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Characterization of Trichoderma polysporum from Spitsbergen, Svalbard archipelago, Norway, with species identity, pathogenicity to moss, and polygalacturonase activity
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Characterization of Trichoderma polysporum from Spitsbergen, Svalbard archipelago, Norway, with species identity, pathogenicity to moss, and polygalacturonase activity Yusuke YAMAZAKIa, Motoaki TOJOa,*, Tamotsu HOSHINOb, Kenichi KIDAa,1, Tatsuji SAKAMOTOa, Hideshi IHARAc, Isao YUMOTOb, Anne Marte TRONSMOd, Hiroshi KANDAe a
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan c Graduate School of Science, Osaka Prefecture University, Osaka, Japan d Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Aas, Norway e Department of Biology, National Institute of Polar Research, Tokyo, Japan b
article info
abstract
Article history:
Six isolates of Trichoderma polysporum-like species isolated from a moss (Sanionia uncinata) in
Received 18 March 2010
high arctic wetlands at Spitsbergen Island, Svalbard, Norway, were examined for species
Revision received 22 June 2010
identification, effects of temperature on growth, pathogenicity to the moss, and
Accepted 22 June 2010
polygalacturonase (PGase) activity. All isolates from Spitsbergen were identified as
Available online 8 September 2010
T. polysporum based on morphology, sequences of the rDNA-ITS regions, and growth
Corresponding editor: Peter Kennedy
response to temperature. In addition, two isolates of T. polysporum from Germany and Japan were used. All isolates from Spitsbergen, Germany and Japan infected epidermal tissues of
Keywords:
the moss, but did not cause any symptoms in in vitro inoculation tests at 0 C. One of the
Arctic region
isolates from Spitsbergen and the German isolate showed high activity of PGase, an enzyme
Endophyte
produced by many saprotrophic and pathogenic fungi, in a pectin liquid medium. The
Sanionia uncinata
enzyme activity was evident for both isolates even at 0 C. The present results document the
Saprotroph
presence of T. polysporum in the high arctic wetlands. The epidermal infection to the moss and PGase production at 0 C of the T. polysporum isolates suggest that the species likely inhabits a niche as a saprotroph or an endophyte in the arctic environments. ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.
Introduction Trichoderma species are common inhabitants of root, soil and foliar environments, and are amongst the most frequently isolated soil fungi (Domsch et al. 1980; Harman et al. 2004). Their rapid growth and the ability to metabolize a wide variety
of substrates enable them to be predominant components of the soil mycobiota in diverse ecosystems such as agricultural fields, pastures, grassland, forests, salt wetlands, deserts (Klein & Eveleigh 1998) and polar regions (Elvebakk et al. 1996). The genus includes a number of species which are important as antagonists and biocontrol agents of plant pathogens
* Corresponding author. Tel.: þ81 (0) 72 254 9411; fax: þ81 (0) 72 254 9918. E-mail address: [email protected] (M. Tojo). 1 Present address: Life Science Research Institute, Kumiai Chemical Industry Co., Ltd., Shizuoka, Japan. 1754-5048/$ e see front matter ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2010.06.002
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Y. Yamazaki et al.
(Antal et al. 2000; Barbosa et al. 2001; Benitez et al. 2004; Verma et al. 2007) and nematodes (Kyalo et al. 2007; Goswami et al. 2008). Some of them are known as pathogens of many crops (Kishi 1998) including vegetable seedlings (Menzies 1993). Trichoderma spp. are also known as producers of cell walldegrading enzymes of plants such as cellulases, xylanases and polygalacturonases (PGases) (Biely & Tenkanen 1998; Mohamed et al. 2003, 2006). PGase is associated with both pathogenicity (Miyairi et al. 1985; ten Have et al. 1998; Oeser et al. 2002) and saprotrophic activity (Go¨tesson et al. 2002; Li et al. 2004) in many fungi. PGases produced by Trichoderma spp. have been extensively studied (Fanelli et al. 1978; Markovic et al. 1985; Mohamed et al. 2003, 2006). Trichoderma polysporum is one of the psycrotrophic Trichoderma species present in temperate (Goldfarb et al. 1989; Lu et al. 2004) and arctic environments (Zabawski 1982). T. polysporum from western Spitsbergen, Svalbard archipelago, Norway is the only example of this species recorded from the arctic environments (Zabawski 1982). However, neither detailed taxonomic information nor herbarium materials of the arctic isolates of this species have been available. In Aug. 2002, we obtained isolates of T. polysporum-like fungus from a moss (Sanionia uncinata) in the high arctic wetlands at Spitsbergen. The purposes of this study were to characterize these Spitsbergen isolates in terms of species identification, growth response to temperature, pathogenicity to moss, and PGase activity, and to compare these isolates with those from Germany and Japan.
