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Detection of Ophiocordyceps sinensis in the roots of plants in alpine meadows by nested-touchdown polymerase chain reaction Xin ZHONG, Qing-yun PENG, Shao-Song LI, Hai CHEN, Hong-Xia SUN, Gu-Ren ZHANG, Xin LIU* Sun Yat-sen University, School of Life Sciences, Food and Health Engineering Research Center of State Education Ministry, Guangzhou 510275, China
article info
abstract
Article history:
Ophiocordyceps sinensis, one of the most important income sources of rural Tibetan families,
Received 23 January 2013
is an entomopathogenic fungus that parasitizes the ghost moth Thitarodes larvae, which
Received in revised form
live in alpine meadows on the Tibetan Plateau and in the Himalayas. The annual yield of
26 November 2013
O. sinensis has gradually declined in recent years. However, there is no effective method
Accepted 11 December 2013
to sustain or increase the yield of O. sinensis artificially because the life cycle of the O. sinen-
Available online 13 February 2014
sis anamorph remains unclear. Here we detected O. sinensis in alpine plant roots by nested-
Corresponding Editor:
touchdown polymerase chain reaction (PCR). Forty-two alpine plant species were screened.
Martin I. Bidartondo
The roots from 23 alpine plant species (54.76 %) tested positive including 13 families and 18
Keywords:
the anamorph life cycle, to deal with harsh conditions in alpine habitats and have an in-
Chinese caterpillar fungus
creased opportunity to infect the larvae. The finding provides new information regarding
Psychrophyte
the biology and ecology of O. sinensis that may be used to sustain this valuable resource.
genera. The detection results indicate that O. sinensis is present in the plant roots during
Entomopathogen
ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Anamorph life cycle Rhizosphere
Introduction Ophiocordyceps sinensis (Berk.) [formerly Cordyceps sinensis (Berk.) Sacc.] (Sung et al. 2007) is an entomopathogenic fungus, which is a specific parasite of the ghost moth Thitarodes larvae (Lepidopterra: Hepialidae) (Cannon et al. 2009; Sun et al. 2011). Thitarodes larvae live underground in soil burrows of the Tibetan Plateau and mainly feed on tender roots of vegetation (Zhang et al. 2009; Maczey et al. 2010; Li et al. 2011; Chen et al. 2010). Ophiocordyceps sinensis is one of the most valuable species in Traditional Chinese Medicine because of its medicinal properties (Zhu et al. 1998a,b; Buenz et al. 2005; Yue et al. 2013). Additionally, it is one of
the most important sources of income for rural households in Tibet (Winkler 2008). However, the annual yield of O. sinensis has gradually declined in recent years (Jiang & Yao 2002; Stone 2008). Therefore, further investigation of the anamorph life cycle of O. sinensis is warranted to maintain a sustainable population. Ophiocordyceps sinensis is an ascomycete. Its life cycle includes two stages: a teleomorph (sexual) and an anamorph (asexual). The anamorph of O. sinensis is Hirsutella like (Liu et al. 2001; Chen et al. 2001). During the teleomorph life cycle, which has been studied in detail, the fungus infects a host larva, forms a hyphal body (Pu & Li 1996) and threadlike
* Corresponding author. Sun Yat-sen University, School of Life Sciences, Food and Health Engineering Research Center of State Education Ministry, Guangzhou 510275, China. Tel.: þ86 20 84112299; fax: þ86 20 84037249. E-mail address: [email protected] (X. Liu). 1878-6146/$ e see front matter ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.12.005
360
hyphae (Zeng et al. 2006), and finally forms one or more sporulating structures that produce ascospores (Sung et al. 2007). Then, the ascospores spread into the environment and germinate quickly to become filamentous mycelium (Li et al. 1998). However, the life cycle of anamorph and the infection mechanism remains unclear (Stone 2008). Ophiocordyceps sinensis is a psychrophilic fungus (Dong & Yao 2005) that grows slowly even at the optimum temperature for O. sinensis hyphae. Thus, culture-based detection of O. sinensis is time consuming, labour-intensive, and requires considerable knowledge of the fungal genera involved. We applied nested-polymerase chain reaction (PCR) to detect O. sinensis, as reported previously (Zhong et al. 2010). Genes of the ribosomal RNA (rDNA) are the most useful taxonomic markers (Hamby & Zimmer 1992; Bruns et al. 1992; Sung et al. 2007). Nested PCR detection has been widely applied in the detection of microbes, such as Gremmeniella abietina (Zeng et al. 2005), Picoa juniperi (Jamali & Banihashemi 2013), and arbuscular mycorrhizal fungi (Stukenbrock & Rosendahl 2005). Some types of entomopathogenic fungi, such as Metarhizium anisopliae, were identified at the rhizosphere, which is the site where insects, such as white grubs, mole crickets, caterpillars, black vine weevils and fire ants, are most likely to interact with the entomopathogen (Roberts and St. Leger 2004; Kepler & Bruck 2006; St. Leger 2008). Another entomopathogenic fungus Beauveria bassiana can artificially colonize plants by inoculation and can be reisolated from other tissues of the inoculated plant (Quesada-Moraga et al. 2006; Posada et al. 2007) and has been detected from several species of plants (Wagner & Lewis 2000; Arnold & Lewis 2005; Fuller-Schaefer et al. 2005). In this context, we hypothesized that the entomopathogenic fungus O. sinensis may colonize the plant roots tissue or rhizosphere during the anamorph life cycle. To validate this hypothesis, we tested the roots of 42 species of high altitude plants for O. sinensis using nested-touchdown PCR with O. sinensis-specific primers.
