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Edaphologia, No. 8e:25-28, December 11, 2006 25

Entomopathogenic Fungi Isolated fi:om Laboratory-Reared Baculentulusdensus (Acerentomidae, Protura)"

Yuko Kurihara'""'*, Ryuichiro Machida*"*, Makiko Fukui"", Toru Okuda", Shigeaki Harayama"""

" tacoiogy & bdetabolic Dive"sity Reseai=h Cente4 7kemagawa blaivenyity Re,s'earch institute, 1lrmagaiva-gakuen 6-1-1,

Mtichidla, 7bkyo 194-8610, Jlrpan

"' NlTIi Biological Resouree Center (?V'BRq, National Institute offechnotogy andEvaluation, 2-5-8 Kbizusakamatari,

Kisarazu, C'hiba292-0818, .lopan

"" Graduate Sthoot ofLijZ] andEnvironmental Scienees, Uhiversity oflkukuha, 1-1-1 fennoudoi, %'ukuba, Ibaraki

305-8572, Japan

Currentadeessr Sugadoira Afontane Research Centeny University oftsukuha, Sugadoira Kbgen 1278-294, Ulado,

Ndgano 386-2204, Jbpan

Received: 17 May 2006; Accepted: 13 November 2006

Abstract Three entomopathogenic fungi, Lecanicillium psalliotae, Lecanicillium sp. and Conidioboltts coronattts were isolated from laboratory-reared proturans (Ba(:ulentulus densus). Lecanicillium psalliotae and C. coronatus are known as general pathogens of various soil insects. This is the first record of these species in association with proturans.

Key words: Baeutentulus densus, Conid)ohotus ceronatus, Lecaniciiiiumpsalliotae, entomopathogenic fungi, Protura

Corc6,ceps (Fr.) Link and 7b"rubiella Boud. species and Vistergaard, 2002; Drornph, 2003). On the other hand, so far their anamorphs (lsaria Fr., Lecaniciltium Gams & Zare, there has been no report on fungi parasitic to proturans,

Simpiicillium Gams & Zare, and Beauveria Vuj]1,, for Protura is the most ancestral group of insects (Kinzelbach, exarnples) are parasites of insects and spiders, and they have 1971; Machida, in press), and proturans are obligate soil been regarded as representatives of entomopathogenic fUngi. dwellers (Tuxen, 1964). It used to be diMcult to maintain them Coftlycqps species show high host specificity fbr pterygote in the laboratory for long periods owing to their minute size and

orders (Aoki, 2003). They are considered to have co-cvolved lifestyle (Machida and Takahashi, 2004). Recently, Machida with host pterygotes with host shifting and jumping (Nikoh and Takahashi (2004) established a method for keeping a and Fukatsu, 2000). To comprehend the co-evolution between proturan, Baculentttlus denstts lmadate (Acerentemidae), tmder host insects and thcir parasitic fungi, however, information on semi-natural conditions in the laboratory. For the discovery

the fungal parasites ofancestral insects is necessary, of ancestral entomopathogenic fungi, investigations of the -as Entomopathogenic fungj of ancestral insects and other pathogens of proturans the most ancestral insect- are

soil arthropods have not been well investigated (Kcller required. Thus, we report entomopathogenic fungi isolated from

and Zimmermann, 1989) except fbr those of mites and B, clensus kept in the laboratory,

collembolans (Keller and Zimmermann, 1989; Bridge et To obtain proturans and other soil arthropods, litter and

al., 2005, for cxarnples). This is because some mites and soil sarnples were oollected from a copse on a hill at Shinko-

collembolans are known to be infected by various parasitic ji temple in Sanada, Ueda City, Nagano Prefecture jn 2003,

fungi of pterygotes withlwithout symptoms, and thus they Arthropods were extracted from the samples using Tullgren

can be used as vectors for spreading entomopathogenic fungi funnels. Proturans were picked up and transferred to plastic in thc biological control of pterygote pests (Dormph and containers. Small numbers of collembolans and mites that

*Co-tributionfrom SugadairaMontanc Rcscarch Ccntcr, No, 205,

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26 VlikoKurihara,RyuichiroMachida,MakikoFukui, ToruOkuda, ShigeakiHarayama

tt' i2itwge,su , i esisuth'e' .,,. /ge.ggva.eteslee

Fig.1-5. Entornopathogenic fungi fbund from Baeulentulus densus. 1: A cadaver infected with Conidioboiu,s' coronatus', 2: Immature conidia (arrows) and mycelia of C. cororeatus within and on the surface ofthe host. 3: The habit ofLecanicillium psalliotae on Miura rnedium. Venicillate phialides bearing sticky conidial masses (arrows) produced on an aerial hypha. 4: A single phialide of L. psallioiae after the separation of mature conidia. 5: Falcatc conidia ofL. psatliotae.

Ilable 1. Frequency of entomopathogenic fungi isolated frem cadavcrs ofBacbllentulus densus and their cohabited arthropods underrearing.'

