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Trichoderma biodiversity in China: Evidence for a North to South distribution of species in East Asia
FEMS Microbiology Letters 251 (2005) 251–257 www.fems-microbiology.org
Trichoderma biodiversity in China: Evidence for a North to South distribution of species in East Asia Chu-long Zhang a, Irina S. Druzhinina b, Christian P. Kubicek
b,*
, Tong Xu
a,*
a
b
Institute of Biotechnology, Zhejiang University, 310029, China Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Getreidemarkt 9/E1665, A-1060 Vienna, Austria Received 19 July 2005; accepted 8 August 2005 First published online 6 September 2005 Edited by M.J. Bidochka
Abstract Towards assessing the biodiversity and biogeography of Trichoderma, we have analyzed the occurrence of Trichoderma species in soil and litter from four areas in China: North (Hebei province), South-East (Zhejiang province), West (Himalayan, Tibet) and South-West (Yunnan province). One hundred and thirty five isolates were grouped according to tentative morphological identification. A representative 64 isolates were verified at the species level by the oligonucleotide barcode program TrichO Key v.1.0 and the custom BLAST server TrichoBLAST, using sequences of the ITS1 and 2 region of the rRNA cluster and from the longest intron of the tef1 (translation elongation factor 1-a) gene. Eleven known species (Trichoderma asperellum, T. koningii, T. atroviride, T. viride, T. velutinum, T. cerinum, T. virens, T. harzianum, T. sinensis, T. citrinoviride, T. longibrachiatum) and two putative new species (T. sp. C1, and T. sp. C2), distinguished from known species both by morphological characters and phylogenetic analysis, were identified. A significant difference in the occurrence of these species was found between the North (Hebei) and South-West (Yunnan) areas, which correlates with previously reported species distributions in Siberia and South-East Asia, respectively. As in previous studies, T. harzianum accounted for almost half of the biodiversity; although, in this study, it was exclusively found in the North, and was predominantly represented by an ITS1 and 2 haplotype, which has so far been rarely found elsewhere. This study therefore reveals a North–South gradient in species distribution in East Asia, and identifies Northern China as a potential center of origin of a unique haplotype of T. harzianum. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hypocrea; ITS1 and 2; tef1; Biogeography; Phylogeny
1. Introduction Fungi are considered the fifth kingdom, characterized as eukaryotic heterotrophic organisms with a low level * Corresponding authors. Tel.: +43 1 58801 17250; fax: +43 1 58801 17299 (C.P. Kubicek), Tel.: +86 571 86971208; fax: +86 571 86046615 (T. Xu). E-mail addresses: [email protected] (C.P. Kubicek), [email protected] (T. Xu). URLs: http://www.vt.tuwien.ac.at, http://www.isth.info (C.P. Kubicek).
of ‘‘tissue’’ differentiation and chitin/b-glucan-containing cell walls. They form spores as a stress resistant and spreading phase of their life cycle (http://www. mycolog.com/index.html). Approximately 120,000 species of fungi have so far been identified worldwide [1], but recent estimates claim about 1.6 million species in total [2]. Despite this high diversity, the biogeography of fungi is still a neglected topic, and molecular studies directed towards an assessment of the global biodiversity of fungi are only slowly emerging [3–8].
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.08.034
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The hymenomycete genus Trichoderma is characterized by species remarkable for their rapid growth, capability of utilizing diverse substrates and resistance to noxious chemicals [9]. They are often the predominant components of the mycoflora in soils of various ecosystems, such as agricultural fields, prairie, forest, salt marshes and deserts, in all climatic zones [10–13]. They make a significant contribution to the decomposition of woody and herbaceous materials and exhibit saprotrophic activities against primary wood decomposers [14]. Some species of this genus have economic importance because of their production of enzymes and antibiotics, or use as biocontrol agents [15–17]. Due to the ecological importance of Trichoderma and its application as a biocontrol agent in the field, it is important to understand its biodiversity and biogeography. Until recently, most of the known species had been isolated from North America and Central Europe [18]. We have previously initiated a survey of the occurrence of Trichoderma spp. in thus far neglected regions such as Siberia and the Himalayas [19], South-East Asia [20], Egypt [21] and Central and South America [22]. These studies led to the identification and description of several new species [23,24], and furthermore revealed a unique species composition in all these regions. This could be the result of a geographic/climatic bias of some species, or be an artifact of the limited number of samples and locations involved. In order to reveal the possibility of a geographic/climatic bias in such studies, we investigated whether a geographic area linking two previously investigated areas would show a gradient in species distribution consistent with the established data. Here we report the biodiversity of Trichoderma from four different regions of China (North, Hebei province; South-East , Zhejiang province; West, Himalayan-Tibet; and South-West, Yunnan province), and compare the resulting species diversity to previous data from Siberia and South Himalaya (Nepal), and South-East Asia (Thailand, Burma) [19,20].
2. Materials and methods 2.1. Geography of sample sites Trichoderma spp. were sampled in four areas of China, which differ in their geographic location, altitude and climate: Xiaowutai Shan, Hebei province (39.5 N/ 115 E; climate continental and arid; 1000 m above sea level); Hangzhou, Zhejiang province (30.3 N/120.2 E; climate subtropical and humid; 500 m above sea level); Kunming, Yunnan province (24.6 N/102.7 E; climate subtropical, warm and humid; 1800 m above sea level); and three different locations in Tibet, Himalaya (29 N/ 85–87 E; about 5000 m above sea level; climate cold and dry).
2.2. Substrates, storage and isolation of pure cultures Samples of soil, pieces of decayed wood, bark, vegetable debris and other substrata were collected in sterile polyethylene bags or kraft envelopes and stored at 5 °C until isolation. Rose Bengal agar [25] was used as a selective medium for Trichoderma, using the soil dilution plating method. Putative Trichoderma colonies were purified by two rounds of subculturing on potato-dextrose agar (PDA). Pure cultures were suspended in 10% (w/v) non-fat milk and in silica gel at 20 °C. 2.3. Morphological analysis For morphological analysis, strains were grown on special nutrient agar (SNA), on 2% (w/v) cornmeal dextrose agar (CMD) and on PDA at 20 °C under ambient daylight conditions. Growth rates were determined at 20, 25, 30, 35 and 40 °C for 72 h on SNA and PDA [26]. Microscopic observations and measurements were made from preparations mounted in lactic acid. Conidiophore structure and morphology were examined on macronematous conidiophores, taken either from the edge of conidiogenous pustules, or from fascicles when conidia were maturing. Conidial morphology and size were recorded after 14 days of incubation. 2.4. DNA sequencing and phylogenetic analysis DNA was isolated using the DNeasy Plant Mini kit (QIAGEN). A region of nuclear rRNA, containing the internal transcribed spacer regions 1 and 2 and the 5.8S rRNA gene, was amplified as described previously [27]. A 0.3 kb fragment of tef1, containing the fourth large intron, was amplified by the primer pair EF1728F (5 0 -CATCGAGAAGTTCGAGAAGG-3 0 ) and EF1-986R (5 0 -TACTTGAAGGAACCCTTACC-3 0 ) as described [28]. The amplicons were sequenced with the aid of a MegaBACE 1000 DNA automatic sequencing system (Pharmacia), using cycle-sequencing with the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Pharmacia). The NCBI GenBank accession numbers for all sequences obtained in this study are given in Table 1. DNA sequences were visually aligned using Genedoc 2.6. The interleaved NEXUS file was formatted using PAUP*4.0b10 and manually edited for recognition by MrBayes v3.0B4 [29–31]. The model of evolution and prior settings for individual loci of Hypocrea/Trichoderma have been described previously [28–30]. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed with four incrementally heated chains that were simultaneously run for 3 millions of generations.
