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Densus(Acerentomidae TheTheJapaneseSociety Japanese Society ofofSoil Soil Zoology 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, NII-Electronic Library Service TheTheJapaneseSociety Japanese Society ofofSoilZoology Soil Zoology 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, NII-Electronic Library Service TheTheJapaneseSociety Japanese Society ofofSoil Soil Zoology 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 were they often exclusively without being to litter also transferredto the containers soil arthropods, grew -15 by other fungal species including saprophytes. without intention.These conta{ners were incubatedat O accompanied 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 NII-Electronic Library Service The Japanese SocietySooiety of Soil Zoology 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
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