Methods and materials Collection of plant root samples Root samples of 42 plant species were collected around the Qinghai-Tibetan Plateau Peculiar Bio-resources Research Station of Sun Yat-sen University, which was established at Mountain Segy La (4156 m altitude, N29 360 , E94 360 ) in Tibet in August 2006. Five replicate plants of each species were examined. Approximately 200 mg of root tissues from each plant sample were used for DNA extraction. Roots were washed in sterilized culture dishes five times, with 10 mL pure water (Millipore Milli-Q Integral 3, Resistivity ¼ 18.2 MU cm@25 C, TOC < 5 ppb) each time. Then each sample was cut into several segments for a single DNA isolation.
X. Zhong et al.
(Nanodrop 2000, Thermo Fisher Scientific, DE, USA) at 260 nm/280 nm. Finally, the DNA samples were stored at 20 C. Before PCR detection, the DNA samples were adjusted to 100 ng ml1 using the Eluent solution of the DNA Kit.
PCR detection and sequencing Ophiocordyceps sinensis DNA was detected in plant roots using O. sinensis-specific PCR primers, OsT-F (50 -GTCAAGAAGCAAG CAAAGGAATC-30 ) and OsT-R (50 -TCAACTGGAGGGTGTGGTG G-30 ), flanked by the universal primers, ITS5 (50 -GGAAGTAA AAGTCGTAACAAGG-30 ), and ITS4 (50 -TCCTCCGCTTATTGA TATGC-30 ), respectively (Chen et al. 2004). DNA from each plant root sample was examined in a single PCR. DNA samples from five replicate plants were examined for each species. Two amplifications of nested-touchdown PCR were conducted for detecting O. sinensis DNA as follows: the first amplification was carried out in a 25 ml reaction mixture consisting of 2.5 ml 10 PCR buffer, 2 ml (2.5 mmol l1) dNTP, 0.5 ml (10 mmol l1) of external primers (ITS5 and ITS4), 0.1 ml (5 U ml1) of rTaq DNA polymerase (Takara, Tokyo, Japan), and 2 ml (100 ng ml1) of DNA template. PCR conditions involved one DNA denaturing cycle at 95 C for 4 min, followed by 30 cycles of 95 C 30s, 55 C 30 s, 72 C 60 s, and a final extension at 72 C for 7 min. The PCR products from the first amplification were analyzed by electrophoresis in 1.5 % agarose gels. The gels were stained with ethidium bromide and visualized under UV light to ensure that the PCR amplification was successful. One microlitre of PCR product from the first round was used as template in the second amplification. The second amplification PCR reaction mix (25 ml) consisted of 2.5 ml 10 PCR buffer, 2 ml (2.5 mmol l1) dNTP, 0.5 ml (10 mmol l1) of internal primers (OsT-F and OsT-R), 0.1 ml (5 U ml1) of rTaq DNA polymerase (Takara, Tokyo, Japan), and 1 ml of DNA template from the PCR product of the first round of amplification. The second amplification followed this profile: 1 cycle at 95 C for 4 min (DNA denaturing), 1 cycle of 95 C for 30 s, 65 C for 30 s, and 72 C for 1 min, followed by 9 cycles lowering the annealing temperature from 65 C to 60 C at 0.5 C steps for each cycle, and finally 20 cycles of 95 C for 30 s, 60 C for 30 s, and 72 C for 1 min, and a final extension at 72 C for 7 min. For the negative result samples, other parallel amplifications were conducted using nested-touchdown PCR on additional 2 ml (100 ng ml-1) samples of Ophiocordyceps sinensis DNA template to ensure that the PCR was not inhibited. One of the PCR products of the five replicates from each plant species was purified at random with an AxyPrep PCR Cleanup Kit (Axygen, CA, USA). The purified products were sequenced directly by Shanghai Invitrogen Biotechnology Corporation. The resulting sequences were assembled, corrected, and exported using ContigExpress software. The nucleotide sequences were determined and deposited in the GenBank, EMBL, and DDBJ nucleotide sequence databases. The accession number, length, and AT content of each fragment are listed in Table 1.
