JOURNAL OF INVERTEBRATE PATHOLOGY 45, 187-194 (1985)

The Infectivity of anisopliae to Two Pests of Coconuts C. PRIOR Cocoa Industry Co. Ltd., P.O. Box 1846. Rabaal, Papaa New, Guinea

AND

M. ARURA Department of Primary Industry, Bubia Agricultural Research Centre, P.O. Box 73, Lae. Papua New Guineu Received May 21, 1984; accepted August 20, 1984

Papua New Guinea isolates of Metarhiziam arzisopliae from the rhinoceros , Scapanes australis and Oryctes rhinoceros; the black palm weevil, Rhynchophorus bilineatus (Coleoptera); and three other insect orders (Hemiptera, Lepidoptera, and Dermaptera) were all short-spored (M. anisopliae var. anisopliae). Isolates from Scapanes were also pathogenic to Rhynchophorus and Oryctes. When a Scapanes isolate was grown on brown rice and released into the frond axils of young palms, 32% of dead Scapanes adults collected during the following 48 weeks were infected compared to 1% in the control nonrelease palms. Some infection of Rhynchophoras also occurred. infection in Scapanes but not Rhynchophoras also increased in a plot of palms adjacent to the release zone. The rice inoculum could be recovered and the reisolated up to 84 days after release, but infection of Scapanes continued to occur after all trace of the inoculum had disappeared. ‘!Z 1985 Academic Press. Inc. KEY WORDS: Metarhizium anisopliue: Scapanes aastralis: Rhyachophoras bilineutus; coconut pests; mycoinsecticide; biological control.

In Papua New Guinea initial damage to (Anonymous, 1983), and the Solomon Is- coconut palms by rhinoceros beetles (Co- lands (Stapley, 1980) is to apply 6% a.i. leoptera: ) is frequently fol- gamma-HCH (Lindane) granules to the lowed by lethal infestations of the black axils of the fronds at 3-month intervals or palm weevil, Rhynchophorus bilineatus less, but control is not complete. Scapanes (Coleoptera: Curculionidae), (Bedford, is now a major pest of coconuts in Papua 1974), which Smee (1965) characterized as New Guinea and, since it attacks mainly the most serious coconut pest in the young palms, its seriousness will probably country. Since Rhynchophorus usually at- increase as more old palms are replanted tacks damaged tissue, control of rhinoceros with new hybrid material. damage is vital for control of the The Metarhi- weevil. The Asiatic rhinoceros beetle, zium anisopliae has been much discussed Oryctes rhinoceros, is effectively con- as a biological agent for controlling rhinoc- trolled by the introduced Bacdovirus of Or- eros beetles (Swan, 1974). In Tonga and yctes in many countries and this virus is Samoa, artificial “trap” breeding sites for now established in Papua New Guinea Oryctes were sprayed with and very (Gorick, 1980). However, Gorick (unpubl.) high levels of larval infection were noted was unable to infect adults of the indige- (Marschall, 1980). Very little spread to nat- nous rhinoceros beetle, Scapanes australis, ural breeding sites occurred, presumably with the virus. The currently recommended because the died before emerging control method in Papua New Guinea from the infected breeding sites. A decline

