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Activity of aphids associated with lettuce and broccoli in Spain and their efficiency as vectors of Lettuce mosaic virus
Virus Research 100 (2004) 83–88
Activity of aphids associated with lettuce and broccoli in Spain and their efficiency as vectors of Lettuce mosaic virus M. Nebreda, A. Moreno, N. Pérez, I. Palacios, V. Seco-Fernández, A. Fereres∗ Departamento de Protección Vegetal, Centro de Ciencias Medioambientales (CSIC), c/Serrano 115 Dpdo, Madrid 28006, Spain
Abstract This research sought to identify the aphid virus vector species associated with lettuce and broccoli crops in Spain, and to determine their population dynamics and ability to transmit Lettuce mosaic virus (LMV). Green tile traps and Moericke yellow water-pan traps were used to monitor aphid flights during the spring and autumn growing seasons of 2001. Aphid species feeding on lettuce were counted weekly. The transmission efficiencies of LMV were determined for the aphid species caught most frequently. The Moericke traps generally caught more aphid species than the tile trap, but the latter was the most suitable to estimate flight activity of species involved in virus spread. Spring aphid catches indicated that the main aphid species landing on lettuce in the regions of Madrid and Murcia was Hyperomyzus lactucae, but Brachycaudus helichrysi was also abundant in both regions. In broccoli in the Navarra region, the most abundant species in spring were Aphis fabae, B. helichrysi and H. lactucae. In autumn-sown crops, the main species landing on lettuce in the Madrid region were Hyadaphis coriandri and Aphis spiraecola. In Murcia, A. spiraecola and Myzus persicae were the most abundant, while in Navarra, Therioaphis trifolii, and various Aphis spp. were the most numerous landing on broccoli. The main aphid species colonising lettuce was Nasonovia ribisnigri, but other less abundant colonising species were Aulacorthum solani and Macrosiphum euphorbiae. The most efficient vectors of LMV were M. persicae, Aphis gossypii and M. euphorbiae, while A. fabae and H. lactucae transmitted with low efficiency, and Rhopalosiphum padi and N. ribisnigri did not transmit. Occurrence of LMV epidemics in central Spain in relation to aphid flights and the role of weeds as virus reservoirs is discussed. © 2003 Elsevier B.V. All rights reserved. Keywords: Lettuce mosaic virus; Virus transmission; Nasonovia ribisnigri; Hyperomyzus lactucae; Myzus persicae
1. Introduction Lettuce and Brassica crops (cauliflower, broccoli and cabbage) are important in the Spanish economy because they are exported, mainly to central and northern Europe. Cauliflower and broccoli exports have more than doubled in the past 5 years (from 90,922 mt in 1995 to 213,476 mt in 2000). A similar situation has occurred with lettuce and exports increased from 295,545 mt in 1995 to 459,568 mt in 2000 (Anonymous, 2002). Lettuce and Brassica spp. require less irrigation than other vegetables because they are grown during rainy seasons of the year (spring and autumn), which is particularly important in countries with limited water resources. The increase in yield of these crops achieved in Spain is due to intensification of crop production and increasing use of pesticides. However, there have been losses
∗
Corresponding author. Tel.: +34-91-5627620; fax: +34-91-5640800. E-mail address: [email protected] (A. Fereres).
