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Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan
Mycoscience xxx (xxxx) xxx
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Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan Kohei Yamamoto a, *, Muneyuki Ohmae b, Takamichi Orihara c a
Tochigi Prefectural Museum, 2-2 Mutsumi-cho, Utsunomiya, Tochigi, 320-0865, Japan Hokken Co. Ltd., 7-3 Ekihigashimachi, Mibu, Shimotsuga-gun, Tochigi, 321-0222, Japan c Kanagawa Prefectural Museum of Natural History, 499 Iryuda, Odawara, Kanagawa, 250-0031, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 June 2019 Received in revised form 3 September 2019 Accepted 6 September 2019 Available online xxx
Ascomata of a Metarhizium species were collected from Tochigi Prefecture, Japan. The ascomata were similar to M. kalasinense in its host preference for elaterids, olive-green clavate stromata, and obliquely immersed perithecia, but the asci and ascospores were significantly shorter in length than those of the latter species. Furthermore, phylogenetic analyses based on the internal transcribed spacer region of nuclear ribosomal DNA and the elongation factor 1-alpha gene showed that the Japanese species was phylogenetically distinctive in the M. anisopliae lineage, including M. kalasinense. Accordingly, we describe a new species, M. brachyspermum. Isolates of this species produced olive-green conidial masses on the surface of the colony. Also, hyphal anastomoses between two metulae were often observed on conidiophores. © 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Anamorph Entomopathogenic fungi Hypocreales Phylogeny Taxonomy Teleomorph
1. Introduction Species of Metarhizium Sorokin (Clavicipitaceae, Hypocreales) are entomopathogenic fungi characterized by phialidic conidiogenesis and generally greenish conidia (Bischoff, Rehner, & Humber, 2009). In Japan, at least 19 species of Metarhizium have been reported (Iwasaki et al., 2019; Japanese Society of Cordyceps Research, 2014; Nishi, Shimizu, & Sato, 2017; Nishi & Sato, 2017). The genus is mostly composed of entomopathogens causing green muscardine (Bischoff et al., 2009), but they also cause opportunistic infections of human and chameleon (Kepler, Humber, Bischoff, & Rehner, 2014; Nourrisson et al., 2017; Sigler, Gibas, Kokotovic, & Bertelsen, 2010). In particular, M. anisopliae (Metschn.) Sorokin lineage (Bischoff et al., 2009) includes several species that can infect major crop pests, i.e., various insects (e.g., Coleoptera, Hemiptera, and Lepidoptera) and Acari, and pests of humans (e.g., malaria-vectoring mosquitoes) (de Faria & Wraight, 2007; Lacey et al., 2015; Scholte et al., 2005). Species of the M. anisopliae lineage are used for crop pest management (de Faria & Wraight, 2007; Lacey et al., 2015). Furthermore, Zhang et al. (2019) suggested that
* Corresponding author. E-mail address: [email protected] (K. Yamamoto).
the entomopathogen Metarhizium robertsii J.F. Bisch., S.A. Rehner & Humber in this lineage evolved from endophytes. Several species of this clade behave as endophytes of crop plants, promoting uptake of soil minerals and root development (Behie, Zelisko, & Bidochka, 2012; Sasan & Bidochka, 2012). Therefore, there is a possibility of using the M. anisopliae lineage as both pest control and crop production agents. Surveys targeting members of this clade as bioresources may be accelerated by further taxonomic study. The M. anisopliae lineage includes at least 13 valid species (Bischoff et al., 2009; Chen, Xu, Yang, Zhang, & Yang, 2018; Kepler et al., 2014; Lopes et al., 2018; Luangsa-ard et al., 2017). Among them, seven species have been described from Asia, especially East and Southeast Asia. It should be noted that a teleomorphic stage is also known in three species in this clade, that is, M. indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, M. guizhouense Q.T. Chen & H.L. Guo, and M. kalasinense Tasan., Khons., Thanakitp., Mongkols. & Luangsa-ard (Bischoff et al., 2009; Kepler et al., 2014; Luangsa-ard et al., 2017). Interestingly, Bidochka and Small (2005) suggested that Asia is the origin of the evolution and diversity of the M. anisopliae lineage: this theory stands on the basis that the largest genetic diversity of this lineage was found in this area and all teleomorphs of M. anisopliae lineage have been reported only from East and Southeast Asia.
https://doi.org/10.1016/j.myc.2019.09.001 1340-3540/© 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Species diversity of the M. anisopliae lineage is remarkably high in Japan; at least eight species, including a teleomorphic species (M. indigoticum), have been identified (Kepler et al., 2014; Nishi & Sato, 2017). During our field surveys of hypocrealean entomopathogens in Japan, we collected a novel teleomorph of Metarhizium growing on cadaver larvae and a pupa of an elaterid. Based on the results of morphological observation of the collection and molecular phylogenetic analyses, we describe M. brachyspermum Koh. Yamam., Ohmae & Orihara, sp. nov. 2. Materials and methods 2.1. Morphological observation of specimens We used one specimen collected from Kyoto Prefecture in Jun 2000 and three from Tochigi Prefecture in Jul 2017. The macroscopic morphology and color of the ascomata were recorded based on fresh materials. For microscopic observation, hand-cut sections of the fresh and dried specimens were mounted in lactoglycerol (lactic acid: glycerol: water, 1:1:1, v/v/v) and observed by brightfield microscopy (BX50, Olympus, Tokyo, Japan). Asci and ascospores were stained with cotton blue (0.5% in lactophenol). The dimensions of mature ascospores were measured from those discharged from fresh ascomata. All measurements were performed with PhotoRuler 1.1.3 (http://inocybe.info/). After the observations, the specimens were air-dried (for specimens from Kyoto Prefecture) or freeze-dried and oven-dried at 60 C overnight and deposited in the National Museum of Nature and Science, Tokyo (TNS) and the Tochigi Prefectural Museum (TPM) in Japan. 2.2. Isolation and observation of mycelial growth and anamorphs Two mature ascomata, KY170702-1 and KY170709-1, were used for isolation. Ascospores discharged from perithecia onto potato dextrose agar (PDA; Difco, Detroit, USA) were used to obtain cultures (Uchiyama, 1999). These plates were incubated at 20 C in the dark until germination, and mycelial growth was observed. The established isolates, CM1 from KY170702-1 and CM2 from KY170709-1, were deposited in the Medical Mycology Research Center, Chiba University, Japan under accession numbers IFM 65744 (for CM1) and IFM 65745 (CM2). The established strains were then transferred to PDA and quarter-strength Sabouraud dextrose agar with yeast extract (SDAY/4: 2.5 g/L Bactopeptone (Difco), 10 g/L dextrose (Wako Pure Chemical Industries, Osaka, Japan), 2.5 g/L yeast extract (Difco), 20 g/L agar (Difco)) (Bischoff et al., 2009) for observation and measurement of their colonies and anamorphs. A mycelial plug (6 mm) punched from the margin of each of the precultured colonies on PDA and SDAY/4 was inoculated onto the center of fresh PDA and SDAY/4 (both with three replicates), respectively. These plates were incubated at 25 C in the dark. Hyphal growth (four replicates per plate) was measured as the distance from the inoculated plug to the margin of the colony at 2 wk. Conidiophores dissected from the colonies were mounted in lactoglycerol with or without staining with cotton blue and observed under a brightfield microscope 3 wk after subculturing. 2.3. Sequencing and phylogenetic analyses of the established strains
