第23巻 第1号 日本線虫学会誌 1993年7月

Infectivity of Entomopathogenic , carpocapsae, on the Mango Shoot Borer, Rhytidodera simulans*

Eizo KONDO** and Abd. Rahman RAZAK***

Infectivity of Steinernema carpocapsae strain All on larvae of the mango shoot borer Rhytidodera simulans, a serious pest of mango trees in , was investigated under laboratory conditions. When the larvae were placed individually in a 6-cm-d petri dish containing 5g of moistened mango sawdust with 0, 10, 100, 1,000 or 10,000 infective juveniles (J3), the nematode caused insect death 2 days or later after inoculation at the inoculum levels higher than 1,000 J3 and then grew and reproduced in the insect cadavers. Most of the which escaped nematode infection or those died later made gallery-like tunnel along the petri dish wall. About 1/2 to 1/6 individuals of insects introduced in the galleries artificially made in mango cut-branches were infected when the branches were either injected through small side holes reaching the galleries or sprayed over the holes with 1 m/ of nematode suspension containing 10,000 or 100,000 J3. The nematode infection on R. simulans larvae in galleries was found to be greatly affected by the insect feeding behavior. Jpn. J. Nematol. 23(1): 28-36. Key words: insect gallery, Malaysia, mango branch, nematode infectivity, shoot borer

Mango is a very important tropical fruit and, in Malaysia, is attacked by various species of insects; colleopterans (Rhytidodera simulans, Deporaus marginatus, Hypomeces squamosus, Sterno- chetus frigidus, S. mangiferae, Anomala cupripes), dipteran (Dacus dorsalis), hemipteran (Mictis longicornis), homopteran (Lepidosaphes euryae), and lepidopterans (Parasa lepida, Setora nitens, transversa, jocosatrix)(8). Of these pests, the most serious one is the mango shoot borer, R. simulans which distributes wherever mango is planted. Rhytidodera simulans larva bores into the shoot and continues to tunnel into the branch (20). Therefore the branch attacked by the insect will die and often break off in storms. So far the most practical control method of the insect is to prune the branches at an initial stage of infestation. Chemical control of the insect pests is generally not so effective due to a cryptic habitat of the insect. The present preliminary studies on the infectivity of entomopathogenic nematode Steinernema carpocapsae were conducted under laboratory conditions to provide a basis for the insect biocontrol by .

MATERIALS AND METHODS

Nematodes: Steinernema carpocapsae strain All was used for all inoculation experiments. A

* This work was conducted as a part of international collaborating study between Japan , Malaysia and U. K. under the title of "Ecological/biological control of agricultural pests in the tropics", organized by Prof. N. ISHIBASHI, Saga University, Japan and Dr. D. J. WRIGHT, Imperial College, England. ** Faculty of Agriculture , Saga University, Saga 840, Japan. *** Faculty of Agriculture , Universiti Pertanian Malaysia, Malaysia.

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preliminary inoculation experiment showed high infection of the nematode which was shipped to Universiti Pertanian Malaysia (UPM) from Biosys Co. (Palo Atlot, CA, USA) via Japan; all of the last instar larvae of the greater wax , Galleria mellonella, were killed or became moribund in 24 hours at 25•Ž after inoculation with ca. 500 infective juveniles (J3) per insect in petri dish inoculation experiments. Insects: The late instar larvae of the mango shoot borer, Rhytidodera simulans were collected from the infested mango trees in UPM campus and used for the nematode inoculation experi- ments noted below. Because of a limited number of insects available and a considerable variation in their body size, weighing from 0.41 to 2.12 g, a consideration was taken to make similar the average body weight of the insects among the treatments in the following inoculation experi- ments.

Inoculation methods: Two inoculation methods were employed for the present study.

1) Petri dish method. A sheet of filter paper (Toyo No.1; 6cm diam.) was placed on the bottom of a 6-cm-d petri dish and then inoculated with 0.4ml of 0.1% formalin solution containing 0 (=control), 10, 100, 1,000, or 10,000 J3. About 30 minutes after inoculation, the larvae were individually introduced into petri dishes which were then added with 5g of water- saturated sawdust of mango branch. The nematode-inoculated and non-inoculated insects were kept at 25•Ž and examined for an insect mortality 1, 2, 3, 4, 6, and 10 days after inoculation. The experiments were performed with 4 insects for each inoculum level of the nematode.