0168-1702/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2003.12.016
caused by aphid-borne viruses, of which Lettuce mosaic virus (LMV) is the most important. Aphids transmit most of the viruses causing disease problems in lettuce and vegetable Brassica crops. Essentially, there are few aphid species that colonise these crops, despite the many species able to transmit viruses to them (Kennedy et al., 1962). Precise information on the population dynamics of aphid vectors alighting on crops is essential to develop appropriate forecasting methods that can predict the timing and extent of virus epidemics (Thresh, 1986). Also, it is essential to develop effective and practical aphid sampling methods to forecast the occurrence of aphid populations. This is especially important for viruses transmitted in a persistent manner. By obtaining sufficient information on aphid abundance and relative transmission efficiencies for a given virus it has been possible to calculate specific indexes that forecast virus epidemics. Examples are the one developed by Plumb et al. (1986) for estimating the incidence of Barley yellow dwarf virus in cereal crops, the vector intensity index used by Ruesink and Irwin (1986) to develop
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a simulation model for epidemics of Soybean mosaic virus in soybean crops, and the one developed by Thackray et al. (2003) for use in a simulation model for epidemics of Cucumber mosaic virus in lupin (this volume). Also, the proportion of viruliferous aphids trapped, as revealed by squash capture-PCR of individual aphids, has been used to forecast the incidence of Citrus tristeza virus (CTV) (Marroquin et al., 2002). Broadbent et al. (1951) studied the temporal and spatial patterns of spread of LMV in lettuce crops in England and identified two major vectors of the virus, Myzus persicae and Macrosiphum euphorbiae. These authors also showed that the use of virus-tested seed is the most effective way of reducing the incidence of LMV. In recent years, outbreaks of LMV have occurred in large areas of central Spain, causing serious yield losses, especially in autumn-grown lettuce (Moreno et al., 2003). The reasons for these outbreaks are unknown, but the recent introduction of ornamentals that act as alternative hosts of LMV has been associated with outbreaks of the virus in the Salinas valley, CA, USA (Zerbini et al., 1997). Also, several weed and ornamental species of the Compositae are potential reservoirs of LMV, which is seed-borne in common weeds including Lactuca serriola and Senecio vulgaris (Tomlinson, 1970). This paper reports, the relative abundance of aphid species alighting on lettuce and broccoli crops in three regions of Spain during the spring and autumn growing seasons in 2001. In addition, aphid species able to colonise lettuce and the transmission efficiencies of LMV by species that alight frequently on lettuce were determined. Finally, the key factors that drive LMV epidemics in lettuce in central Spain during the autumn growing season are discussed.
2. Aphid species associated with lettuce and broccoli crops Aphid species alighting on the crop were monitored in fields of lettuce at two different sites in Navalcarnero (Madrid) and Roldan (Murcia), and in fields of broccoli at one site in Ribaforada (Navarra) during the spring and autumn growing seasons of 2001. Two types of traps were used at each sampling site: a horizontal mosaic-green tile (15.5 cm × 15.5 cm) trap (Irwin, 1980) and a Moericke yellow pan (60 cm × 60 cm surface) water trap. In addition, the aphid species colonising lettuce were sampled directly by visual counting at Navalcarnero. 2.1. Aphid flights in lettuce and broccoli crops The two trapping periods were from date of transplanting until harvest: March–May for spring crops and September–November for autumn crops. Insect samples were collected weekly from the traps and aphids were separated in the laboratory using a stereomicroscope (Leica Microsystems). All aphid specimens were preserved in 70%
ethanol and they were prepared and identified as described previously (Perez et al., 1995). A permanent collection of voucher specimens provided by J.M. Nieto (Universidad de León, Spain) was used to verify identification. The Moericke traps generally caught more aphid species than the tile traps at all sampling locations. That fewer species were caught by the tile trap is probably because of its smaller size (Boiteau, 1990). However, the tile trap is the most suitable to determine which aphids land on the crop because its colour closely resembles that of row crops, based on data from reflectance spectrophotometry (Irwin and Ruesink, 1986). Moreover, the Moericke trap tends to overestimate numbers of certain aphid species, such as M. persicae, which alight preferentially on yellow targets (Boiteau, 1990; Fereres et al., 1999). Fig. 1 shows a 10-fold increase in the numbers of M. persicae trapped in the Moericke trap compared with those caught in the