The effect of host plant-induced stomach precipitate on the ability of Myzus persicae (Hemiptera: Aphididae) to transmit sugarbeet yellowing viruses

Transcription

1 Bulletin of Entomological Research (1997) 87, The effect of host plant-induced stomach precipitate on the ability of Myzus persicae (Hemiptera: Aphididae) to transmit sugarbeet yellowing viruses I.S. Williams 1 *, A.M. Dewar 1 and A.F.G. Dixon 2 1 IACR Broom s Barn, Higham, Bury St Edmunds, Suffolk, IP28 6NP, UK: 2 University of East Anglia, Norwich, NR4 7TJ, UK Abstract When Myzus persicae (Sulzer) feeds on healthy sugarbeet it develops a white precipitate inside its stomach which causes the stomach to enlarge. Infection of sugarbeet plants with beet yellows virus (BYV), but not beet mild yellowing virus (BMYV) results in further increases in stomach size. The influence of the white precipitate on the transmission of BYV and BMYV was investigated by rearing M. persicae on sugarbeet Beta vulgaris, Tetragonia expansa and Capsella bursa-pastoris, which are hosts for both BYV and BMYV, BYV and BMYV respectively, but the latter two hosts do not stimulate the formation of white precipitate in the aphid s stomach. Aphids reared on BYV-infected T. expansa were significantly better vectors of BYV than those reared on BYV-infected sugarbeet, but aphids reared on BMYV-infected C. bursa-pastoris did not transmit BMYV more efficiently than those reared on BMYV-infected sugarbeet. The consequences of these results for the spread of beet yellowing viruses are discussed. Introduction Aphids feeding on sugarbeet, Beta vulgaris, can be adversely affected by the plants as the plants mature. Williams (1995) has shown that the reproductive rate and longevity of Myzus persicae (Sulzer) (Hemiptera: Aphididae) decline on mature plants. Kift & Dewar (1996) have linked the early decline in populations of this species on sugarbeet to the occurrence of a black deposit in the aphid s stomach, which may be the cause of early death. The precursor of the black substance is a white precipitate, first described by Edwards (1964, 1966) and is associated with an enlargement of the aphid s stomach. Elemental analysis has shown the precipitate to be an organic complex dominated by no single compound type. Chemical hydrolysis of the precipitate recovers 25% of the compound the majority of which is glucose and amino acids. Reduction of the white precipitate appears to result in formation of the black deposit which is *Address for correspondence: Population Biology Sector, School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK. insoluble in all common solvents (Kift, 1997). The black deposit appears to form shortly before the death of the aphid. In this study the effects of beet yellowing viruses on the occurrence of the white precipitate, and of the precipitate on the ability of M. persicae to transmit the viruses are investigated. Materials and methods Virus-infected plants and aphid cultures The aphids used were from an insecticide-susceptible clone of M. persicae maintained on Chinese cabbage, Brassica pekinensis, at IACR Broom s Barn. The aphid cultures were raised in acrylic culture boxes ( cm), ventilated with electric fans and kept in greenhouses at 20 5 C. They were given a 16 h day length of natural light supplemented with tungsten lighting. The aphid cultures were raised on: (i) beet yellows virus (BYV)-infected sugarbeet; (ii) BYV-infected Tetragonia expansa (Aizoaceae); (iii) beet mild yellowing virus (BMYV)- infected sugarbeet; (iv) BMYV-infected Capsella bursa-pastoris

2 644 I.S. Williams et al. (Brassicaceae); (v) healthy sugarbeet; and (vi) Chinese cabbage (B. pekinensis). Tetragonia expansa is a host for BYV, but not for BMYV and C. bursa-pastoris is host for BMYV, but not for BYV (Bennett & Costa, 1954; Heathcote et al., 1965). The aphid cultures raised on virus-infected sugarbeet were used both for the transmission studies and for the measurement of stomach size. Those raised on T. expansa and C. bursa-pastoris were used for the virus transmission studies, and for dissection of aphids reared on these hosts to ascertain whether their stomachs contained a white precipitate. The healthy sugarbeet and Chinese cabbage cultures were maintained because M. persicae feeding on these hosts are known to possess large precipitate-filled stomachs or small clear stomachs respectively (Edwards, 1964). Chinese cabbage is not a host for either of the beet yellowing viruses. The effect of virus infection of sugarbeet, T. expansa and C. bursa-pastoris on stomach size of aphids Twelve sugarbeet plants at the 4 6 leaf stage were inoculated with either BYV or BMYV using adult apterous M. persicae. Four weeks after inoculation each plant plus six control plants of the same age were tested for both viruses using polyclonal enzyme-linked immunosorbent assay (ELISA) (Clark & Adams, 1977; Smith, 1983). This was to confirm that