Materials and methods Isolation and identification Six isolates of T. polysporum-like fungus, OPU 1567, OPU 1568, OPU 1569, OPU 1570, OPU 1571 and OPU 1572, were isolated from brown discolored S. uncinata from the land area of wetlands in Longyearbyen (78 150 N, 15 300 E) and Barentsburg (78 040 N, 14 140 E), Spitsbergen, Svalbard archipelago, Norway, in Aug. 2002, using water agar composed of 1.5 % agar (WA) (Wako, Osaka, Japan) (Table 1). Brown discolored stemleaves of S. uncinata were collected from the wetlands. Sections of the stem-leaves were washed in tap water, air dried, and incubated on WA at 4 C for a week. Fungal mycelia
growing on the agar were transferred to corn-meal agar (CMA) (Becton Dickinson and Company, Franklin Lakes, USA). Two T. polysporum isolates, CBS 820.68 from Kiel-Kitzeberg, Germany and TMI 34109 from Tottori Prefecture, Japan, were used for comparison. The isolates were maintained on CMA slant at 13 C in darkness until use. Morphology of these isolates was examined on potato dextrose agar (PDA) (Becton Dickinson, Franklin Lakes, USA). The morphological identification was based on the keys by Gams & Bissett (1998). To confirm the morphological identification of the species, the nucleotide sequences of the ITS regions containing ITS1, ITS2 and intervening 5.8S rDNA were determined by the method described previously (Tojo et al. 2001). Sequences of ITS regions were used for phylogenetic analysis. Initially, these sequences were aligned using Clustal W (Thompson et al. 1997). A phylogenetic tree was constructed by MEGA version 4.0 (Kumar et al. 2008) based on the neighbor-joining (NJ) method (Saitou & Nei 1987), and evolutionary distance was calculated according to Kimura’s two parameter method (Kimura 1980). To determine the support for each clade, a bootstrap analysis was performed with 1 000 replications. All sequences determined in this study were deposited in GenBank (accession numbers as shown in Table 1). The complete alignments and the phylogenetic tree were deposited in TreeBASE as SN4879.
Relationship of growth to temperature All isolates of T. polysporum shown in Table 1 were used. An agar plug (5 mm diameter), taken from edge of each culture of the isolates grown at 19 C for a week on PDA, was placed in the center of a PDA plate. The plates were incubated at 22 C for 24 hr, and then incubated at 0, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 34 C in darkness. Mycelial extension was measured along a line marked beneath the plate. Linear growth rate was calculated in mm d1. There were three replicates for each isolate.
Pathogenicity to S. uncinata All isolates of T. polysporum shown in Table 1 were used. Stemleaf sections (15 mm long, by 0.5 mm wide) of S. uncinata from Spitsbergen were grown in an incubator at 15 C for 3e4 months and placed on a WA plate. A PDA plug (12 mm
Table 1 e Trichoderma polysporum isolates used in this study Isolate no.
Origin
Locality
Year of isolation
GenBank accession no.
References
OPU 1567 OPU 1568 OPU 1569 OPU 1570 OPU 1571 OPU 1572 CBS 820.68 TMI 34109
Sanionia uncinata Sanionia uncinata Sanionia uncinata Sanionia uncinata Sanionia uncinata Sanionia uncinata Wheat field soil Quercus sp. colonized by Lentinula edodes
Barentsburg, Spitsbergen, Norway Barentsburg, Spitsbergen, Norway Barentsburg, Spitsbergen, Norway Longyearbyen, Spitsbergen, Norway Longyearbyen, Spitsbergen, Norway Longyearbyen, Spitsbergen, Norway Kiel-Kitzeberg, Germany Tottori Prefecture, Japan
2002 2002 2002 2002 2002 2002 1963 ea
AB539729 AB539730 AB539731 AB539732 AB539733 AB539734 Z48815 AB542058
This study This study This study This study This study This study Kuhls, unpublished This study
a Unknown.
ND ND NDe ND 1.6 8.5 a b c d e
‘W’ indicates white to cream-colored, and ‘B’ indicates bright greenish-yellow or pale rosy-buff. Not formed on potato dextrose agar (PDA). ‘ST’ indicates spiral-shaped and usually tuberculate, and ‘SS’ indicates spiral-shaped and usually smooth-walled. Extension rate was determined on PDA. Not determined.
0.4 7.6 1.3 10.2
5.0 17.1
1.3 12.6
1.7 10.3
1.4 7.8
1.5 8.9
4e7 2.5e4 4e8 2.5e3.5 3.5e7 2.7e3.6 4e6.8 2.6e3.8 3e3.5 1.5e2.5 3e3.5 2 2.3e3.6 1.4e2.2 2.9e4 2e2.9
B 1e3 2e5 ST W 2e4 1e7 ST W 1e3 1e7 ST W 1e3 1e5 SS e e e e
Rifai (1969) TMI 34109 CBS 820.68 OPU 1572 OPU 1571 OPU 1570 OPU 1569
b a
OPU 1568
Growth rated (mm/d) 4 C 25 C
All isolates from Spitsbergen were morphologically similar to each other with a few exceptions such as the isolate OPU 1572 that produced no conidiophores and conidia, and isolate OPU 1571 that had longer phialides and larger conidia than the others (Table 2). All isolates were also morphologically similar to the T. polysporum isolates from Germany and Japan (Table 2), and formed white to cream colonies (Fig S1aeg). Detailed descriptions are provided here for representative isolate OPU 1570.