Simplicillium Beauveria Samples No. ofsamplesLecanicitlium L. sp.1 psalliotae sp, bassiana

proturans 42 18 1

collembolans 18 6 3

'mltes 4 2 1

e: Saprophytic species found from the samples were not shown in the tab]e,

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Entomopathogenic Fungi Isolated from Laboratery-Reared Baculentulus densors (Acerentornidae, Protura) 27

Tleible 2. Frequency ofentoinopathogenic fungi isolated from Bacutentulus densus and ether soil arthrepeds immcdiate]y after extraction from a soil sample.'

Conidio- Lecani-

Samples No, ofsamples bolus ciiliumpsalliotaeL.sp.1 L.sp.2

coronatus

proturans 4

mites 7 4 1

collembolans 7 2 1

mMipedes 1 1

coleopteran 2 2 larvae ------L------H------litter at the sampling site'" + +

S: Saprophytic species found from the samples were not shewn in the table.

": Entomepathogenic species isolated from 1O litter pieces (5 x 5 mm) shown as present (+) er absent (-). These species were considered to be prcscnt in the 1iner when they were found from a piece or more.

attached to litter were also transferred to the containers soil arthropods, they often grew exclusively without being -15 without intention. These conta{ners were incubated at O accompanied by other fungal species including saprophytes.

relative under conditions an anamorphic of and ℃ , 1OO % humidity dark for6 Lecanicilliumis genus Cord.vcqps

months to a year, as described by Machida & fakahashi (2004). 7brrubiella (Zare and Gams, 2001). Lecanicillium psalliotae Many individuals of the arthropods died whjle rearing at has been regarded as a generalist entomopathogen (Bridge laboratory, Fungal iso]ation was conducted from 42 randornly et al,, 2005), and has been recorded from collembolans selected cadavers ol' proturans on Jan. 9, 2004 (Table 1), Each (Orv,chiurus subtenuis) (Visser et aL, 1987). However, this cadaver was inoculated directly onto a Miura agar medium species would be a facultative parasjte of soil arthropods, ' plate ( di ! 6 cm; glucose 1,O g, KH2P04 1.0 g MgS04 because it was also isolated from litter (Tleible 2), fbund from 7H20 O.2 g, KCI O.2 g, NaN03 2.0 g, yeast extract O.2 g, soit, Agarics (Zare and Garns, 2001), grew well on artificial agar 13.0 g, distilled water 1 L; pH 6,5-7.0), and incubated at media. This is the first record of this species in association = 26 ℃ fbr 10 days under lightfdark conditions (L:D 12:12). ' withproturans.

Fungal spores forrned on and around the cadavers were In this study, two other Corelyceps-7brrubiella lineages,

transferred to a new Miura agar medium with a sterilized fine Simplieillium sp, and B, bassiana were isolated from

ncedle to establish pure cultures. Those fungi were identMed col]embolans or mites, but fiot from proturans. To decide

based on their morphological characteristics. Fungi from 18 the host range of these fungal species, however, infection

co]lernboLans and 4 mites co-habiting wjth the proturans were experiments on proturans with these two fungi should be

also identified for comparison (Table 1). performed. From the laboratory-kept proturans, Lecanicillium Tb compare the pathogen{c rnycoflora under lab-incubation

psalliotae (Treschew) Zare & W. Gams (Figs, 3-5) and an and in nature, entomopathogenic fungal species were were

unidentified species ofLecaniciltium were isolated (Table 1). identified (Table 2), which were isolatcd from soil arthropods

From the co-habiting collembolans and mites, L. psalliotae, (4 proturans and 17 other arthropods) co]lected immediately SimpliciUium sp., and Beauveria hassiana (Bals,-Criv.) Vuill. after Tullgren extraction from a soi[ sample. The sample was fungi were isolated (Table 1), When these fUngi appeared from the collected on 20 July, 2004 after rain, and iso]ation of

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28 狗ko Kuhhara， Ryuichiro Machida， Makiko Fukui， Toru OkUda， ShigeakMarayama

was perfbrmed on 21・22 July，2004 by inoculating arthropods 飼育下 の サ イ コ ク カ マ ア シ ム シ （Baculentulus densus）か ら， ニ onto Miura agar medlum plates（φ 6cm ）． Before 3 種 の 昆 虫病 原 菌 類 ，LecaniciUium psatliotae， Lecanicillium

inocu］ation each arthlopod was dragged onto the surface of の 1 種 と ConidiobolUS coronatUS を し た ． Lecaniciliium ， 属 分離

amedium to remQve conidia of saprophytic fungi adhering psalliotaeとccoronatusは 様 々 な 土 壌昆虫 に 感染す る 多犯性