2 ) Mango branch method. Non-infested branches were harvested from the mango fields and cut into ca. 20cm long. To create a similar inhabiting condition for the insect, an artificial gallery (10cm long, 1cm in diam. roughly corresponding to a diameter of natural gallery of late instar larva) was made using an electric drill from one end of the cut branch, and a small hole

(3mm diam. corresponding to a natural hole from which the insect frass comes out) reaching the artificial gallery was made at 26•}2mm from the cut end mentioned above. To these artificial galleries, insect larvae were individually introduced with heads ahead via the hole mouths, plugged with mango wood discs (ca. 5mm thick) , fastened with cotton sticky tape, and kept at room temperature (18-25•Ž) to habituate the insects to the artificial condition. Three days after insect introduction, the wood discs damaged and/or push off by the insects were replaced with new ones and the both ends of these branches were completely sealed with melted paraffin to prevent penetration of the applied nematodes into the artificial gallery from the cut ends. Another three days after insect introduction, nematodes were applied with two different methods; spraying and injection at the inoculum level of 10,000 and 100,000 J3 which were suspended in 1 m/ of deionized water. For spraying, the nematode suspension was applied using a plastic hand mist sprayer (500 ml in volume) around the small hole on the cut branch. To make an accurate application, a bottle of the sprayer was replaced with a plastic centrifuge tube (15 m/ in volume) containing 1ml of nematode suspension. For injection, the suspension was applied using a 1ml syringe through the hole. The inoculated branches were kept at room temperature in plastic containers (ca. 3.8 1 in capacity) sheeted with a wetted double-folded paper towel and covered with a plastic sheet to keep moist inside. Six cut branches were used for the two inoculation methods with two inoculum levels. For controls, 1 m/ of deionized water was applied with either of the two methods using three cut branches. All branches used were split open 6 days after inoculation with nematodes to examine insect survival and frass deposition in the artificial

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Table 1. Infectivity of Steinernema carpocapsae on larvae of the mango shoot borer, Rhytidodera simulans. (Petri dish method)

galleries. Investigation of natural insect galleries in mango trees: As a basis for a practical biocontrol of R. simulans larvae in fields, the insect galleries in branches of mango trees in UPM campus or orchards were investigated in late August, 1992.

RESULTS AND DISCUSSION

Petri dish tests: The results of petri dish inoculation experiments are shown in Table 1. All R. simulans larvae used were killed in 2 days after inoculation with 10,000 J3, and 1/2 of the insects were killed with 1,000 J3; the larval mortality decreased as the nematode inoculum size decreased. The internal tissues of dead insects were mostly decomposed by the symbiotic bacterium- A B nematode complex, excepting the tracheal sys- Fig. 1. Internal views of healthy (A) and dead (B) tem (Fig. 1). In these insect cadavers, the R. simulans larvae dissected longitudinally along a ventral body axis 7 days after inocu- nematode was assured to grow and reproduce. lation with ca. 1,000 J3 of S. carpocapsae in a Seven days after inoculation, for instance, an 6-cm-d petri dish. Mi, midgut; FB, fat body; insect cadaver weighing 0.68 g had ca. 8,000 Tr, trachea; He, head. nematodes consisting of adults (2.5%) and parasitic juveniles (97.5%) . The newly formed infective juveniles (J3) started to emerge out of the small insect, weighing 0.41 g, 12 days after inoculation with 10,000 J3. The insect behavior in petri dishes was different depending on an inoculum size of nematodes. When the insect was inoculated with ten J3, 1/2 of insects used hid under the mango sawdust as some non-inoculated ones did (Fig. 2A). Of all insects used, half of them made gallery-like tunnel along the side wall of petri dish (Fig. 2B). Contrarily, an insect inoculated with 10,000 J3 died on the surface layer of sawdust before constructing gallery-like tunnel (Fig. 2C). Mango branch tests: After 3-day habituating period of R. simulans larvae, the condition of