Total DNA isolation Total DNA from the plant roots were isolated following the directions of the AxyPrep Multisource Genomic DNA Kit (Axygen, CA, USA). After isolation, the concentrations and quality of the DNA samples were determined by spectrophotometry
Results and discussion Forty-two plant species were identified, as described by Hu (2010) (Fig S1), covering 23 families and 34 genera (Table 1).
Detection of Ophiocordyceps sinensis in the roots of alpine plants
361
Table 1 e Plant samples from Ophiocordyceps sinensis habitats and PCR results using O. sinensis-specific primers. No.
Family
Genus
Species
Results
Access no.
Length (bp)/AT (%)
JD1 JD2 JD3 JD4 JD5 JD8 JD9 JD10 JD11 JD12 JD14 JD16 JD18 JD19 JD20 JD21 JD22 JD23 JD24 JD25 JD26 JD27 JD28 JD29 JD30 JD32 JD33 JD34 JD35 JD36 JD37 JD38 JD39 JD40 JD42 JD43 JD44 JD45 JD46 JD47 JD48 JD49
Onagraceae Poaceae Rosaceae Campanulaceae Ranunculaceae Scrophulariaceae Gentianaceae Scrophulariaceae Cyperaceae Compositae Compositae Ranunculaceae Cruciferae Primulaceae Compositae Rosaceae Polygonaceae Compositae Apiaceae Compositae Apiaceae Compositae Juncaceae Lamiaceae Unknown Juncaceae Polygonaceae Lamiaceae Ranunculaceae Polygonaceae Dryopteridaceae Ranunculaceae Caryophyllaceae Cruciferae Polygonaceae Plantaginaceae Caprifoliaceae Rosaceae Rosaceae Ranunculaceae Ericaceae Salicaceae
Epilobium Deschampsia Fragaria Cyananthus Thalictrum Pedicularis Gentiana Veronica Carex Cremanthodium Dubyaea Ranunculus Draba Primula Ligularia Potentilla Polygonum Saussurea Bupleurum Aster Pachypleurum Saussurea Luzula Phlomis Unknown Juncus Rumex Mentha Ranunculus Polygonum Polystichum Anemone Cerastium Capsella Polygonum Plantago Lonicera Spiraea Potentilla Coptis Rhododendron Salix
Epilobium sikkimense Deschampsia caespitosa Fragaria nubicola Cyananthus macrocalyx Thalictrum uncatum Pedicularis sherriffii Gentiana nyingchiensis Veronica chayuensis Carex lehmanii Cremanthodium linguiatum Dubyaea hispida Ranunculus nephelogens Draba nemorosa Primula alpicola Ligularia nyingchiensis Potentilla cuneata Polygonum macrophyllum Saussurea ovatifolia Bupleurum triadiatum Aster tongolensis Pachypleurum xizangense Saussurea lavrenkoana Luzula multiflora Phlomis tibetica Unknown Juncus leucanthus Rumex nepalensis Mentha canadensis Ranunculus tanguticus Polygonum filicaule Polystichum moupinense Anemone rivularis Cerastium fontanum Capsella bursa-pastoris Polygonum macrophyllum Plantago asiatica Lonicera humilis Spiraea salicifolia Potentilla fruticosa Coptis chinensis Rhododendron vellereum Salix gilashanica
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
JQ936567 JQ936568 JQ936569 JQ936570 JQ936571 JQ936572 JQ936573 JQ936574 JQ936575 JQ936576 JQ936577 JQ936578 JQ936579 JQ936580 JQ936581 JQ936582 JQ936583 JQ936584 JQ936585 JQ936586 JQ936587 JQ936588 JQ936589
359/38.2 359/38.2 359/37.9 359/38.4 359/37.9 358/38.3 359/38.2 359/38.2 359/38.2 359/38.2 359/38.2 359/38.2 359/38.2 359/38.4 291/43 359/38.2 359/38.2 359/38.2 359/49.6 359/38.4 359/38.2 359/38.2 359/37.9
Plant vouchers were deposited in the Food and Health Engineering Research Center of the State Education Ministry, Guangzhou, China (Voucher No. SJLP1021-1063). Most of the alpine plants belonged to Compositae, Ranunculaceae and
Polygonaceae, and each family had six, five, and five species, respectively (Table 1). The nested-touchdown PCR detection results showed that 23 of the 42 species were positive for Ophiocordyceps sinensis in the plant roots, including 13 families
Fig 1 e Results of the nested-touchdown PCR to detect O. sinensis DNA in root samples. PCR was performed using O. sinensisspecific primers, OsT-F and OsT-R, flanked by universal primers ITS5 and ITS4, respectively. M: DNA Marker DL2000; NK: negative control; CK: positive control; 1e49: plant roots samples JD1eJD49.
