re-identification of hypocrea/trichoderma strains

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Microbiol. Cult. Coll. 26（2）：119 ─ 124, 2010

INTRODUCTION　Bissett (1991) classi f ied fungi of the genus Trichoderma into 27 species on the basis of mor-phology. In the last two decades, molecular phyloge-netic techniques have rapidly developed and improved the process of fungal systematics. By using such fungal systematics based on molecular phylogeny, over 100 species of Trichoderma have been validated. In addition, more than 400 species are presently recognized in the teleomorphic genus Hypocrea. Furthermore, even specialists may often encounter difficulties with species identification based on morphology. On the other hand, various gene-coding regions have been used for phylogenic studies and additional gene regions are being exam-ined.　Species of Trichoderma are useful in industries for their cellulase activity and many Trichoderma strains are preserved in several culture collections worldwide. However, even at world-famous culture collections, the names of fungi registered at the time of deposition, which may have been assigned on the basis of morphological identification, are used for these strains. Some of the names are expected to be inconsistent with those assigned according to the modern classification system. Druzhinina et al., (2006) pointed out that approximately 40% of the sequence data of Trichoderma spp. registered in the DNA databanks were derived from misidentified strains at the rank of species or from unidentified strains. To resolve such confusion, some American mycolo-gists have been engaged in the development of web-

based tools such as “Trichoderma Home,” which pro-vides morphological features (Samuels et al., 2010), and “International Subcommission on Trichoderma and Hypocrea Taxonomy” (ISTH; http://www.isth.info/tools/molkey/index.php), which serves as a con-venient identification tool using DNA sequences (Chaverii & Samuels, 2004; Samuels et al., 2006). 　Fungal cultures, including those of Trichoderma strains, maintained at the Institute for Fermentation, Osaka (IFO) were transferred to the NITE Biological Resource Center (NBRC) in 2002. Many of these strains had been primarily identified on the basis of classical morphological taxonomy at the time of deposition to IFO. Recently, several users have questioned the validity of the scientific names of the Trichoderma strains at NBRC. Therefore, we con-ducted a molecular phylogenetic analysis to re-iden-tify all of the 100 strains of Hypocrea/Trichoderma that were preserved at NBRC as of 2009. The sequence data of the NBRC strains were analyzed by 2 different methods available through the ISTH web tools: (1) TrichOKEY (version 2; Druzhinina et al., 2005), a molecular barcode reader that recognizes the species-specificity of several base pairs (known as anchors) within the rDNA ITS (ITS1-5.8S-ITS2) region; and (2) TrichoBLAST (Kopchinskiy et al., 2005), a homology searching tool for analyzing data-sets composed of only sequence data of the ex-type or authentic strains in the regions of ITS, elongation factor 1-a (tef1), and the second largest subunit of RNA polymerase II (RPB2). The results yielded by TrichOKEY and TrichoBLAST are occasionally dis-cordant. Such inconsistencies might be caused by insufficient sequence data and/or a lack of taxa in the web tool systems. In such conflicting cases, the morphological observation and the phylogenetic analysis were also performed. Finally, we deter-

*Corresponding authorE-mail: [email protected]この報告の和文が日本微生物資源学会ホームページからダウンロードできます．

Re-identification of Hypocrea/Trichoderma strains preserved at the NBRC collection

Sayaka Ban*, Kaoru Yamaguchi, Izumi Okane, Akira Nakagiri, Yukiko Tabuchi, Miwako Genra, Kuniko Shimamura, Shinzo Mayuzumi, Fumie Yokoyama,

Rieko Suzuki, Shigeki Inaba and Yasuhisa Tsurumi

NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan

Ban et al.Re-identification of NBRC fungal strains

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mined that the results from the phylogenetic analy-sis based on RPB2 sequences should be given priori-ty over the results obtained using the web tools in cases of discordant identification.

RESULTS　Table 1 provides a list of 30 strains, the species names of which were changed on the basis of the results of this analysis. In the case of NBRC 9062 (former name: H. rufa), NBRC 9065 (former name: T. koningii), and NBRC 9167, 104993 (former name; T. virens), the identified names obtained from the BLAST search on ITS and tef1 in the TrichoBLAST and phylogenetic analysis based on RPB2 were dis-cordant. Therefore, anamorphic structures of these strains were also examined and evaluated (Fig. 1).　Although their former names were found to be disputable and certain strains were highly similar to strains in other taxa, according to the homology search of the ITS region (Table 1), NBRC 5836, 31396, 31397, 31694, 31917, 31920, and 104991 were

not clearly assigned to any of the previously described Trichoderma species. For example, NBRC 5836, 31396, and 31920 are separated solely into a distinct deep branch in the phylogenetic tree (Fig. 2), whereas NBRC 31917 and 31694 were included in complex clusters composed of several species (data not shown). Thus, their names were revised as “Trichoderma sp”.