e e
Identification
17
Mycelium color on agar surface W W W W W Number of branch per conidiophore main axis 1e2 1e3 1e2 1e3 1e3 Number of phialide per terminal branch 1e5 1e5 1e5 1e5 1e5 ST ST ST ST Morphology of upper part of conidiophore STc main axis Size of phialide (mm) 4e8 2e3.5 4e8 2.5e4 3.5e7 2e3 4e8 2.5e4 2e3 5.5e10 Size of conidia (mm) 3e3.5 2.5 3e3.5 2 3e3.5 2 2.5e3 2e2.5 3.5e4 2e3.5
Results
OPU 1567
The isolates OPU 1567, OPU 1568, OPU 1570, CBS 820.68 and TMI 34109 were used (Table 1). They were first cultured for 1 week at 19 C on agar plates with pectin as carbon source, with 1.5 % agar, 0.2 % NH4NO3, 0.1 % K2HPO4, 0.05 % MgSO4$7H2O, 0.05 % KCl, 0.001 % FeSO4$7H2O (all obtained from Wako, Osaka, Japan), 0.1 % peptone (Becton Dickinson, Franklin Lakes, USA) and 0.5 % citrus pectin (pH 5.0) (SigmaeAldrich, St Louis, MO, USA). Five agar plugs (5 mm) were put into 300 ml Erlenmeyer flasks containing 100 ml of liquid medium composed of the same ingredients described above, except for agar. Cultures were grown for 7 weeks at 14 C without shaking and the culture filtrates were dialyzed by using a size 20 kDa dialysis membrane (Wako, Osaka, Japan) against 20 mM sodium acetate buffer (pH 5.0). The pectinase activity in the culture filtrate was assayed at two temperatures, 0 C and 40 C using polygalacturonic acid (SigmaeAldrich, St Louis, MO, USA) as substrate. PGase activity was determined by measuring the release of reducing sugars in a reaction mixture containing 200 ml of 0.1 % polygalacturonic acid in 20 mM sodium acetate buffer (pH 5.0) and 20 ml of the dialyzed enzyme sample at 0 C and 40 C for 30 min. Reducing sugars were quantified by the method of SomogyieNelson (Nelson 1944; Somogyi 1952). One unit (U) was defined as the amount of enzyme that forms reducing sugars corresponding to 1 mmol of D-galacturonic acid in 1 min. When isolates showed pectinase activity at 0 C and 40 C, the enzyme activities were examined at various temperatures between 0 C and 70 C by the method described above.
T. polysporum
PGase activity
Table 2 e Taxonomic features of Trichoderma polysporum isolates used in this study with reference data of the species and related species
diameter) from each fungal culture, grown at 19 C for 1 week, was placed in the center of the plate. These plates were incubated at 0 C in darkness for 2 months. Then, the moss stem-leaf was washed by dipping in distilled water three times to remove mycelia from the surface. The moss was air dried, and transferred to CMA plates. The plates were incubated for 1 week and the number of moss pieces with mycelia was counted. The identity of the recovered mycelia was based on their morphology on PDA. Pathogenicity was also confirmed by microscopic observation. The development of symptoms such as stem-leaf discoloration was observed. There were 24 replicates for each isolate using one moss pieces for each replicate.
T. croceum (Bissett 1991)
Characterization of T. polysporum from Spitsbergen, Svalbard archipelago, Norway
18
Y. Yamazaki et al.
the isolates OPU 1570, OPU 1571 and OPU 1572. T. polysporum isolates from Spitsbergen consisted of phylogenetically two different groups each corresponding to their localities (Fig S2). The sequences of OPU 1567, OPU 1568 and OPU 1569 were highly homologous with those of T. croceum isolate CBS 337.93, while the sequences of OPU 1570, OPU 1571 and OPU 1572 shared high homology with those of T. polysporum isolate CBS 820.68 (Fig S2).
14 OPU 1567 a OPU 1568
12
Extension (mmd-1)
OPU 1569 OPU 1570
10
OPU 1571 OPU 1572
8
TMI 34109 CBS 820.68
6
Growth temperature relations
4 2 0 0
4
7
10
13
16
19
22
25
28
31
Temperature (ºC) Fig 1 e Mycelial extension rate of Trichoderma polysporum isolates on potato dextrose agar at different temperatures in darkness. aIsolate numbers of T. polysporum shown in Table 1.
Conidiophores were hyaline (Fig S1hej), branches were mostly short, usually paired, occasionally in verticils of three, and secondary branches in whorls (Fig S1h). The upper part of the conidiophore main axis was sterile, spiral-shaped, and the conidiophore apex was smooth-walled (Fig S1i). Mature conidiophores were cream-colored, and mostly tuberculate especially toward the apex (Fig S1j). Phialides usually arose in whorls of 2e3 at the apex of the terminal branches (Fig S1h), occasionally five phialides or a solitary terminal phialide was found. Phialides were ampulliform (Fig S1H), 4e8 2.5e4 mm. Conidia were ellipsoid, 2.5e3 2e2.5 mm (Fig S1k), and had a dimple in the center (Fig S1k, arrowheads). The morphological features of the isolate agreed with the species description by Rifai (1969) (Table 2). Morphological features were also similar to those of Trichoderma croceum which is thought to be synonymous with T. polysporum (Lee & Hseu 2002; Lu et al. 2004) except for colony color (Table 2). Analysis of rDNA-ITS sequences demonstrated that all the T. polysporum isolates examined were placed in “clade II”, described by Lee & Hseu (2002), that consist of species T. polysporum, T. croceum, Trichoderma oblongisporum and Trichoderma lignorum (Fig S2). Within this clade, the isolates used were monophyletic that contained only T. polysporum and T. croceum. Based on the morphological and molecular phylogenetical analysis, the isolates from Spitsbergen were identified as T. polysporum. The length of the ITS region was 531 bp in the isolate OPU 1567, 529 bp in the isolates OPU 1568 and OPU 1569 and 526 bp in
All the isolates used had similar mycelial extension rates on PDA at different temperatures, except the isolate CBS 820.68 from Germany that grew slower. This isolate is now 46 years old and reduced growth is not unexpected for cultures of this age. All the isolates grew between 0 C and 25 C, and showed maximum growth at 22 C or 25 C. The growth rate varied from 0.1 mm d1 0 C to 12.8 mm d1 at 28 C, and was nil at 31 C (Fig 1).
