Vol.23 No.2 Japanese Journal of Nematology December, N993

Field Control of the Japanese Pine Sawyer , Monochamus alternatus (Coleoptera: Cerambycidae) Larvae by Steinernema carpocapsae (Nematoda: Rhabditida)

Satoshi YAMANAKA*

Field trials for control of Japanese pine sawyer (Monochamus alternatus) larvae

infesting pine logs were conducted in 1987 with the entomopathogenic nematode , Steinernema carpocapsae (str. Mexican) . Differences in spray volume and application

timing and rate were investigated. The upper surface of horizontally placed logs was

applied with 20 million infective juveniles/m2 in a spray volume of 300, 600 or 1,200 ml/

m2. The 600 ml/m2 rate resulted in the highest larval mortality. Nematodes were

applied on three different dates, March 17, 30 and April 17 with the highest mortality

of Japanese pine sawyer larvae being recorded for the April 17 application . This is attributed to the ambient air temperatures being above 12•Ž during the treatment. At

the April 17 application, rates of 6 x 106 and 12 x 106 nematodes/m2 showed 69 .2 and 72.2% mortality of larvae within the logs. Steinernema carpocapsae appears to be an

effective alternative to the chemical insecticide, fenitrothion, for treatment of pine logs

infested with the pine sawyer larvae. However, this nematodes can not prevent the

spread of pine wilt disease and integrated approach for pine sawyer suppression will

still be needed. Jpn. J. Nematol. 23: 71-78 (1993).

Key words: Biological control, forest entomology, Steinernema carpocapsae , Japanese pine sawyer, entomopathogenic nematode.

The Japanese pine sawyer, Monochamus alternatus, plays an important role in the transmis- sion of the pinewood nematode, Bursaphelenchus xylophilus (4), the causative agent of pine wilt disease. This parasitic nematode is a serious problem in Japan because it kills Japanese red pine, Pinus densiflora, and black pine, P. thunbergii, trees. To minimize the spread of pine wilt disease, two main approaches have been used. One approach is to treat infested pine logs or dead standing trees with chemical pesticides against the beetle vector. For example, control of the adult beetles during emergence from dead pine trees have been accomplished by spraying the chemical insecti- cide, fenitrothion, onto the bark surface (4). However, spraying of chemical insecticide on felled pine trees is less effective, partly because the beetle larvae occur deeply in the wood at the time of treatment. To be effective, the chemical pesticide must be applied during adult emergence. Another approach is to prevent living trees from nematode or beetle infestation . Thus nematicides such as mesulfenofos, leverumysol or morantel tartrate have been injected into trunks of living pine trees and have protected the trees (7) . Finally, the most effective control measure is the aerial application of insecticides to crowns of the living trees to prevent the

* SDS Biotech K . K. Tsukuba Technology Center, Midorigahara, Tsukuba, Ibaraki, 300-26 Japan.

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maturation feeding of M. alternatus from May through June.

Both approaches rely on the use of chemical pesticides in attempts to control the larval or

adult stages of the beetle or to protect the trees against nematode invasion. The perceived threat

of these chemical pesticides to the environment and public safety necessitates evaluation of

alternative control measure. Spraying of dead infested logs with a chemical insecticide is

considered to have less environmental impact, but this approach has not been effective. A

biological control approach, especially entomopathogenic nematodes against the immature stage

in infested logs, may provide an acceptable alternative.

The entomopathogenic nematode, Steinernema carpocapsae, is an obligate parasite of insects . The third stage infective juvenile can survive without feeding for a long time (8). The infective

juvenile enters an insect through the mouth, anus or spiracle, penetrates the gut lining or trachea and then releases its symbiotic bacterium (Xenorhabdus nematophilus) in the insect's hemocoel

(1,9). The bacterial septicemia that ensues causes the death of the insect, usually within 48 hours

(at 25•Ž). Because this nematode exhibits a host searching behavior, it has a distinct advantage

over chemical insecticides for controlling Japanese pine sawyer larvae (5) . A number of studies against beetles attacking trees suggest that entomopathogenic nematode

can be effective biological control agents. For example , FINNEY and MORDUE (2) reported the susceptibility of the elm bark beetle, Scolytus scolytus, to entomopathogenic nematodes in labora-

tory trials. MOORE (6) described the ability of the DD-136 strain of S. carpocapsae to seek out the

southern pine beetle, Dendroctonus frontalis. The smaller elm bark beetle , Scolytus multistriatus, is also susceptible to entomopathogenic nematodes (10) . Must importantly, MAMIYA and SHOJI

(5) reported that entomopathogenic nematodes have potential to control the Japanese pine

sawyer.