inoculations were successful, that no crossinfection between the viruses had occurred and that the healthy plants were still uninfected. Five weeks after infestation, when the plants were at the leaf stage, 30 adult apterous aphids were taken from each culture. An additional 30 aphids were also taken from the Chinese cabbage stock culture. Each aphid was weighed and the stomach was dissected out in phosphate buffered saline (PBS) (ph 7.4). The head of the aphid was carefully removed and the abdominal contents extracted from the body by gently pressing against the abdomen. The stomach was then easy to separate and measure. The longest and shortest axes of the stomach were measured, and the stomach volume, which approximated to a prolate sphere, was calculated using the formula 4/3ab 2 where a is half the long axis and b is half the short axis (Korin & Korin, 1961). Stomach size is an appropriate measure of accumulation as the stomach swells as it accumulates the white precipitate (fig. 1). The effect of the precipitate on transmission of BYV and BMYV Sugarbeet, cv. Saxon, was sown in a field trial at IACR Broom s Barn, Suffolk, UK on the 11 April with a seed spacing of 17.3 cm within rows and 50 cm between rows. Twenty plants within a 6 m 6 row plot were covered at the time of emergence with a 3 1 m field cage. Within each field cage, when the plants were at the 6 10 leaf stage, two aphids were clip-caged to each of ten plants. The clip cages had a diameter of 2.5 cm and were attached to the underside of a mature leaf. The aphids were previously starved overnight at 4 C; starving aphids does not affect their ability to transmit virus (Watson, 1946; Bradley, 1962) but does increase the probability of aphids settling on a new host. The aphids within each field cage came from one of the four virus-infected cultures described above and these were distributed randomly between 36 field cages, resulting in four treatments with each treatment replicated nine times. Two plants within each field cage were not inoculated, and were used as controls to determine whether natural infection from outside the cages or spread from the inoculated plants within the cages had taken place. Inoculations with aphids cultured on the BMYV-infected hosts took place seven days after those from the BYVinfected hosts. The aphids were left on the plants for three days, after which the numbers of aphids feeding, dead or walking around the cage were recorded and each clip cage was removed and the aphids killed. All plants in the field cages Fig. 1. Stomachs of Myzus persicae cultured on Chinese cabbage (upper) and sugarbeet (lower).

3 Influence of Myzus persicae stomach precipitate on virus transmission 645 were then sprayed to run off with pirimicarb (Aphox 500 g/kg; SG; Zeneca Crop Protection) at 0.7 g a.i./l. The plants were left in the field under the field cages for six weeks after inoculation to allow virus symptoms to develop. During this time all plants were sprayed every 14 days with triazamate (Aztec 140 g/l; EC; Cyanamid Agriculture UK), at 0.28 g a.i./l plus 2.5 ml/l of mineral oil, to prevent any further colonization and possible infection. This insecticide was used as it is more persistent than pirimicarb (Dewar et al., 1994). Four and six weeks after inoculation, leaf discs were taken from each of the inoculated plants and from the two control plants. The leaf discs from inoculated plants were tested for the appropriate virus and control plants were tested for both BYV and BMYV. Eight replicates were not included in the analysis because one or both of the control plants in the cage was infected and this indicated that naturally-occurring aphids could have infected plants within the cages. Polyclonal ELISA techniques were used to detect the viruses, as before. Statistical analysis The stomach sizes were compared by an analysis of covariance of aphid weight plotted against stomach volume. The behaviour of the aphids was compared using a two way ANOVA on the arcsine transformed proportion (Sokal & Rolf, 1995) of the number of aphids that were clip-caged on the sugarbeet plants in the field. A comparison of transmission efficiencies of aphids from the different hosts was carried out using a two way ANOVA on the proportion of plants infected (arcsine percent) with the virus per replicate. Results The effect of host plant and virus infection on stomach size White precipitate was found in all M. persicae cultured on sugarbeet, both healthy and virus infected (fig. 1). No white precipitate was found in aphids feeding on Chinese cabbage, T. expansa or C. bursa-pastoris. The aphids reared on healthy and BMYV-infected sugarbeet had significantly larger stomachs than aphids reared on Chinese cabbage for a comparable aphid weight (t = 5.48; d.f. = 56; P < 0.001). There was no significant difference in the stomach size of aphids feeding on BMYV-infected sugarbeet and healthy sugarbeet (t = 