMs) of both species at seven temperatures in darkness in order to get an advanced profile of carbon source utilization by T. Three strains of T. temperature-dependent carbon source utilization profiling. jecorina. 717 and C. jecorina and likely resembles the ancestor of the latter species. parareesei.0002 and n ϭ 67). The Reesei subclade is characterized by relatively short intercladal genetic distances compared to those present between other species in the section. However. similar to those of T. The detailed PM profiles are given in Table S1 in the supplemental material. sativum up to 40%. jecorina (Fig. the variation of inhibition among the T. reesei. parareesei is considerably different from H. Temperature-dependent growth of T. Phytotoxicity assays. parareesei constitutes a cryptic phylogenetic species genetically isolated from H. Results show that in general both species have their growth optimum at temperatures between 28 and 35°C. indeed. D-xylose. based on three of the most polymorphic phylogenetic markers applied previously (4). 3419 inhibited L. lageniform. The topology of the phylogram based on the combined data set (Fig. parareesei.P. it was noticed that T.K. suggesting that soil may be its natural habitat. we found that. parareesei (C. P Ͼ 0. 3426 [Sri Lanka]) are closest to the ancestral node. we have performed a pilot phytotoxicity assay. but no statistically significant differences were found between the species. parareesei. straight. similar patterns were also observed on gentobiose. This was also confirmed on both individual phylograms for rpb2 and chi18-5 (data not shown). Under such conditions. parareesei have been isolated from soil. in particular. Downloaded from http://aem. Larger branches becoming coarsely warted with age. jecorina and Trichoderma sp. and D-fructose.K. D-mannitol. 634 [Ghana]. P ϭ 0. ENVIRON.5 to 5 ␮m wide. D-arabitol. only rarely slightly curved. strain C. C.P. parareesei is conspicuously abundant and is positively correlated with temperatures up to 37°C.7264 ATANASOVA ET AL. which is contemporarily represented by T. growth of the plants with H. jecorina. sativum plants the most conspicuously. it also revealed a monophyletic Reesei subclade combining T. In order to extend our knowledge on the ecology of T. jecorina. 12). 524. APPL. jecorina on all media at all temperatures.K.K. (20) and shows that. which allow alignments with other species of Trichoderma section Longibrachiatum (Fig. we used Bayesian phylogenetic analysis of nucleotide sequences of two relatively conserved phylogenetic markers. In H. MICROBIOL. T. 3692 [Ethiopia]. jecorina. contrary to H. parareesei and H. 1837 [3]) are the next genetic neighbors to the species studied in this work. sometimes with long cylindrical necks. Phialides solitary. We have recently reported that T. parareesei was erroneously ascribed to earlier (1. Conidiation in T. strain C. parareesei is able to produce statistically higher biomass in this temperature range compared to that for H.05). orientalis and Trichoderma sp. 3419 of H. The most distinct difference between PM profiles of the two species was detected at 35°C. and D-galactose than the rate for H. which tests the influences of T. jecorina was strain dependent. D-cellobiose.P. showed that T. D-mannose. D-trehalose. parareesei. C. 7. jecorina exhibit qualitatively similar carbon source utilization profiles under standard temperature conditions (25°C) but respond differently to light (4). jecorina are. Conidia variable. ellipsoidal. resulting in a thick.asm. the anamorph of H. parareesei strains was minimal. jecorina on multiple carbon sources. and phylogenetic analysis resulted in a clear differentiation between T.P. However. rpb2 and chi18-5. several generations of bright yellowgreen pustules form consecutive superposed layers. The genealogical concordance phylogenetic species recognition criterion. straight. jecorina. P ϭ 0. parareesei and H.K. An inspection of the growth pattern on i-erythritol and D-mannitol can be used in the laboratory to distinguish the two species.P.P. or oblong with parallel sides. parareesei. Trichoderma sp. parareesei and T. T. strain C. The phylogram shows that both H. able to inhibit growth of L. jecorina. 3). ␣-D-glucose. as some strains (C. scar indistinct. 3B). This plant was chosen for its rapid growth in soil-free cultivations. T. jecorina. Statistically supported differentiation was detected only between each of the investigated fungal species and the control samples (for T. parareesei does not occupy any separate clade but is most closely related to the hypothetical common ancestor of the whole Reesei subclade. Furthermore. On richer media such as PDA and MEA. 2. parareesei showed a more variable utilization profile compared to that of H. In order to learn about the evolution of these two species with respect to other related taxa. jecorina. P Ͻ 0. parareesei as a new phylogenetic species of Trichoderma section Longibrachiatum to a full taxonomic description. 3A) confirms the result of Samuels et al. jecorina show considerable infraspecific polymorphism and occupy the longest branches of the Reesei subclade. in fact. all strains of T.05). parareesei in comparison to that of H. In addition to the carbon sources listed above. yet the plants were normally developed. parareesei in shrubs.0009 [ANOVA] and n ϭ 59. 3B) revealed that T.K. parareesei. parareesei is distinctly more abundant than in H.