Pathogenicity to S. uncinata All isolates of T. polysporum infected S. uncinata using the in vitro inoculation test but caused no appreciable symptoms (Table 3). Microscopic observations revealed that all isolates colonized dead epidermal cells of the plant, but did not invade living inner cells of the plant (Fig 2). Uninoculated control plants had no symptoms or fungal growth in epidermal cells.
PGase activity The isolates OPU 1570 and CBS 820.68 exhibited distinct PGase activities (Fig 3). The enzyme activity of both isolates was evident even at 0 C. The enzyme activity was very low in other isolates examined. The highest levels of PGases were 40 C and 50 C in OPU 1570 and CBS 820.68, respectively (Fig S3).
Discussion All six isolates of Trichoderma sp. from Spitsbergen were identified as T. polysporum based on their morphology, rDNAITS sequences, and growth temperature relations. The present results confirm the previous report (Zabawski 1982) which recorded this species from western Spitsbergen. The results also support that T. polysporum is widely distributed in low temperature environments (Domsch et al. 1980). The taxonomic features of the isolates from Spitsbergen are similar to those of T. polysporum isolates from Germany and Japan. Some conidial fungi are known to remain viable after long-distance dispersal across polar regions and oceans (Pady & Kapica 1953,
Table 3 e Infectivity of Trichoderma polysporum isolates to Sanionia uncinata in an in vitro inoculation test
Colonization (%)b
OPU 1567a
OPU 1568
OPU 1569
OPU 1570
OPU 1571
OPU 1572
CBS 820.68
TMI 34109
Uninfested control
100
100
100
12.5
45.8
100
50
45.8
0
a Isolate nos. shown in Table 1. b Infectivity was evaluated by the percentages of reisolation of inoculated isolates from S. uncinata.
Characterization of T. polysporum from Spitsbergen, Svalbard archipelago, Norway
Fig 2 e Colonization of dead epidermal cells of Sanionia uncinata by Trichoderma polysporum isolate OPU 1569. Arrowheads indicate penetration of the hyphae into the cells. Bar 10 mm. 1955; Pady & Kelly 1954). Kubicek et al. (2008) suggested that some of the Trichoderma species disperse long distances through air flow. Conidial production of T. polysporum (Rifai 1969) and its aerial transport may contribute to its wide distribution. The rDNA-ITS analysis of T. polysporum isolates demonstrated that the intraspecific variations of the ITS sequence are common in this species. The T. polysporum isolates from Spitsbergen were phylogenetically divided into two groups corresponding to their localities. One of them formed an independent sub-clade and another one formed the same sub-clade with a Canadian isolate of the species. These results suggest that T. polysporum is genetically diversified in its worldwide spread and some of them were distributed in Spitsbergen. The similarity in growth response to temperature among all the isolates of T. polysporum suggests that the isolates from Spitsbergen do not differentiate significantly from the Japanese isolate TMI 34109 in terms of temperature effects. This may also suggest that temperature is not a limiting factor for
19
their survival in the arctic environments. This hypothesis is supported by previous reports, indicating growth response to temperatures of soil fungi isolated from polar regions was not different from those of taxonomically closely related species present in temperate or tropical regions (Mo¨ller et al. 1996; Hoshino et al. 2002). At Spitsbergen, Longyearbyen, the average summer temperature is around 7.7 C, while winter averages around 15.9 C (The Norwegian Meteorological Institute 2010). T. polysporum isolates from Spitsbergen may grow during summer months, and they may over-winter as conidia mixed with plant debris. All T. polysporum isolates colonized dead epidermal cells, but not living inner cells of S. uncinata in the in vitro inoculation tests at 0 C. They did not cause any symptoms on the plant under the experimental condition. T. polysporum has been isolated from various types of soil and rhizospheres of many plants (Domsch et al. 1980). Present results and the previous report suggest that T. polysporum inhabits the high arctic environments as a saprotroph or an endophyte. Two isolates of T. polysporum from Spitsbergen and Germany showed PGase activity at 0 C. The highest levels of PGases were at 40 C and 50 C in the Spitsbergen and German isolates, respectively. Optimum temperature for PGases activity of fungi is generally around 50 C (Pilnik & Rombouts 1981; Parenicova´ 2000). Although the T. polysporum isolates used were limited, present results and the previous data suggest that the arctic isolate of T. polysporum produces PGases having activity at low temperature. In conclusion, we isolated T. polysporum from the high arctic wetlands in Spitsbergen Island. The fungus had traits similar to those of T. polysporum from temperate regions, suggesting that the fungus likely inhabits a niche as a saprotroph in moss colonies on the island.
180 160
We would like to thank Prof. Nitaro Maekawa, Tottori University, for providing the Trichoderma polysporum strain from Tottori Pref., Japan. We thank Dr. Janice Y. Uchida of University of Hawaii at Manoa for her critical reading of the manuscript. This study was supported from the Japan Society for the Promotion of Science by the grant-in-aid for scientific research No. 19510033 to M. Tojo.
0ºC
140
40ºC
120 100 80 60 40
Supplementary material
20 0 9
68
I3 41 0
BS C
TM
82 0.
70 15 PU O
O
O
PU
15 67 PU 15 68
a
Polygalacturonase activity (mUml-1)
Acknowledgements
Fig 3 e Polygalacturonase activity in the liquid culture filtrates of Trichoderma polysporum isolates at 0 and 40 C. The enzyme activities were assayed by measuring the release of reducing sugars using the method of SomogyieNelson assays (Nelson 1944; Somogyi 1952). a Isolate numbers of T. polysporum shown in Table 1. N [ 3, bars are SEM.
The supplementary data associated with this article can be found in the on-line version, at doi:10.1016/j.funeco.2010.06.002.
