Ethane in pine needles preventing the feeding of the beetle, Monochamus alternatus

y. Insect Physiol., 1975, Vol. 21, pp. 713 to 722. Pergamon Press. Printed in Great Britain

ETHANE IN PINE NEEDLES PREVENTING THE FEEDING OF THE BEETLE, MONOCHAMUS ALTERNATUS M. SUMIMOTO,

M. SHIRAGA,

and T. KOPJDO

Faculty of Agriculture, Kyushu University, Fukuoka, Japan (Received 30 May

1974 ; revised 5 September1974)

Abstract-Since the remarkable repulsion activity for Monochamus altevnatus of a gas from freshly ground needles of Pinus densiflora was observed by feeding tests, volatile components in the gas were submitted for further feeding tests. Among six components, five were known monoterpenic hydrocarbons, all of which showed a relatively low activity. The most abundant residual volatile in the gas was ethane, whose presence in gymnosperms has not been reported so far. Ethane showed a strong repulsion activity and was proved to be present also in the gases from other conifer needles of nine species but in less quantity than in the gas from the needles of P. denstxora. However, the order of repulsion due to various conifer needles was found to be roughly consistent with the order of ethane concentration in the gases from the respective needles. Saturated hydrocarbons with straight-chain C5 to Cl0 were also shown to be active for the beetle. INTRODUCTION

KIYOHARAAND TOKU~HIGE(1971) f ound that a group of nematodes, detected by TOKUSHIGEand KIYOHARA (1969) and named as Bumaphelenchus lignicolus by MAMIYA and KIYOHARA(1972), is capable of killing a healthy pine under certain conditions of inoculation. The vector of this nematode, which enters a healthy pine through the feeding wound caused by the vector, was found by MORIMOTO and IWASAKI (1972) and also by MAMIYA and ENDA (1972) to be a cerambycid beetle, Monochamus alternatus Hope, which attacks Pinus dens$ora as well as P. thunbergii. It is now generally believed that the cause of severe damage to pine trees, which has occurred in a wide area of the western part of Japan during the past few decades, is due to attacks by M. altematus in combination with B. lignicolus. In our preliminary feeding tests with M. altemutus, we observed that many adults appeared weakened within a few days when the entrance to a feeding cage had been covered with pine needles. This seemed to indicate the presence of some volatile and toxic constituents. The purpose of the present study was to find, with the aid of feeding tests, volatiles that prevent feeding in pine needles. In this context, repulsion due to the volatiles from some other conifer needles and related hydrocarbons was also examined. MATERIALS AND METHODS M. alternatus usedfor the feeding tests An adult beetle was fed in a test tube (2.7 x 20.0 cm), in which a 2-year-old 713 24

714

M. SUMIMOTO, M. SHIRAGA, AND T. KONDO

pine shoot (15 x ca. 0.8 cm) without needles was placed. Feeding areas on the shoot were noted every day, and healthy beetles of the same sex with a constant feeding habit, which emerged at the same date from dead pine trees laid in a net house, were chosen for further feeding tests. Feeding tests Two pine shoots 2 years old (6 x ca. 0.8 cm) and an adult beetle were put on to a filter paper placed at the bottom of a Petri dish (8.5 x 3.2 cm) with a glass cover. Areas on both shoots’ on which feeding had occurred were measured at different times and the mean values from 4 to 12 adult beetles were represented in cm2 as a standard. In other Petri dishes, 10 g of freshly ground pine needles and those of 9 species of other conifer needles, 20 mg of steam distillates from pine needles, 20 mg of each monoterpene constituent in the distillate, 20 mg of an artificial mixture of monoterpenes in a natural ratio, and 20 mg of each lower hydrocarbon were laid respectively beneath the filter paper in a Petri dish. Areas eaten were measured as described above, and average values from 6 to 12 replicates were compared with the standard. In these treatments 20 mg each corresponds to the total yield of monoterpenic hydrocarbons from 10 g of fresh pine needles. Further feeding tests with 12 mg of mixed monoterpenes and 3.6 ,ul of ethane, corresponding to the amount from 6 g of freshly ground 2-year-old pine needles, were made in a test tube (2.7 x 20.0 cm) with a silicon rubber stopper. These were compared to the standard and also to the test by 6 g of freshly ground 2year-old pine needles in the test tube. Monoterpenes and lower hydrocarbons were commercially available and used after distillation. Steam distillates were prepared from needles of P. densijlora. Freshly harvested 2-year-old needles from 9 conifers, as will be mentioned in the next section, and needles from Metasequoia glyptostroboides were used as plant materials. One-year-old needles of P. densifiora were also added for comparison. Analyses of the gases liberated from the ground needles of pine and other conifers Freshly harvested needles were ground in a iron mortar within 1 min. About 10 g of ground needles were immediately put into a centrifuge tube (15 ml vol.) with a silicon rubber stopper and this was weighed. After standing for 30 min in the tube, 1.0 ml of the gas was extracted by a gas-tight microsyringe and this was submitted to gas chromatographic analyses. A Yanagimoto Model GC-550 equipped with a flame ionization detector was used for analyses under the following conditions: 5% PEG 20M on Chromosorb W, 60 mesh, 2.25 mx 4 mm (i.d.) stainless steel, carrier gas N, 1.95 kg/cm2 at 5O”C, injector llO”C, detector 13O”C, oven 50 to 100°C at 2”C/min. IdentiJLication of P, with ethane P, (R, = 0.7 min) from the pine needles on the gas chromatogram with PEG 20M under the conditions described above was compared by gas chromatography