(3TEF) were performed in accordance with Orihara, Smith, Shimomura, Iwase, and Maekawa (2012). Additionally, a partial sequence of the 50 region of elongation factor 1-alpha (5TEF), a suitable species-level phylogenetic marker for Metarhizium (Bischoff et al., 2009; Luangsa-ard et al., 2017), was amplified under the same cycle conditions as 3TEF. The PCR primer pairs were ITS1F (Gardes & Bruns, 1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990) for ITS, LR0R and LR5 (Vilgalys & Hester, 1990) for 28S, 983F and 2218R (Rehner & Buckley, 2005) for 3TEF, and EF1T (Rehner & Buckley, 2005) and EF2T (Bischoff, Rehner, & Humber, 2006) for 5TEF. Cycle sequencing of the amplicons and Sanger sequencing were performed in accordance with Orihara et al. (2012). The resulting bidirectional sequences were edited using 4 Peaks 1.8 (http://nucleobytes.com/4peaks) and assembled with MEGA 7 (Kumar, Stecher, & Tamura, 2016). Newly generated sequences were deposited in the DNA Data Bank of Japan (DDBJ; http://www. ddbj.nig.ac.jp) under accession numbers LC469747eLC469753. DNA sequence similarity was examined by National Center for Biotechnology Information (NCBI) nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE¼BlastSearch). For phylogenetic analyses of the combined dataset of ITS and 5TEF, which is suitable for species-level phylogenetic analyses of Metarhizium (Luangsa-ard et al., 2017), 32 sequences of 10 species in the PARB-MGT clade (Bischoff et al., 2009) within the M. anisopliae lineage were retrieved from NCBI and included in the dataset (Supplementary Table S1) because the Japanese specimens were suggested to belong to that clade based on the result of the BLAST search before performing phylogenetic analyses. Metarhizium lepidiotae (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber and M. alvesii R.B. Lopes, M. Faria, C. Montalva & R.A. Humber, which were placed outside of the PARB-MGT clade (Bischoff et al., 2009; Lopes et al., 2018), were selected as the outgroup. Sequences of each region were aligned individually by MUSCLE (Edgar, 2004) operated on MEGA 7 for the multialignment, with final manual adjustment of the dataset. Alignment gaps were treated as missing data. Ambiguously aligned sites in the ITS dataset were detected and removed using the Gblocks Server (http://molevol.cmima.csic.es/castresana/Gblocks_server. html) under the least stringent setting. Topological conflicts between the two trees (ITS: 523 bp; 5TEF: 713 bp) were checked directly by topological comparison of those preliminary maximum likelihood (ML) trees. Next, the two datasets were combined into a single dataset of 1236 bp, and deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S24184). A phylogenetic analysis of the combined dataset was conducted using the ML and maximum parsimony (MP) methods based on Yamamoto, Degawa, Takashima, Fukuda, and Yamada (2017). Briefly, ML and MP analyses were conducted with raxmlGUI 1.31 (Silvestro & Michalak, 2012) and MEGA 7, respectively, under the general time reversible model of nucleotide substitution, with a discrete gamma distribution (þG), selected by MEGA 7, for ML and the tree bisection-reconnection search for MP. Branch support was evaluated by 1000 replicates of bootstrap analysis. Different ashape parameters, GTR rates, and empirical base frequencies were assigned to each partition (i.e., ITS and exons and introns of 5TEF) for ML. 3. Results and discussion 3.1. Taxonomy
Total DNA was extracted from cultured mycelia of CM1 and CM2 on PDA based on Izumitsu et al. (2012). Polymerase chain reaction (PCR) amplifications of partial sequences of the internal transcribed spacer region (ITS) and 28S (containing the D1eD2 region) nuclear ribosomal DNA and of the 30 region of elongation factor 1-alpha
Metarhizium brachyspermum Koh. Yamam., Ohmae & Orihara, sp. nov. Fig. 1. MycoBank no.: MB 830553. Diagnosis: This species is most similar to M. kalasinense in
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Fig. 1. Morphological characteristics of Metarhizium brachyspermum (A, B, DeG: KY170702-1; C: KY170709-1; HeJ: CM1 on SDAY/4; KeO: CM1 on PDA). A: Stroma arising from a pupa. Exuvia of a last-instar larva attached to a host. B: Above-ground portion of the stroma composed of a sterile stipe (left) and fertile part (right). C: Stroma arising from a larva. D: Longitudinal section of the fertile tissue. Obliquely buried perithecia are shown. E, F: Whole asci (E) and apical caps (F), stained with cotton blue. G: Ascospore with 15 septa (arrowheads). H, K: Upper (left) and reverse (right) sides of colonies after 3 wk of incubation. I, L: Conidiophores, stained with (L) and without (I) cotton blue. J, O: Conidia. Arrowhead indicates adhesion of two conidial chains. M, N: Anastomoses between two metulae (arrowheads), stained with cotton blue. Bars: A, C, H 10 mm; B 5 mm; D 500 mm; E, G 50 mm; F, M 5 mm; I, J 10 mm. Images in H and K, I and L, J and O, and M and N are at the same magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
morphology of stroma but the ascospores are significantly shorter in length than the latter. Holotype: JAPAN, Tochigi Prefecture, Utsunomiya-shi, Nagaokacho, ca. 170 m above sea level, on a pupa of an elaterid in the underground pupal-chamber, under Cryptomeria japonica (Thunb. ex L.f.) D. Don, Carpinus tschonoskii Maxim., Quercus myrsinifolia Blume, and Quercus serrata Murray, 2 Jul 2017, K. Yamamoto, KY170702-1 (TNS-F-70755; ex-type strain, CM1 (IFM 65744)). Gene sequences from ex-type strain: LC469747 (ITS), LC469749 (28S), LC469751 (3TEF), LC469752 (5TEF). Etymology: From the Greek brachy ¼ short and spermum ¼ spore, referring to the characteristic that the ascospore is shorter in length than that of M. kalasinense, a relative of this species. Stromata on larva or pupa of elaterid, single or multiple; upper, epigeous portion clavate, sometimes branched, pale green when young, turning olive green, up to 3 cm long; fertile head 17e20 mm long, 2.5e4.2 mm wide, slightly wider than sterile stipe; ostiole dark olive-green, up to 11 per 1 mm2; underground part cylindrical, irregular in width, lateral short branch present, yellow with partial green pigmentation, up to 29 mm long, 1.1e2.3 mm wide; host surface covered with pale yellow subiculum. Perithecia
