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Pochonia Cordycepisociata, a New Species Associated with Chinese Cordyceps in Tibet, China

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Pochonia Cordycepisociata, a New Species Associated with Chinese Cordyceps in Tibet, China Phytotaxa 208 (4): 278–286 ISSN 1179-3155 (print edition) www.mapress.com/phytotaxa/ PHYTOTAXA Copyright © 2015 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.208.4.3 Pochonia cordycepisociata, a new species associated with Chinese cordyceps in Tibet, China HAI HUANG1, *, ZHANG LUO, HAI MEI YUE, WEN FENG GONG, LEI CAI2 & MU WANG1* 1Agriculture and Animal Husbandry College of Tibet University, Lyingchi, Tibet 860000, China 2State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, Beichen West Road, Chaoy- ang District, Beijing 100101, China *Corresponding author’s email: [email protected] Abstract During a survey of fungi associated with the Chinese cordyceps (Ophiocordyceps sinensis) collected from Tibet, China, a new species, Pochonia cordycepisociata was isolated from the sclerotium of O. sinensis. It is characterized and differ- ent from other Pochonia species by its aggregated compact hyphae, slender phialides, global to ellipsoid conidia and the absence of dictyochlamydospores. A multi-locus (ITS, SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analysis supported its independence from other species. A synopsis of the morphological characters of the new species and its related taxa is provided. Key words: Metacordyceps, morphology, Ophiocordyceps sinensis, phylogeny, systematics Introduction The genus Pochonia Bat. & O.M. Fonseca was resurrected based on the phylogenetic investigation of Verticillium sec. Prostrata which was re-classified into Lecanicillium W. Gams & Zare, Pochonia, Haptocillium W. Gams & Zare and Simplicillium Zare & W. Gams (Zare et al. 2001). Pochonia was circumstantiated based on the type species of P. chlamydosporia (Goddard) Zare & W. Gams, and currently 13 taxa are recognized (Zare et al. 2001; Sung et al. 2007; Zare and Gams 2007; Nonaka et al. 2013). All species of Pochonia are characterized by dictyochlamydospores or swollen hyphae cells, and phialidic conidia (Zare and Gams 2001, 2004). Moreover, phylogenetic studies by Kepler et al. (2012) supported that the genus Pochonia belongs to Claviciptaceae, but did not constitute a monophyletic clade. It is well-known that Pochonia species usually isolated from soil are able to parasitize nematode cysts (Barron et al. 1996), such as P. chlamydosporia, which is a potential bio-control agent (Zare & Gams 2003, Esteves et al. 2009). Zare & Gams (2004) recognized six species in this genus, which were acquired from cysts and eggs of Heterodera spp., leaf litter and soil. In a survey of fungi associated with Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora collected from Miling, Tibet, China, several isolates had moderately slow growth, slender flask-shaper phialides clustering in regular terminal or intercalary whorls, fitting the generic concept of Pochonia. However, the isolates could be easily distinguished from other species of Pochonia by the shape of the mature conidia and aggregated compact hyphae submerged in the medium. A multi-locus phylogenetic analysis also showed that these isolates represent an independent species of Pochonia. Herewith we described and illustrated the new species. Materials and Methods Strains, media and morphological observation 278 Accepted by Jian-Kui Liu: 2 Apr. 2015; published: 25 May 2015 A NEW SPECIES TABLE 1. Taxa used in phylogenetic analysis of Pochonia and related species Species Strain Locality Substrate of isolation ITS SSU LSU TEF RPB1 RPB2 associated Claviceps fusiformis ATCC 26019 Africa Pennisetum typhoideum JN049817 DQ522539 U17402 AF543778 AY489648 DQ522417 C. purpurea GAM 12885 - - U57669 AF543765 AF543789 DQ522320 DQ522366 Underside of Hypocrella discoidea BCC 8237 dicotyledonous Nam Nao National Park JN049840 DQ384937 EF469056 EF469085 EF469104 leaf WITH H. raciborskii BCC 7869 Thailand Homoptera, scale insect JN049842 EF469121 EF469074 EF469056 EF469085 EF469104 Metacordyceps atrovirens TNM-F 10184 Yamagata, Japan Larva of Coleoptera JN049882 JF415950 JF415966 JN049884 C HINESE M. chlamydosporia CBS 101244 Brazil Egg of slug JN049821 DQ522544 DQ518758 DQ522327 DQ522372 DQ522424 M. indigotica TNS F18553 Yamagata, Japan Lepidoptera JN049874 JF415952 JF415968 JF416010 JN049886 JF415992 M. indigotica TNS F18554 Yamagata, Japan Lepidoptera JN049875 JF415953 JF415969 JF416011 JN049887 JF415993 CORDYCEPS M. khaoyaiensis BCC 12687 Thailand Lepidoptera JN049868 JF415971 JF416013 JN049889 M. khaoyaiensis BCC 14290 Thailand Lepidoptera JN049869 JF415970 JF416012 JN049888 M. kusanagiensis TNS F18494 Yamagata, Japan Coleoptera pupa JN049873 JF415954 JF415972 JF416014 JN049890 M. liangshanensis EFCC 1523 