tile trap at Navalcarnero in spring 2001. In our study, although the tile trap was more selective than the Moericke trap, its catches represented well the most abundant aphid species present, including Hyperomyzus lactucae and Aphis fabae. H. lactucae was the most abundant aphid species caught in lettuce during spring, 2001. Cumulative catches of H. lactucae at Navalcarnero were 750 and 600 individuals per metre square in the tile and Moericke trap, respectively (Table 1). Cumulative catches of H. lactucae at Roldan were 1292 and 1381 individuals per metre square in the tile and Moericke traps, respectively. Other abundant species landing on lettuce were A. fabae and Brachycaudus helichrysi at Navalcarnero, and B. helichrysi and Aploneura lentisci at Roldan. In broccoli, A. fabae was most abundant. Cumulative catches of A. fabae at Ribaforada were 8083 and 506 individuals per metre square in the tile and Moericke traps, respectively (Table 1). Other abundant aphid species landing in broccoli plantings were B. helichrysi and H. lactucae. During the spring growing season at Navalcarnero, there was a peak of M. persicae landing in the week of 22–29 May (125 aphids per metre square in the tile trap and 958 aphids per metre square in the Moericke trap). At Roldan, H. lactucae and Hayhurstia atriplicis were most numerous during the week of 10–17 April, when catches in the tile trap were 625 and 208 aphids per metre square, respectively. At Ribaforada, the peak density occurred during the week of 10–17 May when there were many A. fabae (3083 aphids per metre square in the tile trap). The most abundant aphid species present in autumn-sown lettuce or broccoli differed from those found in spring of the same year. H. lactucae was most numerous in spring and was almost absent in the autumn season. During the autumn heavy rains were frequent and an unknown number of aphids landing on the tile traps were washed out of the trap and lost before the collection date. Therefore, Moericke trap captures were considered more reliable in the autumn because they had four drainage holes protected by nets that retain aphids inside the trap. The most abundant species captured in Moericke traps in Navalcarnero during autumn
M. Nebreda et al. / Virus Research 100 (2004) 83–88
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Fig. 1. Comparison between the cumulative number of aphids caught in each type of trap located in a lettuce field at Navalcarnero, Madrid during spring, 2001.
were Hyadaphis coriandri and Aphis spiraecola (Table 2). Peak flights were in the 1-week of October for H. coriandri (97 aphids per metre square) and mid-September for A. spiraecola (83 aphids per metre square). Other Aphis spp. were
Table 1 Cumulative numbers of aphids per metre square and percentage (in parentheses) of total catches of the most abundant aphid species landing in two lettuce fields located at Navalcarnero (Madrid) and Roldan (Murcia), and one broccoli field located at Ribaforada (Navarra) during spring, 2001 Location
Aphid speciesa
Navalcarnero (Madrid)
H. lactucae
750 (24)
600 (17)
A. fabae B. helichrysi Brachycaudus rumexicolens M. persicae
375 (12) 292 (9) 167 (5)
253 (7) 178 (5) 228 (7)
Roldan (Murcia)
Ribaforada (Navarra)
125 (4)
Moericke trap
Table 2 Cumulative numbers of aphids per metre square and percentage (in parentheses) of total catches of the most abundant aphid species landing in two lettuce fields located at Navalcarnero (Madrid) and Roldan (Murcia), and one broccoli field located at Ribaforada (Navarra) during autumn, 2001 Location
Aphid speciesa
Navalcarnero (Madrid)
H. coriandri A. spiraecola M. persicae A. fabae Aphis sp.
1,028 (30)
Total
3,125
H. lactucae B. helichrysi Aploneura lentisci H. atriplicis Hyalopterus pruni
1,292 875 500 42 0
Total
3,833
A. fabae
8,083 (30)
506 (11)
B. helichrysi H. lactucae Aphis sp. Brachycaudus cardui
4,000 1,958 1,750 1,500
225 273 202 297
3,447 (34) (23) (13) (1) (0)
1,381 750 411 517 583
Total (27) (15) (8) (10) (12)
Roldan (Murcia)
5,061 Ribaforada (Navarra)
Total a
Tile trap
also very common at the end of September along with M. persicae that reached a peak of 55 aphids per metre square during the 1-week of October. At Roldan, the most abundant species landing on autumn-sown lettuce were A. spiraecola and M. persicae (Table 2). In Ribaforada, the most abundant aphid species landing in broccoli fields were Therioaphis
26,541
(15) (7) (7) (6)
(5) (6) (5) (7)
4,412
The species shown were those most abundant in the tile traps.
a
A. spiraecola M. persicae Aphis sp Pemphigus sp. Sitobion avenae
Tile trap 42 0 42 0 84
(17) (0) (17) (0) (33)
252 42 42 84 84 84
Total
417
Therioaphis trifolli A. craccivora Aphis sp A. spiraecola A. fabae
208 84 84 125 125
Total
917
Moericke trap 278 183 156 128 125
(21) (14) (12) (10) (10)
1,300 (10) (10) (20) (20) (20)
492 200 22 5 0
(60) (24) (3) (1)
822 (23) (9) (9) (14) (14)
61 56 19 14 8
(15) (14) (5) (3) (2)
411
The species shown were those most abundant in the Moericke traps.