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flask-shaped, obliquely immersed with slightly protuberant ostiole, 500e795 190e300 mm, mean 650 250 mm (n ¼ 26); walls composed of about 5 layers of plectenchymatous tissue, 9e14 mm thick, hyphae 1e4.5 mm wide. Asci hyaline, cylindrical, 8-spored, 230e400 2.5e4.5 mm, mean 320 3.4 mm (n ¼ 15); apical cap prominent, 1.5e2.5 mm high, 3e3.5 mm wide, mean 2 3.2 mm (n ¼ 12). Ascospores filiform, hyaline, 190e270 1 mm, mean 220 1 mm (n ¼ 20); septa up to 15, sometimes obscure, not fragmenting into part-spores; length between adjacent septa 11e42 mm, mean 19 mm (n ¼ 88). Colonies on SDAY/4: Colonies growing 10e16 mm within 14 d at 25 C; surface floccose with abundant aerial hyphae, at first white, becoming greenly pigmented due to conidiation within 10 d; reverse pale yellow. Conidiophores dense, erect, terminating in branches, with 2e3 phialides per metula, forming a palisadelike layer; hyphae bridge formation by anastomosis between adjacent metulae relatively abundant; without formation of synnemata. Phialides hyaline, cylindrical, tapering apices, 7.5e10.5 1.5e2.5 mm, mean 9.3 2.1 mm (n ¼ 10), forming conidial chains. Conidia unicellular, cylindrical, smooth, olive green, 6e8 2.5e3 mm, mean 6.9 2.8 mm (n ¼ 50), Q ¼ 2.1e3.0, mean 2.5 (n ¼ 50). Colonies on PDA: Colonies growing 10e18 mm within 14 d at 25 C; surface relatively floccose with aerial hyphae, at first white, becoming green-pigmented due to conidiation within 6 d; reverse pale yellow. Conidiophores dense, erect, terminating in branches, with mostly 2 phialides per metula, forming a palisade-like layer; without formation of synnemata. Phialides hyaline, cylindrical, tapering apices, 7e13 2e2.5 mm, mean 9.5 2.4 mm (n ¼ 15), forming conidial chains. Conidia unicellular, cylindrical, smooth, olive green, 5.5e7.5 2e3 mm, mean 6.8 2.6 mm (n ¼ 50), Q ¼ 2.2e3.2, mean 2.6 (n ¼ 50). Habitat, distribution and fruiting season: On larva or pupa of Elateridae buried in broadleaved tree-dominated forests in the temperate zone of Honshu, Japan; summer (Jun to Sep). Other specimens examined: Kyoto Prefecture, Uji-shi, Uji, Kousai, ca. 40 m above sea level, on a larva of Elateridae, buried under Castanopsis cuspidata (Thunb.) Schottky, 18 Jun 2000, T. Yamamoto, no. 81 (TNS-F-70754). Tochigi Prefecture, Utsunomiya-shi, Nagaoka-cho, ca. 170 m above sea level, on a larva of Elateridae, buried under C. japonica, C. tschonoskii, Q. myrsinifolia, and Q. serrata, 9 Jul 2017, K. Yamamoto, KY170709-1 (TNS-F-70756; living culture, CM2 (IFM 65745)). Ibid., on a larva of Elateridae buried in decayed wood of a broadleaf tree, 2 Jul 2017, T. Shoda (TPM-M-9238). Nom. Jap.: Kusairo-kometsukimusi-take (from the Japanese kusairo ¼ glass-green, kometsukimusi ¼ elaterids, and take ¼ fungi)
3.2. Morphological comparison of teleomorphs of M. brachyspermum and its relatives parasitic on Elateridae Hosts of the four specimens examined in this study were larvae (Fig. 1C) and pupae (Fig. 1A) of the Elateridae. Metarhizium brachyspermum is morphologically similar to M. kalasinense described from Thailand: both species grow on larvae of the Elateridae, and their stroma is composed of a clavate, greenish fertile head, and rhizoidal stipe appearing yellowish below ground (Fig. 1A, C; Luangsa-ard et al., 2017); however, asci and ascospores of the latter are significantly longer in length than the former (Table 1). Metarhizium campsosterni (W.M. Zhang & T.H. Li) Kepler, S.A. Rehner & Humber from China similarly grows on larvae of the Elateridae and forms a green-yellow stroma but its perithecia are vertically buried
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Table 1 Morphological characteristics of teleomorphic stages of Metarhizium spp. in the MGT clade. Species
Hosts
Fertile part of stromata
Perithecia
Asci
Ascospores
M. brachyspermum
Larvae and pupae of Elateridaef Larvae and adult of Elateridaec Larvae of Hepialidae or Cossidaead Larvae of Elateridaee
clavate; olive greenf
Obliquely immersed; 500 e795 190e300 mmf Vertically immersed; 275 e433 165e276 mmc Obliquely immersed; 700 e750 275e325 mma Obliquely immersed; 700 e800 250e350 mme Vertically immersed; 767 e940(e1100) mmb
230e400 2.5 e4.5 mmf 175 e349 3.9 mmc 300e350 3 e4 mmd 500e650 4 e5 mme 305e480 3.3 e4.5 mmb
190e270 1 mm; nonfragmentingf ND; fragmenting into part sporesc 250e360 1 mm; fragmenting into part sporesd 400e500 1e1.5 mm; nonfragmentinge ND; fragmenting into part sporesb
M. campsosterni M. indigoticum M. kalasinense M. guizhouense (¼ Metacordyceps taii) a b c d e f
Larvae of Noctuidaeb
cylindrical, tapering; greenish yellowc fusiform, slightly compressed, corniculate tip; blueish greenad cylindrical or clavate; olive green to greenish browne cylindrical, tapering; yellowishb
Kobayasi and Shimizu (1978). Liang, Liu, and Liu (1991). Zhang et al. (2004). Japanese Society of Cordyceps Research (2014). Luangsa-ard et al. (2017). This study.
in stroma and asci are shorter in length than that of M. brachyspermum (Zhang, Li, Chen, & Qu, 2004, Table 1). 3.3. Phylogenetic relationship between M. brachyspermum and its relatives in the MGT clade Four and three sequences were successfully obtained from CM1 (ITS: LC469747; 28S: LC469749; 3TEF: LC469751; 5TEF: LC469752) and CM2 (ITS: LC469748; 28S: LC469750; 3TEF: LC469753), respectively. Phylogenetic trees inferred from the individual ITS and 5TEF datasets showed that no significant topological conflicts were present. Therefore, a phylogenetic analysis of the combined dataset was conducted. The scores of the resultant trees (Fig. 2) were as follows: parsimony informative sites ¼ 52; tree length ¼ 146 steps; consistency index ¼ 0.848; retention index ¼ 0.917; rescaled consistency index ¼ 0.778. This tree showed that M. brachyspermum belonged to the MGT clade within the M. anisopliae lineage. Former studies showed that M. indigoticum, M. guizhouense, M. kalasinense, and M. majus (J.R. Johnst.) J.F. Bisch., S.A. Rehner & Humber placed within this clade (Kepler et al., 2014;
Fig. 2. Maximum likelihood (ML) phylogenetic tree of the combined dataset of the ITS and 5TEF sequences of Metarhizium brachyspermum and its allies (ln L ¼ 2381.899044). Metarhizium alvesii and M. lepidiotae were used as outgroups. Bootstrap (BS) values (1000 replicates) > 50% from ML (left) and maximum parsimony (MP) (right) are shown near nodes. Branches supported by a BS of ML/MP of 70% are depicted as thick black lines. Black circles indicate teleomorphic species. T: ex-type strain; ET: ex-epitype strain.