Sichuan, China Lepidoptera EF468961 EF468814 EF468755 EF468918 M. liangshanensis EFCC 1452 Sichuan, China Lepidoptera EF468962 EF468815 EF468756 Phytotaxa M. martialis TTZ070716-04 - Lepidoptera JN049871 JF415956 JF415974 JN049891 M. martialis EFCC 6863 - Lepidoptera JF415975 JF416016 JF415995 M. martialis HMAS 197472 (S) - Lepidoptera JN049881 JF415955 JF415973 JF416015 JN049892 JF415994 208(4)©2015 MagnoliaPress M. owariensis NBRC 33258 Japan Nymph of cicada JN049883 JF415976 JF416017 JF415996 M. pseudoatrovirens TNS F16380 Aomori, Japan Coleoptera larva JN049870 JF415977 JN049893 JF415997 M. sp. HMAS 199601 - Hemiptera JN049879 JF415957 JF415978 JF416018 JN049894 JF415998 M. taii ARSEF 5714 - JN049829 AF543763 AF543787 AF543775 DQ522383 DQ522434 M. yongmunensis EFCC 2131 South Korea Lepidopteran pupa JN049856 EF468833 EF468770 EF468876 M. yongmunensis EFCC 2135 South Korea Lepidopteran pupa EF468979 EF468834 EF468769 EF468877 Pochonia boninensis JCM 18597 Tokyo, Japan Soil AB709858 AB758255 AB709831 AB758463 AB758666 AB758693 ...Continue on the next page • 279 280 • Phytotaxa TABLE 1. (Continued) Species Strain Locality Substrate of isolation ITS SSU LSU TEF RPB1 RPB2 208(4)©2015 MagnoliaPress P. bulbillosa CBS 145.70 Denmark Root of Picea abies AJ292410 AF339591 AF339542 EF468796 EF468902 EF468943 P. bulbillosa JCM 18596 Nagano, Japan Soil under Q. serrata AB709836 AB758252 AB709809 AB758460 AB758663 AB758690 P. chlamydosporia var. CBS 504.66 Canada Soil AJ292398 AF339593 AF339544 EF469069 EF469098 EF469120 catenulata P. c. var. catenulata JCM 18598 Okinawa, Japan Soil AB709837 AB758248 AB709810 AB758456 AB758659 AB758686 P. c. var. catenulata JCM 18600 Japan Soil AB709839 AB758266 AB709812 AB758474 AB758677 AB758704 P. c.var. chlamydosporia JCM 18605 Tokyo, Japan Soil under Phoenix sp. AB709844 AB758261 AB709817 AB758469 AB758672 AB758699 P. c.var. chlamydosporia JCM 18607 Okinawa, Japan Soil AB709846 AB758270 AB709819 AB758478 AB758681 AB758708 P. c.var. ellipsopsora JCM 18609 Tokyo, Japan Soil under Morus sp. AB709848 AB758257 AB709821 AB758465 AB758668 AB758695 P. c.var. ellipsopsora JCM 18611 Japan Soil under Alocasia odora AB709850 AB758265 AB709823 AB758473 AB758676 AB758703 P. c.var. spinulospora JCM 18613 Tokyo, Japan Soil under Morus sp. AB709854 AB758258 AB709827 AB758466 AB758669 AB758696 Soil under Cyathea P. c.var. spinulospora JCM 18619 Okinawa, Japan AB709857 AB758272 AB709830 AB758480 AB758683 AB758710 lepifera Sclerotium of Chinese P. cordycepisociata CGMCC 3.17365 Tibet, China KM263569 KM263572 KM263573 KM263584 KM263576 KM263579 cordyceps Sclerotium of Chinese P. cordycepisociata CGMCC 3.17366 Tibet, China KM263567 KM263570 KM263574 KM263582 KM263578 KM263580 cordyceps Sclerotium of Chinese P. cordycepisociata CGMCC 3.17367 Tibet, China KM263568 KM263571 KM263575 KM263583 KM263577 KM263581 cordyceps P. globispora CBS 203.86 Netherlands Root of P. menziesii DQ516079 P. gonioides CBS 891.72 Germany Pulcherricium caeruleum AJ292409 AF339599 AF339550 DQ522354 DQ522401 DQ522458 P. microbactrospora CBS 101433 Ibaraki, Japan Rotifers in pine litter AJ292408 P. parasitica ATCC 52203 Canada Manure pile Z54112 Eggs of Heterodera P. rubescens CBS 464.88 U. K. AJ292400 AF339615 AF339566 EF468797 EF468903 EF468944 avenae P. rubescens JCM 18620 Kagoshima, Japan Soil AB709859 AB758247 AB709832 AB758455 AB758658 AB758685 HUANG P. suchlasporia var. catenata CBS 248.83 Sweden eggs of Heterodera avenae AJ292406 KJ398789 KJ398648 P. s. var. suchlasporia CBS 251.83 Sweden eggs of Heterodera avenae AJ292402 KJ398790 KJ398600 ET AL. ET The Chinese cordyceps (Ophiocordyceps sinensis) was collected from Miling County (E: 93°11'; N: 29°31''; h: 3080 m), Tibet, China, in April 2014. The fungus was isolated from a sclerotium tissue within dead insect part of the O. sinensis specimen. Isolation was made on a Potato Dextrose Agar (PDA) supplemented with 5 % wheat bran and 0.5 % peptone at 18 ºC. After the fungus grew out from the sclerotium tissue, individual conidium was picked up from phialide and inoculated onto agar plate for single spore isolation. The isolates were incubated on the agar plates containing different media such as potato dextrose agar (PDA), Malt extract agar (MEA), Cornmeal agar (CMA), and Oatmeal agar (OA) at 20 ºC. Colony morphology and microscopic characteristics were examined, measured and photographed after incubation for 15 days. All measurements were conducted from more than 50 individuals in water mounts. The ex-type living cultures were deposited in China General Microbiological Culture Collection Center (CGMCC) and the dried culture and microscope slide were deposited in Herbarium Mycologicum, Academica Sinica, Beijing, China (HMAS). DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted from the fungal mycelia on PDA plates following the protocol
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