Journal of Chemical Ecology, VoL 20, No. 10, 1994
WINTER HOST COMPONENT REDUCES
COLONIZATION BY BIRD-CHERRY-OAT APHID, Rhopalosiphum padi (L.) (HOMOPTERA, APHIDIDAE),
A N D OTHER APHIDS IN CEREAL FIELDS
J. PETTERSSON, I J.A. PICKETT, z'* B.J. PYE, 2 A. QUIROZ, 3 L.E. SMART, z L.J. WADHAMS, 2 and C.M. WOODCOCK z
~ Swedish University of Agricultural Sciences P. O, Box 7044, S-750 07 Uppsala, Sweden
21ACR Rothamsted Experimental Station Harpenden, Hertfordshire, AL5 2JQ, U.K.
3Laboratorio de Qulmica Ecol6gica, Departamento de Quimic~l Facultad de Ciencias, Universidad de Chile
Casilla 653, Santiago, Chile
(Received February 16, 1994; accepted May 23, 1994)
Abstract--Methyl salicylate, a volatile component of Prunus padus, the win- ter host of Rhopalosiphum padi, was found to reduce colonization of the summer host by this aphid. The compound was identified by gas chromato- graphic analysis coupled with recordings from cells in the primary rhinarium on the sixth antennal segment of the aphid. Methyl saticylate eliminated the attractancy of oat leaves to spring migrants in olfactometer tests. In Sweden, this compound significantly decreased colonization of field grown cereals by R. padi and in the U.K., populations of Sitobion avenae and Metopolophium dirhodum were significantly lower on treated plots.
Key Words--Aphid, Rhopalosiphum padi, Homoptera, Aphididae, cereal, electrophysiology, methyl salicylate, behavior, field study.
INTRODUCTION
The bird-cherry-oat aphid, Rhopalosiphum padi (L.) (Homoptera, Aphididae), is an host alternating species: bird-cherry, Prunus padus L. (Rosaceae), is the
*To whom correspondence should be addressed.
2565
0098-0331/94f 1000-2565507.00/0 Cq} 1994 Plenum Publishing Corp~lr:ation
2566 PETTERSSON ET AL.
winter or primary host and a wide range of cereals and grasses (Poaceae = Gramineae) are summer or secondary hosts. R. padi is an important pest that causes economic damage to cereals both as a vires vector and as a phloem feeder (MAFF, 1982).
The behavioral ecology of R. padi is strongly influenced by olfactory stim- uli of various types (Pettersson, 1994). Interactions with the winter host have been studied, particularly with regard to the autumn migration and the factors affecting reproductive success during this process (Leather 1981, 1986; Petters- son, 1993, 1994). There are differences in attractiveness of P. padus for ovi- position, and the immigrants select among individual plants, thus allowing the survival and reproduction of their offspring to be optimized. The effects on population size in the succeeding year have been analyzed using suction trap data and direct estimates of the feeding population (Wiktelius et al., 1990).
Spring migration of R. padi is not well understood. Aggregation behavior on cereal plants has been observed, and the existence of an aggregation pher- omone, associated with migration onto the summer host, has been demonstrated (Pettersson, 1994). It was also shown that leaves of P. padus inhibited the activity of the aggregation pheromone. This effect is comparable with a plant resistance factor decreasing a settling response and could have value in control strategies for masking host attractancy or as a repellent, since even a 20% decrease in settling would have an economic impact on damage by this pest (Wiktetius and Pettersson, 1985).
In this study, volatiles from P. padus are investigated using high-resolution gas chromatography coupled with single cell recordings from R. padi olfactory receptors. One component is identified as having significant electrophysiological activity. Behavioral responses of R. padi spring migrants to this compound are examined in olfactometer assays and field trials, and effects on the other major cereal aphid pests, the grain aphid Sitobion avenae (Fab.) and the rose-grain aphid Metopolophium dirhodum (Walk.), are also described.