The purpose of my study was to investigate the possibility of surface applications of

commercially produced S. carpocapsae to infested pine logs to control M . alternatus. Nematode

dosage, spray volume of nematode suspension , and application timing were evaluated in field trials conducted in 1987.

MATERIALS AND METHODS

Nematode

The entomopathogenic nematode, Steinernema carpocapsae (str . Mexican) was obtained from

Biotechnology Australia Pty., Ltd., Roseville , Australia, and shipped by air to Japan on polyureth- ane sponge. The infective juveniles of the nematode were stored for no more than 6 weeks at 5•Ž

until used.

Influence of spray volume on efficacy

Field trials were conducted on April 7 at the Ito Country Club (Shizuoka , Japan) . Mono- chamus alternatus infested pine logs (8 logs of 1.2 m long by 10-12 cm diameter / plot) were placed parallel on the ground. The following treatments were made: untreated, 20 x 106 infective juveniles in 300, 600 or 1,200 ml of water, and 1% fenitrothion (80% EC) solution in 600 ml of water. The nematodes were sprayed on the upper surface of the exposed logs , whereas the fenitrothion was sprayed over the entire log surfaces . The application rate was 20•~106 nematodes/m2 and 6 g a.i. (active ingredient) fenitrothion/m2 . On May 13, the number of living and dead larvae were counted by destructive sampling of the logs . Counts of larvae were

―72― Vol.23 No.2 Japanese Journal of Nematology December, 1993 separated into those under the bark and those in tunnels in the wood. Influence of application timing on efficacy Nematode applications were made on three different dates, March 17 and 30 and April 17, 1987 at the SDS Biotech K. K. Minori Experimental Station (Ibaraki, Japan). Monochamus alternatus infested pine logs (20 logs of 0.8 m long by 6-10 cm diameter / plot) were placed parallel on the ground. Nematode suspensions containing either 5,000, 10,000 or 20,000 nematodes/ml in 600 ml (equal to 3 x 106, 6 x 106, or 12 x 106 nematodes/m2 of log surface, respectively) of water were sprayed on 1 m2 of the upper surface of the logs. Thirty days following application, the number of living and dead beetle larvae on the treated upper and untreated lower surfaces were counted separately. All insects on each date were placed individu- ally in plastic cups and dead insects were dissected to determine nematode infection. Tempera- ture and relative humidity during the trials were recorded.

Table 1. Effect of the entomopathogenic nematode, Steinernema carpocapsae, (str. Mexican) on pine sawyer larvae in infested pine logs at three different spray volumes.

*: nematodes were applied only on the exposed upper surface of the log whereas fenitrothion was sprayed on all surfaces of logs. **: (6/8) =6 dead larvae of 8 examined .

fi g. 1. Monochamus alternatus fourth instar larva containing large first generation adult nematodes (Steinernema carpocapsae).

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RESULTS

Influence of spray volume on efficacy The overall M. alternatus mortality in the S. carpocapsae treatment under the bark or within the tunnels at the three spray volumes showed no differences (Table 1). However, at 600 ml/m2,

fi rst trial March 17 - April 17

Second trial March 30 - April 30

Third trial April 17 - May 18

fi g. 2. Average temperature and relative humidity during three field trials with S. carpocapsae and pine sawyer at Minori Experimental Station (17 and 30 March, 17 April 1987).

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Table 2. Average temperature and relative humidity (R. H.) for each trial at Minori Experimental Station in 1987.

100% mortality of larvae in the tunnels was observed, but the larval mortality under the bark was low. At the 300 ml/m2 rate, larval mortality under the bark was higher than that in the tunnels.

Of the dead larvae, confirmed nematode mobility (Fig. 1.) was 92, 73 and 50% for the 300, 600, and 1,200 ml/m2 rate, respectively. The 1% fenitrothion solution application caused 50% total mortality of the larvae.

