第23巻 第1号 日本線虫学会誌 1993年7月 Infectivity of Entomopathogenic Nematode, Steinernema 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 Malaysia, was investigated under laboratory conditions. When the insect 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 insects 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, Chlumetia transversa, Penicillaria 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 nematodes. 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. ―28― Vol.23 No.1 Japanese Journal of Nematology July, 1993 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 moth, 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 ―29― 第23巻 第1号 日本線虫学会誌 1993年7月 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 ―30― Vol.23 No.1 Japanese Journal of Nematology July,19% 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.