METHODS AND MATERIALS
Aphids. For olfactometer studies, spring morphs of R. padi were raised from eggs laid on twigs of P. padus, collected at the Swedish University of Agricultural Sciences, Uppsala, Sweden. The twigs were placed in wet sand in a greenhouse at a temperature of 18-22°C and a 12:12 hr light-dark regime. Leaves produced on the twigs supported aphids after egg hatch long enough for alate spring migrants to develop. Alate virginoparous cereal aphids for electro- physiological studies were reared on oats, Avena sativa L. (Poaceae), c.v. Com- mander, at 20°C and a 16:8 hr light-dark regime.
Entrainment of Volatiles. Volatiles were entrained from P. padus twigs
WINTER HOST REDUCES APHID COLONIZATION 2567
with foliage standing in water and from pots of oat seedlings (ca. 10 cm) with or without colonies of R. padi. Air, dried and purified by passage through an activated 5 ~. molecular sieve and charcoal, was drawn at I liter/min through a glass culture vessel (Quickfit FV range, 11) containing plant material. Volatiles were entrained onto Porapak Q, which had been purified by washing with ether (5 ml) and heating for 12 hr at 150°C under a stream of nitrogen. Collected volatiles were desorbed from the Porapak by elution with freshly distilled diethyl ether. The resulting extract was concentrated under a stream of nitrogen and stored in sealed glass ampoules at - 2 0 ° C .
Gas Chromatography (GC). Air entrainment volatiles were separated on an AI 93 gas chromatograph equipped with a cold on-column injector, a flame ionization detector (FID), and a 50 m x 0.32 mm id HP-1 bonded phase fused silica capillary column. The oven temperature was maintained at 40°C for 1 min and then programmed at 5°/min to 100°C and then at 10°/min to 250°C. The carrier gas was hydrogen.
Coupled Gas Chromatography-Mass Spectrometry (GC-MS). A capillary GC column (50 m × 0.32 mm id HP-1) was directly coupled to the MS and integrated data system (70-250 VG Analytical). Ionization was by electron impact at 70 eV, 230°C. The GC was maintained at 30°C for 5 min and then programmed at 5°/min to 180°C. Tentative identifications by GC-MS were confirmed by comparison with authentic samples and then by peak enhancement on GC (Pickett, 1990). Methyl salicylate was obtained from Aldrich and diluted in hexane for electrophysiological and behavioral assays.
Single Cell Recording (SCR). Electrophysiological recordings from olfac- tory cells associated with the distal primary rhinaria of cereal aphid virginoparae were obtained using tungsten microelectrodes. The indifferent electrode was positioned in the first antennal segment, and the recording electrode was then brought into contact with the rhinarium until impulses were recorded. Permanent copies of the action potentials generated by the olfactory ceils were obtained by standard methods (Wadhams et al., 1982). The stimulus was delivered into a purified airstream (1 liter/min) flowing continuously over the preparation. The delivery system, employing a filter paper in a disposable Pasteur pipet cartridge, has been described previously (Wadhams et al., 1982). The impulse frequency was determined as the number of impulses elicited during the first I sec after stimulus application.
Coupled Gas Chromatography-Single Cell Recording (GC-SCR). The GC- SCR system, in which the antennal preparation is directly coupled to the cap- illary column gas chromatograph, has been described previously (Wadhams, 1990). Simultaneous records of the FID response and of the action potential frequencies were obtained by detecting with a level discriminator and plotting by means of a voltage/frequency convertor.
Olfactometry. Behavioral assays on R. padi spring migrants were carried
2 5 6 8 PETTERSSON ET AL.
out in a Perspex olfactometer (100 mm diam.) as described by Pettersson (1970), with a weak airstream directed towards the center from each of four side arms. The test stimulus was placed at the end of one of the arms, with oat leaves in the other three arms as a control. Treatments comprised (1) oat leaves + 5 spring migrants; (2) as (1) + 2 /xl eluate from P. padus entrainment; and (3) as (1) + methyl salicylate applied in a 10-#1 capillary sealed at one end. Ten aphids were introduced into the center of the chamber and their positions were noted every 2 min for 20 min. Each experiment was replicated five or six times, and results were analyzed by paired t test; the number of visits into the treatment arm were compared with the mean of the visits into the three control arms. If aphids in the arena showed little activity, the experiment was terminated and the aphids changed.