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Table 1 Trichoderma spp. from China identified in this study Location
Species
Isolate
Tibet
T. harzianum
ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT
Hebei
T. harzianum
T. longibrachiatum T. atroviride
T. citrinoviride T. velutinum T. sp. nov. C2 Zhejiang
T. harzianum T. asperellum
T. longibrachiatum Yunnan
T. atroviride
T. koningii
T. longibrachiatum T. virens T. cerinum T. sinensis T. viride T. sp. nov. C1 a
256 255 251 253 221 219 209 235 239 220 240 212 214 233 245 248 249 202 203 225 227 230 231 232 241 205 215 216 217 222 223 224 236 237 242 243 250 244 238 204 208 039 002 152 153 154 040 041 286 302 318 288 289 290 310 314 287 275 284 308 312 304 291 337
Origin
Substrate
GenBank Accession Nos.a
Jilong
Cattle faeces
Dingjie Nielamu Xiaowutai mountain
Soil Pasture soil Timber scraps Soil
AF408086 AF408093 AF408078 AF408070 AF408085 AF408087 AF408075 AF408073 AF408068 AF408088 AF408076 AF408077 AF408071 AF408069 AF408073 AF408066 AF408067 AF408095 AF408090 AF408096 AF408089 AF408092 AF408094 AF408097 AF408091 AY864320 AY864320 AF408137 AF408137 AY864321 AF408137 AF408129 AF408133 AF408134 AF408131 AF408137 AF408137 AF408112 AF408098 AF408063, AF408064, AF408074 AF408121 AF408126 AF408126 AF408126 AF408113 AF408113 AF408135 AF408136 AF408132 AF408139, AF408141, AF408142, AF408140, AF408143, AF408113 AF408101 AF408100 AF408081 AF408082 AF408116 AF408138, AF408065,
Decayed Feather Decayed Decayed Decayed
agarics bark vegetable timber
Sheep faeces Defoliation
Timber scraps Decayed sundries Feather Defoliation Decayed pine needle Timber scraps Soil
Decayed peach stone
Hangzhou
Decayed apricot stone Decayed paper Soil Cattle faeces Herbaceous plant Soil
Kunming
Soil
AY864325, DQ056746 AY864325
AY864326 AY864326 AY864326 AY864326 AY864328
AY864327 AY864329, DQ056745
When more then one accession number is given, they are in the order: ITS1 and 2; tef1 short intron (fifth); tef1 long intron (forth), for definition see [34].
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3. Results 3.1. Species identification A total of 135 strains of Trichoderma were isolated from four geographically diverse areas of China. North and South-West (Hebei and Yunnan provinces, respectively) were represented by most samples (41 and 61, respectively), followed by 28 for Zhejiang (South-East) and 5 for Tibet (West). From Yunnan and Zheijang, only soil samples were used, but many of the samples from Hebei consisted of decaying leaves or woody material, due to the aridity of the area. All isolates were preliminarily identified at the species level by the analysis of morphological characters [26,32]. Species identity of a group of 64 representative isolates (at least one of two to four isolates which were originated from the same sample and displayed identical morphological characters) was detected by use of ITS1 and 2 oligonucleotide barcode integrated in TrichO Key v.1.0 (www.isth.info/tools/ molkey/; [22]). In a few cases, where ITS1 and 2 did not provide unambiguous identification (e.g. T. longibrachiatum, T. koningii, T. viride), the large intron of tef1 was sequenced and used to search for the best match in TrichoBLAST (www.isth.info/tools/blast/; [33]). This approach was applied to 7, 37, 16 and 4 strains, respectively, from Zhejiang, Hebei, Yunnan and Tibet. Thereby, the following species (Table 1) were identified (number of strains given after comma): from section Trichoderma: T. viride, 1; T. asperellum, 4; T. koningii, 5; T. atroviride, 13; from section Pachybasium: T. velutinum, 1; T. cerinum, 2; T. virens, 2; and T. harzianum, 26: and from section Longibrachiatum: T. sinensis, 1; T. citrinoviride, 1; and T. longibrachiatum, 5. Three further isolates yielded ITS1 and 2 sequences, which did not correspond to any previously described species: T. sp. ZAUT 337 from Yunnan (=Trichoderma sp. C1), and T. sp. ZAUT 204 and ZAUT 208 from Hebei (T. sp. C2). The ITS 1 and 2 sequences of both isolates from Hebei were identical so these two isolates are likely to belong to the same taxon. T. sp. C1 and C2 may represent putative new species as they also exhibit morphological and/or physiological characters that are not displayed by any of the recognized species (Zhang, C., and Xu, T., unpublished data). In order to support this assumption, we also analyzed sequences of the large intron of the tef1 gene encoding translation elongation factor 1-a and used it to assess the phylogenetic position of Trichoderma sp. C1 and C2. The result is shown in Fig. 1: the combined ITS 1 and 2 and tef1 gene tree consistently placed T. sp. C1 and T. sp. C2 in the Catoptron-Lixii clade of species [22,34]. Interestingly, T. sp. C2 formed a sister clade to T. cf. aureoviride DAOM 175924 [27,35], another still
Fig. 1. Bayesian tree of ITS1 and 2 and the long tef1 intron of selected ex-type strains of the Catoptron-Lixii Clade of Trichoderma, showing the phylogenetic position of putative new Trichoderma sp. C1 and C2. Posterior probabilities of >0.95 are mapped on the tree. ITS1 and 2 and tef1 sequences of the ex-type strains in the tree are available at the Multiloci Database of Phylogenetic Markers for Hypocrea and Trichoderma (http://www.isth.info/tools/blast/show_all_seq.php).
unnamed new species for which several world-wide isolates are available. 3.2. Species distribution Grouping of the species according to their geographic origin revealed significant differences (Fig. 2). The 37 isolates from the province of Hebei comprised 6 species: T. longibrachiatum, T. citrinoviride, T. harzianum, T. velutinum, T. atroviride, and the putative new species T. sp. C2; T. harzianum being the dominant species in this area. On the other hand, the group of eight species found in Yunnan comprised T. longibrachiatum, T. sinensis, T. virens, T. cerinum, T. atroviride, T. koningii, T. viride and the putative new species T. sp. C1. In the limited sample (four) from Tibet, only T. harzianum was found. In samples from the province of Zhejiang (seven isolates), three species were identified: T. harzianum, T. asperellum and T. longibrachiatum. We have recently proposed that T. harzianum isolates can be grouped into several different ITS 1 and 2 haplotypes (then termed ‘‘genotypes’’; [19]). Strains identified in this study belonged to haplotypes 1 (which is confirmed for the ex-type strain of T. harzianum CBS 226.95), 2a and 5, and a new haplotype variant of 5
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Fig. 2. Summary of species distribution as assessed in this study. Individual species are abbreviated as follows: a – T. harzianum; b – T. longibrachiatum; c – T. atroviride; d – T. citrinoviride; e – T. velutinum; f – Trichoderma sp. C2; g – T. asperellum; h – T. koningii; i – T. virens; j – T. cerinum; k – T. sinensis; l – T. viride; m – Trichoderma sp. C1.
not previously described. The latter two accounted for the vast majority of isolates (20 of 26).