Influence of application timing on efficacy

Climatic data during the three trials are presented in Figure 2 and Table 2. Temperatures during the first ten days of the first trial was below 10•Ž and began to rise slightly above 10•Žby the end of the test period. Relative humidity ranged between 60 and 90%. Weather conditions were much the same for the second trial. In the third trial, temperature was over 10•Ž throughout the test period and averaged 14.1•Ž. Relative humidity ranged between 50 and 90%.

M. alternatus larval mortalities for each of the three trials are shown in Table 3. In the first trial, the overall larval mortality varied from 47 to 60%. In the second trial on March 30, overall efficacy did not exceeded 48%. For the third trial on April 17, larval mortality ranged from 44 to 72%. Nematode caused mortality in the treatments ranged from 17 - 100% (Table 3). None

Table 3. Effects of application time of the entomopathogenic nematode, Steinernema carpocap- sae, on the mortality of Monochamus alternatus larvae at Minori Experimental Station in 1987.

*: (4/6)= 4 dead larvae of 6 examined .

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of the larvae in the control logs was dead. Larval mortality for all trials due to nematode infection also occurred in the lower untreated surface. However, in general, the mortality was lower than that observed for the upper treated surface (Table 3).

DISCUSSION

The spray volume appeared to influence the efficacy of the nematode. The 600 ml spray volume provided the highest mortality (Table 1) although there appeared to be no significant differences among the three volumes tested. Based on these results, the 600 ml spray volume/m2 appears to be the most feasible. It is a sufficient volume to wet the logs whereas the 300 ml volume did not provide good coverage and the 1,200 ml volume resulted in excessive run off and loss of nematodes. However, if the logs are sufficiently moist, the 300 ml volume may be acceptable.

Nematode concentration is an important aspect for controlling pine sawyer larvae in logs.

The 6 x 106 and 12 x 106 nematodes/m2 provided the good results in the third trial (Table 3).

The earlier trials conducted when temperatures were not favorable for the nematode infection provided lower efficacy. These results suggest that the 6 x 106 nematodes/m2 at temperature exceeding 13•Ž would provide good efficacy. The influence of temperature on nematode efficacy has been documented by MAMIYA and SHOJI (5) . They described temperature effects on the pathogenicity of S. carpocapsae (str. Mexican) to M. alternatus larvae. Control efficacy was reduced when temperature dropped below 10•Ž. MAMIYA and SHOJI (5) also reported that S.

carpocapsae had the ability to kill M. alternatus larvae at 10•Ž, but the nematode did not reproduce in the cadaver in the laboratory bioassay. My data confirm the results obtained by MAMIYA and

SHOJI (5) under field conditions. That is, although some larvae appeared to be killed by nematodes, these cadavers did not contain nematodes upon dissection.

Conventional chemical insecticide applications require that all surfaces of the logs be treated.

Data from my study suggest that only the upper surface of the horizontally placed logs need to be treated with nematodes, thus making nematode treatment more convenient. The increased efficacy of the nematode can be explained by its searching ability. The nematodes migrated to the nontreated area through the pine sawyers' galleries and/or space between the bark and the wood, killing many of the larvae. However, the mortality of the pine sawyer larvae in relation to their position in the log provided mixed results. It would appear that the larvae under the bark would be most vulnerable to nematode infection because the nematodes would have greater opportunity to contact these larvae before they entered the wood. At present, the reason for the mixed results are unknown but may be related to the insect's behavior under the bark.

If measures to control the Japanese pine sawyer larvae are not maintained, emerging adults will move onto healthy trees and assist in the spread of pine wilt disease. Aerial application of chemical insecticides to kill adults during maturation feeding has provided effective control but is not sufficient to prevent pine wilt disease. Reduction of larval populations in conjunction with aerial application against adults may be the key to minimize pine wilt disease. The chemical insecticide is timed for spring and fall applications. Chemical insecticide applications are most effective in the fall when the larvae are young and feeding under the bark. Once they burrow into the logs, they are difficult to control with conventional chemical insecticides. Entomopathogenic nematodes applied in the fall or spring when temperatures are favorable have an advantage over

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chemical insecticide because of their host finding ability (5). The nematode concentrations used in this study are much higher than those used commercially for control of other coleopteran pests (3, 11). Further studies are needed to test nematodes at lower concentrations under warmer temperature conditions. Accordingly, the future for the use of entomopathogenic nematodes against pine sawyer larvae may be more efficacious and economically and environmentally acceptable. Moreover, new nematode species and strains are being discovered which may be more effective against the pine sawyer larvae. Although chemical insecticides are still needed for the control of pine sawyer adults, the integration of entomopathogenic nematode to control the larvae will reduce chemical insecticide usage.