Field Experiment: Sweden 1992. A commercial field of barley, Hordeum sativum Pers. (Poaceae), c.v. Pemilla, was sown to give five replicates of five treatments in a randomized block arrangement: plot size was 2 x 5 m, with 1 m between plots. Methyl salicylate was applied in two ways. A: an emulsion (1% methyl salicylate, 2% xylene, 0.3% Ethylan B.V. wetter, 96.7% water) was sprayed directly onto the plants using an electrostatic rotary atomizer (Arnold and Pye, 1981); the plots were treated every day at noon during the experimental period, and aphid counting was started on the day after the first application. B: six slow-release polyethylene vials (WP/5, Fisons), having four top holes (I mm diam.) and containing 50 mg methyl salicylate, were positioned on 40-cm- high sticks in three pairs evenly distributed in each plot. Three control treatments were also included. These were--C: emulsion as described for A, omitting methyl salicylate; D: empty vials distributed as in B; and E: no treatment. The experiment was situated 200 m from a 12-m suction trap and was started when R. padi were found regularly in the trap. Total numbers of alate R. padi on the plots were determined by inspecting each plant for three consecutive days. On the fourth day, alatae were counted on a randomly selected l-m 2 area in each plot. Analysis of variance (ANOVA) was done on the raw and log-transformed data for each day. The log transformation used was x = log~0cy ÷ ~, where x and y represent the transformed and untransformed data, respectively. The least significant difference test (LSD) was applied to the results at P = 0.05.
Field Experiment: U.K. 1993. Treatments were set out in a 5 x 5 quasi complete Latin square in spring-sown wheat, Triticum aestivum L. (Poaceae), c.v. Canon. Each plot was 3 x 5 m with 3 m between plots, the guard strips comprising 0.5 m fallow, 2 m wheat, and 0.5 m fallow. Methyl salicylate was released from three point sources provided by polyethylene bags suspended 5-10 cm above the crop and spaced 1 m apart in the center of each plot. Four different release rates were tested, calculated by weighing the sources every 10- 11 days to be 1.0, 4.9, 46.5 and 125.4 mg/ptot/day. Total numbers of aphids
WINTER HOST REDUCES APHID COLONIZATION 2569
along a 1-m line in the center of each plot were recorded twice a week over the period May 11 to July 26 and analyzed by ANOVA.
RESULTS
Coupled GC-SCR analysis of volatiles from undamaged P. padus, using recordings from the distal primary rhinaria of alate R. padi virginoparae, showed a peak with high electrophysiological activity (Figure 1). The active compound was tentatively identified from GC-MS as methyl salicylate, and this was con- firmed by peak enhancement on GC with authentic compound. GC-MS of vol- atiles from oats, either with or without colonies of R. padi, by mass fragmentography of the diagnostic ions at m/z 92, 120, and 152, showed no trace of methyl salicylate at the appropriate retention time. The dose-response
FIG. 1. GC-SCR of volatiles from Prunus padus. Upper trace: FIB response; lower trace: action potential summation from cells in the distal primary rhinarium of Rhopa- losiphum padi. Arrow: peak from methyl salicylate.
2570 PETTERSSON El" AL.
curve for the methyl saticylate cell (Figure 2A) showed a high level of sensi- tivity, with a threshold concentration of 10-9g. Similar methyl salicylate-specific cells were found in the distal primary rhinaria of S. avenae (Figure 2B) and M. dirhodum (Figure 3). For R. padi, olfactometer tests (Table 1) showed that spring migrants feeding on oats released volatiles that attracted other spring migrants. This activity was inhibited by volatiles from P. padus leaves, and a similar effect was observed with methyl salicylate.