D-cellobiose. Vertical bars correspond to standard deviations. a species that has lost its ability to reproduce sexually. parareesei and H. 2010 TRICHODERMA PARAREESEI ANCESTOR OF T. i-erythritol. parareesei. parareesei. D-arabitol. D-xylose. ␣-D-glucose. conidiation on PDA is usually yellow and turns green only slowly. the . D-melezitose. jecorina on different carbon sources. Phialide length only rarely exceeds 10 ␮m in T. Conidiophores of H. maltotriose. but they are generally straighter. L-arabinose. The formation of a yellow pigment is variable among isolates of both species.VOL. likely closely resembles the anamorphic stage of a common ancestor of all three species detected in the Reesei subclade. jecorina. Our data show that T. D-mannitol. ␥-aminobutyric acid). D-trehalose. parareesei possesses both the MAT1-1 and MAT1-2 loci. parareesei. We have recently demonstrated that although T. Reasons for genetic isolation and speciation. (2) for H. while on CMD and SNA. dextrin. jecorina on individual carbon sources. parareesei and are formed in shrubs. D-fructose. jecorina are comparable to those of T. while this is common in H. (A) Temperature-dependent growth rates of both species calculated for the 16 best carbon sources (cluster I. Temperature-dependent growth of T. REESEI 7265 Downloaded from http://aem. as estimated by Druzhinina et al. 2.org/ on February 11. jecorina.asm. respectively. parareesei and H. Colored labels highlight the differences. D-mannose. The complete carbon source utilization profiles are given in Table S1 in the supplemental material. with slightly longer phialides and conidia that vary in shape and size more conspicuously than those of T. jecorina. gentobiose. parareesei and H. calculated on the basis of the profiles of six and five strains for T. 76. 2012 by guest FIG. (B) Temperature-dependent growth of T. only shrubs and no welldefined pustules are formed.

html). it is interesting to speculate on reasons which led to the survival of T. we have also observed that T. and FWF P22081-B17 to W. MICROBIOL.org/Trive1/Trive1. T. parareesei and H. ENVIRON. we speculate that the inability of sexual reproduction likely originated from mutations in the MAT1-2 gene (4). and C. T. while T. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea.K. The ex type strain of T. (B) phylogenetic relations between species of the Reesei subclade (enlarged from panel A). Fungal Genet. strain C. parareesei is well adapted to various lighting conditions. which is one of the most sustainable and ancient ecosystems on the planet. 2005. G. . Bayesian circular phylogram inferred from the concatenated data set of partial exons of rpb2 and chi18-5 phylogenetic markers. parareesei is the oldest taxon which apparently nearly stopped its evolutionary development and that H. 524. where speciation occurred by restriction to a narrow habitat and lifestyle.K.7266 ATANASOVA ET AL. P. ACKNOWLEDGMENTS This work was supported partly by Austrian Science Fund grant FWF P-19340-MOB to C.P. jecorina and Trichoderma sp. Moreover the two species also have differences in their antagonistic potential in dual confrontations with fungi pathogenic for plants. Szakacs. parareesei is a relict agamospecies which resembles the ancestor of H. e. hamatum. jecorina has an unusual small genome compared to the sizes of the genomes of other ascomycetes and Trichoderma species (16. These considerations call for a comparison of the genomes of T.html and http://genome. APPL.J. also http://genome.home. longibrachiatum. jecorina.. G. latter is essentially altered compared to that of H. Downloaded from http://aem. Here we used conservative phylogenetic markers (coding fragments of the chi18-5 and rpb2 genes) to infer the evolution of the group of species rather than to differentiate T.org/ on February 11. reesei QM 6a (C. Thus. 524 arose from it. jecorina extensive production of propagules and growth on certain carbon sources was reduced during evolution. parareesei was to a large extent possible due to the long-term stability of its habitat. Symbols at the nodes correspond to PPs of Ͼ94%. and T. Arrows point to clades/nodes of phylogenetic species.. Biol.P. cf. (4) also showed that H. which theoretically makes the species vulnerable to changing environments. Kopchinskiy. M.K. In this case. jecorina.K. 917) is underlined. We think that the survival of T. the tropical forest.P. parareesei from the rest. However. showing that T. which showed abundant molecular footprints of sexual recombination for H.home. parareesei (4). We express special thanks to Aquino Benigno for his help with the DNA sequences. Komon. The analyses show that.jgi-psf. This hypothesis is well supported by the observations that H. I.jgi-psf. Bissett. we would rather suggest that in H. 42:813–828. where both mating-type loci are functional even in vitro. A.P. in fact. parareesei is generally more competitive. 2012 by guest FIG. extensive conidiation. jecorina is photoinhibited to a certain extent.org/Triat1/Triat1. J. strain C. T. suggesting that its overall phenotype likely reflects some losses in the genome. Druzhinina. jecorina and none for T. because this may provide insights into the original genetic potential of this important industrial species. Kubicek. As it is hard to imagine which mechanisms could be exploited by the agamospecies to gain new genetic/phenetic properties (6). parareesei.M. REFERENCES 1. parareesei has a versatile phenotype in a broad range of temperatures. (A) Phylogenetic position of the Reesei subclade with respect to other species from Trichoderma section Longibrachiatum. which altogether are characteristics in line with strongly opportunistic members of the genus.asm. The reduction of sexual recombination is reflected in the low level of infraspecific polymorphism of T. jecorina and Trichoderma sp. This is in perfect agreement with our previous results.g. Therefore. parareesei. 3. we conclude that T. Druzhinina et al. S. asperellum. and fast growth rates.

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