4. Discussion We have carried out a survey of the occurrence of Trichoderma spp. in China which aimed to complement previous studies in Siberia, the Himalayas and SouthEast Asia [19,20] in order to obtain a more complete picture of the biodiversity of this genus in Asia. From a total sample of 135 isolates, 64 were subjected to species identification by sequence analysis. We cannot completely rule out that the grouping of isolates according to a putative species identification by the analysis of morphological characters may in some cases have overlooked the presence of a different species. Although, our experience from previous work [19–22] showed that different isolates from the same sample, and exhibiting identical morphology, indeed are the same species in 19 of 20 cases (unpublished data). Thus, it is sufficient to verify only one of them by sequence analysis. We therefore consider this strategy a valid and economic strategy for studies where a large number of isolates need to be identified. However, in order to consider this approach, we will further concentrate the discussion only on the number of strains identified by sequence analysis. 4.1. Two potential new species This survey led to the identification of 61 of 64 isolates at the species level, and the detection of two potential new species which were also distinctive in molecular characters. Among the latter, Trichoderma sp. C2 has high ITS1 and 2 sequence similarity with T. cf. aureoviride DAOM 175924 [27,35], yet differs
in some diagnostic nucleotides. We have recently encountered a further isolate with the same ITS1 and 2 haplotype in soil of Trivandrum, South India (Szakacs, G., and Kubicek, C.P., unpublished data), thus documenting a broader distribution of this haplotype in Asia. Although the tef1 tree placed C2 in a sister clade to T. cf. aureoviride DAOM 175924, it is unclear whether it is a new species closely related to it, or an allopatric subform. Trichoderma sp. C1 is also closely related to the Catoptron-Lixii Clade, but not to any of the known species, and we are not aware of any other report of an isolate with this characteristic ITS1 and 2 and tef1 sequence.
4.2. North to South distribution of T. harzianum in China Among the 61 isolates verified by sequence analysis, 26 were identified as T. harzianum. This is in accordance with results from previous studies in Asia [19,20], Central Europe [36], Egypt [21] and South America [22]. However, a closer inspection of these strains shows some interesting details. First, almost all T. harzianum strains came from the North of China (Hebei province), and all four isolates obtained from Tibet were T. harzianum, whereas only a single isolate obtained from Zhejiang was identified as T. harzianum and none of isolates were identified as this species in Yunnan. This indicates a clear North to South gradient in the distribution of T. harzianum in China. Furthermore, only 4 isolates corresponded to the haplotype of the ex-type strain of T. harzianum, whereas – with the exception of isolate ZAUT 251 – all other isolates belonged to haplotype 5 of ITS1 and 2 [19,20] and a new variant of it. The finding of strains with this haplotype accounted for only a minor portion in previous studies, suggests that China could be their center of origin.
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4.3. Species difference between North and South China Apart of T. harzianum, there was also a clear difference in the distribution of species between North and South in China: whereas T. velutinum, T. citrinoviride, T. sp. C2, and the majority of strains of T. atroviride were found in Hebei province, T. koningii, T. virens, T. cerinum, T. sinensis, T. viride and the putative new species T. sp. C1 were only found in soils from Yunnan. This finding is in a perfect accordance with results from earlier studies in Siberia and the Himalaya [19] and South-East Asia [20], as the species found in Hebei were the same as those predominantly occurring in Siberia. Whereas those from Yunnan were the same as those found in South-East Asia. This is probably due to climatic preferences of these isolates. As an example, T. velutinum was described as a new species on the basis of an isolate detected in Nepal [23], but strains with identical ITS1 and 2 sequences were found in Iran (Zafari, D., personal communication), Japan and Korea, which suggested its occurrence in a temperate climate, which was further proven in this paper. In contrast, T. sinensis has been isolated from tree bark from Taiwan, China [23], and has also been found in Vietnam (Samuels, G.J., personal communication). Its identification in Yunnan confirms a common distribution of this species in South-East Asia. The finding of T. longibrachiatum in China was surprising, because a previous biogeographic survey of species of section Longibrachiatum failed to detect any isolate of this species in East Asia, whereas T. citrinoviride was abundant [37]. Subsequent studies also failed to find T. longibrachiatum in Asia [19,20]. Turner et al. [37] hypothesized that the geographic occurrence of T. citrinoviride and T. longibrachiatum may be complementary. The present finding of T. longibrachiatum in China is consistent, however, with the occurrence of H. orientalis in Yunnan, which has been proposed as a potential teleomorph of T. longibrachiatum [38]. The high number of species of Trichoderma found in the province of Yunnan confirms that this is one of ChinaÕs biologically most diverse regions with a large portion of endemic species (http://www.biodiversityhotspots.org/xp/Hotspots/China). Earlier studies [39,40] stressed the abundance of Trichoderma spp. in the South-West of China. Unfortunately, identification of these strains was based on a now outdated morphological key, and thus the species compositions of these studies are uncertain. Further research on Trichoderma biodiversity from Yunnan province may therefore lead to the eventual discovery of further new taxa.
Acknowledgements This work was supported by NSFC grants to Xu Tong, and grant FWF P-16601 from the Austrian Sci-
ence Foundation to Christian P. Kubicek. The authors acknowledge the skillful technical help of Monica Komon´ and fruitful discussions with Walter M. Jaklitsch.