In the Swedish field experiment on barley in 1992, methyl salicylate applied either as an emulsion or from slow-release vials significantly reduced settling by R. padi spring migrants (Table 2). ANOVA of the log-transformed data showed no differences between the three control treatments. Therefore, the data for these treatments were combined and reanalyzed. Back-transformed means are included for clarity. In the U.K. field trial on wheat in 1993, total numbers of aphids on each treatment were recorded over a two-month period (Figure 4). Although numbers of R. padi were too low to analyze statistically, there was a significant reduction in populations of S. avenae and M. dirhodum associated
80 A B
i 60 6(~ /
40 40 _E
20 2C
So -; -~ -'T -; -4o -; -; -~ Stlmulus (log g / filter paper)
Flo. 2. Dose-response curves of olfactory cells in the distal primary rhinaria to methyl salicylate (each point is the mean of two stimulations): (A) Rhopatosiphum padi, (B) Sitobion avenae.
fITl [ I q Ill " I1] T I- I ' lll [l T lF ln,fPl[IiNImII M[[IllIl[[[IIIIl rIllrll IrllfIrr]lrll[lll f lll r'
Fie, 3 Olfactory cell in the distal primary rhinafium of Metopolophium dirhodum: re- sponse to methyl salicylate at 10 -7 g (bar = 1-see stimulation).
WINTER HOST REDUCES APHID COLONIZATION 2571
TABLE t . RESPONSES OF Rhopatosiphurn padi SPRING MIGRANTS TO DIFFERENT
STIMULI IN OLFACTOMETER a
Mean number aphids Replicates
Stimulus Treated arm Each control arm ~' P (N)
Oats + 5 spring migrants 27,2 16,3 <0.001 5 Oats + 5 spring migrants +
Prunus padus volatiles 22.2 23,2 NS 6 Oats + 5 spring migrants +
methyl salicylate 21.5 23.1 NS 6
"Cumulative counts over 20 min. NS = not significantly different at P = <0 .05 , paired t test, /'Control = oat leaves alone,
TABLE 2. FIELD TRIAL, SWEDEN 1992: EFFECT OF METHYL SALICYLATE ON
COLONIZATION OF BARLEY BY Rhopalosiphum padi
Log transformed mean no. spring migrants per plot"
Treatment Day 1 Day 2 Day 3 Day 4
Methyl salicylate 0.407ab 0~ 156a 0~739a 0.709a (emulsion) (1,55) (0.43) (4,48) (4, 12)
Methyl salicylate 0.216a 0.120a 0.658a 0.756a (vials) (0.64) (0.32) (3.55) (4.70)
Combined controls I' 0,576b 0.426b 1,025b 1.028b (2.77) (1.67) (9,59) (9.67)
SE ' 0.101 0.107 0,097 0.057
"Values in the same column followed by different letters differ significantly (LSD test, P < 0.05). Values in parentheses are back-transformed means.
hThe control treatments (blank emulsion, blank vials, no treatment) were not significantly different from each other, so are combined here for brevity.
"SE = standard error of difference between means.
with the methyl salicylate treatments. Overall reductions of alatae and apterae for both species were of the order of 40-50% (P < 0.001). No significant differences were observed between the four release rates.
DISCUSSION
Olfactory cells in the distal primary rhinarium responding specifically to methyl salicylate have now been found in many aphid species and morphs (Pickett et al., in preparation). The ubiquity of such receptors suggests that this
2572 PETTERSSON ET AL,
BB R, padi ~ S.avenae rqM.dirhodum BBTotal aphids
1000 I a
® ~= 800 l .o E 600 b b
~a 400
200
0 0 1 5 50 125
Release rate (mgs]plot]day)
FIG. 4. Field trial, U.K., May 1 l-July 26, 1993: total aphid numbers along a 1-m line in the center of each plot (difference a from b, P < 0.001).
compound may be involved in several signaling processes in aphid chemical ecology. For R. padi, the absence of methyl salicylate from summer host vol- atiles, in addition to its abundance in the winter host P. padus, would point strongly to a role in host alternation.