0.279; d.f. = 56; P > 0.05). Infection of sugarbeet with BYV further increased the stomach size of feeding aphids with these aphids having significantly larger stomachs than those Fig. 2. Analysis of covariance between stomach volume and adult weight of Myzus persicae reared on healthy sugarbeet, virus-infected sugarbeet, or Chinese cabbage. (Regression equations. BYV-infected sugarbeet, y = 0.001x 0.004; healthy sugarbeet and BMYV-infected sugarbeet, y = 0.001x 0.011; Chinese cabbage, y = 0.000x ). No significant difference between aphid stomach volume on healthy and BMYV-infected sugarbeet t = 0.279; d.f. = 56; P > Stomach volume on healthy and BMYV-infected sugarbeet significantly greater than on Chinese cabbage t = 5.48; d.f. = 56; P < and stomach volume on BYV-infected sugarbeet significantly greater than on BMYV-infected sugarbeet and healthy sugarbeet t = 2.31; d.f. = 56; P < Table 1. Effect of previous host plant on the behaviour of Myzus persicae on sugarbeet. Previous BYV-infected BYV-infected BMYV-infected BMYV-infected host sugarbeet T. expansa sugarbeet C. bursa-pastoris Feeding Mean F = F = 0.60 se p < n.s. Walking Mean F = F = 0.48 on cage se n.s. n.s. Dead Mean F = F = 2.78 se p < 0.05 n.s. Missing Mean F = F = 2.12 se p < 0.01 n.s. Percentage (arcsine transformed) of aphids, means standard error; n.s. = not significant (P > 0.05).

4 646 I.S. Williams et al. feeding on healthy and BMYV-infected sugarbeet (t = 2.31; d.f. = 56; P < 0.05) (fig. 2). The effect of the white precipitate on the transmission of BYV and BMYV Aphid behaviour Three days after infestation the numbers of M. persicae found feeding, walking on the cage, dead or missing were recorded. Significantly more of the aphids reared on BYV-infected T. expansa were observed feeding (F = 16.47; d.f. = 17; P < 0.001) than the aphids reared on BYV-infected sugarbeet. Significantly more of the aphids cultured on BYV-infected sugarbeet were missing (F = 8.97; d.f. = 17; P < 0.01) or dead (F = 4.20; d.f. = 17; P < 0.05) than the aphids reared on BYV-infected T. expansa (table 1). There was no significant difference in the number of aphids found walking in the cage. There were no significant differences between the behaviour of aphids cultured on BMYV-infected sugarbeet and those cultured on BMYV-infected C. bursa-pastoris. Virus transmission A higher proportion of sugarbeet plants became infected with BYV following infestation with aphids from BYVinfected T. expansa than with aphids from BYV-infected sugarbeet at both four (F = 5.39; d.f. = 13; P < 0.05) and six weeks (F = 5.03; d.f. = 12; P < 0.05) after inoculation (fig. 3a). However there was no difference in the proportion of sugarbeet plants that became infected whether infested with aphids from BMYV-infected sugarbeet or with those from BMYV-infected C. bursa-pastoris (fig. 3b). Discussion The results confirm those of Edwards (1964) that M. persicae reared on sugarbeet, regardless of virus infection, have significantly larger stomachs, which contain a white precipitate, than aphids reared on Chinese cabbage, T. expansa and C. bursa-pastoris. Myzus persicae reared on BYV-infected sugarbeet accumulated larger quantities of precipitate which consequently increased their stomach size more than aphids reared on BMYV-infected sugarbeet and healthy sugarbeet. This is somewhat surprising as BYV infection of sugarbeet increases the aphids growth rate, reproduction rate, nymphal survival and adult weight (Baker, 1960; Akers, 1988; Williams, 1995). However, the precipitate is the precursor to the black deposit which is associated with the premature death of the aphids (Kift & Dewar, 1996). Myzus persicae feeding on BYV-infected sugarbeet possibly have increased amounts of precipitate because these plants have a higher sucrose concentration in the phloem than healthy sugarbeet (Watson & Watson, 1951). Sucrose is a phagostimulant and an increased rate of feeding may result in more rapid accumulation of precipitate. Alternatively the precipitate itself may be composed of sucrose, or compounds derived from sucrose, and hence accumulates faster in the aphids feeding on BYV-infected sugarbeet. The lower transmission efficiency of the aphids cultured on BYV-infected sugarbeet compared with those cultured on T. expansa could be due to the precipitate either influencing virus transmission from within the aphid s body, or influencing the feeding behaviour of the aphid which, in turn, affects virus transmission. The former is unlikely since Fig. 3. (a) The percentage (arcsine transformed) of sugarbeet plants infected with BYV four and six weeks after inoculation by Myzus persicae reared on BYV-infected sugarbeet and BYVinfected Tetragonia expansa. (b) The percentage (arcsine transformed) of sugarbeet plants infected with BMYV four and six weeks after inoculation by Myzus persicae reared on BMYVinfected sugarbeet and BMYV-infected Capsella bursa-pastoralis.