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Characterization of T. polysporum from Spitsbergen, Svalbard archipelago, Norway
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Characterization of Trichoderma polysporum from Spitsbergen, Svalbard archipelago, Norway, with species identity, pathogenicity to moss, and polygalacturonase activity Yusuke YAMAZAKIa, Motoaki TOJOa,*, Tamotsu HOSHINOb, Kenichi KIDAa,1, Tatsuji SAKAMOTOa, Hideshi IHARAc, Isao YUMOTOb, Anne Marte TRONSMOd, Hiroshi KANDAe a
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan c Graduate School of Science, Osaka Prefecture University, Osaka, Japan d Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Aas, Norway e Department of Biology, National Institute of Polar Research, Tokyo, Japan b
article info
abstract
Article history:
Six isolates of Trichoderma polysporum-like species isolated from a moss (Sanionia uncinata) in
Received 18 March 2010
high arctic wetlands at Spitsbergen Island, Svalbard, Norway, were examined for species
Revision received 22 June 2010
identification, effects of temperature on growth, pathogenicity to the moss, and
Accepted 22 June 2010
polygalacturonase (PGase) activity. All isolates from Spitsbergen were identified as
Available online 8 September 2010
T. polysporum based on morphology, sequences of the rDNA-ITS regions, and growth
Corresponding editor: Peter Kennedy
response to temperature. In addition, two isolates of T. polysporum from Germany and Japan were used. All isolates from Spitsbergen, Germany and Japan infected epidermal tissues of
Keywords:
the moss, but did not cause any symptoms in in vitro inoculation tests at 0 C. One of the
Arctic region
isolates from Spitsbergen and the German isolate showed high activity of PGase, an enzyme
Endophyte
produced by many saprotrophic and pathogenic fungi, in a pectin liquid medium. The
Sanionia uncinata
enzyme activity was evident for both isolates even at 0 C. The present results document the
Saprotroph
presence of T. polysporum in the high arctic wetlands. The epidermal infection to the moss and PGase production at 0 C of the T. polysporum isolates suggest that the species likely inhabits a niche as a saprotroph or an endophyte in the arctic environments. ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.
Introduction Trichoderma species are common inhabitants of root, soil and foliar environments, and are amongst the most frequently isolated soil fungi (Domsch et al. 1980; Harman et al. 2004). Their rapid growth and the ability to metabolize a wide variety
of substrates enable them to be predominant components of the soil mycobiota in diverse ecosystems such as agricultural fields, pastures, grassland, forests, salt wetlands, deserts (Klein & Eveleigh 1998) and polar regions (Elvebakk et al. 1996). The genus includes a number of species which are important as antagonists and biocontrol agents of plant pathogens
* Corresponding author. Tel.: þ81 (0) 72 254 9411; fax: þ81 (0) 72 254 9918. E-mail address: [email protected] (M. Tojo). 1 Present address: Life Science Research Institute, Kumiai Chemical Industry Co., Ltd., Shizuoka, Japan. 1754-5048/$ e see front matter ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2010.06.002
16
Y. Yamazaki et al.
(Antal et al. 2000; Barbosa et al. 2001; Benitez et al. 2004; Verma et al. 2007) and nematodes (Kyalo et al. 2007; Goswami et al. 2008). Some of them are known as pathogens of many crops (Kishi 1998) including vegetable seedlings (Menzies 1993). Trichoderma spp. are also known as producers of cell walldegrading enzymes of plants such as cellulases, xylanases and polygalacturonases (PGases) (Biely & Tenkanen 1998; Mohamed et al. 2003, 2006). PGase is associated with both pathogenicity (Miyairi et al. 1985; ten Have et al. 1998; Oeser et al. 2002) and saprotrophic activity (Go¨tesson et al. 2002; Li et al. 2004) in many fungi. PGases produced by Trichoderma spp. have been extensively studied (Fanelli et al. 1978; Markovic et al. 1985; Mohamed et al. 2003, 2006). Trichoderma polysporum is one of the psycrotrophic Trichoderma species present in temperate (Goldfarb et al. 1989; Lu et al. 2004) and arctic environments (Zabawski 1982). T. polysporum from western Spitsbergen, Svalbard archipelago, Norway is the only example of this species recorded from the arctic environments (Zabawski 1982). However, neither detailed taxonomic information nor herbarium materials of the arctic isolates of this species have been available. In Aug. 2002, we obtained isolates of T. polysporum-like fungus from a moss (Sanionia uncinata) in the high arctic wetlands at Spitsbergen. The purposes of this study were to characterize these Spitsbergen isolates in terms of species identification, growth response to temperature, pathogenicity to moss, and PGase activity, and to compare these isolates with those from Germany and Japan.