The general importance of volatile semiochemicals in aphid chemical ecol- ogy is now being recognized, and evidence is accumulating for the role of nonhost plant volatiles as repellents and agents capable of masking host kairo- mones (Pickett et al., 1992). Thus, the black bean aphid, Aphis fabae Scop., and the damson-hop aphid, Phorodon humuli (Schrank), are repelled by organic isothiocyanates released by members of the Brassicaceae (= Cruciferae), upon which these insects do not feed (Nottingham et al, 1991; Pickett et al., 1992; Isaacs et al., 1993). This is analogous to the role of methyl salicylate for R. padi, as a component of the winter host acting as a repellent for the spring morphs. Methyl salicylate may fulfil a similar function for M. dirhodurn in the migration from the rosaceous winter host. However, its role in the life-cycle of S. avenae is not clear, as this species remains throughout the year on cereals and grasses. The response to this compound could relate to an earlier host alternation that no longer exists. Alternatively, the methyl salicylate may be acting as a volatile signal related to host plant defense since it is the methylated metabolite of salicylic acid, a systemic plant component inducing a range of defense mechanisms. Although such a role for salicylic acid is not well char- acterized in the Poaceae, it is biosynthesized as part of the inducible phenylal-
WINTER HOST REDUCES APHID COLONIZATION 2573
anine ammonia- lyase (PAL) system that is responsible for plant phenolic-based defense (Ward et al. , 1991), known to be active against some aphids (Grayer et al., 1992). Indeed, the predatory mite Phytoseiulus persimilis Athias-Henriot (Acarina: Phytoseiidae) is reported to employ methyl salicylate, released from Lima bean, Phaseolus lunatus L. (Fabaceae = Leguminosae) on feeding by the phytophagous mite Tetranychus urticae Koch (Acari: Tetranychidae), as a for- aging stimulus (Dicke and Sabelis, 1988; Takabayashi et al., 1991; Bruin et al. , 1992). Furthermore, methyl salicylate eliminates attraction of A. fabae to its host Vicia faba L. (Hardie et al., 1994), which is also in the Fabaceae, a family with a well-established inducible chemical defense based on the PAL system (Dixon and Paiva, 1993). In addition, methyl salicylate is present in hops, Humulus lupulus (L.) (Cannabaceae), at higher levels when colonized by P. humuli, and in the olfactometer this compound eliminated the attractancy of other host-derived compounds (Campbell et al., 1993).
The significant activity of methyl salicylate against cereal aphids in the field trials indicates its potential in the development of novel control strategies. The overall reductions observed in these trials would be sufficient to allow the population to be controlled by natural enemies (Wiktelius and Pettersson, 1985). Methyl salicylate is a common constituent of foods, naturally and as a flavoring agent, which should facilitate registration if it is to be developed for agricultural use.
Acknowledgments--Part of this work was supported by the United Kingdom Ministry of Agriculture, Fisheries and Food. Financial support was also given by the International Program in the Chemical Sciences-Uppsala University, and the Swedish Agency for Research Cooperation with Developing Countries (SAREC). We would like to thank C. Bodin and G. Wadhams for technical assistance.
REFERENCES
ARNOLD, A.J., and PYE, B.J. 198t. Electrostatic spraying of crops with the APE 80. Proceedings, 1981 British Crop Protection Conference--Pests and Diseases, pp. 661-666.
BRUIN, J., DICKE, M. and SABEUS, M.W. 1992. Plants are better protected against spider-mites after exposure to volatiles from infested conspecifics. Erperientia 48:525-529.
CAMPBELl., C.A.M., PErTERSSON, J., PICKE'I-F, J.A., WADHAMS, L.J., and WOODCOCK, C.M. 1993. Spring migration of the damson-hop aphid, Phorodon humuli (Homoptera, Aphididae), and summer host plant-derived semiochemicals released on feeding. J. Chem. Ecol. 19:1569-1576.
DICKE, M., and SABELlS, M.W. 1988. HOW plants obtain predatory mites as bodyguards. Neth. J. Zool. 38:148-165.
DIXON, R.A., and PAIVA, N.L. 1993. Prospects for the genetic manipulation of antimicrobiat plant secondary products. 1993 BCPC Monograph No. 55: Opportunities for Molecular Biology in Crop Production. pp. 113-118.
GRAYER, R.J., KIMMINS, F.M., PADGHAM, D.E., HARBORNE, J.B,, and RANGA RAO, D.V, 1992. Condensed tannin levels and resistance of groundnuts (Arachis hypogaea) against Aphis crac- civora. Phytochemistry 31:3795-3800.