362
and 18 genera (Fig 1, Table 1). The PCR-positive plant samples were herbs, whereas the woody plants samples (JD44eJD49) were PCR negative. Following positive detection, the PCR products were sequenced directly and compared using BLAST in GenBank, EMBL, and DDBJ nucleotide sequence databases. All sequences from plant root samples were highly analogous (96e100 %) to O. sinensis ribosomal DNA. These results suggested that O. sinensis was present in the alpine herb roots. Although O. sinensis colonize the plant roots as shown by nested PCR detection, further histological evidence is necessary to confirm these results. The observation that Ophiocordyceps sinensis associates with plant roots is in line with other entomopathogenic fungi, such as those of the genera Metarhizium, Beauveria, and Pochonia, which are able to derive nutrition from plant sources in the absence of insect hosts (Posada & Vega 2005; Akello et al. -Vicente et al. 2009; Wyrebek et al. 2007; Vega 2008; Macia 2011; Behie et al. 2012). The growth of O. sinensis in the roots or rhizosphere may be supported by plants, and O. sinensis may benefit plants in a manner similar to Metarhizium anisopliae (Kepler & Bruck 2006). When we consider the life cycle and ecology of O. sinensis and Thitarodes larvae, Thitarodes larvae live underground on the Tibetan Plateau and in the Himalayas soil burrows and mainly feed on tender roots of vegetation. According to dietary preference studies on Thitarodes larvae, root fragments of the genera Cyananthus, Veronica, Ranunculus, Juncus, Deschampsia, Ligularia, Pachypleurum, Polygonum, and Cerastium were detected in the foregut of Thitarodes larvae using rDNA nested PCR assays with primers that targeted ITS regions (Chen et al. 2010; Hu 2010). These plant root tissues, which are preferred by Thitarodes larvae, were also found to be positive for O. sinensis according to PCR detection. As a result, when O. sinensis colonizes the alpine plant roots or rhizosphere, it could increase transmission to Thitarodes larvae. As far as we know, O. sinensis cannot complete the teleomorph life cycle without Thitarodes larvae because it requires unknown factors or the nutrition from the larval tissue (Holliday & Cleaver 2008). This study fills in gaps in understanding about how O. sinensis hyphae are present in plant tissue or the rhizosphere during the anamorph life cycle to cope with harsh conditions in alpine habitats and have an increased opportunity to infect Thitarodes larvae. These results may help manage plant communities to support O. sinensis populations by increasing the number of plants preferred by Thitarodes larvae and colonized by O. sinensis, such as Cyananthus, Veronica, Ranunculus, and Juncus herbs, at the fungal habitats. Future research will focus on reconstructing the colonization of O. sinensis mycelium in the aseptic seedling roots and rhizosphere of alpine herbs, such as Polygonum macrophyllum to reveal interactions between the fungus and plants. These studies will determine whether O. sinensis actually grow in or on the plant roots and whether this association is a parasitic or mutualistic relationship. Ophiocordyceps sinensis is believed to distribute on the Tibetan Plateau and in the Himalayas through ascospores produced in the teleomorph pathway (Sung et al. 2007; Cannon et al. 2009). However, the detection results in this study indicate that O. sinensis also spreads to plants via the anamorph conidia or hypha, forming an abundant and stable population by colonizing plant roots. This finding may help develop a new
X. Zhong et al.
strategy to sustain O. sinensis, which is a valuable and rare resource of the Tibetan Plateau and the Himalayas.