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A B C Fig. 2. Larvae of R. simulans (I) inoculated with infective juveniles (J3) of Steinernema carpocapsae (str. All) in petri dishes containing moistened sawdust of mango branch (SM). Pictures were taken 3 days after inoculation. A: sawdust covering an active larva which escaped nematode attack. Note insect feces (arrows) on top layer of sawdust. B: a dead larva in a gallery-like tunnel along petri dish wall. C: a dead larva on sawdust without making gallery-like tnnel. branch was considerably variable in frass production through a small artificial hole, in plugging the hole with chewed wood fiber, and in making new small hole. These observations indicated that the feeding behavior of insect in artificial galleries was so variable that the insect activity could not be evaluated by the amount of frass evacuated out of the branches. The larvae in the artificial galleries were not easily infected with the nematodes: only 1/6 to 1/2 of the insects used were killed in 6 days when the cut mango branches were treated with 10,000 or 100,000 J3 by spraying or injection methods (Table 2). No positive relations were observed between the nematode application method and insect mortality. In order to make clear the cause of rather low nematode infection against the insects in artificial galleries, comparison was made on the conditions of mango galleries having dead and living insects. Excepting one occasion, the galleries with dead insects were almost empty (Fig. 3) probably because the infection was established in short time after nematode application, resulting in early stop of feeding and in turn decrease in frass production. The following features were recognized in the galleries with living/surviving insects and

Table 2. Effects of Steinernema carpocapsae application on Rhytidodera simulans larvae inhabiting in the artificial galleries made in mango cut branches.

1) Data were recorded 6 days after nematode inoculation.

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A B C D E Fig. 3. Dead larvae of R. simulans (I) in galleries artificially made in cut branches of mango. Photographs were taken 6 days after nematode inoculation by spraying or injection of 10,000 or 100,000 J3 of S. carpocapsae. Photographs were arranged due to a distance of insects from cut-ends of branches. A, C, and E; sprayed with 10,000 J3: B, injected with 100,000 J3; D, sprayed with 100,000 J3. f, frass.

A B C D E F G H Fig. 4. Living larvae of R. simulans (I) in galleries artificially made in cut mango branches. Photographs were taken 5 days after spraying or injection of 10,000 or 100,000 J3 of S. carpocapsaea and arranged due to a distance of insects from cut-ends of branches. B, C and D, injected with 100,000 J3; E, sprayed with 10,000 J3; F, injected with 10,000 J3; A, G and H, sprayed with 100,000 J3. Arrow indicate a whitish plug made in a gallery. f, frass.

―32― Vol.23 No.1 Japanese Journal of Nemato logy July,1993 tentatively categorized into three cases (Fig. 4). Case 1: rather hard whitish plug, not known the origin of it, was formed in a gallery. Case 2: frass was deeply deposited in the galleries. Case 3: the insects fed actively and made deep galleries beyond the end of artificially-made gallery locating themselves far away from the nematode entrance hole. Cases 1 and 2 were common to the non-inoculated healthy insects. All of these characteristics were considered to decrease, at least partly, the chance of nematode infection. Characteristics of natural galleries in mango trees: The surface of infested mango branches, in most cases, became black (Fig. 5A) due to exudation and/or excretion of the plant from small holes made at rather regular intervals by R. simulans larvae. The internal structure and moisture of galleries were greatly variable among galleries as shown in Fig. 5. The steinernematid entomopathogenic nematodes are naturally inhabiting in the soil and so susceptible to environmental extremes such as desiccation, high temperature, or solar radiation (7). Therefore, the efficacy of nematodes are mostly low for the control of insect pests attacking above-ground parts of plants in fields, although these insects are generally more susceptible than those living in soil environment. An exception is cryptic insects which are living inner parts of plants where direct sunlight is excluded and the moisture is kept high enough to ensure survival and infective activity of applied nematodes (2, 22). Actually, controls have been conducted by steinernematid and heterorhabditid nematodes on such cryptic insects as leafrollers or borers (4, 5, 6, 12, 15, 16, 18, 21, 22, 24, 25).