Luangsa-ard et al., 2017; Nishi & Sato, 2017), and this relationship was also recovered in this study. Likewise, our phylogenetic analysis showed that M. baoshanense Z.H. Chen, Ling Xu, X.N. Yang, Y.G. Zhang & Y.M. Yang, a recently described anamorphic species, and M. campsosterni were also members of the MGT clade. Teleomorphs of M. indigoticum and M. guizhouense (¼ Metacordyceps taii (Z.Q. Liang & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora) are clearly distinguishable from M. brachyspermum by their lepidopteran hosts and ascospores that break into part spores (Table 1). 3.4. Growth of M. brachyspermum isolates and characteristics of its anamorph Colonies of CM1 grew 11e14 mm on SDAY/4 and 16e18 mm within 2 wk on PDA. CM2 grew 12e16 mm and 10e15 mm; therefore, CM2 tends to grow more slowly than CM1 on PDA. Colonies on SDAY/4 formed slightly larger conidia than those of PDA (Table 2). In contrast, phialides were larger on PDA (Table 2). Anastomoses between two metulae were often observed on conidiophores of M. brachyspermum (Fig. 1M, N). Similar anastomosis was also observed on conidiophores of M. minus (Rombach, Humber & D.W. Roberts) Kepler, S.A. Rehner & Humber (Glare, Milner, & Beaton, 1996) which belongs to M. flavoviride W. Gams & Rozsypal lineage (Kepler et al., 2014). In addition, conidial chains on dense phialides are laterally adhered to each other (Fig. 1O) as observed in other species of Metarhizium (Bidochka & Small, 2005; Rombach, Humber, & Evans, 1987). Compared with the colony of M. kalasinense, that of M. brachyspermum tends to be paler and the aerial hyphae in the colony are often abundant (Fig. 1H, K); however, it is unclear whether this tendency is useful for classification because of the limited number of comparisons of isolates. In addition, differences in the size of phialides and conidia between M. brachyspermum and M. kalasinense are unclear (Table 2) even though their teleomorphs clearly differ in the size of asci and ascospores (Table 1). In fact, colony color and conidial size among the isolates within phylogenetically supported species in the M. anisopliae lineage sometimes overlap with the variations in other species (Bischoff et al., 2009). Thus, clarification of the teleomorphic stages of further species may be beneficial for defining the species concept of Metarhizium. Other species in the MGT clade (i.e., M. baoshanense, M. guizhouense, M. indigoticum, and M. majus) form slightly or much larger phialides and/or conidia than M. brachyspermum (Table 2). Recently, Kepler, Chen, Kilcrease, Shao, and Rehner (2016) identified stable diploid lineages formed by the union of strains with opposite mating types in M. majus species complex. They
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Table 2 Size of phialides and conidia of Metarhizium spp. in the MGT clade on SDAY/4 and PDA media. Species
Phialides (mm)
Conidia (mm)
SDAY/4 M. M. M. M. M. M. a b c d e f
brachyspermum baoshanense guizhouense indigoticum kalasinense majus
7.5e10.5 1.5e2.5 ND 7e12 2e3c ND 8e12 2e3d 10e18 2.5e3.5c
PDA f
SDAY/4 f
7e13 2e2.5 8.2e19.2 2.1e3.5e ND 8.5e20.5 1.7e3.5e ND 6.7e17.7 2.1e3.3b
PDA f
6e8 2.5e3 ND 7e10 2e3c ND 6e8 2e3d 10.5e13 2.5e4c
5.5e7.5 2e3f 6.7e8.5 2.6e3.3e 6.7e7.3 3e3.4a 8.2e12.3 2.6e4.2e 6e7 2e3d 10.5e12.5 2.5e3.7b
Guo, Ye, Yue, Chen, and Fu (1986). Glare et al. (1996). Bischoff et al. (2009). Luangsa-ard et al. (2017). Chen et al. (2018). This study.
suggested that this mechanism enables to acquire or maintain diversity in the absence of frequent sexual reproduction. Further field sampling and mating-type determinations of anamorphic stages of M. brachyspermum and other teleomorphic species will promote understand the function of this unique reproductive strategy and whether diploidy is widespread among the MGT clade. Disclosure The authors declare no conflicts of interest. Acknowledgments We are grateful to Dr. Janet Jennifer Luangsa-ard for providing information on M. kalasinense. We are also grateful to Mr. Tsutomu Yamamoto and Mr. Tatsuhiko Shoda for sampling specimens. We thank Dr. Kentaro Hosaka for depositing specimens and Dr. Sayaka Ban for depositing cultures. This study was supported in part by KAKENHI Grant-in-Aid for Young Scientists (B) (no. 17K15184) from the Japan Society for the Promotion of Science (JSPS) and a Grantin-Aid from the Institute for Fermentation, Osaka (IFO). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.myc.2019.09.001. References Behie, S. W., Zelisko, P. M., & Bidochka, M. J. (2012). Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science, 336, 1576e1577. https://doi.org/10.1126/science.1222289. Bidochka, M. J., & Small, C. L. (2005). Phylogeography of Metarhizium, and insect pathogenic fungus. In F. E. Vega, & M. Blackwell (Eds.), Insectefungal associations: Ecology and evolution (pp. 28e50). New York: Oxford University Press. Bischoff, J. F., Rehner, S. A., & Humber, R. A. (2006). Metarhizium frigidum sp. nov.: A cryptic species of M. anisopliae and a member of the M. flavoviride complex. Mycologia, 98, 737e745. https://doi.org/10.1080/15572536.2006.11832645. Bischoff, J. F., Rehner, S. A., & Humber, R. A. (2009). A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia, 101, 512e530. https://doi.org/10. 3852/07-202. Chen, Z., Xu, L., Yang, X., Zhang, Y., & Yang, Y. (2018). Metarhizium baoshanense sp. nov., a new entomopathogen fungus from Southwestern China. Pakistan Journal of Zoology, 50, 1739e1746. https://doi.org/10.17582/journal.pjz/2018.50.5.1739. 1746. Edgar, R. C. (2004). Muscle: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792e1797. https://doi.org/10.1093/ nar/gkh340. de Faria, M. R., & Wraight, S. P. (2007). Mycoinsecticides and mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types. Biological Control, 43, 237e256. https://doi.org/10.1016/j. biocontrol.2007.08.001. Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts.