ETHANE

IN

PINE

NEEDLES

PREVENTING

BEETLE

71.5

FEEDING

with ethylene, ethane, and propane under the following conditions: (a) activated alumina, 60 to 80 mesh, 2.25 m x 4 mm (i.d.) stainless steel, carrier gas N, 1.0 kg/cm2 at 6O”C, injector 6O”C, detector 80°C oven 60°C; (b) active carbon, 60 to 80 mesh, 1.5 m x 4 mm (i.d.) stainless steel, carrier gas N, 1-O kg/cm2 at 80°C injector 8O”C, detector lOO”C, oven 80 to 100°C at 2”C/min; (c) molecular sieve 13X, 60 to 80 mesh, 1.5 m x 4 mm (i.d.) stainless steel, carrier gas N, 1.0 kg/cm2 at 60°C injector 7O”C, detector 120°C oven 60 to 120°C at 2”C/min. Ethylene was synthesized, and pure ethane and propane were provided by Dr. Nishimura of this university. Determimation of ethane from pine and other con$eer needles Gases from the ground pine and other conifer needles for determination were prepared as described above. Gas chromatographic determination of ethane was done by using a column packed with active carbon under the conditions mentioned above, followed by comparing with a calibration curve of ethane. RESULTS

AND DISCUSSION

Feeding tests with ground conifer needles of 10 species revealed that 2-yearold needles of P. densiflora and needles of Me&sequoia glyptostroboides were constantly the most repulsive to M. alternates. These could attract nothing in all the tests after being placed for 12 to 24 hr except in tests at the end of October, e.g. as shown in Figs. 1 and 2.. Needles of the other 8 species also showed various

-0 --O

Standard

A.firma -P. densiflora(two _P

Time, hr -+ t? thunbergiictwo ......A M. glyptostraboides yea;

years old) -0

old)

FIG. 1. Feeding tests under gases from various conifer needles. 1973). Mean values of 12 replicates.

I (12-29

June

degrees of activity for the beetle. However, the order of activity was not aIways so certain in spite of repeated tests, and only rough estimates of relative activities could be made as follows: P. ~dem@ora (2 years old), M. glyptostroboides$ Abies

716

M. SUMIMOTO,

M.

SHIRAGA, ANDT. KONDO

jirma, P. thunbergii, P. taeda > Cedrus deodora, P. densajlora (1 year old), Chamaecyparis obtusa, Cryptomeria japonica, Seiadopytis verticillata, Podocarpus macrophylus. These results, however, indicated the presence of some volatile and toxic repellants in P. densijloora and other conifers and, therefore, gases from ground

needles were subjected to gas chromatographic analyses. A gas chromatogram of the gas from pine needles on PEG ‘20M consists of six peaks, namely P, (unknown), P, (a+pinene), Pa (camphene), P, (/?-pinene), P, (myrcene), and P,

5.0

"E 0

4.0

d E 0

3.0

2

2.0 IO 0

IO

20

30

Time, hr -0 Standard -4 C. deodora F! densifloro(one year old) ‘..w --O f? densifloro(two years old) -~ FIG.

2.

40 4 _p tm

Feeding tests under gases from various conifer needles. 1973). Mean values of 6 replicates.