References [1] Mu¨ller, E. and Lo¨ffler, W. (1992) Mycology. Georg Thieme Verlag, Stuttgart, FRG. [2] Hawksworth, D.L. (1991) The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol. Res. 95, 641– 655. [3] James, T.Y., Porter, D., Hamrick, J.L. and Vilgalys, T. (2001) Evidence for restricted intercontinental flow in the cosmopolitan mushroom Schizophyllum commune. Evolution 53, 1665–1677. [4] Coetzee, M.P.A., Wingfield, B.D., Harrington, T.C., Dalevi, D., Coutinho, T.A. and Wingfield, M.J. (2002) Geographical diversity of Armillaria mellea s.s. based on phylogenetic analysis. Mycologia 92, 105–113. [5] Lickey, E.B., Hughes, K.W. and Petersen, R.H. (2002) Biogeographical patterns in Artomyces pyxidatus. Mycologia 94, 461– 471. [6] Shen, Q., Geiser, D.M. and Royse, D.J. (2002) Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition separating Eastern North America and Asian isolates. Mycologia 94, 472–482. [7] Oda, T., Tanaka, C. and Tsuda, M. (2004) Molecular phylogeny and biogeography of the widely distributed Amanita species, A. muscaria and A. pantherina. Mycol. Res. 108, 885–896. [8] Zervakis, G.I., Moncalvo, J.M. and Vilgalys, R. (2004) Molecular phylogeny, biogeography and speciation of the mushroom species Pleurotus cystidiosus and allied taxa. Microbiol UK 150, 715–726. [9] Klein, D. and Eveleigh, D.E. (1998) Ecology of Trichoderma In: Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics (Kubicek, C.P. and Harman, G.E., Eds.), pp. 57–74. Taylor & Francis Ltd., London, UK. [10] Danielson, R.M. and Davey, C.B. (1973) The abundance of Trichoderma propagules and the distribution of species in forest soils. Soil Biol. Biochem. 5, 486–494. [11] Roiger, D.J., Jeffers, S.N. and Caldwell, R.W. (1991) Occurrence of Trichoderma species in apple orchard and woodland soils. Soil Biol. Biochem. 23, 353–359. [12] Smith, W.H. (1995) Forest occurrence of Trichoderma species: emphasis on potential organochlorine (xenobiotic) degradation. Ecotoxicol. Environ. Safety 32, 179–183. [13] Wardle, D.A., Parkinson, D. and Waller, J.E. (1993) Interspecific competitive interactions between pairs of fungal species in natural substrates. Oecologia 94, 165–172. [14] Rossman, A.Y. (1996) Morphological and molecular perspectives on systematics of Hypocreales. Mycologia 88, 1–19. [15] Kubicek, C.P. and Penttila¨, M.E. (1998) Regulation of production of plant polysaccharide degrading enzymes by Trichoderma In: Trichoderma and Gliocladium. Enzymes, Biological Control and Commercial Applications (Harman, G.E. and Kubicek, C.P., Eds.), pp. 49–71. Taylor & Francis, London, UK. [16] Hjeljord, L. and Tronsmo, A. (1998) Trichoderma and Gliocladium in biological control: an overview In: Trichoderma and Gliocladium. Enzymes, Biological Control and Commercial Applications (Harman, G.E. and Kubicek, C.P., Eds.), pp. 131– 151. Taylor & Francis Ltd., London, UK. [17] Sivasithamparam, K. and Ghisalberti, E.L. (1998) Secondary metabolism in Trichoderma and Gliocladium In: Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics (Kubicek, C.P. and Harman, G.E., Eds.), pp. 139–191. Taylor & Francis Ltd., London, UK.
C.-l. Zhang et al. / FEMS Microbiology Letters 251 (2005) 251–257 [18] Bissett, J. (1991) A revision of the genus Trichoderma. III. Section Pachybasium. Can. J. Bot. 69, 2373–2417. [19] Kullnig, C.M., Szakacs, G. and Kubicek, C.P. (2000) Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya. Mycol. Res. 104, 1117–1125. [20] Kubicek, C.P., Bissett, J., Druzhinina, I., Kullnig-Gradinger, C. and Szakacs, G. (2003) Genetic and metabolic diversity of Trichoderma: a case study on South East Asian isolates. Fungal Genetics Biol. 38, 310–319. [21] Gherbawy, Y., Druzhinina, I., Shaban, G.M., Wuczkowsky, M., Yaser, M., El-Naghy, M.A., Prillinger, H.J. and Kubicek, C.P. (2004) Trichoderma populations from alkaline agricultural soil in the Nile valley, Egypt, consist of only two species. Mycol. Prog. 3, 211–218. [22] Druzhinina, I., Koptchinski, A., Komon, M., Bissett, J., Szakacs, G., Kubicek, C.P. An oligonucleotide barcode for species identification in Hypocrea and Trichoderma, Fungal Genet. Biol., in press. [23] Bissett, J., Szakacs, G., Nolan, C.A., Druzhinina, I., KullnigGradinger, C. and Kubicek, C.P. (2003) New species of Trichoderma from Asia. Can. J. Bot. 81, 570–586. [24] Kraus, G., Druzhinina, I., Bissett, J., Prillinger, H.J., Szakacs, G., Gams, W. and Kubicek, C.P. (2004) Trichoderma brevicompactum sp. nov.. Mycologia 96, 1057–1071. [25] Martin, J.P. (1950) Use of acid, Rose Bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69, 215–232. [26] Samuels, G.J., Chaverri, P., Farr, D.F., McCray, E.B. (2004) Trichoderma Online. http://nt.ars-grin.gov/taxadescriptions/keys/ TrichodermaIndex.cfm, 2004. [27] Kullnig-Gradinger, C.M., Szakacs, G. and Kubicek, C.P. (2002) Phylogeny and evolution of the fungal genus Trichoderma – a multigene approach. Mycol. Res. 106, 757–767. [28] Druzhinina, I., Chaverri, P., Fallah, P., Kubicek, C.P. and Samuels, G.J. (2004) Hypocrea flaviconidia, a new species with yellow conidia from Costa Rica. Stud. Mycol. 50, 401–407. [29] Rannala, B. and Yang, Z. (1997) Probability distribution of molecular evolutionary trees: a new method of phylogenetic interference. J. Mol. Evol. 43, 304–311.
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[30] Yang, Z. and Rannala, B. (1997) Bayesian phylogenetic interference using DNA sequences: a Markov chain Monte Carlo method. Mol. Biol. Evol. 14, 717–724. [31] Huelsenbeck, J.P. and Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. [32] Gams, W. and Bissett, J. (1998) Morphology and identification of Trichoderma In: Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics (Kubicek, C.P. and Harman, G.E., Eds.), pp. 3–34. Taylor & Francis Ltd., London. [33] Kopchinskiy, A., Komon´, M., Kubicek, C.P. and Druzhinina, I.S. (2005) Tricho BLAST: a multiloci database of phylogenetic markers for Trichoderma and Hypocrea powered by sequence diagnosis and similarity search tools. Mycol. Res. 109, 557– 560. [34] Druzhinina, I. and Kubicek, C.P. (2005) Species concepts and biodiversity in Trichoderma and Hypocrea: from species aggregates to species clusters? J. Zeihjang Univ. Sci. B 6, 100–112. [35] Lieckfeldt, E., Kullnig, C.M., Kubicek, C.P., Samuels, G.J. and Bo¨rner, T. (2001) Trichoderma aureoviride: phylogenetic position and characterization. Mycol. Res. 105, 313–322. [36] Wuczkowski, M., Druzhinina, I., Gherbawy, Y., Klug, B., Prillinger, H.J. and Kubicek, C.P. (2003) Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. Microbiol. Res. 158, 125–133. [37] Turner, D., Kovacs, W., Kuhls, K., Lieckfeldt, E., Peter, B., Arisan-Atac, I., Strauss, J., Samuels, G.J., Bo¨rner, T. and Kubicek, C.P. (1996) Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species. Mycol. Res. 101, 449–459. [38] Samuels, G.J., Petrini, O., Kuhls, K., Lieckfeldt, E. and Kubicek, C.P. (1998) The Hypocrea schweinitzii complex and Trichoderma sect. Longibrachiatum. Stud. Mycol. 41, 1–54. [39] Wen, C.J., Tao, J.F. and Cheng, W.R. (1993) Studies on the taxonomy of the genus Trichoderma in southwestern China. Acta Mycol. Sin. 12, 118–130. [40] Tai, F.L. (1979) Sylloge fungorum sinicorum. Science Press, Academia Sinica, Peking, China.
