Morphological and genetic characteristics of the entomopathogenic fungus Ophiocordyceps nutans and its host insects

mycological research 112 (2008) 1241–1244

journal homepage: www.elsevier.com/locate/mycres

Morphological and genetic characteristics of the entomopathogenic fungus Ophiocordyceps nutans and its host insects Fumito SASAKIa, Toshizumi MIYAMOTOa,*, Aki YAMAMOTOb, Yutaka TAMAIa, Takashi YAJIMAa a

Laboratory of Forest Resource Biology, Graduate School of Agriculture, Hokkaido University, N9-W9, Kita-ku, Sapporo 060-8589, Japan Otaru Museum, 1-3-6 Temiya, Otaru 047-0041, Japan

b

article info

abstract

Article history:

We examined the morphology, genetic variation, and insect host species of the fungus

Received 11 April 2007

Ophiocordyceps nutans. Fifty-two fruit bodies collected in Hokkaido, Japan, were grouped

Received in revised form

by host species, and 19 samples were analysed. The ranges of the lengths and breadths

19 March 2008

of all fruit body parts were similar among host groups. In the genetic analysis, the 5.8S

Accepted 1 April 2008

rDNA region showed completely identical sequences, although differences of up to six

Corresponding Editor:

nucleotides were recognized in the ITS regions. The distance values between our samples

Richard A. Humber

using the Kimura two-parameter model were lower than 0.007. Thus, the O. nutans examined here were concluded to form a closely related group with little detectable variability

Keywords: Biological control

that parasitized nine hemipteran species. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Entomophagous Host specificity Intraspecific variation Vegetable wasps and plant worms

Introduction Cordyceps species (in the broad sense) are entomopathogenic fungi belonging to the order Hypocreales (Ascomycota). They infect insect larvae or imagoes, kill them, and form fruit bodies on the corpses (Ito & Hirano 1997). Some Cordyceps species are used as herbal medicines (Shimizu 1994; Kinjo & Zang 2001). Because members of the Cordyceps spp. usually have host specificity, their anamorphs are likely useful as selective biological pest control agents (Ito & Hirano 1997; Sato et al. 1997; Evans et al. 1999; Nikoh & Fukatsu 2000).

Ophiocordyceps nutans, which specifically parasitizes stinkbugs (Hemiptera) (Hywel-Jones 1995; Fukatsu 1999), occurs in Japan, Taiwan, China, and New Guinea, as well as other locations (Shimizu 1994). This species is among the most common of all Cordyceps species in Korea (Sung 1996). In China, it is thought to have medicinal value (Mao 1998; Liu & Xu 2000). Stinkbugs cause considerable damage to agriculture and forestry, and the anamorph of O. nutans, Hymenostilbe nutans, is a potential selective biocontrol agent in these cases. Cordyceps species, O. nutans included, are mainly classified morphologically by their colour, fruit body shape, and host insect species (Shimizu 1994; Ito & Hirano 1996). Three known

* Corresponding author. Tel.: þ81 11 706 2536. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.04.008

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species and unidentified insect species in five families of Hemiptera serve as hosts of O. nutans (Moureau 1949; Samson & Evans 1975; Sung et al. 1993). Few investigations have examined the fungal host species among hemipterans; without this knowledge, however, the development and application of biological controls cannot proceed. Moreover, the variation of O. nutans among different hosts has not been examined, although morphological and/or genetic variation related to host species has been reported in other entomopathogenic fungi (Fegan et al. 1993; Jensen et al. 2001; Wada et al. 2003). Therefore, we examined the host insect species for O. nutans, as well as the morphological and genetic characteristics of the fungus.

Materials and methods From July to September 2002, we collected fruit bodies of Ophiocordyceps nutans in the Tomakomai Experimental Forest, Hokkaido University, Japan (42 390 3300 –42 430 0600 N, 141 320 4500 – 141 380 3600 E; 2.7 ha), and identified the host insect species (Tomokuni et al. 1993). Fruit bodies were grouped by host species, and three fruit bodies from each group were examined. For groups that had fewer than three fruit bodies, we used one or two. The strains used are listed in Table 1. Isolation was conducted according to the surface sterilization methods of Sasaki et al. (2004). The following isolates were obtained: T23, T24, T30, T35, T39, T41, and T71. These isolates were incubated using Sabouraud–glucose agar medium (pH 8) at 20  C about 3 months in the dark according to Sasaki et al. (2005). All samples we examined were deposited in the Laboratory of Forest Resource Biology, Hokkaido University, Hokkaido, Japan.

Morphological observation Fruit bodies were oven-dried for 60–72 h at 60  C. Perithecial heads were rehydrated in 0.05 % Triton X-100 solution (Hywel-Jones 1995) and observed using differential interference microscopy. The lengths and breadths of 20 perithecia, 20 asci, and 30 partspores were measured for each fruit body. The measurements of the fruit bodies, each with a maximum of three replicates, were compared among host insect species groups using one-way analysis of variance (ANOVA; SPSS 10.0 J for Windows, SPSS, Chicago, IL).

Sequencing and phylogenetic analysis Isolates or specimens of each sample were used for DNA extraction. Total DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden) according to the manufacturer’s instructions. The extracted DNA was dissolved in 50 ml TE buffer; 1 ml of the DNA solution was used as template DNA. The primer pair ITS1f (Gardes & Bruns 1993) and ITS4 (White et al. 1990) was used for amplification. The samples were run using an initial denaturation for 5 min at 94  C; followed by 25 cycles of denaturation for 1 min at 94  C, annealing for 1 min at 55  C, and extension for 3 min at 72  C; and a final extension for 7 min at 72  C. The abovementioned primers were used for the cycle sequencing reaction. After the samples were purified, we used an ABI Auto Sequencer 3730 (Applied Biosystems, Foster City, CA) to determine the ITS1–5.8S rDNA–ITS2 regions of the rDNA sequences. The sequences obtained were aligned and compared using CLUSTALW (Thompson et al. 1994). Phylogenetic analysis was performed using MEGA version 3.1 (Kumar et al. 2004), using Kimura two-parameter models (Kimura 1980).