C A B D

F E G H

Fig. 5. Outer and inner views of mango branches attacked by R. simulans larvae. A, blacken surface of infested branch. B: small holes (arrows) made on the underside of branch. C: longitudinal section of the branch shown in Fig. 5B. Note an internal structure under the small holes (arrows) connecting to a gallery. D: a thin wall (TW) separating two insect galleries. E: brown frass packed in a gallery. F: whitish frass deposited in a gallery. G: a wet gallery. H: very dried gallery in which a larva wa detected. GI, gallery of the insect.

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Among cryptic insects, borers are the most difficult pests to be controlled by chemical pesticides, so much trials by entomopathogenic nematodes have been requested (1, 3, 6, 9, 10, 11, 13, 14, 17, 19). As was briefly studied by SHEN and HAN (23) on Rhytidodera bowringi, the mango shoot borer Rhytidodera simulans was also infected by Steinernema carpocapsae in the present petri dish inoculation experiments, however, the nematode infection was not so high when it was applied against borers introduced into the artificial galleries in cut mango branches. Through our careful inspections on R. simulans larvae in petri dishes and in galleries, it is concluded that behavior of host insect and environmental factors are more important for establishing nematode infection than having been expected so far for cryptic insects. Namely, the insect feeding behavior was greatly related to frass production and to distance between the nematode entry hole and insect position in the galleries, both of these may play as mechanical and/or spacial obstructions for establishing nematode infection. In addition, substance (s) in the water-extract of mango branch had about five times more toxic to the nematodes than that of breadfruit, mulberry, and monkey-jack (local name, cempedak) bearing Apriona borer; the nematode mortality was 43% after being incubated at 25•Ž for 24 hr in the filtrate (ca. 2 mm in depth) of

10 g of mango sawdust in 50 ml water (unpublished data) . These behavioral and chemical factors are considered to be possible reasons why the nematode infection was rather low on R. simulans larvae in petri dishes containing moistened mango sawdust and in artificial galleries in mango cut branches. In mango fields, the structural and moisture conditions of insect galleries are greatly variable; some galleries were very wet and the others very dry, some galleries were deep and the others not so much, some galleries were close to each other and the others were isolated, and so on. These laboratory experiments and field observations may indicate that a more detailed ecological study on R. simulans is required to develop an effective biocontrol of the mango shoot borer by entomopathogenic nematodes in tropical countries.

ACKNOWLEDGEMENT

The authors express sincere thanks to Prof. N. ISHIBASHI, Saga University, for his valuable comments on this manuscript, to Mr. SHAMSUDIN,UPM, for his technical assistance in Malaysia, and to JSPS (Japan Society for the Promotion of Science) for auspice.

LITERATURE CITED

1) ARRIGORI, E. B., DINARDO, L. L. CONDE, A. J. & TERAN, F. O. (1986) Field applications of Neoa- plectana carpocapsae WEISER, 1955 to control Migdolus spp. (Coleoptera, Cerambycidae) Nematologia Brasileira 10, 181-189. 2) BEGLEY, J. W. (1990) Efficiency against insects in habitats other than soil. pp.215-132. In: Entomopathogenic nematodes in biological control (GAUGLER, R. & KAYA, H. K., eds.), CRC Press, Boca Raton, Florida. 3) CHAUDHRY, M. I. & GUL, H.(1986) Some observations on entomophagous Neoaplectana on poplar stem borer, Apriona cinerea CHEV. in NWFP. Pak. J. For. 36, 119-123. 4) DESEO, K. V. & MILLER, L. A.(1985) Efficacy of entomogenous nematodes, Steinernema spp., against clearwing , Synanthedon spp., in north Italian apple orchards. Nematologica 31, 100-108. 5) FINNEY, J. R. & WALKEY, C. (1979) Assessment of a field trial using the DD-136 strain of Neoaplectana sp. for the control of Scolytus scolytus. J. Invertebr. Pathol. 33, 239-241. 6) FORSCHLER, B. T. & NORDIN, G. L. (1988) Comparative pathogenicity of selected entomogenous