5 Influence of Myzus persicae stomach precipitate on virus transmission 647 BYV is transmitted semi-persistently (Sylvester, 1956), with viable virus particles remaining in the foregut of the aphid (Watson & Plumb, 1972). The reduction in virus transmission was more likely to have been caused by differences in aphid behaviour. More aphids from the BYV-infected sugar beet culture were missing from the clip cages compared with those cultured on T. expansa, suggesting greater restlessness of the former. Also a significantly greater proportion of the aphids cultured on T. expansa settled to feed on the sugarbeet compared with those cultured on BYV-infected sugarbeet. This greater restlessness of the aphids reared on BYVinfected sugarbeet could have been caused by the presence of the precipitate. The white precipitate had no effect on BMYV transmission. In this case viable virus particles would have come into contact with the white precipitate as BMYV is a circulative virus (Russell, 1962) which passes through the fore and mid gut to the hind gut, where particles migrate through the hind gut wall into the haemocoel (Gildow, 1985) and ultimately to the accessory salivary glands (Gildow & Rochow, 1980). All aphids feeding on sugarbeet have the white precipitate in their stomach (mid gut), but only small quantities of precipitate are occasionally found in the hind gut (personal observations). As there was no difference in the transmission efficiency of BMYV by aphids reared on sugarbeet and those reared on C. bursa-pastoris, it suggests the precipitate does not inhibit the passage of the virus particles through the mid gut. Also, the small quantities observed in the hind gut are probably insufficient to inhibit the passage of virus particles into the hind gut cell wall and on into the haemocoel. The larger quantity of precipitate in the stomachs of M. persicae cultured on BYV-infected sugarbeet compared with aphids cultured on BMYV-infected sugarbeet may have resulted in their increased restlessness. Insufficient precipitate may have accumulated in the aphids cultured on BMYV-infected sugarbeet to increase this restlessness over and above that of the aphids cultured on C. bursa-pastoris. Therefore the amount of precipitate accumulated appears to influence the behaviour of M. persicae and consequently its efficiency in transmitting virus. Although in this trial the greater restlessness of aphids reared on BYV-infected sugarbeet was associated with a reduction in virus transmission to sugarbeet plants under cages, it is possible that under natural conditions it could result in more rapid dispersal of the aphids and consequently an increased spread of virus throughout the crop. Acknowledgements We are grateful for John Webb and his team for drilling the sugarbeet crop and applying the basal treatments in the field and for the technical assistance of Lisa Haylock. This work was funded by the Biotechnology and Biological Sciences Research Council and Sugar Beet Research and Education Fund. References Akers, D.E. (1988) Colonisation of sugar beet by Myzus persicae. PhD thesis, University of East Anglia, pp Baker, P.F. (1960) Aphid behaviour on healthy and on yellows-virus-infected sugar beet. Annals of Applied Biology 48, Bennett, C.W. & Costa, A.S. (1954) Observations and studies of virus yellows of sugar beet in California. Proceedings of the American Society of Sugar Beet Technologists 8, 230. Bradley, R.H.E. (1962) Effect of keeping viruliferous aphids on plants or glass on their transmission of sugar beet yellows virus. Virology 17, Clark, M.F. & Adams, A.N. 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