Trichoderma biodiversity in China: Evidence for a North to South distribution of species in East Asia Chu-long Zhang a, Irina S. Druzhinina b, Christian P. Kubicek
b,*
, Tong Xu
a,*
a
b
Institute of Biotechnology, Zhejiang University, 310029, China Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Getreidemarkt 9/E1665, A-1060 Vienna, Austria Received 19 July 2005; accepted 8 August 2005 First published online 6 September 2005 Edited by M.J. Bidochka
Abstract Towards assessing the biodiversity and biogeography of Trichoderma, we have analyzed the occurrence of Trichoderma species in soil and litter from four areas in China: North (Hebei province), South-East (Zhejiang province), West (Himalayan, Tibet) and South-West (Yunnan province). One hundred and thirty five isolates were grouped according to tentative morphological identification. A representative 64 isolates were verified at the species level by the oligonucleotide barcode program TrichO Key v.1.0 and the custom BLAST server TrichoBLAST, using sequences of the ITS1 and 2 region of the rRNA cluster and from the longest intron of the tef1 (translation elongation factor 1-a) gene. Eleven known species (Trichoderma asperellum, T. koningii, T. atroviride, T. viride, T. velutinum, T. cerinum, T. virens, T. harzianum, T. sinensis, T. citrinoviride, T. longibrachiatum) and two putative new species (T. sp. C1, and T. sp. C2), distinguished from known species both by morphological characters and phylogenetic analysis, were identified. A significant difference in the occurrence of these species was found between the North (Hebei) and South-West (Yunnan) areas, which correlates with previously reported species distributions in Siberia and South-East Asia, respectively. As in previous studies, T. harzianum accounted for almost half of the biodiversity; although, in this study, it was exclusively found in the North, and was predominantly represented by an ITS1 and 2 haplotype, which has so far been rarely found elsewhere. This study therefore reveals a North–South gradient in species distribution in East Asia, and identifies Northern China as a potential center of origin of a unique haplotype of T. harzianum. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hypocrea; ITS1 and 2; tef1; Biogeography; Phylogeny
1. Introduction Fungi are considered the fifth kingdom, characterized as eukaryotic heterotrophic organisms with a low level * Corresponding authors. Tel.: +43 1 58801 17250; fax: +43 1 58801 17299 (C.P. Kubicek), Tel.: +86 571 86971208; fax: +86 571 86046615 (T. Xu). E-mail addresses: [email protected] (C.P. Kubicek), [email protected] (T. Xu). URLs: http://www.vt.tuwien.ac.at, http://www.isth.info (C.P. Kubicek).
of ‘‘tissue’’ differentiation and chitin/b-glucan-containing cell walls. They form spores as a stress resistant and spreading phase of their life cycle (http://www. mycolog.com/index.html). Approximately 120,000 species of fungi have so far been identified worldwide [1], but recent estimates claim about 1.6 million species in total [2]. Despite this high diversity, the biogeography of fungi is still a neglected topic, and molecular studies directed towards an assessment of the global biodiversity of fungi are only slowly emerging [3–8].
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.08.034
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The hymenomycete genus Trichoderma is characterized by species remarkable for their rapid growth, capability of utilizing diverse substrates and resistance to noxious chemicals [9]. They are often the predominant components of the mycoflora in soils of various ecosystems, such as agricultural fields, prairie, forest, salt marshes and deserts, in all climatic zones [10–13]. They make a significant contribution to the decomposition of woody and herbaceous materials and exhibit saprotrophic activities against primary wood decomposers [14]. Some species of this genus have economic importance because of their production of enzymes and antibiotics, or use as biocontrol agents [15–17]. Due to the ecological importance of Trichoderma and its application as a biocontrol agent in the field, it is important to understand its biodiversity and biogeography. Until recently, most of the known species had been isolated from North America and Central Europe [18]. We have previously initiated a survey of the occurrence of Trichoderma spp. in thus far neglected regions such as Siberia and the Himalayas [19], South-East Asia [20], Egypt [21] and Central and South America [22]. These studies led to the identification and description of several new species [23,24], and furthermore revealed a unique species composition in all these regions. This could be the result of a geographic/climatic bias of some species, or be an artifact of the limited number of samples and locations involved. In order to reveal the possibility of a geographic/climatic bias in such studies, we investigated whether a geographic area linking two previously investigated areas would show a gradient in species distribution consistent with the established data. Here we report the biodiversity of Trichoderma from four different regions of China (North, Hebei province; South-East , Zhejiang province; West, Himalayan-Tibet; and South-West, Yunnan province), and compare the resulting species diversity to previous data from Siberia and South Himalaya (Nepal), and South-East Asia (Thailand, Burma) [19,20].
2. Materials and methods 2.1. Geography of sample sites Trichoderma spp. were sampled in four areas of China, which differ in their geographic location, altitude and climate: Xiaowutai Shan, Hebei province (39.5 N/ 115 E; climate continental and arid; 1000 m above sea level); Hangzhou, Zhejiang province (30.3 N/120.2 E; climate subtropical and humid; 500 m above sea level); Kunming, Yunnan province (24.6 N/102.7 E; climate subtropical, warm and humid; 1800 m above sea level); and three different locations in Tibet, Himalaya (29 N/ 85–87 E; about 5000 m above sea level; climate cold and dry).
2.2. Substrates, storage and isolation of pure cultures Samples of soil, pieces of decayed wood, bark, vegetable debris and other substrata were collected in sterile polyethylene bags or kraft envelopes and stored at 5 °C until isolation. Rose Bengal agar [25] was used as a selective medium for Trichoderma, using the soil dilution plating method. Putative Trichoderma colonies were purified by two rounds of subculturing on potato-dextrose agar (PDA). Pure cultures were suspended in 10% (w/v) non-fat milk and in silica gel at 20 °C. 2.3. Morphological analysis For morphological analysis, strains were grown on special nutrient agar (SNA), on 2% (w/v) cornmeal dextrose agar (CMD) and on PDA at 20 °C under ambient daylight conditions. Growth rates were determined at 20, 25, 30, 35 and 40 °C for 72 h on SNA and PDA [26]. Microscopic observations and measurements were made from preparations mounted in lactic acid. Conidiophore structure and morphology were examined on macronematous conidiophores, taken either from the edge of conidiogenous pustules, or from fascicles when conidia were maturing. Conidial morphology and size were recorded after 14 days of incubation. 2.4. DNA sequencing and phylogenetic analysis DNA was isolated using the DNeasy Plant Mini kit (QIAGEN). A region of nuclear rRNA, containing the internal transcribed spacer regions 1 and 2 and the 5.8S rRNA gene, was amplified as described previously [27]. A 0.3 kb fragment of tef1, containing the fourth large intron, was amplified by the primer pair EF1728F (5 0 -CATCGAGAAGTTCGAGAAGG-3 0 ) and EF1-986R (5 0 -TACTTGAAGGAACCCTTACC-3 0 ) as described [28]. The amplicons were sequenced with the aid of a MegaBACE 1000 DNA automatic sequencing system (Pharmacia), using cycle-sequencing with the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Pharmacia). The NCBI GenBank accession numbers for all sequences obtained in this study are given in Table 1. DNA sequences were visually aligned using Genedoc 2.6. The interleaved NEXUS file was formatted using PAUP*4.0b10 and manually edited for recognition by MrBayes v3.0B4 [29–31]. The model of evolution and prior settings for individual loci of Hypocrea/Trichoderma have been described previously [28–30]. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed with four incrementally heated chains that were simultaneously run for 3 millions of generations.