Acknowledgements This work was supported by grants from the National Natural Science Foundation (31300427), National Key Technology R&D Program in the 12th Five-year Plan of China (2011BAI13B06), and China Postdoctoral Science Foundation (2012M511861).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2013.12.005
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journal homepage: www.elsevier.com/locate/funbio
Detection of Ophiocordyceps sinensis in the roots of plants in alpine meadows by nested-touchdown polymerase chain reaction Xin ZHONG, Qing-yun PENG, Shao-Song LI, Hai CHEN, Hong-Xia SUN, Gu-Ren ZHANG, Xin LIU* Sun Yat-sen University, School of Life Sciences, Food and Health Engineering Research Center of State Education Ministry, Guangzhou 510275, China
article info
abstract
Article history:
Ophiocordyceps sinensis, one of the most important income sources of rural Tibetan families,
Received 23 January 2013
is an entomopathogenic fungus that parasitizes the ghost moth Thitarodes larvae, which
Received in revised form
live in alpine meadows on the Tibetan Plateau and in the Himalayas. The annual yield of
26 November 2013
O. sinensis has gradually declined in recent years. However, there is no effective method
Accepted 11 December 2013
to sustain or increase the yield of O. sinensis artificially because the life cycle of the O. sinen-
Available online 13 February 2014
sis anamorph remains unclear. Here we detected O. sinensis in alpine plant roots by nested-
Corresponding Editor:
touchdown polymerase chain reaction (PCR). Forty-two alpine plant species were screened.
Martin I. Bidartondo
The roots from 23 alpine plant species (54.76 %) tested positive including 13 families and 18
Keywords:
the anamorph life cycle, to deal with harsh conditions in alpine habitats and have an in-
Chinese caterpillar fungus
creased opportunity to infect the larvae. The finding provides new information regarding
Psychrophyte
the biology and ecology of O. sinensis that may be used to sustain this valuable resource.
genera. The detection results indicate that O. sinensis is present in the plant roots during
Entomopathogen
ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Anamorph life cycle Rhizosphere
Introduction Ophiocordyceps sinensis (Berk.) [formerly Cordyceps sinensis (Berk.) Sacc.] (Sung et al. 2007) is an entomopathogenic fungus, which is a specific parasite of the ghost moth Thitarodes larvae (Lepidopterra: Hepialidae) (Cannon et al. 2009; Sun et al. 2011). Thitarodes larvae live underground in soil burrows of the Tibetan Plateau and mainly feed on tender roots of vegetation (Zhang et al. 2009; Maczey et al. 2010; Li et al. 2011; Chen et al. 2010). Ophiocordyceps sinensis is one of the most valuable species in Traditional Chinese Medicine because of its medicinal properties (Zhu et al. 1998a,b; Buenz et al. 2005; Yue et al. 2013). Additionally, it is one of
the most important sources of income for rural households in Tibet (Winkler 2008). However, the annual yield of O. sinensis has gradually declined in recent years (Jiang & Yao 2002; Stone 2008). Therefore, further investigation of the anamorph life cycle of O. sinensis is warranted to maintain a sustainable population. Ophiocordyceps sinensis is an ascomycete. Its life cycle includes two stages: a teleomorph (sexual) and an anamorph (asexual). The anamorph of O. sinensis is Hirsutella like (Liu et al. 2001; Chen et al. 2001). During the teleomorph life cycle, which has been studied in detail, the fungus infects a host larva, forms a hyphal body (Pu & Li 1996) and threadlike
* Corresponding author. Sun Yat-sen University, School of Life Sciences, Food and Health Engineering Research Center of State Education Ministry, Guangzhou 510275, China. Tel.: þ86 20 84112299; fax: þ86 20 84037249. E-mail address: [email protected] (X. Liu). 1878-6146/$ e see front matter ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.12.005
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hyphae (Zeng et al. 2006), and finally forms one or more sporulating structures that produce ascospores (Sung et al. 2007). Then, the ascospores spread into the environment and germinate quickly to become filamentous mycelium (Li et al. 1998). However, the life cycle of anamorph and the infection mechanism remains unclear (Stone 2008). Ophiocordyceps sinensis is a psychrophilic fungus (Dong & Yao 2005) that grows slowly even at the optimum temperature for O. sinensis hyphae. Thus, culture-based detection of O. sinensis is time consuming, labour-intensive, and requires considerable knowledge of the fungal genera involved. We applied nested-polymerase chain reaction (PCR) to detect O. sinensis, as reported previously (Zhong et al. 2010). Genes of the ribosomal RNA (rDNA) are the most useful taxonomic markers (Hamby & Zimmer 1992; Bruns et al. 1992; Sung et al. 2007). Nested PCR detection has been widely applied in the detection of microbes, such as Gremmeniella abietina (Zeng et al. 2005), Picoa juniperi (Jamali & Banihashemi 2013), and arbuscular mycorrhizal fungi (Stukenbrock & Rosendahl 2005). Some types of entomopathogenic fungi, such as Metarhizium anisopliae, were identified at the rhizosphere, which is the site where insects, such as white grubs, mole crickets, caterpillars, black vine weevils and fire ants, are most likely to interact with the entomopathogen (Roberts and St. Leger 2004; Kepler & Bruck 2006; St. Leger 2008). Another entomopathogenic fungus Beauveria bassiana can artificially colonize plants by inoculation and can be reisolated from other tissues of the inoculated plant (Quesada-Moraga et al. 2006; Posada et al. 2007) and has been detected from several species of plants (Wagner & Lewis 2000; Arnold & Lewis 2005; Fuller-Schaefer et al. 2005). In this context, we hypothesized that the entomopathogenic fungus O. sinensis may colonize the plant roots tissue or rhizosphere during the anamorph life cycle. To validate this hypothesis, we tested the roots of 42 species of high altitude plants for O. sinensis using nested-touchdown PCR with O. sinensis-specific primers.