187 0022-2011185 $1.50 Copyright 10 1985 by Academic Press. Inc. All rights of reproduction rn any form reserved. 188 PRIOK AND AKURA in palm damage was noted after release of pawes in laboratory trials. Brown rice was the fungus in Tonga (Marschall, 1980) and precooked in the ratio of 1 kg rice:580 ml Samoa (Young, 1974), but it was not clear water until all the water was absorbed. It if this was due to fungus-induced mortality was then autoclaved for 30 min in 25 x 40- or to the recently released Baculoviras cm “Glad” oven bags at 150°C. The bags (Swan, 1974; Young, 1974). Swan (1974) were inoculated with an aqueous suspen- concluded that Metarhizirm had failed to sion of spores and incubated for 7 days be- initiate epizootics in Oryctes populations fore use. and had no future as a biological Field release. Two- to five-year-old against Oryctes larvae. However, the pos- palms growing at Vudal Agricultural Col- sibility of applying the fungus to palms to lege near Rabaul were divided into five control Scapanes or Oryctes adults has not zones of unequal size containing a total of been tested. In the work described in this 563 palms. All recorded palms were num- paper, a locally collected isolate from Sca- bered. Scapanes and Rhynchophorus panes was applied to young coconut palms damage at this site was severe and had al- to investigate its effect on Scapanes under ready led to the loss of many palms, with field conditions. Some laboratory inocula- some replanting. The release zone, con- tion experiments are also described. These taining 127 palms, was located at one end were carried out with a view to increasing of the site. One hundred control palms were the pathogenicity of the fungus to the target also numbered in a 6-year-old block at the insects as described by Timonin et al. Lowlands Agricultural Experiment Station, (1980). Keravat, 5 km distant from the release zone. Damage was very severe in this MATERIALS AND METHODS block. Collection und culture of isolates. Col- Two surveys were made in the preceding lections of sporulating infected insects were year to check the natural level of Metarhi- made in East New Britain and New Ireland ,-iurn infection. In addition a pretreatment Provinces in the Papua New Guinea Islands survey of all the palms at the College was Region. Isolations were made initially onto made on the day before the release. All 2% malt extract agar (Oxoid), with strep- dead insects were collected and microscop- tomycin at 100 units/ml. In subsequent ically checked for infection. These insects work a modification of Doberski and were not replaced in the palms. The control Tribe’s (1980) medium was found to be block at Keravat was not included in this better. This consisted of 20 g/liter dextrose, survey. 5 g/liter peptone, 5 mg/liter crystal violet, The fungus was released in December 0.3 g/liter chloramphenicol, and 0.14 g/liter 1981, by distributing 100 g rice inoculum cycloheximide (autoclaved separately), and per palm in the axils of every frond on 12 g/liter agar. Isolates were stored on every palm in the release zone. Fortnightly slopes of 2% malt agar at 26°C. recordings were made of all live, dead, and lengths of 14 isolates were measured in dis- infected adult insects in every palm in the tilled water. release zone, the adjacent nonrelease zone, For spore production, a modification of the other nonrelease zones, and the control Latch’s (1976) medium was used: 20 g/liter zone for 48 weeks after release. Dead and dextrose, 10 g/liter peptone, 10 g/liter infected insects were marked with different casein hydrolysate, and 12 g/liter agar. colored pins. This enabled recorders to de- For field release, an isolate from Scu- tect very recent infections which had not panes was used that had already been been visible at the time the insect was first shown to be pathogenic to Oryctes and Sca- recorded. During the postrelease counts, all INFECTIVITY OF Mcturhiziwn TO TWO COCONUT PESTS 189 dead and infected insects were replaced RESULTS where they were found so as not to remove the infection source from the field. One Collection and Culture of isolates large section of the “other nonrelease Metarhizium was readily isolated onto zones” was not recorded from week 34 on- modified Doberski and Tribe’s agar. Colo- ward. nies grew slowly and sporulated after 7-9 The fortnightly data were blocked into days. The only contaminants that were 12-week periods for analysis. The propor- commonly encountered were Penicillium tions of dead to infected insects in the three SPP. zones at the release site were compared All Papua New Guinea isolates from four with the proportion in the control zone insect orders (Coleoptera, Lepidoptera, using the chi-square test (Snedecor and Hemiptera and Dermaptera) were “short- Cochran, 1967). spored” by Latch’s (1976) definition. Mean Viability of the fungus inoculum after re- spore lengths ranged from 5.5 to 7.5 km lease. Three rice grains from each of 10 ran- (4.5-8 km). All these isolates conformed to domly chosen palms were collected at in- Tulloch’s (1976) definition of Metarhizium tervals after release and visually checked anisopliae var. anisopliae. for Metarhizium. One grain from each palm Cultures remained viable for I year on was plated on modified Doberski and Tribe 2% malt agar. The fungus sporulated abun- agar for confirmation. dantly on modified Latch’s medium, pro- Laboratory inoculation experiments on ducing a thick spore layer after 7 days. Scapanes, Oryctes and Rhynchophorus. However, several isolates lost the ability to Inoculation experiments were carried out sporulate after one or two subculturings on to check the pathogenicity of isolates and agar. to try to increase pathogenicity and extend it to other target insects. Isolations were made from two infected Scapanes and two Field Release infected Rhynchophorus collected from the Only a single infected Scapanes and no release zone after release of the original iso- infected Rhynchophorus were found at late. These isolates were used for reinocu- Vudal College in the two surveys made in lation, and the fungus was then reisolated the year preceding release, and none were from infected weevils and used to inoculate found in the prerelease survey made the beetles and vice versa. It was never pos- day before release. The 12-weekly totals of sible to obtain enough insects for replicated live, dead, and infected adults in the re- experiments. lease, adjacent nonrelease, other nonre- Adult Scapanes and Rhynchophorus lease, and control zones are given in Tables (and Oryctes in one experiment) were in- 1 (Scapanes) and 2 (Rhynchophorrrs). The oculated by dipping them for 5- 10 set into data are blocked from the fortnightly aqueous spore suspensions prepared from counts, and are actual numbers of insects cultures on modified Latch’s medium by rather than percentages because of the flooding the colonies with 20 ml water plus small numbers of insects recorded at each one drop of Tween 20, brushing off the count. spores, and diluting to 500 ml. Spore con- The results in Table I show that, in the centrations were in the range 0.5- 1.5 x lo7 release zone, significantly more dead Sca- spores/ml. In one experiment, weevils were panes adults were infected than in the con- inoculated by scattering rice grains covered trol zone up to 36 weeks after release of the with sporulating fungus on the floor of the fungus. In the first 12 weeks, significantly cage. more dead Scapanes were infected in the 190 PRIOR i2ND ARURA