I

0

I

2

I 4

I

6 Time,

FIG. 3. Gas chromatogram

I IO

I 8

II (30 June-2 July

I 12

min

of gas from freshly ground 2-year-old P. den$Gra on PEG 20M.

needles of

ETHANR

(limonene)

IN PINE NEEDLES PREVRNTINGBEETLE

as shown in Fig. 3.

717

FEEDING

Since at least three peaks, P,, P,, and P, from

P. densifloora,were also found in nearly equal amounts (besides P, in a different amount on gas chromatograms from P. thunbergii, A, Jirma, and M. glyptostroboides), some of the volatiles P, to P, were supposed to be repelIants. Feeding tests under the atmosphere of each individual monoterpene and a mixture of monoterpenes as in the natural ratio of needle oil of P. den#ora actually showed relatively lower activities in comparison to those of freshly ground needles as represented in Fig. 4. No distinct difference in activities among five monoterpenes as well as mixed monoterpenes was observed. ol-Pinene and oleoresin from P. den.$ora are known to function also as attractants for M. alternatus. Therefore, it is highly probable that a compound like cl-pinene works for the beetle as an attractant when diluted, but as an anti-feedant when more concentrated. A similar relation was also described for some scolytid pine beetles by SMITH (1961, 1965). However, relatively low repulsion activities due to monoterpenes suggested some important contribution of the unknown compound P, to the whole repulsion activity revealed by the freshly ground pine needles, Comparison of the whole activity due to fresh needles with the activity due to a gas lacking most of P, but not P, to P,, was made by the following simple method. Before putting a beetle into a Petri dish, freshly ground pine needles had been placed in it for 2 hr, this being enough time to remove most of P, but not others as shown in Table 1. Eaten areas given by this method were compared to those on the freshly ground needles, as shown in Fig. 5. The result supports the significant r8le of P, as a repellant. TARLE~--RELATIVE AMOUNT OF P1 AND~,LIBERATEDFROM GROUND AFTBRSTANDINGFORDIFFRRBNTPRRIODSOFTIMRINANOPBNPLACE

0

2

P, (z,/P1(0)xlOO*

100

3

1

P,(,,/P,~~,XlOO’ .~

100

87

44

Time

standing

PINENEEDLES

6

*P 1 (0j and PZ (,-,):P, and P, from needles after standing for 0 hr. Plczj and P 2 (;2j: P1 and P, from needles after standing for 0, 2, and 6 hr, respectively. Lack of success in the isolation of P, by the usual methods, however, suggested that the volatile might be a gas with a very low boiling point. Identification of P, was therefore made by gas chromatography on activated alumina, active carbon, and molecular sieve 13X through comparison of the retention times of P, with those of ethylene, ethane, and propane, as shown in Table 2. The result and co-gas chromatography of P, with ethane by using the above three adsorbents confirmed it to

718

M.SUMIMOTO,

(I

M. SHIRAGA, ANDT.KONDO

IStandard

Time, hr (2) Monoteraenes

FIG. 4. Feeding test in an atmosphere of 5 monoterpenes. (17 July-14 August 1973). Mean values of 12 replicates were all included in the narrow area (2).

4-ol

3-o"E U 2

./

4

2.0-

_/;a

///

5

/,/" /&.J"

B LL I.0 -

/:....'" .Q"

0

;__:.q.:&" IO

, 20

I 30

Time, --o

Standard

-,-A

I 40

hr

Steam

distillates

,..+a Needles after standing for two hr in a petri-dish --o Freshly ground needles

FIG. 5. Feeding

test for comparison of the effect due to component PI from October 1973). Mean values of 10 replicates.

P. densiflora (28 September-3

be ethane. In addition, the presence of ethylene in the gas was also suggested by a very small peak with a retention time corresponding to that of ethylene. When gas chromatographic determination of ethane was made after standing ground pine needles in a sealed tube for 15, 30, and 60 min, a rapid increase in the relative amount of ethane of 1.0, 1.6, and 3.1, respectively, was observed. The result indicates that the major part of ethane from the needles might be produced by grinding and mixing the leaf-tissue. It may be worth noting again the fact that