Methods and materials Collection of plant root samples Root samples of 42 plant species were collected around the Qinghai-Tibetan Plateau Peculiar Bio-resources Research Station of Sun Yat-sen University, which was established at Mountain Segy La (4156 m altitude, N29 360 , E94 360 ) in Tibet in August 2006. Five replicate plants of each species were examined. Approximately 200 mg of root tissues from each plant sample were used for DNA extraction. Roots were washed in sterilized culture dishes five times, with 10 mL pure water (Millipore Milli-Q Integral 3, Resistivity ¼ 18.2 MU cm@25 C, TOC < 5 ppb) each time. Then each sample was cut into several segments for a single DNA isolation.
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(Nanodrop 2000, Thermo Fisher Scientific, DE, USA) at 260 nm/280 nm. Finally, the DNA samples were stored at 20 C. Before PCR detection, the DNA samples were adjusted to 100 ng ml1 using the Eluent solution of the DNA Kit.
PCR detection and sequencing Ophiocordyceps sinensis DNA was detected in plant roots using O. sinensis-specific PCR primers, OsT-F (50 -GTCAAGAAGCAAG CAAAGGAATC-30 ) and OsT-R (50 -TCAACTGGAGGGTGTGGTG G-30 ), flanked by the universal primers, ITS5 (50 -GGAAGTAA AAGTCGTAACAAGG-30 ), and ITS4 (50 -TCCTCCGCTTATTGA TATGC-30 ), respectively (Chen et al. 2004). DNA from each plant root sample was examined in a single PCR. DNA samples from five replicate plants were examined for each species. Two amplifications of nested-touchdown PCR were conducted for detecting O. sinensis DNA as follows: the first amplification was carried out in a 25 ml reaction mixture consisting of 2.5 ml 10 PCR buffer, 2 ml (2.5 mmol l1) dNTP, 0.5 ml (10 mmol l1) of external primers (ITS5 and ITS4), 0.1 ml (5 U ml1) of rTaq DNA polymerase (Takara, Tokyo, Japan), and 2 ml (100 ng ml1) of DNA template. PCR conditions involved one DNA denaturing cycle at 95 C for 4 min, followed by 30 cycles of 95 C 30s, 55 C 30 s, 72 C 60 s, and a final extension at 72 C for 7 min. The PCR products from the first amplification were analyzed by electrophoresis in 1.5 % agarose gels. The gels were stained with ethidium bromide and visualized under UV light to ensure that the PCR amplification was successful. One microlitre of PCR product from the first round was used as template in the second amplification. The second amplification PCR reaction mix (25 ml) consisted of 2.5 ml 10 PCR buffer, 2 ml (2.5 mmol l1) dNTP, 0.5 ml (10 mmol l1) of internal primers (OsT-F and OsT-R), 0.1 ml (5 U ml1) of rTaq DNA polymerase (Takara, Tokyo, Japan), and 1 ml of DNA template from the PCR product of the first round of amplification. The second amplification followed this profile: 1 cycle at 95 C for 4 min (DNA denaturing), 1 cycle of 95 C for 30 s, 65 C for 30 s, and 72 C for 1 min, followed by 9 cycles lowering the annealing temperature from 65 C to 60 C at 0.5 C steps for each cycle, and finally 20 cycles of 95 C for 30 s, 60 C for 30 s, and 72 C for 1 min, and a final extension at 72 C for 7 min. For the negative result samples, other parallel amplifications were conducted using nested-touchdown PCR on additional 2 ml (100 ng ml-1) samples of Ophiocordyceps sinensis DNA template to ensure that the PCR was not inhibited. One of the PCR products of the five replicates from each plant species was purified at random with an AxyPrep PCR Cleanup Kit (Axygen, CA, USA). The purified products were sequenced directly by Shanghai Invitrogen Biotechnology Corporation. The resulting sequences were assembled, corrected, and exported using ContigExpress software. The nucleotide sequences were determined and deposited in the GenBank, EMBL, and DDBJ nucleotide sequence databases. The accession number, length, and AT content of each fragment are listed in Table 1.