Section Rufa/Viride　The phylogenet ic analysis based on RPB2 revealed that the 28 strains belonged to the section Rufa/Viride (Fig. 2). Within this group, 12 strains were renamed as indicated in Table 1. Although T. ovalisporum NBRC 101778T was found to match with T. koningiopsis with 100% similarity in the TrichoBLAST search of the ITS region, the sequence analysis of tef1 indicated a match with T. ovalisporum strain DIS172h. Moreover, the phyloge-netic analysis based on RPB2 sequences indicated that the strain represents an isolated lineage (Fig. 2).

Table 1 Hypocrea/Trichoderma strains that were re-identified in this workNBRC No. Former name Re-identified name Highly matched taxon based on ITS *

5720 T. viride T. longibrachiatum T. longibrachiatum ATCC 18648 (100)8436 H. lactea H. atroviridis H. atroviridis CBS 142.95 (100)9062 H. rufa H. minutispora H. stellata GJS 99-222 (98.8)9065 T. koningii T. rossicum T. stromaticum CBS 101.875 (97.6)9066 T. viride T. harzianum T. harzianum DAOM 231412 (99.8)9165 H. gelatinosa H. rufa H. rufa GJS 91-62 (100)9167 T. virens T. crassum T. virens CBS 249.59 (99.8)30498 T. viride T. asperellum T. asperellum CBS 433.97 (100)30543 T. harzianum T. atroviride T. atroviride CBS 142.95 (100)30544 T. koningii T. spirale T. spirale CBS 346.93 (99.5)30545 T. pseudokoningii T. citrinoviride T. citrinoviride CBS 258.85 (100)30547 T. viride T. virens T. virens CBS 249.59 (100)31137 T. viride T. citrinoviride T. citrinoviride CBS 258.85 (100)31291 T. hamatum T. stramineum T. stramineum GJS 02-84 (99.3)31932 T. aureoviride T. hamatum T. hamatum DAOM 167057 (99.8)31976 T. harzianum T. viride T. viride GJS 91-62 (100)100100 T. viride T. harzianum T. harzianum DAOM 231412 (99.5)100586 T. harzianum T. atroviride T. atroviride CBS 142.95 (100)100846 T. longibrachiatum T. crassum T. virens CBS 249.59 (99.8)104569 H. cf. atroviridis H. voglmayrii H. voglmayrii CBS 117711 (100)104570 H. cf. atroviridis H. voglmayrii H. voglmayrii CBS 117711 (100)104993 T. virens T. crassum T. virens CBS 249.59 (99.8)5836 T. viride Trichoderma sp. T. koningii CBS 459.96 (99.5)31396 T. koningii Trichoderma sp. T. rogersonii GJS 90-125 (96.9)31397 T. viride Trichoderma sp. H. hunua CBS 238.63 (99.5)31694 T. aureoviride Trichoderma sp. T. harzianum NR5555 (99.8)31917 T. aureoviride Trichoderma sp. T. harzianum NR5555 (100)31920 T. pseudokoningii Trichoderma sp. T. koningiopsis GJS 93-20 (100)104991 T. koningii Trichoderma sp. H. semiorbis DAOM 167636 (99.3)7711 Hypocrea splendens Cosmospora matuoi Not Hypocrea/Trichoderma species

*The results by using TrichoBLAST based on ITS and their similarities (%) are shown.

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Microbiol. Cult. Coll. Dec. 2010 Vol. 26, No. 2

a b c

d e f

g h i

j k l

Fig. 1 Anamorphic structures of NBRC strains whose scientific names were corrected in this work. Scale bars: 20 mm. The accession number of NBRC, their former names and re-identified names were shown as follows; a: NBRC 5720 T. viride → T. longibrachiatum b: NBRC 9066 T. viride → T. harzianum c: NBRC 100100 T. viride → T. harzianum d: NBRC 31137 T. viride → T. citrinoviride e: NBRC 30547 T. viride → T. virens f: NBRC 9062 H. rufa → H. minutispora g: NBRC 30543 T. harzianum → T. atroviride h, i: NBRC 100586 T. harzianum → T. atroviride j: NBRC 100846 T. longibrachiatum → T. crassum k: NBRC 104993 T. virens → T. crassum l: NBRC 31932 T. aureoviride → T. hamatum


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Because no ITS and RPB2 sequences of T. ovalispo-rum were included in the ISTH dataset, a definitive conclusion could not be made.