ETHANE

IN PINENEEDLES PREVENTING BEETLEFEEDING

719

TABLE%--RELATIVERETENTION TIMEOF P1 TO ETHYLENE

Ethylene Ethane Propane P1

Activated alumina

Active carbon

Molecular sieve 13X

1.00 0.47 1.54 0.47

1.00 1.40 4.58 l-40

1.00 0.32 l-07 0.32

emission of ethane from the needles after standing for 2 hr in an open place decreases rapidly, as shown in Table 1. Since determination of ethane from needles without grinding has not been tried in the present experiments, confirmation that the residual part of ethane is either a natural product or an artefact will be discussed elsewhere. However, to save time, determinations of ethane and monoterpenes in gases were all performed after standing for 30 min in a sealed tube. The amount of ethane evolved from 1-O g of pine needles thus determined was estimated to be 0.60 ~1. Production of a minute quantity of ethane during apple ripening in storage was first reported in the course of his study on ethylene formation by MEIGH (1959). For later work on ethane from plant tissue see MEIGH (1962), CURTIS (1969), and SCOTT et al. (1971). No work on ethane from gymnosperms has so far appeared. Liberation of a remarkable quantity of ethane in contrast to that of traces of ethylene from some ground conifer needles may therefore be of significance not only from the viewpoint of plant physiology but also from that of the physiology of the insect. The relative volumes of ethane from other conifer needles when that from pine needles is 100 is shown in Table 3. This was followed, in late October, by the TABLE BASED

%-%LATIVE ON THAT

FROM

VOLUME

OF ETHANE

CORRESPONDING

EVOLVED

AMOUNT

FROM

VARIOUS

OF %-YEAR-OLD

Relative amount Pinus Cdrus Abies Pinus Pinus

de&flora deodora jrirma thunbergii taeda

lOO+O 73-6 60.6 36.9 14.7

CONIFER

NEEDLES

OF P.

NEEDLES

dens$wa

Relative amount Chamaecyparis obtusa Sciaddpytis verticillata Podocarpus macrophylus _%niperus chine&s Cryptomeria japonica

7-O 4.9 4.4 0.8

Trace

feeding tests with ethane by using the quantity corresponding to that liberated from 6 g of 2-year-old pine needles. Its comparison to the standard and to mixed monoterpenes and the ground pine needles is shown in Fig. 6. By the end of September or early October, only a few adult beetles are known to be able to

720

M. SUMIMOTO, M. SHIRAGA, ANDT. KONDO

Time, hr -* Standard -.* Needles after standing for two hr in CIpetri-dish --@ Mixed monoterpenes ....a Ethane --o Freshly ground pine needles

FIG. 6. Feeding test with ethane (27-30 October 1973). Mean values of 4 replicates. survive in the field. Beetles fed from the end of June under air-conditioning at 20°C were therefore used for thetests. However, only a few respond at this stage, as shown in Fig. 6. The result shows their insensitivity to factors to which they had always responded well until the beginning of October. In spite of these difficulties, repulsion activity caused by ethane was proved to be strong enough, and this confirmed the importance of ethane in repulsion by pine needles. Of 9 species of conifers, liberation of the largest quantity of ethane from needles was observed in 2-year-old needles of P. densiflora, as shown in Table 3. Unfortunately, needles of M. glyptostroboides with a strong repulsion activity had all fallen by late October when determination of ethane from other conifer needles was made. No accurate estimate of ethane from M. glyptostroboides could therefore be made. However, in a preliminary experiment. carried out at the end of July, the quantity of ethane was shown to be almost similar or even a little greater in comparison to that from 2-year-old pine needles. In the same experiment, the two species, i.e. P. densi$ora 2 years old and M. glyptostroboides, have more ethane than A. jkna and P. thunbergii. Moreover, monoterpene concentrations in the gases from the 4 species were shown to be similar. These findings suggest that the major difference in repulsion activities among the 4 species as shown in Fig. 1 may be attributed mainly to the difference in ethane concentration in the gases. In other words, the order of activity of the 4 species may be consistent with the order of ethane concentration in the gases. Such a relation was further supported by the fact that the order of repulsion activities due to conifer needles of 10 species as described at the beginning of this section, almost corresponds to the order of

ETHANE IN PINENEEDLESPREVENTINGBEETLEFEEDING

721

ethane concentration as given in Table 3 except that of C. deodora. The importance of ethane in repelling the beetle was thus established. However, the contribution of monoterpenes and other minor constituents to the degree of repulsion should not be disregarded. The possibility of a synergistic effect of ethane with monoterpenes may not be excluded, although no tests of this have been made. It is, however, noteworthy that needles of P. densi$ora, the host for M. alternatus, showed the strongest repulsion activity and the highest quantity of ethane among the conifers tested. It is interesting to note the fact that the beetle usually cuts off pine needles from their base in advance of feeding on a pine shoot. It is also uncertain how the natural emission of ethane from various conifer needles differs in degree from that by grinding the needles.