ACKNOWLEDGMENT

I thank Miss Sayoko KAWAGUCHIfor valuable technical assistance and Dr. Kirk A. SMITH, Biosys, Palo Alto, CA., for critically reading the initial draft of this manuscript.

REFERENCES

1) AKHURST, R. J.(1980) Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes, Neoaplectana and Heterorhabditis. J. Gen. Micro. 121, 303-309. 2) FINNEY, J. R. & MORDUE,W.(1976) The susceptibility of the elm bark beetle Scolytus scolytus to the DD-136 strain of Neoaplectana sp. Ann. appl. Biol. 83, 311-312. 3) GEORGIS, R. & GAUGLER, R.(1991) Predictability in biological control using entomopathogenic nematodes. J. Econ. Entomol. 84, 713-720. 4) MAMIYA, Y.(1984) The pine wood nematode. In: Plant and insect nematodes (NicKLE W. R., ed.). Marcel Dekker, New York, 589-626. 5) 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.). Saga Univ. Saga, 31-43.(in Japanese with English summary). 6) MOORE, G. E.(1970) Dendroctonus frontalis infection by the DD- 136 strain of Neoaplectana carpocap- sae and its bacterium complex. J. Nematol. 2, 341-344. 7) OGAWA, S.(1988) Biological and physical control of the pine wilt disease. Bull. Fukuoka Forest Exper. Sta. 35, 56-73 (In Japanese). 8) POINAR, G. O., Jr.(1979) Nematodes for Biological Control of Insects. CRC Press, Boca Raton, 167 pp. 9) POINAR, G. O., Jr. & THOMAS, G. M.(1967) The nature of Achromobacter nematophilus as an insect pathogen. J. Invertebr. Pathol. 9, 510-514. 10) POINAR, G. O., Jr. & DESCHAMPS,N.(1981) Susceptibility of Scolytus multistriatus to neoaplectanid and heterorhabditid nematodes. Environ. Entomol. 10, 85-87. 11) PYE, A. E. & PYE, N. L.(1985) Different applications of the insect parasitic nematode Neoaplectana carpocapsae to control the large pine weevil, Hylobius abietis. Nematologica 31, 109-116. Accepted for publication: August 19,1993

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和文摘要

Steinernema carpocapsaeに よ る マ ツ ノ マ ダ ラ カ ミ キ リ (Monochamus alternatus)幼 虫 の 防 除 試 験

山中 聡

昆 虫 寄 生 性 線 虫Steinernema carpocapsaeメ キ シ カ ン 系 統 を 用 い て 、 マ ツ ノ マ ダ ラ カ ミ キ リ幼 虫 に 加 害 さ れ た マ ツ の 伐 倒 木 駆 除 試 験 を1987年 に 実 施 し た 。 本 研 究 に お い て 、 線 虫 懸 濁 液 量 、 線 虫 散 布 量 及び散布時期の検討を行った。 最 適 散 布 量 の 検 討 に は 、1m2当 り300,600及 び1200m1(線 虫 施 用 量:20×106Ij/m2)を 水 平 に 並 べ た 伐 倒 木 上 面 に 散 布 し た 。 こ の 試 験 で は 、1m2当 り600mlの 散 布 で の 防 除 効 果 が 他 に 比 べ て 高 か っ た 。散 布 時 期 の 検 討 で は 、3月17日 、3月30日 及 び4月17日 の 異 な る 時 期 に 散 布 し た 。 最 も 高 い 死 亡 率 が 観 察 さ れ た の は 、4月17日 の 散 布 で あ っ た 。 これ は 、 こ の 試 験 期 間 中 に 平 均 気 温 が12QC以 上 で あ っ た こ と に よ る と 考 え られ た 。4月17日 の 散 布 で は 、6×106及 び12×1061j/m2の 散 布 量 で 、 伐 倒 木 内 の 幼 虫

は 、 各 々69.2、72.2%の 死 亡 率 を 示 し た 。 散 布 し た 伐 倒 木 上 面 と 散 布 し な い 下 面 で の 幼 虫 の 死 亡 率 は 、 異 な っ て い た 。

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