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Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan Kohei Yamamoto a, *, Muneyuki Ohmae b, Takamichi Orihara c a
Tochigi Prefectural Museum, 2-2 Mutsumi-cho, Utsunomiya, Tochigi, 320-0865, Japan Hokken Co. Ltd., 7-3 Ekihigashimachi, Mibu, Shimotsuga-gun, Tochigi, 321-0222, Japan c Kanagawa Prefectural Museum of Natural History, 499 Iryuda, Odawara, Kanagawa, 250-0031, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 June 2019 Received in revised form 3 September 2019 Accepted 6 September 2019 Available online xxx
Ascomata of a Metarhizium species were collected from Tochigi Prefecture, Japan. The ascomata were similar to M. kalasinense in its host preference for elaterids, olive-green clavate stromata, and obliquely immersed perithecia, but the asci and ascospores were significantly shorter in length than those of the latter species. Furthermore, phylogenetic analyses based on the internal transcribed spacer region of nuclear ribosomal DNA and the elongation factor 1-alpha gene showed that the Japanese species was phylogenetically distinctive in the M. anisopliae lineage, including M. kalasinense. Accordingly, we describe a new species, M. brachyspermum. Isolates of this species produced olive-green conidial masses on the surface of the colony. Also, hyphal anastomoses between two metulae were often observed on conidiophores. © 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Anamorph Entomopathogenic fungi Hypocreales Phylogeny Taxonomy Teleomorph
1. Introduction Species of Metarhizium Sorokin (Clavicipitaceae, Hypocreales) are entomopathogenic fungi characterized by phialidic conidiogenesis and generally greenish conidia (Bischoff, Rehner, & Humber, 2009). In Japan, at least 19 species of Metarhizium have been reported (Iwasaki et al., 2019; Japanese Society of Cordyceps Research, 2014; Nishi, Shimizu, & Sato, 2017; Nishi & Sato, 2017). The genus is mostly composed of entomopathogens causing green muscardine (Bischoff et al., 2009), but they also cause opportunistic infections of human and chameleon (Kepler, Humber, Bischoff, & Rehner, 2014; Nourrisson et al., 2017; Sigler, Gibas, Kokotovic, & Bertelsen, 2010). In particular, M. anisopliae (Metschn.) Sorokin lineage (Bischoff et al., 2009) includes several species that can infect major crop pests, i.e., various insects (e.g., Coleoptera, Hemiptera, and Lepidoptera) and Acari, and pests of humans (e.g., malaria-vectoring mosquitoes) (de Faria & Wraight, 2007; Lacey et al., 2015; Scholte et al., 2005). Species of the M. anisopliae lineage are used for crop pest management (de Faria & Wraight, 2007; Lacey et al., 2015). Furthermore, Zhang et al. (2019) suggested that
* Corresponding author. E-mail address: [email protected] (K. Yamamoto).
the entomopathogen Metarhizium robertsii J.F. Bisch., S.A. Rehner & Humber in this lineage evolved from endophytes. Several species of this clade behave as endophytes of crop plants, promoting uptake of soil minerals and root development (Behie, Zelisko, & Bidochka, 2012; Sasan & Bidochka, 2012). Therefore, there is a possibility of using the M. anisopliae lineage as both pest control and crop production agents. Surveys targeting members of this clade as bioresources may be accelerated by further taxonomic study. The M. anisopliae lineage includes at least 13 valid species (Bischoff et al., 2009; Chen, Xu, Yang, Zhang, & Yang, 2018; Kepler et al., 2014; Lopes et al., 2018; Luangsa-ard et al., 2017). Among them, seven species have been described from Asia, especially East and Southeast Asia. It should be noted that a teleomorphic stage is also known in three species in this clade, that is, M. indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, M. guizhouense Q.T. Chen & H.L. Guo, and M. kalasinense Tasan., Khons., Thanakitp., Mongkols. & Luangsa-ard (Bischoff et al., 2009; Kepler et al., 2014; Luangsa-ard et al., 2017). Interestingly, Bidochka and Small (2005) suggested that Asia is the origin of the evolution and diversity of the M. anisopliae lineage: this theory stands on the basis that the largest genetic diversity of this lineage was found in this area and all teleomorphs of M. anisopliae lineage have been reported only from East and Southeast Asia.
https://doi.org/10.1016/j.myc.2019.09.001 1340-3540/© 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Species diversity of the M. anisopliae lineage is remarkably high in Japan; at least eight species, including a teleomorphic species (M. indigoticum), have been identified (Kepler et al., 2014; Nishi & Sato, 2017). During our field surveys of hypocrealean entomopathogens in Japan, we collected a novel teleomorph of Metarhizium growing on cadaver larvae and a pupa of an elaterid. Based on the results of morphological observation of the collection and molecular phylogenetic analyses, we describe M. brachyspermum Koh. Yamam., Ohmae & Orihara, sp. nov. 2. Materials and methods 2.1. Morphological observation of specimens We used one specimen collected from Kyoto Prefecture in Jun 2000 and three from Tochigi Prefecture in Jul 2017. The macroscopic morphology and color of the ascomata were recorded based on fresh materials. For microscopic observation, hand-cut sections of the fresh and dried specimens were mounted in lactoglycerol (lactic acid: glycerol: water, 1:1:1, v/v/v) and observed by brightfield microscopy (BX50, Olympus, Tokyo, Japan). Asci and ascospores were stained with cotton blue (0.5% in lactophenol). The dimensions of mature ascospores were measured from those discharged from fresh ascomata. All measurements were performed with PhotoRuler 1.1.3 (http://inocybe.info/). After the observations, the specimens were air-dried (for specimens from Kyoto Prefecture) or freeze-dried and oven-dried at 60 C overnight and deposited in the National Museum of Nature and Science, Tokyo (TNS) and the Tochigi Prefectural Museum (TPM) in Japan. 2.2. Isolation and observation of mycelial growth and anamorphs Two mature ascomata, KY170702-1 and KY170709-1, were used for isolation. Ascospores discharged from perithecia onto potato dextrose agar (PDA; Difco, Detroit, USA) were used to obtain cultures (Uchiyama, 1999). These plates were incubated at 20 C in the dark until germination, and mycelial growth was observed. The established isolates, CM1 from KY170702-1 and CM2 from KY170709-1, were deposited in the Medical Mycology Research Center, Chiba University, Japan under accession numbers IFM 65744 (for CM1) and IFM 65745 (CM2). The established strains were then transferred to PDA and quarter-strength Sabouraud dextrose agar with yeast extract (SDAY/4: 2.5 g/L Bactopeptone (Difco), 10 g/L dextrose (Wako Pure Chemical Industries, Osaka, Japan), 2.5 g/L yeast extract (Difco), 20 g/L agar (Difco)) (Bischoff et al., 2009) for observation and measurement of their colonies and anamorphs. A mycelial plug (6 mm) punched from the margin of each of the precultured colonies on PDA and SDAY/4 was inoculated onto the center of fresh PDA and SDAY/4 (both with three replicates), respectively. These plates were incubated at 25 C in the dark. Hyphal growth (four replicates per plate) was measured as the distance from the inoculated plug to the margin of the colony at 2 wk. Conidiophores dissected from the colonies were mounted in lactoglycerol with or without staining with cotton blue and observed under a brightfield microscope 3 wk after subculturing. 2.3. Sequencing and phylogenetic analyses of the established strains