の て マ ア シ ム シ tothe surface of the arthropod ， and thentransfαred to a new 昆 虫 病 原 菌 と し て 知 ら れ お り，今 回 カ 類 か ら

agar plate． Most of these arthropods were kiUed by dragging． 初 め て 記録 さ れ た ．

For comparison ， fUngi were also isolated from the litter of References the s phng site （Table 2）． Ten small pieces of li賃er （ca ．5x ’ 5mm ）were inoculated onto a Miura agar medium plate（φ Aoki， J，，2003，KeJ．s to 五ntomopathogenic Fungi， revised レersion ． 三 − − − 9cm ）． All these platcs were inoubated at 26 ℃ 負）r l O days Zenkoku NosQn Kyoiku Kyokai，］「bkyo．（in Japanese） ＝ under light！dark conditions （L ：D 12：12）． Bridge， PD ．， Clark， M ．S． and Pearce， DA ，2005． A new species As a result， Conidiobolus coronatus （Costantin）A ． Batko of Paecilomyees isolated from the Antarctic springtail

was prevalent among the arthropods tested． This species C 「p／ptopygzls antarcticus ・Mycotα xon ，92 ：213−222．

apPeared exclusively fi℃ rn 90f the 17 arthropods ， but not Domsch ， K ．H ．， Gams， W ， and Anderson，工H ．，1980． Co〃ependium

丘 om would of ∫oil Fungi vo 〃 ． Academic Press London． proturans（Table2）；however， proturans be ， ，

susceptible to C ． coronatus ． This is because the species Dromph ， K ．M ．，2003 ． Collembolans as vectors of

） − appeared exclusively from 50f 7 dead proturans collected at entomopathogenlG fUngi．1ed（｝ろガo’〔，g’α，47；245256．

the same time and reared f（〕r a month in a rearing container Dromph ， K ，M and Vestergaard， S．，2 02． P 飢 hogcnicity and

in the laboratory that had died during lncubation（Figs．1， attractiveness of entomopathgenlc hyphomycete fungi to − 2）．Conidiobolus coronatus （Zygomycetes ）is known as a collembolans ，！4ppiiedSoil Ecotogy，21 ： 197 210． pathogen of various orders of lnsects（Aoki，2003）． This Keller， S． and Zimmcrmann， G ．，1989． Mycopathogens of soil

species can also be isolated丘om soil as a saprophyte Domsch i且 sects 」 n ： lnsect− interactions，eds N ． Wi 亅ding （ fungus （ ，

et al ． usually not dominate as described N ．M Collins RM ． Hammond and J．F． Webber 240−27 ， ， 1980）， but itdoes ， ），pp

here． The species was also newly recorded in association with Academic Press， London．

proturans． Kinzelbach ， R 、K ．，1971 ，Mvrphotogische Befunde an

砲 肋 醇 観9ε伽 und ihre phytogenetisc乃e Bedeutung （rnsecta： Acknowledgements Strepsiptera）． Zoologica （119）．

This work was partly supported by a grant from NEDO ， Machida， R ．，2006． Evidenoe丘om embryology fbr reconstruction

japan（PO2038）． Mr I． Takahash．i and Mr T ． Shirouzu provided hexapod basal clades ， Entomologische Abh α ndlungen ．（in us with samples and isolates． Mr T ． Kamijyo ， Mr N ． Goto， press）

Ms K ． Anzai， and Ms R． Suzuki preserved the isolates， Dr K ． Machida， R ． alld Takahashl，1．，2004． Rearing technique ibr − Isono read and contributed to the improvement of drafts， protUrans（Hexapoda：Protura＞． Pedobio∬ogia ，48：227 229 ，

Nikoh N ． and Fukatsu T．2000 ． lntcrkingdom host ， ， ， jumping 摘 要 underground ： phylogenetic analysis of entomopa エasitic 且mgi ・ 栗 原 祐 了 （モ 川 大 学 学 術 研 究 所 菌 学 応 用 研 究 施 設 of tho Corcly〔eps ルft丿lecular Biologr； and Evo∬ution genus ， ， − ． − − − 〒 194 8610 東京都 町 田 市玉 川 学園 6 1 L （独）製品評価技 17：629 638． ー 術基盤機構バ イ オ テ ク ノ ロ ジ 本部 〒 292−0818 千葉県木 Tuxen， S．L．1964， The ．Protura． Hermann ， Paris． − − 一 史津市 か ず さ 鎌足 2 5 9），町 田 龍 郎．福 井 生 子 筑 波 大 Visser S， Paエ 1son D 、 and Hassall M ．1987． Fungi assoclated 眞 （ ， ， ， ， ， − 学大学院生命環境科学研 究科 〒 3058572 茨城県 つ くば 市 with On ．vchiurus subtenuis （Co 囗embola ）in an aspen 』 − − ： ー − 天 王 台 1 1 1 〒 現住所 筑波 大 学菅平高原実験 セ ン タ woodland ． Canadian Journal o．〆B‘）tany，65：635642． − − 〒 386 2204 長野県．ヒ田市菅平高原 1278294），奥 田 徹 （玉 Zare， R ， and Gams， W ，，2001．A revision of Vertic〃ium section

） 川大学学術研究所菌学応用研究施 設），原 山 重明 （（独）製品 ノrostrata ． IV． Thc genera LecaniciUium and Simp 〜ici〃ium

バ ノ ロ ー ： コ − 評 価技 術 基 盤 機 構 イ オ テ ク ジ 本部） 飼育下 の サ イ gen， nov 」 1＞と丿v α Hedwigia，73：1 50． ク カ マ ア シ ム シ 〔カ マ ァ シ ム シ 凵，ク シ カ マ ア マ ム シ 科） か

ら分離 さ れ た 昆虫病 原 菌

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