Adjacent Other Release nonrelease nonrelease Control Zone ZOlle Lanes zone (127) (96) (340156)” f 100) Weeks l-12 Live I87 146 293 262 Dead 97 39 78 97 Infected 39**r g*** p I Weeks 13-24 Live I28 80 89 I I9 Dead 36 20 I4 24 Infected 20** 3”‘ I”’ I Weeks 25-36 Live 49 76 49 56 Dead 13 8 3 II Infected II** I”‘ oN1 0 Weeks 37-48 Live 40 56 18 69 Dead 6 4 2 I2 Infected 1”s ONT ONT 0 Total (Weeks I-48) Live 404 358 449 506 Dead I52 71 97 144 Infected 7t*** 12*** 3”’ 2 Note. Fortnightly counts blocked into 12.week periods. Numbers in parentheses are the number of palms/ zone. “ One of those zones was not recorded after week 34. *.**.***. Significantly higher proportion of dead to infected insects compared to the control zone at P < 0.05; P < 0.01: P < 0.001, using chi-square test. ns, not significantly different: NT, not tested. adjacent nonrelease zone (50 m distant) as totals of live insects would have been lower well. No increase in infection occurred in and the proportion of total insects infected the more distant nonrelease zones. The in- would have been higher. Because of this crease in infection in Rhynchophorus (Table uncertainty, no analysis was carried out on 2) was much smaller, but in the 48-week the live insect data. total the proportion of dead adults infected There was a gradual decline in the was significantly higher in the release zone number of live and dead (uninfected) in- than in the control zone. sects recorded in all zones during the year. For both Scapanes and Rhynchophorus, This may have been an artifact due to in- a large number of live adults was recorded terference with the habitat caused by fre- at every count. These were not marked, quent sampling. and it was not possible to determine how After 26 weeks, the release zone records many of them contributed to the subse- were analyzed to determine the proportion quent totals of dead and infected insects of palms that had been attacked by Sca- and how many escaped, but it is likely that panes (and Oryctes) and/or Rhynchoph- some did escape. In preparing the live in- orus, and the proportion of palms in which sect totals for Tables 1 and 2 it was neces- Meturhizium infection had occurred (Table sary to assume that no live insects were 3). Seventy-six palms (60% of the total) counted twice. If this did happen, the real were visited by at least one Scapanes INFECTIVITY OF Merarhbim~ TO TWO COCONUT PESTS 191