(3TEF) were performed in accordance with Orihara, Smith, Shimomura, Iwase, and Maekawa (2012). Additionally, a partial sequence of the 50 region of elongation factor 1-alpha (5TEF), a suitable species-level phylogenetic marker for Metarhizium (Bischoff et al., 2009; Luangsa-ard et al., 2017), was amplified under the same cycle conditions as 3TEF. The PCR primer pairs were ITS1F (Gardes & Bruns, 1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990) for ITS, LR0R and LR5 (Vilgalys & Hester, 1990) for 28S, 983F and 2218R (Rehner & Buckley, 2005) for 3TEF, and EF1T (Rehner & Buckley, 2005) and EF2T (Bischoff, Rehner, & Humber, 2006) for 5TEF. Cycle sequencing of the amplicons and Sanger sequencing were performed in accordance with Orihara et al. (2012). The resulting bidirectional sequences were edited using 4 Peaks 1.8 (http://nucleobytes.com/4peaks) and assembled with MEGA 7 (Kumar, Stecher, & Tamura, 2016). Newly generated sequences were deposited in the DNA Data Bank of Japan (DDBJ; http://www. ddbj.nig.ac.jp) under accession numbers LC469747eLC469753. DNA sequence similarity was examined by National Center for Biotechnology Information (NCBI) nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE¼BlastSearch). For phylogenetic analyses of the combined dataset of ITS and 5TEF, which is suitable for species-level phylogenetic analyses of Metarhizium (Luangsa-ard et al., 2017), 32 sequences of 10 species in the PARB-MGT clade (Bischoff et al., 2009) within the M. anisopliae lineage were retrieved from NCBI and included in the dataset (Supplementary Table S1) because the Japanese specimens were suggested to belong to that clade based on the result of the BLAST search before performing phylogenetic analyses. Metarhizium lepidiotae (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber and M. alvesii R.B. Lopes, M. Faria, C. Montalva & R.A. Humber, which were placed outside of the PARB-MGT clade (Bischoff et al., 2009; Lopes et al., 2018), were selected as the outgroup. Sequences of each region were aligned individually by MUSCLE (Edgar, 2004) operated on MEGA 7 for the multialignment, with final manual adjustment of the dataset. Alignment gaps were treated as missing data. Ambiguously aligned sites in the ITS dataset were detected and removed using the Gblocks Server (http://molevol.cmima.csic.es/castresana/Gblocks_server. html) under the least stringent setting. Topological conflicts between the two trees (ITS: 523 bp; 5TEF: 713 bp) were checked directly by topological comparison of those preliminary maximum likelihood (ML) trees. Next, the two datasets were combined into a single dataset of 1236 bp, and deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S24184). A phylogenetic analysis of the combined dataset was conducted using the ML and maximum parsimony (MP) methods based on Yamamoto, Degawa, Takashima, Fukuda, and Yamada (2017). Briefly, ML and MP analyses were conducted with raxmlGUI 1.31 (Silvestro & Michalak, 2012) and MEGA 7, respectively, under the general time reversible model of nucleotide substitution, with a discrete gamma distribution (þG), selected by MEGA 7, for ML and the tree bisection-reconnection search for MP. Branch support was evaluated by 1000 replicates of bootstrap analysis. Different ashape parameters, GTR rates, and empirical base frequencies were assigned to each partition (i.e., ITS and exons and introns of 5TEF) for ML. 3. Results and discussion 3.1. Taxonomy
Total DNA was extracted from cultured mycelia of CM1 and CM2 on PDA based on Izumitsu et al. (2012). Polymerase chain reaction (PCR) amplifications of partial sequences of the internal transcribed spacer region (ITS) and 28S (containing the D1eD2 region) nuclear ribosomal DNA and of the 30 region of elongation factor 1-alpha
Metarhizium brachyspermum Koh. Yamam., Ohmae & Orihara, sp. nov. Fig. 1. MycoBank no.: MB 830553. Diagnosis: This species is most similar to M. kalasinense in
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Fig. 1. Morphological characteristics of Metarhizium brachyspermum (A, B, DeG: KY170702-1; C: KY170709-1; HeJ: CM1 on SDAY/4; KeO: CM1 on PDA). A: Stroma arising from a pupa. Exuvia of a last-instar larva attached to a host. B: Above-ground portion of the stroma composed of a sterile stipe (left) and fertile part (right). C: Stroma arising from a larva. D: Longitudinal section of the fertile tissue. Obliquely buried perithecia are shown. E, F: Whole asci (E) and apical caps (F), stained with cotton blue. G: Ascospore with 15 septa (arrowheads). H, K: Upper (left) and reverse (right) sides of colonies after 3 wk of incubation. I, L: Conidiophores, stained with (L) and without (I) cotton blue. J, O: Conidia. Arrowhead indicates adhesion of two conidial chains. M, N: Anastomoses between two metulae (arrowheads), stained with cotton blue. Bars: A, C, H 10 mm; B 5 mm; D 500 mm; E, G 50 mm; F, M 5 mm; I, J 10 mm. Images in H and K, I and L, J and O, and M and N are at the same magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
morphology of stroma but the ascospores are significantly shorter in length than the latter. Holotype: JAPAN, Tochigi Prefecture, Utsunomiya-shi, Nagaokacho, ca. 170 m above sea level, on a pupa of an elaterid in the underground pupal-chamber, under Cryptomeria japonica (Thunb. ex L.f.) D. Don, Carpinus tschonoskii Maxim., Quercus myrsinifolia Blume, and Quercus serrata Murray, 2 Jul 2017, K. Yamamoto, KY170702-1 (TNS-F-70755; ex-type strain, CM1 (IFM 65744)). Gene sequences from ex-type strain: LC469747 (ITS), LC469749 (28S), LC469751 (3TEF), LC469752 (5TEF). Etymology: From the Greek brachy ¼ short and spermum ¼ spore, referring to the characteristic that the ascospore is shorter in length than that of M. kalasinense, a relative of this species. Stromata on larva or pupa of elaterid, single or multiple; upper, epigeous portion clavate, sometimes branched, pale green when young, turning olive green, up to 3 cm long; fertile head 17e20 mm long, 2.5e4.2 mm wide, slightly wider than sterile stipe; ostiole dark olive-green, up to 11 per 1 mm2; underground part cylindrical, irregular in width, lateral short branch present, yellow with partial green pigmentation, up to 29 mm long, 1.1e2.3 mm wide; host surface covered with pale yellow subiculum. Perithecia