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Table 1 Trichoderma spp. from China identified in this study Location
Species
Isolate
Tibet
T. harzianum
ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT ZAUT
Hebei
T. harzianum
T. longibrachiatum T. atroviride
T. citrinoviride T. velutinum T. sp. nov. C2 Zhejiang
T. harzianum T. asperellum
T. longibrachiatum Yunnan
T. atroviride
T. koningii
T. longibrachiatum T. virens T. cerinum T. sinensis T. viride T. sp. nov. C1 a
256 255 251 253 221 219 209 235 239 220 240 212 214 233 245 248 249 202 203 225 227 230 231 232 241 205 215 216 217 222 223 224 236 237 242 243 250 244 238 204 208 039 002 152 153 154 040 041 286 302 318 288 289 290 310 314 287 275 284 308 312 304 291 337
Origin
Substrate
GenBank Accession Nos.a
Jilong
Cattle faeces
Dingjie Nielamu Xiaowutai mountain
Soil Pasture soil Timber scraps Soil
AF408086 AF408093 AF408078 AF408070 AF408085 AF408087 AF408075 AF408073 AF408068 AF408088 AF408076 AF408077 AF408071 AF408069 AF408073 AF408066 AF408067 AF408095 AF408090 AF408096 AF408089 AF408092 AF408094 AF408097 AF408091 AY864320 AY864320 AF408137 AF408137 AY864321 AF408137 AF408129 AF408133 AF408134 AF408131 AF408137 AF408137 AF408112 AF408098 AF408063, AF408064, AF408074 AF408121 AF408126 AF408126 AF408126 AF408113 AF408113 AF408135 AF408136 AF408132 AF408139, AF408141, AF408142, AF408140, AF408143, AF408113 AF408101 AF408100 AF408081 AF408082 AF408116 AF408138, AF408065,
Decayed Feather Decayed Decayed Decayed
agarics bark vegetable timber
Sheep faeces Defoliation
Timber scraps Decayed sundries Feather Defoliation Decayed pine needle Timber scraps Soil
Decayed peach stone
Hangzhou
Decayed apricot stone Decayed paper Soil Cattle faeces Herbaceous plant Soil
Kunming
Soil
AY864325, DQ056746 AY864325
AY864326 AY864326 AY864326 AY864326 AY864328
AY864327 AY864329, DQ056745
When more then one accession number is given, they are in the order: ITS1 and 2; tef1 short intron (fifth); tef1 long intron (forth), for definition see [34].
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3. Results 3.1. Species identification A total of 135 strains of Trichoderma were isolated from four geographically diverse areas of China. North and South-West (Hebei and Yunnan provinces, respectively) were represented by most samples (41 and 61, respectively), followed by 28 for Zhejiang (South-East) and 5 for Tibet (West). From Yunnan and Zheijang, only soil samples were used, but many of the samples from Hebei consisted of decaying leaves or woody material, due to the aridity of the area. All isolates were preliminarily identified at the species level by the analysis of morphological characters [26,32]. Species identity of a group of 64 representative isolates (at least one of two to four isolates which were originated from the same sample and displayed identical morphological characters) was detected by use of ITS1 and 2 oligonucleotide barcode integrated in TrichO Key v.1.0 (www.isth.info/tools/ molkey/; [22]). In a few cases, where ITS1 and 2 did not provide unambiguous identification (e.g. T. longibrachiatum, T. koningii, T. viride), the large intron of tef1 was sequenced and used to search for the best match in TrichoBLAST (www.isth.info/tools/blast/; [33]). This approach was applied to 7, 37, 16 and 4 strains, respectively, from Zhejiang, Hebei, Yunnan and Tibet. Thereby, the following species (Table 1) were identified (number of strains given after comma): from section Trichoderma: T. viride, 1; T. asperellum, 4; T. koningii, 5; T. atroviride, 13; from section Pachybasium: T. velutinum, 1; T. cerinum, 2; T. virens, 2; and T. harzianum, 26: and from section Longibrachiatum: T. sinensis, 1; T. citrinoviride, 1; and T. longibrachiatum, 5. Three further isolates yielded ITS1 and 2 sequences, which did not correspond to any previously described species: T. sp. ZAUT 337 from Yunnan (=Trichoderma sp. C1), and T. sp. ZAUT 204 and ZAUT 208 from Hebei (T. sp. C2). The ITS 1 and 2 sequences of both isolates from Hebei were identical so these two isolates are likely to belong to the same taxon. T. sp. C1 and C2 may represent putative new species as they also exhibit morphological and/or physiological characters that are not displayed by any of the recognized species (Zhang, C., and Xu, T., unpublished data). In order to support this assumption, we also analyzed sequences of the large intron of the tef1 gene encoding translation elongation factor 1-a and used it to assess the phylogenetic position of Trichoderma sp. C1 and C2. The result is shown in Fig. 1: the combined ITS 1 and 2 and tef1 gene tree consistently placed T. sp. C1 and T. sp. C2 in the Catoptron-Lixii clade of species [22,34]. Interestingly, T. sp. C2 formed a sister clade to T. cf. aureoviride DAOM 175924 [27,35], another still
Fig. 1. Bayesian tree of ITS1 and 2 and the long tef1 intron of selected ex-type strains of the Catoptron-Lixii Clade of Trichoderma, showing the phylogenetic position of putative new Trichoderma sp. C1 and C2. Posterior probabilities of >0.95 are mapped on the tree. ITS1 and 2 and tef1 sequences of the ex-type strains in the tree are available at the Multiloci Database of Phylogenetic Markers for Hypocrea and Trichoderma (http://www.isth.info/tools/blast/show_all_seq.php).
unnamed new species for which several world-wide isolates are available. 3.2. Species distribution Grouping of the species according to their geographic origin revealed significant differences (Fig. 2). The 37 isolates from the province of Hebei comprised 6 species: T. longibrachiatum, T. citrinoviride, T. harzianum, T. velutinum, T. atroviride, and the putative new species T. sp. C2; T. harzianum being the dominant species in this area. On the other hand, the group of eight species found in Yunnan comprised T. longibrachiatum, T. sinensis, T. virens, T. cerinum, T. atroviride, T. koningii, T. viride and the putative new species T. sp. C1. In the limited sample (four) from Tibet, only T. harzianum was found. In samples from the province of Zhejiang (seven isolates), three species were identified: T. harzianum, T. asperellum and T. longibrachiatum. We have recently proposed that T. harzianum isolates can be grouped into several different ITS 1 and 2 haplotypes (then termed ‘‘genotypes’’; [19]). Strains identified in this study belonged to haplotypes 1 (which is confirmed for the ex-type strain of T. harzianum CBS 226.95), 2a and 5, and a new haplotype variant of 5
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Fig. 2. Summary of species distribution as assessed in this study. Individual species are abbreviated as follows: a – T. harzianum; b – T. longibrachiatum; c – T. atroviride; d – T. citrinoviride; e – T. velutinum; f – Trichoderma sp. C2; g – T. asperellum; h – T. koningii; i – T. virens; j – T. cerinum; k – T. sinensis; l – T. viride; m – Trichoderma sp. C1.
not previously described. The latter two accounted for the vast majority of isolates (20 of 26).