Materials and methods Isolation and identification Six isolates of T. polysporum-like fungus, OPU 1567, OPU 1568, OPU 1569, OPU 1570, OPU 1571 and OPU 1572, were isolated from brown discolored S. uncinata from the land area of wetlands in Longyearbyen (78 150 N, 15 300 E) and Barentsburg (78 040 N, 14 140 E), Spitsbergen, Svalbard archipelago, Norway, in Aug. 2002, using water agar composed of 1.5 % agar (WA) (Wako, Osaka, Japan) (Table 1). Brown discolored stemleaves of S. uncinata were collected from the wetlands. Sections of the stem-leaves were washed in tap water, air dried, and incubated on WA at 4 C for a week. Fungal mycelia
growing on the agar were transferred to corn-meal agar (CMA) (Becton Dickinson and Company, Franklin Lakes, USA). Two T. polysporum isolates, CBS 820.68 from Kiel-Kitzeberg, Germany and TMI 34109 from Tottori Prefecture, Japan, were used for comparison. The isolates were maintained on CMA slant at 13 C in darkness until use. Morphology of these isolates was examined on potato dextrose agar (PDA) (Becton Dickinson, Franklin Lakes, USA). The morphological identification was based on the keys by Gams & Bissett (1998). To confirm the morphological identification of the species, the nucleotide sequences of the ITS regions containing ITS1, ITS2 and intervening 5.8S rDNA were determined by the method described previously (Tojo et al. 2001). Sequences of ITS regions were used for phylogenetic analysis. Initially, these sequences were aligned using Clustal W (Thompson et al. 1997). A phylogenetic tree was constructed by MEGA version 4.0 (Kumar et al. 2008) based on the neighbor-joining (NJ) method (Saitou & Nei 1987), and evolutionary distance was calculated according to Kimura’s two parameter method (Kimura 1980). To determine the support for each clade, a bootstrap analysis was performed with 1 000 replications. All sequences determined in this study were deposited in GenBank (accession numbers as shown in Table 1). The complete alignments and the phylogenetic tree were deposited in TreeBASE as SN4879.
Relationship of growth to temperature All isolates of T. polysporum shown in Table 1 were used. An agar plug (5 mm diameter), taken from edge of each culture of the isolates grown at 19 C for a week on PDA, was placed in the center of a PDA plate. The plates were incubated at 22 C for 24 hr, and then incubated at 0, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 34 C in darkness. Mycelial extension was measured along a line marked beneath the plate. Linear growth rate was calculated in mm d1. There were three replicates for each isolate.
Pathogenicity to S. uncinata All isolates of T. polysporum shown in Table 1 were used. Stemleaf sections (15 mm long, by 0.5 mm wide) of S. uncinata from Spitsbergen were grown in an incubator at 15 C for 3e4 months and placed on a WA plate. A PDA plug (12 mm
Table 1 e Trichoderma polysporum isolates used in this study Isolate no.
Origin
Locality
Year of isolation
GenBank accession no.
References
OPU 1567 OPU 1568 OPU 1569 OPU 1570 OPU 1571 OPU 1572 CBS 820.68 TMI 34109
Sanionia uncinata Sanionia uncinata Sanionia uncinata Sanionia uncinata Sanionia uncinata Sanionia uncinata Wheat field soil Quercus sp. colonized by Lentinula edodes
Barentsburg, Spitsbergen, Norway Barentsburg, Spitsbergen, Norway Barentsburg, Spitsbergen, Norway Longyearbyen, Spitsbergen, Norway Longyearbyen, Spitsbergen, Norway Longyearbyen, Spitsbergen, Norway Kiel-Kitzeberg, Germany Tottori Prefecture, Japan
2002 2002 2002 2002 2002 2002 1963 ea
AB539729 AB539730 AB539731 AB539732 AB539733 AB539734 Z48815 AB542058
This study This study This study This study This study This study Kuhls, unpublished This study
a Unknown.
ND ND NDe ND 1.6 8.5 a b c d e
‘W’ indicates white to cream-colored, and ‘B’ indicates bright greenish-yellow or pale rosy-buff. Not formed on potato dextrose agar (PDA). ‘ST’ indicates spiral-shaped and usually tuberculate, and ‘SS’ indicates spiral-shaped and usually smooth-walled. Extension rate was determined on PDA. Not determined.
0.4 7.6 1.3 10.2
5.0 17.1
1.3 12.6
1.7 10.3
1.4 7.8
1.5 8.9
4e7 2.5e4 4e8 2.5e3.5 3.5e7 2.7e3.6 4e6.8 2.6e3.8 3e3.5 1.5e2.5 3e3.5 2 2.3e3.6 1.4e2.2 2.9e4 2e2.9
B 1e3 2e5 ST W 2e4 1e7 ST W 1e3 1e7 ST W 1e3 1e5 SS e e e e
Rifai (1969) TMI 34109 CBS 820.68 OPU 1572 OPU 1571 OPU 1570 OPU 1569
b a
OPU 1568
Growth rated (mm/d) 4 C 25 C
All isolates from Spitsbergen were morphologically similar to each other with a few exceptions such as the isolate OPU 1572 that produced no conidiophores and conidia, and isolate OPU 1571 that had longer phialides and larger conidia than the others (Table 2). All isolates were also morphologically similar to the T. polysporum isolates from Germany and Japan (Table 2), and formed white to cream colonies (Fig S1aeg). Detailed descriptions are provided here for representative isolate OPU 1570.