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flask-shaped, obliquely immersed with slightly protuberant ostiole, 500e795 190e300 mm, mean 650 250 mm (n ¼ 26); walls composed of about 5 layers of plectenchymatous tissue, 9e14 mm thick, hyphae 1e4.5 mm wide. Asci hyaline, cylindrical, 8-spored, 230e400 2.5e4.5 mm, mean 320 3.4 mm (n ¼ 15); apical cap prominent, 1.5e2.5 mm high, 3e3.5 mm wide, mean 2 3.2 mm (n ¼ 12). Ascospores filiform, hyaline, 190e270 1 mm, mean 220 1 mm (n ¼ 20); septa up to 15, sometimes obscure, not fragmenting into part-spores; length between adjacent septa 11e42 mm, mean 19 mm (n ¼ 88). Colonies on SDAY/4: Colonies growing 10e16 mm within 14 d at 25 C; surface floccose with abundant aerial hyphae, at first white, becoming greenly pigmented due to conidiation within 10 d; reverse pale yellow. Conidiophores dense, erect, terminating in branches, with 2e3 phialides per metula, forming a palisadelike layer; hyphae bridge formation by anastomosis between adjacent metulae relatively abundant; without formation of synnemata. Phialides hyaline, cylindrical, tapering apices, 7.5e10.5 1.5e2.5 mm, mean 9.3 2.1 mm (n ¼ 10), forming conidial chains. Conidia unicellular, cylindrical, smooth, olive green, 6e8 2.5e3 mm, mean 6.9 2.8 mm (n ¼ 50), Q ¼ 2.1e3.0, mean 2.5 (n ¼ 50). Colonies on PDA: Colonies growing 10e18 mm within 14 d at 25 C; surface relatively floccose with aerial hyphae, at first white, becoming green-pigmented due to conidiation within 6 d; reverse pale yellow. Conidiophores dense, erect, terminating in branches, with mostly 2 phialides per metula, forming a palisade-like layer; without formation of synnemata. Phialides hyaline, cylindrical, tapering apices, 7e13 2e2.5 mm, mean 9.5 2.4 mm (n ¼ 15), forming conidial chains. Conidia unicellular, cylindrical, smooth, olive green, 5.5e7.5 2e3 mm, mean 6.8 2.6 mm (n ¼ 50), Q ¼ 2.2e3.2, mean 2.6 (n ¼ 50). Habitat, distribution and fruiting season: On larva or pupa of Elateridae buried in broadleaved tree-dominated forests in the temperate zone of Honshu, Japan; summer (Jun to Sep). Other specimens examined: Kyoto Prefecture, Uji-shi, Uji, Kousai, ca. 40 m above sea level, on a larva of Elateridae, buried under Castanopsis cuspidata (Thunb.) Schottky, 18 Jun 2000, T. Yamamoto, no. 81 (TNS-F-70754). Tochigi Prefecture, Utsunomiya-shi, Nagaoka-cho, ca. 170 m above sea level, on a larva of Elateridae, buried under C. japonica, C. tschonoskii, Q. myrsinifolia, and Q. serrata, 9 Jul 2017, K. Yamamoto, KY170709-1 (TNS-F-70756; living culture, CM2 (IFM 65745)). Ibid., on a larva of Elateridae buried in decayed wood of a broadleaf tree, 2 Jul 2017, T. Shoda (TPM-M-9238). Nom. Jap.: Kusairo-kometsukimusi-take (from the Japanese kusairo ¼ glass-green, kometsukimusi ¼ elaterids, and take ¼ fungi)
3.2. Morphological comparison of teleomorphs of M. brachyspermum and its relatives parasitic on Elateridae Hosts of the four specimens examined in this study were larvae (Fig. 1C) and pupae (Fig. 1A) of the Elateridae. Metarhizium brachyspermum is morphologically similar to M. kalasinense described from Thailand: both species grow on larvae of the Elateridae, and their stroma is composed of a clavate, greenish fertile head, and rhizoidal stipe appearing yellowish below ground (Fig. 1A, C; Luangsa-ard et al., 2017); however, asci and ascospores of the latter are significantly longer in length than the former (Table 1). Metarhizium campsosterni (W.M. Zhang & T.H. Li) Kepler, S.A. Rehner & Humber from China similarly grows on larvae of the Elateridae and forms a green-yellow stroma but its perithecia are vertically buried
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Table 1 Morphological characteristics of teleomorphic stages of Metarhizium spp. in the MGT clade. Species
Hosts
Fertile part of stromata
Perithecia
Asci
Ascospores
M. brachyspermum
Larvae and pupae of Elateridaef Larvae and adult of Elateridaec Larvae of Hepialidae or Cossidaead Larvae of Elateridaee
clavate; olive greenf
Obliquely immersed; 500 e795 190e300 mmf Vertically immersed; 275 e433 165e276 mmc Obliquely immersed; 700 e750 275e325 mma Obliquely immersed; 700 e800 250e350 mme Vertically immersed; 767 e940(e1100) mmb
230e400 2.5 e4.5 mmf 175 e349 3.9 mmc 300e350 3 e4 mmd 500e650 4 e5 mme 305e480 3.3 e4.5 mmb
190e270 1 mm; nonfragmentingf ND; fragmenting into part sporesc 250e360 1 mm; fragmenting into part sporesd 400e500 1e1.5 mm; nonfragmentinge ND; fragmenting into part sporesb
M. campsosterni M. indigoticum M. kalasinense M. guizhouense (¼ Metacordyceps taii) a b c d e f
Larvae of Noctuidaeb
cylindrical, tapering; greenish yellowc fusiform, slightly compressed, corniculate tip; blueish greenad cylindrical or clavate; olive green to greenish browne cylindrical, tapering; yellowishb
Kobayasi and Shimizu (1978). Liang, Liu, and Liu (1991). Zhang et al. (2004). Japanese Society of Cordyceps Research (2014). Luangsa-ard et al. (2017). This study.
in stroma and asci are shorter in length than that of M. brachyspermum (Zhang, Li, Chen, & Qu, 2004, Table 1). 3.3. Phylogenetic relationship between M. brachyspermum and its relatives in the MGT clade Four and three sequences were successfully obtained from CM1 (ITS: LC469747; 28S: LC469749; 3TEF: LC469751; 5TEF: LC469752) and CM2 (ITS: LC469748; 28S: LC469750; 3TEF: LC469753), respectively. Phylogenetic trees inferred from the individual ITS and 5TEF datasets showed that no significant topological conflicts were present. Therefore, a phylogenetic analysis of the combined dataset was conducted. The scores of the resultant trees (Fig. 2) were as follows: parsimony informative sites ¼ 52; tree length ¼ 146 steps; consistency index ¼ 0.848; retention index ¼ 0.917; rescaled consistency index ¼ 0.778. This tree showed that M. brachyspermum belonged to the MGT clade within the M. anisopliae lineage. Former studies showed that M. indigoticum, M. guizhouense, M. kalasinense, and M. majus (J.R. Johnst.) J.F. Bisch., S.A. Rehner & Humber placed within this clade (Kepler et al., 2014;
Fig. 2. Maximum likelihood (ML) phylogenetic tree of the combined dataset of the ITS and 5TEF sequences of Metarhizium brachyspermum and its allies (ln L ¼ 2381.899044). Metarhizium alvesii and M. lepidiotae were used as outgroups. Bootstrap (BS) values (1000 replicates) > 50% from ML (left) and maximum parsimony (MP) (right) are shown near nodes. Branches supported by a BS of ML/MP of 70% are depicted as thick black lines. Black circles indicate teleomorphic species. T: ex-type strain; ET: ex-epitype strain.