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nematodes to the hardwood borers, Prionoxystus robinidae (: Cossidae) and Megacyul- lene robiniae (Coleoptera: Cerambycidae). J. Invertebr. Pathol. 52, 343-347. 7) GAUGLER,R. & KAYA,H. K.(1990) Entomopathogenic nematodes in biological control.(GAUGLER, R. & KAYA,H. K., eds.) CRC Press, Boca Raton, Florida, 365 pp. 8) IBRAHIM,Y. B. & CHONG,K. K.(1989) Insects pests on plants of Malaysia. Dewan Bahasa Pustaka Kementerian Pendidikan Malaysia, Kuala Lumpur, 157 pp.(in Malay) 9) KASHIO,T.(1982) Laboratory evaluation of entomogenous nematodes, Neoaplectana carpocapsae WEISER,as a biological control agent of the white spotted longicorn beetle, Anoplophora malasiaca THOMASON.Proc. Plant Prot. Assoc. Kyushu 28, 194-197. (in Japanese with English summary) 10) KASHIO,T.(1986) Application of bark compost containing entomogenous nematodes, Steinernema feltiae DD-136, for the control of white spotted longicorn beetle, Anoplophora malasiaca. Proc. Plant Prot. Assoc. Kyushu 32, 175-178. (in Japanese with English summary) 11) KATAGIRI,K., MAMIYA,Y., SHIMAZU,M., TAMURA,H. & KUSHIDA,T.(1984) A spray application of Steinernema feltiae on pine logs infested with the pine sawyer, Monochamus alternatus, and its mortality induced by the nematode. Trans. 95th Ann. Meet. Jap. For. Soc., 479-480. (in Japanese) 12) KAYA,H. K. & LINDEGREN,J. E.(1983) Parasitic nematode controls western poplar clearwing moth. Calif. Agric. 37, 31-32. 13) KOBAYASHI,M., KIRIHARA,S. & OZAKI, K.(1987) The pathogenic activity of entomogenous nematodes against Psacothea hilaris larvae. In: Recent advances in biological control of insect pests by entomogenous nematodes in Japan (ISHIBASHI,N., ed.), Ministry of Education, Culture and Science, Japan. pp. 54-59.(in Japanese with English summary) 14) LI, P. S., ZHANG,S. G., ZHANG,G. Y., ZHANG,Z., WANG,Z. M., QIN, X. X. & GAO,R. T.(1987) Tests of controlling wood borers and tenthredinids with entomopathogenic nematodes. Chinese J. Biol. Control. 3, 139-140.(in Chinese) 15) LINDEGREN,J. E., CURTIS,C. E. & POINAR,G. O., Jr.(1978) Parasitic nematode seeks out Navel orange worm in almond orchards. Calif. Agric. 32, 10-11. 16) LINDEGREN,J. E. & BARNETT,W. W.(1982) Applying parasitic nematodes to control carpenter- worms in fig orchards. Calif. Agri. 36, 7-8. 17) MAMIYA,Y. & SHOJI,T.(1987) Application of entomogenous nematodes on pine logs infested with the pine sawyer, Monochamus alternatus. In: Recent advances in biological control of insect pests by entomogenous nematodes in Japan (ISHIBASHI,N., ed.) , Ministry of Education, Culture and Science, Japan. pp. 31-42. (in Japanese with English summary) 18) MILLER, L. A. & BEDDING,R. A.(1982) Field testing of the insect parasitic nematode, Neoaplectana bibionis (Nematoda: ) against currant borer moth, Synanthedon tipuliformis (Lep. Sesiidae) in black currants. Entomophaga 27, 109-114. 19) OGAWA,S.(1987) Biological control of cryptomeria bark borer (Semanotus japonicus) by inocula- tion of entomogenous nematodes (Steinernema feltiae). In: Recent advances in biological control of insect pests by entomogenous nematodes in Japan (ISHIBASHI,N., ed.), Ministry of Education, Culture and Science, Japan. pp. 43-45. (in Japanese with English summary) 20) Ooi, P. A. C.(1988) Insects in Malaysian Agriculture. Tropical Press Sdn. Bhd., Jalan Riong and Kuala Lumpur, Malaysia. 106 pp. 21) POINAR,G. 0., Jr. & DESCHAMPS,N.(1981) Susceptibility of Scotylus multistriatus to neoaplectanid and heterorhabditid nematodes. Environ. Entomol. 10, 85-87. 22) SCHMIEGE,D. C. (1963) The feasibility of using a neoaplectanid nematode for control of some forest insect pests. J. Econ. Entomol. 56, 427-431. 23) SHEN, J. D.& HAN, Q. Y.(1985) A preliminary study of controlling Rhytidodera bowrigii with DD- 136 nematode. Nat. Enemies Insects. 7, 28-29. (in Chinese) 24) YAMANAKA,K., SETA, K. & YASUDA,M.(1986) Evaluation of the use of entomogenous nematode, Steinernema feltiae (str. Mexican) for the biological control of the , Hyphantria cunea,