2574 PETTERSSON ET AL.
HARDIE, J., ISAACS, R., PICKETT, J.A., WADHAMS, L.J., and WOODCOCK, C,M. 1994. Methyl salicylate and (-)-(1R,5S)-myrtenal as repellent semiochemicals of the black bean aphid, Aphis fabae Scop. (Homoptera, Aphididae). accepted.
ISAACS, R., HARDIE, J., HICK, A.J., PYE, B.J., SMART, L.E., WADHAMS, L.J., and WOODCOCK, C.M. 1993. Behavioural responses of Aphisfobae to isothiocyanates in the laboratory and field. Pestic. Sci. 39:349-355.
LEATHER, S.R. 1981. Factors affecting egg survival in the bird-cherry-oat aphid Rhopalosiphum padi. Entomol. Exp. Appl. 30:197-199.
LEATHER, S.R. 1986, Host monitoring by aphid migrants: Do gynoparae maximise offspring fitness? Oecologia 68:367-369.
MAFF. 1982. Reference Book 186. Cereal pests. HMSO, London. 124 pp. NOTTINGHAM, S.F., HARDIE, J., DAWSON, G.W., HICK, A.J., PICKETT, J.A., WADHAMS, L.J., and
WOODCOCK, C.M. 1991. Behavioral and electrophysiological responses of aphids to host and nonhost plant volatiles. J. Chem. Ecol. 17:1231-1242.
PETrERSSON, J. 1970. An aphid sex attractant. 1. Biological studies. Entomol. Scand. 1:63-73. PETTERSSON, J. 1993. Odour stimuli affecting autumn migration of Rhopalosiphum padi (L.) (He-
miptera: Homoptera). Ann. Appl. Biol. 122:417-425. PETTERSSON, J. 1994. The bird-cherry-oat aphid, Rhopalosiphum padi (HOM. : APH,) and odours,
pp. 3-12, in S.R. Leather, A. Wyatt, N.A.C. Kidd, and K.F.A. Waiters (eds.), Individuals, Population and Patterns in Ecology. Intercept Ltd., Andover.
PICKETT, J.A. 1990. Gas chromatography-mass spectrometry in insect pheromone identification: three extreme case histories, pp. 299-309, in A.R. McCaffery and I.D. Wilson (eds.). Chro- matography and Isolation of Insect Hormones and Pheromones. Plenum Press, New York.
PICKETT, J.A., WADHAMS, L.J., WOODCOCK, C.M., and HARD1E, J. 1992. The chemical ecology of aphids. Annu. Rev. Entomol. 37:67-90.
TAKABAYASHI, J., DICKIE, i . , and POSTHUMOS, M.A. 1991. Variation in composition of predator- attracting alleclochemicals emitted by herbivore-infested plants: Relative influence of plant and herbivore. Chemoecology 2:1-6.
WADHAMS, L.J. 1990. The use of coupled gas chromatography: electrophysiological techniques in the identification of insect pheromones, pp. 289-298, in A.R. McCaffery and I.D. Wilson (eds.). Chromatography and Isolation of Insect Hormones and Pheromones. Plenum Press, New York.
WADHAMS, L.J., ANGST, M.E., and BLIGHT, M.M. 1982. Responses of the olfactory receptors of Scolytus scolytus (F.) (Coleoptera: Scotytidae) to the stereoisomers of 4-methyl-3-heptanol. J. Chem. Ecol. 8:477-492.
WARD, E.R., UKNES, S.J., WILLIAMS, S.C., D1NCHER, S.S., WIEDERHOLD, D.L., ALEXANDER, D.C., AHL-GOY, P., MI~TRAUX, J.-P., and RYALS, J.A. 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3:1085-1094.
WIKTELIUS, S., and PETTERSSON, J. 1985. Simulations of bird-cherry-oat aphid population dynam- ics: A tool for developing strategies for breeding aphid-resistant plants. Agriculture, Ecosyst. Environ. 14:159-170.
WIKTELIUS, S., WEIBULL, J., and PETrERSSON, J. 1990. Aphid host plant ecology: the bird cherry- oat aphid as a model, pp. 21-36, in R.K. Campbell and R.D. Eikenbary (eds.). Aphid-Plant Genotype Interactions. Elsevier, Amsterdam.