4. Discussion We have carried out a survey of the occurrence of Trichoderma spp. in China which aimed to complement previous studies in Siberia, the Himalayas and SouthEast Asia [19,20] in order to obtain a more complete picture of the biodiversity of this genus in Asia. From a total sample of 135 isolates, 64 were subjected to species identification by sequence analysis. We cannot completely rule out that the grouping of isolates according to a putative species identification by the analysis of morphological characters may in some cases have overlooked the presence of a different species. Although, our experience from previous work [19–22] showed that different isolates from the same sample, and exhibiting identical morphology, indeed are the same species in 19 of 20 cases (unpublished data). Thus, it is sufficient to verify only one of them by sequence analysis. We therefore consider this strategy a valid and economic strategy for studies where a large number of isolates need to be identified. However, in order to consider this approach, we will further concentrate the discussion only on the number of strains identified by sequence analysis. 4.1. Two potential new species This survey led to the identification of 61 of 64 isolates at the species level, and the detection of two potential new species which were also distinctive in molecular characters. Among the latter, Trichoderma sp. C2 has high ITS1 and 2 sequence similarity with T. cf. aureoviride DAOM 175924 [27,35], yet differs
in some diagnostic nucleotides. We have recently encountered a further isolate with the same ITS1 and 2 haplotype in soil of Trivandrum, South India (Szakacs, G., and Kubicek, C.P., unpublished data), thus documenting a broader distribution of this haplotype in Asia. Although the tef1 tree placed C2 in a sister clade to T. cf. aureoviride DAOM 175924, it is unclear whether it is a new species closely related to it, or an allopatric subform. Trichoderma sp. C1 is also closely related to the Catoptron-Lixii Clade, but not to any of the known species, and we are not aware of any other report of an isolate with this characteristic ITS1 and 2 and tef1 sequence.
4.2. North to South distribution of T. harzianum in China Among the 61 isolates verified by sequence analysis, 26 were identified as T. harzianum. This is in accordance with results from previous studies in Asia [19,20], Central Europe [36], Egypt [21] and South America [22]. However, a closer inspection of these strains shows some interesting details. First, almost all T. harzianum strains came from the North of China (Hebei province), and all four isolates obtained from Tibet were T. harzianum, whereas only a single isolate obtained from Zhejiang was identified as T. harzianum and none of isolates were identified as this species in Yunnan. This indicates a clear North to South gradient in the distribution of T. harzianum in China. Furthermore, only 4 isolates corresponded to the haplotype of the ex-type strain of T. harzianum, whereas – with the exception of isolate ZAUT 251 – all other isolates belonged to haplotype 5 of ITS1 and 2 [19,20] and a new variant of it. The finding of strains with this haplotype accounted for only a minor portion in previous studies, suggests that China could be their center of origin.
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4.3. Species difference between North and South China Apart of T. harzianum, there was also a clear difference in the distribution of species between North and South in China: whereas T. velutinum, T. citrinoviride, T. sp. C2, and the majority of strains of T. atroviride were found in Hebei province, T. koningii, T. virens, T. cerinum, T. sinensis, T. viride and the putative new species T. sp. C1 were only found in soils from Yunnan. This finding is in a perfect accordance with results from earlier studies in Siberia and the Himalaya [19] and South-East Asia [20], as the species found in Hebei were the same as those predominantly occurring in Siberia. Whereas those from Yunnan were the same as those found in South-East Asia. This is probably due to climatic preferences of these isolates. As an example, T. velutinum was described as a new species on the basis of an isolate detected in Nepal [23], but strains with identical ITS1 and 2 sequences were found in Iran (Zafari, D., personal communication), Japan and Korea, which suggested its occurrence in a temperate climate, which was further proven in this paper. In contrast, T. sinensis has been isolated from tree bark from Taiwan, China [23], and has also been found in Vietnam (Samuels, G.J., personal communication). Its identification in Yunnan confirms a common distribution of this species in South-East Asia. The finding of T. longibrachiatum in China was surprising, because a previous biogeographic survey of species of section Longibrachiatum failed to detect any isolate of this species in East Asia, whereas T. citrinoviride was abundant [37]. Subsequent studies also failed to find T. longibrachiatum in Asia [19,20]. Turner et al. [37] hypothesized that the geographic occurrence of T. citrinoviride and T. longibrachiatum may be complementary. The present finding of T. longibrachiatum in China is consistent, however, with the occurrence of H. orientalis in Yunnan, which has been proposed as a potential teleomorph of T. longibrachiatum [38]. The high number of species of Trichoderma found in the province of Yunnan confirms that this is one of ChinaÕs biologically most diverse regions with a large portion of endemic species (http://www.biodiversityhotspots.org/xp/Hotspots/China). Earlier studies [39,40] stressed the abundance of Trichoderma spp. in the South-West of China. Unfortunately, identification of these strains was based on a now outdated morphological key, and thus the species compositions of these studies are uncertain. Further research on Trichoderma biodiversity from Yunnan province may therefore lead to the eventual discovery of further new taxa.
Acknowledgements This work was supported by NSFC grants to Xu Tong, and grant FWF P-16601 from the Austrian Sci-
ence Foundation to Christian P. Kubicek. The authors acknowledge the skillful technical help of Monica Komon´ and fruitful discussions with Walter M. Jaklitsch.