e e
Identification
17
Mycelium color on agar surface W W W W W Number of branch per conidiophore main axis 1e2 1e3 1e2 1e3 1e3 Number of phialide per terminal branch 1e5 1e5 1e5 1e5 1e5 ST ST ST ST Morphology of upper part of conidiophore STc main axis Size of phialide (mm) 4e8 2e3.5 4e8 2.5e4 3.5e7 2e3 4e8 2.5e4 2e3 5.5e10 Size of conidia (mm) 3e3.5 2.5 3e3.5 2 3e3.5 2 2.5e3 2e2.5 3.5e4 2e3.5
Results
OPU 1567
The isolates OPU 1567, OPU 1568, OPU 1570, CBS 820.68 and TMI 34109 were used (Table 1). They were first cultured for 1 week at 19 C on agar plates with pectin as carbon source, with 1.5 % agar, 0.2 % NH4NO3, 0.1 % K2HPO4, 0.05 % MgSO4$7H2O, 0.05 % KCl, 0.001 % FeSO4$7H2O (all obtained from Wako, Osaka, Japan), 0.1 % peptone (Becton Dickinson, Franklin Lakes, USA) and 0.5 % citrus pectin (pH 5.0) (SigmaeAldrich, St Louis, MO, USA). Five agar plugs (5 mm) were put into 300 ml Erlenmeyer flasks containing 100 ml of liquid medium composed of the same ingredients described above, except for agar. Cultures were grown for 7 weeks at 14 C without shaking and the culture filtrates were dialyzed by using a size 20 kDa dialysis membrane (Wako, Osaka, Japan) against 20 mM sodium acetate buffer (pH 5.0). The pectinase activity in the culture filtrate was assayed at two temperatures, 0 C and 40 C using polygalacturonic acid (SigmaeAldrich, St Louis, MO, USA) as substrate. PGase activity was determined by measuring the release of reducing sugars in a reaction mixture containing 200 ml of 0.1 % polygalacturonic acid in 20 mM sodium acetate buffer (pH 5.0) and 20 ml of the dialyzed enzyme sample at 0 C and 40 C for 30 min. Reducing sugars were quantified by the method of SomogyieNelson (Nelson 1944; Somogyi 1952). One unit (U) was defined as the amount of enzyme that forms reducing sugars corresponding to 1 mmol of D-galacturonic acid in 1 min. When isolates showed pectinase activity at 0 C and 40 C, the enzyme activities were examined at various temperatures between 0 C and 70 C by the method described above.
T. polysporum
PGase activity
Table 2 e Taxonomic features of Trichoderma polysporum isolates used in this study with reference data of the species and related species
diameter) from each fungal culture, grown at 19 C for 1 week, was placed in the center of the plate. These plates were incubated at 0 C in darkness for 2 months. Then, the moss stem-leaf was washed by dipping in distilled water three times to remove mycelia from the surface. The moss was air dried, and transferred to CMA plates. The plates were incubated for 1 week and the number of moss pieces with mycelia was counted. The identity of the recovered mycelia was based on their morphology on PDA. Pathogenicity was also confirmed by microscopic observation. The development of symptoms such as stem-leaf discoloration was observed. There were 24 replicates for each isolate using one moss pieces for each replicate.
T. croceum (Bissett 1991)
Characterization of T. polysporum from Spitsbergen, Svalbard archipelago, Norway
18
Y. Yamazaki et al.
the isolates OPU 1570, OPU 1571 and OPU 1572. T. polysporum isolates from Spitsbergen consisted of phylogenetically two different groups each corresponding to their localities (Fig S2). The sequences of OPU 1567, OPU 1568 and OPU 1569 were highly homologous with those of T. croceum isolate CBS 337.93, while the sequences of OPU 1570, OPU 1571 and OPU 1572 shared high homology with those of T. polysporum isolate CBS 820.68 (Fig S2).
14 OPU 1567 a OPU 1568
12
Extension (mmd-1)
OPU 1569 OPU 1570
10
OPU 1571 OPU 1572
8
TMI 34109 CBS 820.68
6
Growth temperature relations
4 2 0 0
4
7
10
13
16
19
22
25
28
31
Temperature (ºC) Fig 1 e Mycelial extension rate of Trichoderma polysporum isolates on potato dextrose agar at different temperatures in darkness. aIsolate numbers of T. polysporum shown in Table 1.
Conidiophores were hyaline (Fig S1hej), branches were mostly short, usually paired, occasionally in verticils of three, and secondary branches in whorls (Fig S1h). The upper part of the conidiophore main axis was sterile, spiral-shaped, and the conidiophore apex was smooth-walled (Fig S1i). Mature conidiophores were cream-colored, and mostly tuberculate especially toward the apex (Fig S1j). Phialides usually arose in whorls of 2e3 at the apex of the terminal branches (Fig S1h), occasionally five phialides or a solitary terminal phialide was found. Phialides were ampulliform (Fig S1H), 4e8 2.5e4 mm. Conidia were ellipsoid, 2.5e3 2e2.5 mm (Fig S1k), and had a dimple in the center (Fig S1k, arrowheads). The morphological features of the isolate agreed with the species description by Rifai (1969) (Table 2). Morphological features were also similar to those of Trichoderma croceum which is thought to be synonymous with T. polysporum (Lee & Hseu 2002; Lu et al. 2004) except for colony color (Table 2). Analysis of rDNA-ITS sequences demonstrated that all the T. polysporum isolates examined were placed in “clade II”, described by Lee & Hseu (2002), that consist of species T. polysporum, T. croceum, Trichoderma oblongisporum and Trichoderma lignorum (Fig S2). Within this clade, the isolates used were monophyletic that contained only T. polysporum and T. croceum. Based on the morphological and molecular phylogenetical analysis, the isolates from Spitsbergen were identified as T. polysporum. The length of the ITS region was 531 bp in the isolate OPU 1567, 529 bp in the isolates OPU 1568 and OPU 1569 and 526 bp in
All the isolates used had similar mycelial extension rates on PDA at different temperatures, except the isolate CBS 820.68 from Germany that grew slower. This isolate is now 46 years old and reduced growth is not unexpected for cultures of this age. All the isolates grew between 0 C and 25 C, and showed maximum growth at 22 C or 25 C. The growth rate varied from 0.1 mm d1 0 C to 12.8 mm d1 at 28 C, and was nil at 31 C (Fig 1).