Total DNA isolation Total DNA from the plant roots were isolated following the directions of the AxyPrep Multisource Genomic DNA Kit (Axygen, CA, USA). After isolation, the concentrations and quality of the DNA samples were determined by spectrophotometry
Results and discussion Forty-two plant species were identified, as described by Hu (2010) (Fig S1), covering 23 families and 34 genera (Table 1).
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Table 1 e Plant samples from Ophiocordyceps sinensis habitats and PCR results using O. sinensis-specific primers. No.
Family
Genus
Species
Results
Access no.
Length (bp)/AT (%)
JD1 JD2 JD3 JD4 JD5 JD8 JD9 JD10 JD11 JD12 JD14 JD16 JD18 JD19 JD20 JD21 JD22 JD23 JD24 JD25 JD26 JD27 JD28 JD29 JD30 JD32 JD33 JD34 JD35 JD36 JD37 JD38 JD39 JD40 JD42 JD43 JD44 JD45 JD46 JD47 JD48 JD49
Onagraceae Poaceae Rosaceae Campanulaceae Ranunculaceae Scrophulariaceae Gentianaceae Scrophulariaceae Cyperaceae Compositae Compositae Ranunculaceae Cruciferae Primulaceae Compositae Rosaceae Polygonaceae Compositae Apiaceae Compositae Apiaceae Compositae Juncaceae Lamiaceae Unknown Juncaceae Polygonaceae Lamiaceae Ranunculaceae Polygonaceae Dryopteridaceae Ranunculaceae Caryophyllaceae Cruciferae Polygonaceae Plantaginaceae Caprifoliaceae Rosaceae Rosaceae Ranunculaceae Ericaceae Salicaceae
Epilobium Deschampsia Fragaria Cyananthus Thalictrum Pedicularis Gentiana Veronica Carex Cremanthodium Dubyaea Ranunculus Draba Primula Ligularia Potentilla Polygonum Saussurea Bupleurum Aster Pachypleurum Saussurea Luzula Phlomis Unknown Juncus Rumex Mentha Ranunculus Polygonum Polystichum Anemone Cerastium Capsella Polygonum Plantago Lonicera Spiraea Potentilla Coptis Rhododendron Salix
Epilobium sikkimense Deschampsia caespitosa Fragaria nubicola Cyananthus macrocalyx Thalictrum uncatum Pedicularis sherriffii Gentiana nyingchiensis Veronica chayuensis Carex lehmanii Cremanthodium linguiatum Dubyaea hispida Ranunculus nephelogens Draba nemorosa Primula alpicola Ligularia nyingchiensis Potentilla cuneata Polygonum macrophyllum Saussurea ovatifolia Bupleurum triadiatum Aster tongolensis Pachypleurum xizangense Saussurea lavrenkoana Luzula multiflora Phlomis tibetica Unknown Juncus leucanthus Rumex nepalensis Mentha canadensis Ranunculus tanguticus Polygonum filicaule Polystichum moupinense Anemone rivularis Cerastium fontanum Capsella bursa-pastoris Polygonum macrophyllum Plantago asiatica Lonicera humilis Spiraea salicifolia Potentilla fruticosa Coptis chinensis Rhododendron vellereum Salix gilashanica
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
JQ936567 JQ936568 JQ936569 JQ936570 JQ936571 JQ936572 JQ936573 JQ936574 JQ936575 JQ936576 JQ936577 JQ936578 JQ936579 JQ936580 JQ936581 JQ936582 JQ936583 JQ936584 JQ936585 JQ936586 JQ936587 JQ936588 JQ936589
359/38.2 359/38.2 359/37.9 359/38.4 359/37.9 358/38.3 359/38.2 359/38.2 359/38.2 359/38.2 359/38.2 359/38.2 359/38.2 359/38.4 291/43 359/38.2 359/38.2 359/38.2 359/49.6 359/38.4 359/38.2 359/38.2 359/37.9
Plant vouchers were deposited in the Food and Health Engineering Research Center of the State Education Ministry, Guangzhou, China (Voucher No. SJLP1021-1063). Most of the alpine plants belonged to Compositae, Ranunculaceae and
Polygonaceae, and each family had six, five, and five species, respectively (Table 1). The nested-touchdown PCR detection results showed that 23 of the 42 species were positive for Ophiocordyceps sinensis in the plant roots, including 13 families
Fig 1 e Results of the nested-touchdown PCR to detect O. sinensis DNA in root samples. PCR was performed using O. sinensisspecific primers, OsT-F and OsT-R, flanked by universal primers ITS5 and ITS4, respectively. M: DNA Marker DL2000; NK: negative control; CK: positive control; 1e49: plant roots samples JD1eJD49.