References [1] Mu¨ller, E. and Lo¨ffler, W. (1992) Mycology. Georg Thieme Verlag, Stuttgart, FRG. [2] Hawksworth, D.L. (1991) The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol. Res. 95, 641– 655. [3] James, T.Y., Porter, D., Hamrick, J.L. and Vilgalys, T. (2001) Evidence for restricted intercontinental flow in the cosmopolitan mushroom Schizophyllum commune. Evolution 53, 1665–1677. [4] Coetzee, M.P.A., Wingfield, B.D., Harrington, T.C., Dalevi, D., Coutinho, T.A. and Wingfield, M.J. (2002) Geographical diversity of Armillaria mellea s.s. based on phylogenetic analysis. Mycologia 92, 105–113. [5] Lickey, E.B., Hughes, K.W. and Petersen, R.H. (2002) Biogeographical patterns in Artomyces pyxidatus. Mycologia 94, 461– 471. [6] Shen, Q., Geiser, D.M. and Royse, D.J. (2002) Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition separating Eastern North America and Asian isolates. Mycologia 94, 472–482. [7] Oda, T., Tanaka, C. and Tsuda, M. (2004) Molecular phylogeny and biogeography of the widely distributed Amanita species, A. muscaria and A. pantherina. Mycol. Res. 108, 885–896. [8] Zervakis, G.I., Moncalvo, J.M. and Vilgalys, R. (2004) Molecular phylogeny, biogeography and speciation of the mushroom species Pleurotus cystidiosus and allied taxa. Microbiol UK 150, 715–726. [9] Klein, D. and Eveleigh, D.E. (1998) Ecology of Trichoderma In: Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics (Kubicek, C.P. and Harman, G.E., Eds.), pp. 57–74. Taylor & Francis Ltd., London, UK. [10] Danielson, R.M. and Davey, C.B. (1973) The abundance of Trichoderma propagules and the distribution of species in forest soils. Soil Biol. Biochem. 5, 486–494. [11] Roiger, D.J., Jeffers, S.N. and Caldwell, R.W. (1991) Occurrence of Trichoderma species in apple orchard and woodland soils. Soil Biol. Biochem. 23, 353–359. [12] Smith, W.H. (1995) Forest occurrence of Trichoderma species: emphasis on potential organochlorine (xenobiotic) degradation. Ecotoxicol. Environ. Safety 32, 179–183. [13] Wardle, D.A., Parkinson, D. and Waller, J.E. (1993) Interspecific competitive interactions between pairs of fungal species in natural substrates. Oecologia 94, 165–172. [14] Rossman, A.Y. (1996) Morphological and molecular perspectives on systematics of Hypocreales. Mycologia 88, 1–19. [15] Kubicek, C.P. and Penttila¨, M.E. (1998) Regulation of production of plant polysaccharide degrading enzymes by Trichoderma In: Trichoderma and Gliocladium. Enzymes, Biological Control and Commercial Applications (Harman, G.E. and Kubicek, C.P., Eds.), pp. 49–71. Taylor & Francis, London, UK. [16] Hjeljord, L. and Tronsmo, A. (1998) Trichoderma and Gliocladium in biological control: an overview In: Trichoderma and Gliocladium. Enzymes, Biological Control and Commercial Applications (Harman, G.E. and Kubicek, C.P., Eds.), pp. 131– 151. Taylor & Francis Ltd., London, UK. [17] Sivasithamparam, K. and Ghisalberti, E.L. (1998) Secondary metabolism in Trichoderma and Gliocladium In: Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics (Kubicek, C.P. and Harman, G.E., Eds.), pp. 139–191. Taylor & Francis Ltd., London, UK.
C.-l. Zhang et al. / FEMS Microbiology Letters 251 (2005) 251–257 [18] Bissett, J. (1991) A revision of the genus Trichoderma. III. Section Pachybasium. Can. J. Bot. 69, 2373–2417. [19] Kullnig, C.M., Szakacs, G. and Kubicek, C.P. (2000) Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya. Mycol. Res. 104, 1117–1125. [20] Kubicek, C.P., Bissett, J., Druzhinina, I., Kullnig-Gradinger, C. and Szakacs, G. (2003) Genetic and metabolic diversity of Trichoderma: a case study on South East Asian isolates. Fungal Genetics Biol. 38, 310–319. [21] Gherbawy, Y., Druzhinina, I., Shaban, G.M., Wuczkowsky, M., Yaser, M., El-Naghy, M.A., Prillinger, H.J. and Kubicek, C.P. (2004) Trichoderma populations from alkaline agricultural soil in the Nile valley, Egypt, consist of only two species. Mycol. Prog. 3, 211–218. [22] Druzhinina, I., Koptchinski, A., Komon, M., Bissett, J., Szakacs, G., Kubicek, C.P. An oligonucleotide barcode for species identification in Hypocrea and Trichoderma, Fungal Genet. Biol., in press. [23] Bissett, J., Szakacs, G., Nolan, C.A., Druzhinina, I., KullnigGradinger, C. and Kubicek, C.P. (2003) New species of Trichoderma from Asia. Can. J. Bot. 81, 570–586. [24] Kraus, G., Druzhinina, I., Bissett, J., Prillinger, H.J., Szakacs, G., Gams, W. and Kubicek, C.P. (2004) Trichoderma brevicompactum sp. nov.. Mycologia 96, 1057–1071. [25] Martin, J.P. (1950) Use of acid, Rose Bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69, 215–232. [26] Samuels, G.J., Chaverri, P., Farr, D.F., McCray, E.B. (2004) Trichoderma Online. http://nt.ars-grin.gov/taxadescriptions/keys/ TrichodermaIndex.cfm, 2004. [27] Kullnig-Gradinger, C.M., Szakacs, G. and Kubicek, C.P. (2002) Phylogeny and evolution of the fungal genus Trichoderma – a multigene approach. Mycol. Res. 106, 757–767. [28] Druzhinina, I., Chaverri, P., Fallah, P., Kubicek, C.P. and Samuels, G.J. (2004) Hypocrea flaviconidia, a new species with yellow conidia from Costa Rica. Stud. Mycol. 50, 401–407. [29] Rannala, B. and Yang, Z. (1997) Probability distribution of molecular evolutionary trees: a new method of phylogenetic interference. J. Mol. Evol. 43, 304–311.
257
[30] Yang, Z. and Rannala, B. (1997) Bayesian phylogenetic interference using DNA sequences: a Markov chain Monte Carlo method. Mol. Biol. Evol. 14, 717–724. [31] Huelsenbeck, J.P. and Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. [32] Gams, W. and Bissett, J. (1998) Morphology and identification of Trichoderma In: Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics (Kubicek, C.P. and Harman, G.E., Eds.), pp. 3–34. Taylor & Francis Ltd., London. [33] Kopchinskiy, A., Komon´, M., Kubicek, C.P. and Druzhinina, I.S. (2005) Tricho BLAST: a multiloci database of phylogenetic markers for Trichoderma and Hypocrea powered by sequence diagnosis and similarity search tools. Mycol. Res. 109, 557– 560. [34] Druzhinina, I. and Kubicek, C.P. (2005) Species concepts and biodiversity in Trichoderma and Hypocrea: from species aggregates to species clusters? J. Zeihjang Univ. Sci. B 6, 100–112. [35] Lieckfeldt, E., Kullnig, C.M., Kubicek, C.P., Samuels, G.J. and Bo¨rner, T. (2001) Trichoderma aureoviride: phylogenetic position and characterization. Mycol. Res. 105, 313–322. [36] Wuczkowski, M., Druzhinina, I., Gherbawy, Y., Klug, B., Prillinger, H.J. and Kubicek, C.P. (2003) Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. Microbiol. Res. 158, 125–133. [37] Turner, D., Kovacs, W., Kuhls, K., Lieckfeldt, E., Peter, B., Arisan-Atac, I., Strauss, J., Samuels, G.J., Bo¨rner, T. and Kubicek, C.P. (1996) Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species. Mycol. Res. 101, 449–459. [38] Samuels, G.J., Petrini, O., Kuhls, K., Lieckfeldt, E. and Kubicek, C.P. (1998) The Hypocrea schweinitzii complex and Trichoderma sect. Longibrachiatum. Stud. Mycol. 41, 1–54. [39] Wen, C.J., Tao, J.F. and Cheng, W.R. (1993) Studies on the taxonomy of the genus Trichoderma in southwestern China. Acta Mycol. Sin. 12, 118–130. [40] Tai, F.L. (1979) Sylloge fungorum sinicorum. Science Press, Academia Sinica, Peking, China.