Section Longibrachiatum　Trichoderma pseudokoningii NBRC 31977, 30545 (teleomorph: H. pseudokoningii) and H. schweinitzii NBRC 9063 (anamorph: T. citrinoviride) were clus-tered (Fig. 3). NBRC 31137 (the former name; T. viri-de) was also included in this section. This indicates that the strain should be re-named because T. viri-de belongs to the section Rufa/Viride. This strain

was re-identified as a strain of T. citrinoviride on the basis of the high similarity of its ITS sequence to that of T. citrinoviride. Trichoderma pseudokon-ingii NBRC 30902 and 30903 (represented as dashed squares in Fig. 3) were included in a single clade supported by high bootstrap values, but the basal nodes diverging from T. reesei, T. longibrachiatum, T. pseudokoningii, and T. citrinoviride were not sig-nificantly supported and the topologies were changed according to DNA regions (RPB2 vs. tef1). We renamed the 2 strains as Trichoderma cf. pseudokoningii because they represent a sister

H. minutispora AY481588H. voglmayrii DQ086150

＊H. cf. atroviridis NBRC 104569

＊H. cf. atroviridis NBRC 104570H. pezizoides AF545564

H. brevipes NBRC 101780H. cerebriformis NBRC 30610

T. asperellum NBRC 101777T

＊T. viride NBRC 30498

100

10067

T. pubescens AF545552T. hamatum AF545548T. hamatum NBRC 104990

＊T. aureoviride NBRC 31932100

＊T. koningii NBRC 31396T. strigosum AF545556

＊T. pseudokoningii NBRC 31920T. ovalisporum NBRC 101778T

H. albofulva NBRC 30609H. sulphurea NBRC 8437H. sulphurea NBRC 8438H. sulphurea NBRC 843999

10083

H. stilbohypoxyli NBRC 101774TH. rufa AF545521T. viride NBRC 30546H. rufa NBRC 104903

＊H. gelatinosa NBRC 9165

＊T. harzianum NBRC 31976

98


100

H. muroiana NBRC 31293H. muroiana NBRC 31287

H. muroiana NBRC 31288

＊T. harzianum NBRC 100586

＊H. lactea NBRC 8436

H. atroviridis NBRC 101776T＊T. harzianum NBRC 3054394

99

100

83

51

95

95

100

52

83

67

87

92

100

95

0.01Knuc

H. atroviridis/T. atroviride

→ Trichoderma sp. ?

H. rufa/T. viride

T. hamatum

T. asperellum

T. voglmayrii

→ Trichoderma sp.

→ Trichoderma sp.

Fig. 2　 A neighbor-joining tree showing the phylogenetic positions of the section Rufa/Viride based on RPB2 gene sequence. Bootstrap values greater than 50% are shown above the branches. The scientific names of the strains denoted by asterisks were revised after re-identification.

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Microbiol. Cult. Coll. Dec. 2010 Vol. 26, No. 2

group of T. pseudokoningii in the tef1 analysis.

Hypocrea strains (Section Hypocreanum)　Sequence data of the 12 species of Hypocrea spp. (23 strains of these species are preserved at NBRC) were not ava i lab le in ISTH dataset . In the TrichOKEY analysis, 6 strains of the 5 species (H. lacteal NBRC 8434 and 8435, H. austro-grandis NBRC 9183, H. sublutea NBRC 30156, H. albocornea NBRC 30608, and H. brevipes NBRC 101780) were found to have either no anchor sequences or only some of the species-specific anchor sequences. As a result, the comment, “The query sequence belongs to an unidentified species of Hypocrea/Trichoderma” was displayed. The retention of these species as strain names is not expected to cause any problems.　The phylogenet ic analysis based on RPB2 revealed that the 6 strains of H. nigricans (NBRC 30611, 31285, 31286, 31289, 31290, and 31294) are clus-tered with H. lixii/T. harzianum (data not shown). The BLAST search on ITS sequences also showed high similarities (99-100%) among the 6 strains and H. lixii/T. harzianum. Among them, NBRC 31285 is

identical to H. lixii/T. harzianum with respect to the tef1 sequence homology (total length is approxi-mately 300 bps). These results support Hypocrea nig-ricans is a synonym of H. lixii (Chaverri & Samuels, 2004). The tef1 sequences of the other 5 strains showed only 91-95% similarity with H. lixii/T. har-zianum. Thus, the 6 strains should be retained as H. nigricans until further taxonomical investigations are undertaken.　Hypocrea muroiana NBRC 31287, 31288, and 31293 were revealed to be closely related to H. atrovirid-is/T. atroviride in the homology search of both the ITS and the tef1 regions. The phylogenetic analysis based on RPB2 (Fig. 2) also showed the relatedness of these taxa. There have been no reports on the relationship between H. muroiana and H. atroviridis or H. muroiana and T. atroviride. We retained the name H. muroiana because H. muroiana I. Hino & Katum was described in 1958 and this pre-dates the description of H. atroviridis Dodd, Lieckf. & Samuels in 2003.