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(Lepidoptera: Arctiidae). Jpn. J. Nematol., 16, 26-31. 25) WASSINK, H. & POINAR, G. 0., Jr.(1984) Use of the entomogenous nematode, Neoaplectana carpocap- sae WEISER (Steinernematidae: ), in Latin America. Nematropica 14, 97-109. Accepted for publication: May 17, 1993

和文摘要

マ ン ゴ ー を 加 害 す る カ ミ キ リ ム シRhytidodera simulans

に 対 す るSteinernema carpocapsaeの 感 染 性

近 藤 栄 造 ・Abd. Rahman RAZAK

マ レー シアのマ ンゴー に大 きな被害 を もた らしてい るカ ミキ リム シ(R.simulans)幼 虫 に対 す る昆 虫寄 生性線虫S.carpocapsae(AII系 統)の 感染性 を室 内試験 で調べ た。[実 験1]円 形濾紙 を1枚 敷 いた直径6cmの ペ トリ皿 にカ ミキ リ幼虫 を1頭 つつ放飼 した後 に0.4m1の0.1%ホ ル マ リン液 に懸濁 した感 染態幼虫 を10、100、1,000あ るい は10,000頭 の密度 で接種 し、 マ ンゴー枝 の粉砕 物 を5gづ つ 加 えてか ら25℃ に保 った。 その結果、1,000頭 以上 の接種密度 で接種2日 後 か ら感染死亡 が認 め られ、 昆虫死体 内で の線 虫の発育 と増殖 が確認 された。 その際、感 染 を免 れた カ ミキ リ幼 虫 は、ペ トリ皿 の 壁面 に沿 って枝粉砕物 を用 いて坑 道 を作 ったが、接種 密度が1,000頭 を越 える と坑道 形成前 に死亡 す る ものが増 加 した。[実 験2]長 さ約20cmの マ ンゴー の切 り枝の一端 よ り長 さ約10cmの 人工 的な坑道(直 径10mm)を あ け、 この坑道 に達す る小孔(直 径3mm)を 切 り枝の側面 につ くった。 この よ うな人 工坑 道 内にカ ミキ リ幼虫 を1頭 つ つ入 れた後、坑道 末端部 を木 片 とパ ラフ ィンを用 いて封 じ、脱 イオ ン水 1m1に 懸濁 した線虫 を小 孔の周囲 に噴霧 ない し小孔 か ら注入 し、湿室 に保 った。その結 果、カ ミキ リ 幼 虫 の死亡 数 は、供試 虫数の1/6か ら1/2程 度 と少 な く、 また、線虫 の施 用方法 や施 用量 とカ ミキ リの死亡率 との間 に明瞭 な関係 は認 め られ なかった。 その原 因 を探 るため に人工坑道 中 にお けるカ ミ キ リ幼虫 の存在 状態 を調 べた ところ、坑 道 内に蓄 えられた プラス量が多 く、 小孔か ら幼 虫存在部位 ま での距離 が長 くな るにつれて感染死亡率 が低下 する傾 向が認 め られた。

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