( I ) Standard (3)

Time, (2)Pentane,

hr nonane and decane.

Hexane, heptane and octane.

FIG.7. Feeding test in an atmosphere of saturated hydrocarbons (30 September4 October 1973).

Mean values of 6 replicates.

The facts found above indicated that liquid hydrocarbon analogues might be expected to have similar activities. Therefore, the activities of saturated hydrocarbons with straight-chain C, to C,, were examined. When a test tube containing about 100 mg of pentane and a piece of cotton wool to enhance evaporation were placed under the filter paper in a Petri dish, the beetle, on the filter paper was immediately knocked down. However, it soon revived if removed from the Petri dish within a few minutes. Therefore 20 mg of the respective hydrocarbon without adding cotton wool was used for the test. All the hydrocarbons tested, as shown in Fig. 7, were active in the following order: n-pentane, n-nonane, n-decane < n-hexane, n-heptane, n-octane. Activities of the former three nearly corresponded to those of monoterpenes. The presence of hydrocarbons as plant metabolites, e.g. isoprene (RASMUSSEN and JONES, 1973),heptane,and others(MIROV, 1967; MEIGH et al., 1973) is often reported.

722

M. SUMIMOTO,M. SHIRAGA,AND T. KONDO

In conclusion, repulsion of M. akernatus by gases from pine and other conifer needles may be attributed primarily to the ethane concentration and secondly to the monoterpene concentration. Unknown factors may also be present. Related hydrocarbons of C, to C,, were also shown to have activities in connexion with the beetle. Acknowledgements-We express our gratitude to Professor K. KINASHI, Associate Professors T. KATO and T. AOKI of the University Forest of this school for supplying the insects and the plant materials, and to Professor H. MIYAJIMA of this University for plant taxonomical guidance. Authentic gas samples were kindly provided by Associate Professor K. NISHIMURAof this university, to whom our thanks are also due. REFERENCES CURTIS R. W. (1969) Oxygen requirement for ethane production in vitro by Phaseolus vulgaris. Plant Physiol. 44, 1368-1370. KIYOHARA T. and TOKUSHIGEY. (1971) Inoculation experiments of a nematode, Bursaphelenchus sp., onto pine trees. J. rap. For. Sot. 53, 210-218. MAMIYA Y. and ENDA N. (1972) Transmission of Bursaphelenchus ZignicoZus (Nematoda: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae). Nemutologica 18, 159-162. MAMIYA Y. and KIYOHARA T. (1972) Description of Bursaphelenchus lignicolus N.SP. (Nematoda: Aphelenchoididae) from pine wood and histopathology of nematodeinfested trees. Nematologica 18,120-124. MEIGH D. F. (1959) Nature of the olefins prodiced by apples. Nature, Land. 184, 10721073. MEIGH D. F. (1962) Problems of ethylene metabolism. Nature, Lond. 196, 345-347. MEIGH D. F., FILMERA. A. E., and SELF R. (1973) Growth-inhibitory volatile aromatic compounds produced by Solanum tuberosum tubers. Phytochem. 12, 987-993. MIROV N. T. (1967) The Genus Pinus, pp. 513-515. Ronald Press, New York. MORIMOTO K. and IWASAKIA. (1972) Role of Monochamus alternatus (Coleoptera: Cerambycidae). J. rap. For. Sot. 54, 177-183. RASMUSSEN R. A. and JONESC. A. (1973) E mission of isoprene from leaf discs of Hamamelis. Phytochem. 12, 15-I 9. SCOTT K. J., WILLS R. B. H., and PATTERSON B. D. (1971) Removal by ultra-violet lamp of ethylene and other hydrocarbons produced by bananas. J. Sci. Fd Agr. 22, 496-497. SMITH R. H. (1961) The fumigant toxicity of three pine resins to Dendroctonus brevicomis and D. jeffreyi. y. econ. Ent. 54, 365-369. SMITH R. H. (1965) Effect of monoterpene vapours on the western pine beetle. J. econ. Ent. 58, 509-510. TOKUSHIGEY. and KIYOHARAT. (1969) B ursaphelenchus sp. in wood of dead pine trees. J. Jap. For. Sot. 51, 193-195.