Pathogenicity to S. uncinata All isolates of T. polysporum infected S. uncinata using the in vitro inoculation test but caused no appreciable symptoms (Table 3). Microscopic observations revealed that all isolates colonized dead epidermal cells of the plant, but did not invade living inner cells of the plant (Fig 2). Uninoculated control plants had no symptoms or fungal growth in epidermal cells.
PGase activity The isolates OPU 1570 and CBS 820.68 exhibited distinct PGase activities (Fig 3). The enzyme activity of both isolates was evident even at 0 C. The enzyme activity was very low in other isolates examined. The highest levels of PGases were 40 C and 50 C in OPU 1570 and CBS 820.68, respectively (Fig S3).
Discussion All six isolates of Trichoderma sp. from Spitsbergen were identified as T. polysporum based on their morphology, rDNAITS sequences, and growth temperature relations. The present results confirm the previous report (Zabawski 1982) which recorded this species from western Spitsbergen. The results also support that T. polysporum is widely distributed in low temperature environments (Domsch et al. 1980). The taxonomic features of the isolates from Spitsbergen are similar to those of T. polysporum isolates from Germany and Japan. Some conidial fungi are known to remain viable after long-distance dispersal across polar regions and oceans (Pady & Kapica 1953,
Table 3 e Infectivity of Trichoderma polysporum isolates to Sanionia uncinata in an in vitro inoculation test
Colonization (%)b
OPU 1567a
OPU 1568
OPU 1569
OPU 1570
OPU 1571
OPU 1572
CBS 820.68
TMI 34109
Uninfested control
100
100
100
12.5
45.8
100
50
45.8
0
a Isolate nos. shown in Table 1. b Infectivity was evaluated by the percentages of reisolation of inoculated isolates from S. uncinata.
Characterization of T. polysporum from Spitsbergen, Svalbard archipelago, Norway
Fig 2 e Colonization of dead epidermal cells of Sanionia uncinata by Trichoderma polysporum isolate OPU 1569. Arrowheads indicate penetration of the hyphae into the cells. Bar 10 mm. 1955; Pady & Kelly 1954). Kubicek et al. (2008) suggested that some of the Trichoderma species disperse long distances through air flow. Conidial production of T. polysporum (Rifai 1969) and its aerial transport may contribute to its wide distribution. The rDNA-ITS analysis of T. polysporum isolates demonstrated that the intraspecific variations of the ITS sequence are common in this species. The T. polysporum isolates from Spitsbergen were phylogenetically divided into two groups corresponding to their localities. One of them formed an independent sub-clade and another one formed the same sub-clade with a Canadian isolate of the species. These results suggest that T. polysporum is genetically diversified in its worldwide spread and some of them were distributed in Spitsbergen. The similarity in growth response to temperature among all the isolates of T. polysporum suggests that the isolates from Spitsbergen do not differentiate significantly from the Japanese isolate TMI 34109 in terms of temperature effects. This may also suggest that temperature is not a limiting factor for
19
their survival in the arctic environments. This hypothesis is supported by previous reports, indicating growth response to temperatures of soil fungi isolated from polar regions was not different from those of taxonomically closely related species present in temperate or tropical regions (Mo¨ller et al. 1996; Hoshino et al. 2002). At Spitsbergen, Longyearbyen, the average summer temperature is around 7.7 C, while winter averages around 15.9 C (The Norwegian Meteorological Institute 2010). T. polysporum isolates from Spitsbergen may grow during summer months, and they may over-winter as conidia mixed with plant debris. All T. polysporum isolates colonized dead epidermal cells, but not living inner cells of S. uncinata in the in vitro inoculation tests at 0 C. They did not cause any symptoms on the plant under the experimental condition. T. polysporum has been isolated from various types of soil and rhizospheres of many plants (Domsch et al. 1980). Present results and the previous report suggest that T. polysporum inhabits the high arctic environments as a saprotroph or an endophyte. Two isolates of T. polysporum from Spitsbergen and Germany showed PGase activity at 0 C. The highest levels of PGases were at 40 C and 50 C in the Spitsbergen and German isolates, respectively. Optimum temperature for PGases activity of fungi is generally around 50 C (Pilnik & Rombouts 1981; Parenicova´ 2000). Although the T. polysporum isolates used were limited, present results and the previous data suggest that the arctic isolate of T. polysporum produces PGases having activity at low temperature. In conclusion, we isolated T. polysporum from the high arctic wetlands in Spitsbergen Island. The fungus had traits similar to those of T. polysporum from temperate regions, suggesting that the fungus likely inhabits a niche as a saprotroph in moss colonies on the island.
180 160
We would like to thank Prof. Nitaro Maekawa, Tottori University, for providing the Trichoderma polysporum strain from Tottori Pref., Japan. We thank Dr. Janice Y. Uchida of University of Hawaii at Manoa for her critical reading of the manuscript. This study was supported from the Japan Society for the Promotion of Science by the grant-in-aid for scientific research No. 19510033 to M. Tojo.
0ºC
140
40ºC
120 100 80 60 40
Supplementary material
20 0 9
68
I3 41 0
BS C
TM
82 0.
70 15 PU O
O
O
PU
15 67 PU 15 68
a
Polygalacturonase activity (mUml-1)
Acknowledgements
Fig 3 e Polygalacturonase activity in the liquid culture filtrates of Trichoderma polysporum isolates at 0 and 40 C. The enzyme activities were assayed by measuring the release of reducing sugars using the method of SomogyieNelson assays (Nelson 1944; Somogyi 1952). a Isolate numbers of T. polysporum shown in Table 1. N [ 3, bars are SEM.
The supplementary data associated with this article can be found in the on-line version, at doi:10.1016/j.funeco.2010.06.002.
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