Results Fifty-two fruit bodies were collected, and their host hemipterans were classified into three families, five genera, and nine species (Table 1). More than three O. nutans fruit bodies were found on each of the other hemipteran species. Accordingly, 19 samples were used for morphological observations and DNA analysis. The samples are listed in Table 1. The ranges of the lengths and breadths of all fruit body parts were similar among the host groups (Table 1). No significant differences were observed in the morphologies of the fruit body components among the host insect species (ANOVA, P > 0.05). The 19 samples had high similarity in ITS1–5.8S rDNA–ITS2 sequences. There was no genetic variation in this sequence region related to the host species, although differences of up to six nucleotides were recognized. In particular, in the 5.8S rDNA region, all samples had completely identical sequences. The distance values between the strains were lower than 0.007. All samples were registered in the DNA Databank of Japan (DDBJ) under accession numbers AB176462, AB176463, and AB366618–AB366634.

Table 1 – Host species and the parts in fruit in bodies of o. nutans Host families Acanthosomatidae

Pentatomidae

Urostylidae

Host species Acanthosoma denticaudum Acanthosoma forficula Acanthosoma haemorrhoidale angulatum Acanthosoma labiduroides Elasmucha putoni Lelia decempunctata Pentatoma japonica Pentatoma rufipes Urostylis annulicornis

Strains T21, T38, T32, T41, T70 T33, T39, T62 T23, T35, T42 T24, T30, T37

T46 T43 T71 T82 T63

Perithecia (mm)

Asci (mm)

610–1170200–500 720–1070240–490 780–1030200–380 700–1080210–400 610–880190–370 660–1140290–560 890–1150260–370 550–1110200–400 720–1170220–360

295–7555–9 305–7756–9 355–8306–8 200–8755–9 305–6555–9 225–7155–9 375–7705–8 270–8055–9 275–7405–9

There was no significant difference in each host which has three replicates (ANOVA, p > 0.05).

Partspores (mm) 4–121.5–2.5 4–14.51.5–2 8–14.51.5–2 3.5–12.51–2.5 4–101.5– 2 4–14.51.5–2 4–12.51–1.5 6.5–201.5–2 7–111– 2

Morphological and genetic characteristics of the entomopathogenic fungus

Table 2 – Fruit body sizes of Ophiocordyceps nutans Strains

Perithecial head

Fruit body

length (mm)

breadth (mm)

length (mm)

9.1 12.7 6 7.2 11 10.6 12.8 5.4 8.5 9.8 11.1 13 10.1 10 2.5 11.5 8.4 14 13.3

2.4 2.6 2.5 2.4 2.6 3.7 2.4 2.5 2.9 2.9 2.8 3.6 2.4 2.5 1.5 3 2.9 2.9 3.4

78 104 100 87 88 32 82 112 63 100 81 83 83 65 82 104 93 98 78

T21 T23 T24 T30 T32 T33 T35 T37 T38 T39 T41 T42 T43 T46 T62 T63 T70 T71 T82

Discussion Previously reported hosts of Ophiocordyceps nutans include Pentatomidae, the Plataspidae Coptosoma sp. and Reduviidae (Mouerau 1949); Pyrrhocoridae (Samson & Evans 1975); and the Coreidae Molipteryx fuliginosa, the Pentatomidae Lelia decempunctata, and Palomena angulosa (Sung et al. 1993). Our results corroborated earlier findings (Moureau 1949; Samson & Evans 1975; Sung et al. 1993) indicating that Pentatomidae is the only common host family, within which we found one common species, L. decempunctata (Sung et al. 1993). Because the former studies examined O. nutans from Africa, which is very far from Japan, the host species may differ. Previous research has reported genetic variation related to host species in some entomopathogenic fungi. For example, Jensen et al. (2001) and Wada et al. (2003) showed variation in the ITS1–5.8S rDNA–ITS2 regions of Entomophthora muscae (a fly pathogen) and Beauveria brongniartii (a beetle pathogen), respectively, using PCR-RFLP analysis. B. brongniartii shows variation related to its host species in the 5.8S rDNA region, which is a very short, stable region (Wada et al. 2003). In contrast, our O. nutans samples had completely identical 5.8S rDNA sequences, and there was no variation in the ITS1– 5.8S rDNA–ITS2 sequences related to the use of different host species. In fungi, the distance values of ITS1–5.8S rDNA–ITS2 sequences from one species are generally between 0.000 and 0.050 (Chen et al. 2001, 2004). The distance values between our O. nutans samples were lower than 0.007. In morphological comparisons, we observed no significant differences in perithecia, asci, and partspores among O. nutans using different host insect species, although the sizes of the fruit bodies varied (Table 2). We did not consider geographical variation because we sampled within the same area. Accordingly, from both

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morphological and molecular biological results, the O. nutans strains analysed here were concluded to form a closely related group with little detectable variability that parasitized nine hemipteran species. Previous studies of O. nutans did not identify host insect species or comprehensively investigate the morphological and genetic characteristics and host species. Thus, this is the first study to investigate these in O. nutans.

Acknowledgements This work was supported by Research Fellowships for Young Scientists (No. 17$9011) from the Japan Society for the Promotion of Science (JSPS).

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