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and 18 genera (Fig 1, Table 1). The PCR-positive plant samples were herbs, whereas the woody plants samples (JD44eJD49) were PCR negative. Following positive detection, the PCR products were sequenced directly and compared using BLAST in GenBank, EMBL, and DDBJ nucleotide sequence databases. All sequences from plant root samples were highly analogous (96e100 %) to O. sinensis ribosomal DNA. These results suggested that O. sinensis was present in the alpine herb roots. Although O. sinensis colonize the plant roots as shown by nested PCR detection, further histological evidence is necessary to confirm these results. The observation that Ophiocordyceps sinensis associates with plant roots is in line with other entomopathogenic fungi, such as those of the genera Metarhizium, Beauveria, and Pochonia, which are able to derive nutrition from plant sources in the absence of insect hosts (Posada & Vega 2005; Akello et al. -Vicente et al. 2009; Wyrebek et al. 2007; Vega 2008; Macia 2011; Behie et al. 2012). The growth of O. sinensis in the roots or rhizosphere may be supported by plants, and O. sinensis may benefit plants in a manner similar to Metarhizium anisopliae (Kepler & Bruck 2006). When we consider the life cycle and ecology of O. sinensis and Thitarodes larvae, Thitarodes larvae live underground on the Tibetan Plateau and in the Himalayas soil burrows and mainly feed on tender roots of vegetation. According to dietary preference studies on Thitarodes larvae, root fragments of the genera Cyananthus, Veronica, Ranunculus, Juncus, Deschampsia, Ligularia, Pachypleurum, Polygonum, and Cerastium were detected in the foregut of Thitarodes larvae using rDNA nested PCR assays with primers that targeted ITS regions (Chen et al. 2010; Hu 2010). These plant root tissues, which are preferred by Thitarodes larvae, were also found to be positive for O. sinensis according to PCR detection. As a result, when O. sinensis colonizes the alpine plant roots or rhizosphere, it could increase transmission to Thitarodes larvae. As far as we know, O. sinensis cannot complete the teleomorph life cycle without Thitarodes larvae because it requires unknown factors or the nutrition from the larval tissue (Holliday & Cleaver 2008). This study fills in gaps in understanding about how O. sinensis hyphae are present in plant tissue or the rhizosphere during the anamorph life cycle to cope with harsh conditions in alpine habitats and have an increased opportunity to infect Thitarodes larvae. These results may help manage plant communities to support O. sinensis populations by increasing the number of plants preferred by Thitarodes larvae and colonized by O. sinensis, such as Cyananthus, Veronica, Ranunculus, and Juncus herbs, at the fungal habitats. Future research will focus on reconstructing the colonization of O. sinensis mycelium in the aseptic seedling roots and rhizosphere of alpine herbs, such as Polygonum macrophyllum to reveal interactions between the fungus and plants. These studies will determine whether O. sinensis actually grow in or on the plant roots and whether this association is a parasitic or mutualistic relationship. Ophiocordyceps sinensis is believed to distribute on the Tibetan Plateau and in the Himalayas through ascospores produced in the teleomorph pathway (Sung et al. 2007; Cannon et al. 2009). However, the detection results in this study indicate that O. sinensis also spreads to plants via the anamorph conidia or hypha, forming an abundant and stable population by colonizing plant roots. This finding may help develop a new
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strategy to sustain O. sinensis, which is a valuable and rare resource of the Tibetan Plateau and the Himalayas.
Acknowledgements This work was supported by grants from the National Natural Science Foundation (31300427), National Key Technology R&D Program in the 12th Five-year Plan of China (2011BAI13B06), and China Postdoctoral Science Foundation (2012M511861).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2013.12.005
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