Luangsa-ard et al., 2017; Nishi & Sato, 2017), and this relationship was also recovered in this study. Likewise, our phylogenetic analysis showed that M. baoshanense Z.H. Chen, Ling Xu, X.N. Yang, Y.G. Zhang & Y.M. Yang, a recently described anamorphic species, and M. campsosterni were also members of the MGT clade. Teleomorphs of M. indigoticum and M. guizhouense (¼ Metacordyceps taii (Z.Q. Liang & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora) are clearly distinguishable from M. brachyspermum by their lepidopteran hosts and ascospores that break into part spores (Table 1). 3.4. Growth of M. brachyspermum isolates and characteristics of its anamorph Colonies of CM1 grew 11e14 mm on SDAY/4 and 16e18 mm within 2 wk on PDA. CM2 grew 12e16 mm and 10e15 mm; therefore, CM2 tends to grow more slowly than CM1 on PDA. Colonies on SDAY/4 formed slightly larger conidia than those of PDA (Table 2). In contrast, phialides were larger on PDA (Table 2). Anastomoses between two metulae were often observed on conidiophores of M. brachyspermum (Fig. 1M, N). Similar anastomosis was also observed on conidiophores of M. minus (Rombach, Humber & D.W. Roberts) Kepler, S.A. Rehner & Humber (Glare, Milner, & Beaton, 1996) which belongs to M. flavoviride W. Gams & Rozsypal lineage (Kepler et al., 2014). In addition, conidial chains on dense phialides are laterally adhered to each other (Fig. 1O) as observed in other species of Metarhizium (Bidochka & Small, 2005; Rombach, Humber, & Evans, 1987). Compared with the colony of M. kalasinense, that of M. brachyspermum tends to be paler and the aerial hyphae in the colony are often abundant (Fig. 1H, K); however, it is unclear whether this tendency is useful for classification because of the limited number of comparisons of isolates. In addition, differences in the size of phialides and conidia between M. brachyspermum and M. kalasinense are unclear (Table 2) even though their teleomorphs clearly differ in the size of asci and ascospores (Table 1). In fact, colony color and conidial size among the isolates within phylogenetically supported species in the M. anisopliae lineage sometimes overlap with the variations in other species (Bischoff et al., 2009). Thus, clarification of the teleomorphic stages of further species may be beneficial for defining the species concept of Metarhizium. Other species in the MGT clade (i.e., M. baoshanense, M. guizhouense, M. indigoticum, and M. majus) form slightly or much larger phialides and/or conidia than M. brachyspermum (Table 2). Recently, Kepler, Chen, Kilcrease, Shao, and Rehner (2016) identified stable diploid lineages formed by the union of strains with opposite mating types in M. majus species complex. They
Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001
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Table 2 Size of phialides and conidia of Metarhizium spp. in the MGT clade on SDAY/4 and PDA media. Species
Phialides (mm)
Conidia (mm)
SDAY/4 M. M. M. M. M. M. a b c d e f
brachyspermum baoshanense guizhouense indigoticum kalasinense majus
7.5e10.5 1.5e2.5 ND 7e12 2e3c ND 8e12 2e3d 10e18 2.5e3.5c
PDA f
SDAY/4 f
7e13 2e2.5 8.2e19.2 2.1e3.5e ND 8.5e20.5 1.7e3.5e ND 6.7e17.7 2.1e3.3b
PDA f
6e8 2.5e3 ND 7e10 2e3c ND 6e8 2e3d 10.5e13 2.5e4c
5.5e7.5 2e3f 6.7e8.5 2.6e3.3e 6.7e7.3 3e3.4a 8.2e12.3 2.6e4.2e 6e7 2e3d 10.5e12.5 2.5e3.7b
Guo, Ye, Yue, Chen, and Fu (1986). Glare et al. (1996). Bischoff et al. (2009). Luangsa-ard et al. (2017). Chen et al. (2018). This study.
suggested that this mechanism enables to acquire or maintain diversity in the absence of frequent sexual reproduction. Further field sampling and mating-type determinations of anamorphic stages of M. brachyspermum and other teleomorphic species will promote understand the function of this unique reproductive strategy and whether diploidy is widespread among the MGT clade. Disclosure The authors declare no conflicts of interest. Acknowledgments We are grateful to Dr. Janet Jennifer Luangsa-ard for providing information on M. kalasinense. We are also grateful to Mr. Tsutomu Yamamoto and Mr. Tatsuhiko Shoda for sampling specimens. We thank Dr. Kentaro Hosaka for depositing specimens and Dr. Sayaka Ban for depositing cultures. This study was supported in part by KAKENHI Grant-in-Aid for Young Scientists (B) (no. 17K15184) from the Japan Society for the Promotion of Science (JSPS) and a Grantin-Aid from the Institute for Fermentation, Osaka (IFO). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.myc.2019.09.001. References Behie, S. W., Zelisko, P. M., & Bidochka, M. J. (2012). Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science, 336, 1576e1577. https://doi.org/10.1126/science.1222289. Bidochka, M. J., & Small, C. L. (2005). Phylogeography of Metarhizium, and insect pathogenic fungus. In F. E. Vega, & M. Blackwell (Eds.), Insectefungal associations: Ecology and evolution (pp. 28e50). New York: Oxford University Press. Bischoff, J. F., Rehner, S. A., & Humber, R. A. (2006). Metarhizium frigidum sp. nov.: A cryptic species of M. anisopliae and a member of the M. flavoviride complex. Mycologia, 98, 737e745. https://doi.org/10.1080/15572536.2006.11832645. Bischoff, J. F., Rehner, S. A., & Humber, R. A. (2009). A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia, 101, 512e530. https://doi.org/10. 3852/07-202. Chen, Z., Xu, L., Yang, X., Zhang, Y., & Yang, Y. (2018). Metarhizium baoshanense sp. nov., a new entomopathogen fungus from Southwestern China. Pakistan Journal of Zoology, 50, 1739e1746. https://doi.org/10.17582/journal.pjz/2018.50.5.1739. 1746. Edgar, R. C. (2004). Muscle: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792e1797. https://doi.org/10.1093/ nar/gkh340. de Faria, M. R., & Wraight, S. P. (2007). Mycoinsecticides and mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types. Biological Control, 43, 237e256. https://doi.org/10.1016/j. biocontrol.2007.08.001. Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts.
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Please cite this article as: Yamamoto, K et al., Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.001