TABLE 2 LIVE, DEAD, AND INFECTED Rhynchophorus ADULTS RECORDEDIN FOUR ZONES AFTER RELEASE OF Merarhiziwn Adjacent Other Release nonrelease nonrelease zone zone zones (l-27) (96) (340156)” Weeks l-12 Live 101 61 249 Dead 15 6 43 Infected 10”s 1”’ 4nz Weeks 13-24 Live 135 122 144 100 Dead 12 9 12 15 Infected 3”’ ONT (-jNl 0 Weeks 25-36 Live 90 188 176 21 Dead 13 16 23 0 Infected 2NT INT 2N” 0 Weeks 37-48 Live 32 101 15 24 Dead 3 6 0 2 Infected ONT ONT ON’ 0 Totals (Weeks I-48) Live 358 472 584 250 Dead 43 37 78 26 Infected 15* 2”’ 6”‘ I Note. Fortnightly counts blocked into 12-week periods. See Table I for numbers and superscripts. a One of these zones was not recorded after week 34. during the 26-week period, and infected Laboratory Inoculation Experiments on Scupane.s were recorded in nearly half of Scapanes, Oryctes and Rhynchophorus these. Ninety-nine percbnt of all the rhinoceros Peaks in mortality due to infection oc- beetles recorded in the course of the trial curred at 10 (6-24) days after inoculation were Scapanes, further evidence for the (Scapanes) and 14 (7-30) days (Rhyn- importance of this pest in young palms. chophorus). One isolate caused infection in 1.5 out of 22 Scapanes adults. Mortality to Rhynchophorus was low, ~20% for both Viability of the Fungus Inoculunz isolates. after Release In the experiment where Scapanes iso- Rice grains were easily found in the frond lates were inoculated onto Rhynchophorus axils up to 49 days after release, but were and vice versa, both isolates were patho- difficult to find after 84 days. By this time genic to both target insects. One Rhyn- the wetter grains were disintegrating but chophorus isolate caused mortality in 8 out the exposed and drier grains still had visible of 14 adult Scapanes, but mortality was fungus on them. No grains were found after again low in Rhynchophorus. Slightly 140 days. The fungus was still visible in higher mortality was obtained in Rhyncho- 70% of the grains collected at 84 days, and phorus when adults were inoculated by was fully viable in 100% of the grains plated scattering rice inoculum on the floor of the on modified Doberski and Tribe’s agar. cages. 192 PRIOR AND AKUKA

TABLE 3 why high mortality (>75%) was not ob- PERCENTAGE INFESTATION OF PALMS IN THE RELEASE tained with any of the target insects in the ZONE AFTER 26 WEEKS inoculations. It appeared that mortality in Rhynchophorrrs was higher when the in- Type of insect attack sects were continually exposed to the (within the 26 weeks) fungus in the cage as opposed to a single Palms with at least one recorded exposure by dipping. Scnpanes but no Rlrynchophotxs 31 For field release, a preparation of the Palms with at least one recorded fungus is required which will preserve it in Rhynchophortrs but no Sccrpanes 9 Palms with at least one recorded a sporulating and infective state for as long Rhynchophorus and one as possible; requirements which are well recorded Scaparws 39 met in nature by infected cadavers. Rice Palms with no recorded attack by autoclaved in heat-resistant plastic bags Rhynchophoms or Scapanes 31 has been used as a substrate for Metarhi- Frequency of infection (within the 26 weeks) zillm in Brazil (Aquino et al.. 1977). In Palms with at least one Tonga, Latch and Falloon (1976) found that Metarhizium-infected Scapanes 28 the fungus remained infective in Oryctes Palms with at least one breeding sites for up to 2 years when re- Mernrhizilr,n-infected leased in an oat grain substrate. The brown 8 Rhynchophotm rice inoculum released in this study was hard to detect after 12 weeks, and was un- detectable after 20 weeks. However. infec- DISCUSSION tion continued to occur in the release zone Latch (1976) found that “long-spored” after all traces of the substrate had disap- isolates of M. arzisopliae (Tulloch’s M. un- peared, and these infections could not al- isop[iae var. major) were more pathogenic ways be traced to infected cadavers. so it to Oryctes than most “short-spored” iso- appears that the spores could still be lates (var. anisopliae), but some of the present and infective after the substrate had latter were pathogenic even though they disintegrated. This indicates that infection were obtained from other insects. In Papua may occur from few spores, which is sup- New Guinea all isolates collected from co- ported by the demonstration that Scdytus leopterans and other orders were var. uni- adults can be infected by

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