T. longibrachiatum

This species have not been available at ISTH

H. jecorina/T. reesei

H. schweinitzii/T. citrinoviride

T. konilangbra DQ083021T. sinensis DQ083012

T. longibrachiatum EU401556T. longibrachiatum NBRC 31918

＊T. viride NBRC 5720H. spinulosa NBRC 9064T. longibrachiatum NBRC 31919T. longibrachiatum NBRC 4847

57

57

61

0.005Knuc

Out group

H. pseudokoningii EU280097 DAOM 167678TH. schweinitzii NBRC 9063

＊T. pseudokoningii NBRC 30545


＊T. pseudokoningii NBRC 31977T. citrinoviride Z31017 CBS 258.85T

56

T. saturnisporum Z48726T. reesei NBRC 31326T. reesei NBRC 31327T. reesei NBRC 31328T. reesei NBRC 31329

90

T. pseudokoningii NBRC 30903T. pseudokoningii NBRC 3090293

Fig. 3　 A neighbor-joining tree showing the phylogenetic positions of the section Longibrachiatum based on ITS region. Bootstrap values greater than 50% are shown above the branches. The sci-entific names of the strains denoted by asterisks were revised after re-identification.


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CONCLUSIONS　Although T. viride NBRC 31137 is well known to produce cellulase and as one of the most salable strains of the NBRC, the orders for this strain by users have decreased after the name was changed to T. citrinoviride. According to the results of the sequence analysis, it was determined that all strains of T. viride in the NBRC collection originated from European countries and from Russia. All of the NBRC strains designated as “T. viride,” which were isolated from Japan, were re-identified as other spe-cies. Though many isolates of “T. viride” from Japan have been deposited at NBRC, there are no strains which genetically coincide with T. viride (Tokumasu, 2009; Kageyama, in press).　Changes in the scientific names of strains that have been well distributed have the potential to cause confusion among users. A scientific name adapted at the time of deposition into BRCs might be subsequently used in the scientific papers pub-lished by the depositor or by other researchers who examined the strain. In cases where the name does not represent a mis-identification (such as synony-mous names), there is no need to change the name. For example, some NBRC strains are also preserved at the Centraalbureau voor Schimmelcultures (CBS) and/or the American Type Culture Collection (ATCC), under different names. The names of some strains of fungi have been corrected in NBRC but not yet in other BRCs. Some of the major groups of fungi are actively being re-classified on the basis of various molecular phylogenetic methods. Therefore, it is unreasonable and impossible to keep abreast of the latest taxonomic system for all microbial strains, which have been deposited at different culture col-lections. However, periodic updates to the latest sys-tem would be required for continuous control of the quality of strains and for satisfying users’ needs and

reliance on culture collection systems. The species names used in the second edition of the NBRC cata-logue (2010) are examined by DNA sequence analy-sis, and the DNA sequence data of strains are pro-vided by the NBRC online catalogue (http://www.nbrc.nite.go.jp/e/).

REFERENCESBissett, J. 1991. A revision of the genus Trichoderma.

II. Infrageneric classification. Canad. Jour. Bot. 69: 2357-2372.

Chaverii , P. & Samuels , G.J . 2004. Hypocrea/Trichoderma (Ascomycota , Hypocrea les , Hypocreaceae): species with green ascospores. Stud. Mycol. 48: 1-116.

Druzhinina, I.S., Kopchinskiy, A.G. & Kubicek, C.P. 2006. The first 100 Trichoderma species charac-terized by molecular data. Mycoscience 47: 55-64.

Kageyama, K. Mycobiota in the subtropical and the cool temperate areas in Japan. IFO Res. Commun. (in press, in Japanese).

Kopchinskiy, A.G., Komon, M., Kubicek, C.P. & Druzhinina, I. 2005. TrichoBLAST: A multilocus database for Trichoderma and Hypocrea identifica-tions. Mycol. Res. 109: 657-660.

Samuels, G.J., Chaverri, P., Farr, D.F., & McCray, E.B. 2010. Trichoderma Online, Systematic Mycology and Microbiology Laboratory, ARS, USDA. from http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm

Samuels, G.J., Dodd, S.L., Lu, B.-S., Petrini, O., Schroers, H.J . & Druzhinina, I .S. 2006. The Trichoderma koningii morphological species. Stud. Mycol. 56: 67-135.

Tokumasu, S. 2009. An intensive investigation on the species diversity of microfungi within a small place. IFO Res. Commun. 23: 73-98 (in Japanese).

(Associate editor: Takayuki Aoki)

re-identification of hypocrea/trichoderma strains

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