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trifolii and Aphis craccivora, probably flying from fields of alfalfa located near the sampling area (Table 2). 2.2. Aphid species colonising lettuce fields Aphid species feeding on lettuce were also sampled weekly in 2001 during spring and autumn at Navalcarnero. Spring crops were sampled from 24 April to 29 May and autumn crops from 21 September to 25 October. Each lettuce field was divided into four quadrants and 10 plants per quadrant were selected randomly following a zig-zag sampling pattern. No pesticides were used in the selected quadrants of the lettuce field during the entire crop growth cycle. All leaves of each plant were sampled by counting the number of aphids and morphs of each species. Aphid species and morphs were identified directly in the field using a magnifying hand lens. The most abundant aphid species in both spring and autumn crops was Nasonovia ribisnigri. Other species that formed small colonies on lettuce were M. euphorbiae in spring and Aulacorthum solani in autumn. Peak populations of N. ribisnigri occurred at the end of each crop growth cycle: on 29 May (80 individuals per plant) for the spring crop and on 25 October (50 individuals per plant) for the autumn crop. Most of the morphs recorded were nymphs followed by adult apterae and alatae, which were far fewer. Population densities of the two other species were much lower. In spring, M. euphorbiae reached a peak of two individuals per plant just after transplanting on 21 April, but was displaced by N. ribisnigri 2–3 weeks later. In autumn, A. solani reached a maximum density of 11 individuals per plant on 18 October, but its numbers were always much smaller than those of N. ribisnigri.
3. Estimation of the transmission efficiency of LMV by different aphid species LMV was the virus most frequently detected in lettuce and vegetable Brassica crops surveyed in Spain in 2001 and 2002 (Moreno et al., 2003). The virus was particularly abundant in lettuce crops and weeds in the Madrid region. LMV occurred frequently in lettuce at Navalcarnero during October of 2001 and 2002. LMV was detected in Sonchus tenerrimus and S. vulgaris collected at Navalcarnero in early March 2002 and in several weed species (Sonchus oleraceus, Malva sp., S. vulgaris, Gallinsoga parviflora and Chenopodium quinoa) collected at Navalcarnero and Villa del Prado (Madrid) during August–October, 2002. Therefore, the aphid species found landing frequently on lettuce in Navalcarnero were tested to assess their potential as vectors of LMV. Also, the aphid species found colonising lettuce, together with Aphis gossypii, which is another vector of LMV (Kennedy et al., 1962) were included in the tests. All transmission experiments were repeated three times for each aphid species. Aphid clones used for the
study were from different Spanish localities and maintained in the following host plants: M. persicae (from El Encin, Madrid) on sweet pepper, A. gossypii (El Ejido, Almeria) on melon, M. euphorbiae and N. ribisnigri (Villa del Prado, Madrid) on lettuce, A. fabae (Murcia) on faba bean, H. lactucae (Matalpino, Madrid) on sowthistle (S. oleraceus) and Rhopalosiphum padi (El Encin, Madrid) on barley. Transmission experiments were conducted under laboratory conditions (21 ± 2 ◦ C) using a procedure that provides a good estimate of aphid propensity (sensu Irwin and Ruesink, 1986). Accordingly, groups of 25–30 aphids were placed inside empty plastic cages for a 1 h pre-acquisition starvation period. The youngest expanded leaf of a young lettuce (cv. Cazorla) plant aphid-inoculated 3–4 weeks earlier was used as a source for virus acquisition by the aphids. The leaf was detached from the plant after completion of the aphid starvation period. The petiole of the infected leaf was then placed in an Eppendorf tube previously filled with water to keep the leaf turgid during the test and the whole set-up was placed in a plastic cage. Twenty young apterous adult aphids were released at a time on the upper surface of the infected leaf. After a 5 min acquisition access period, groups of five aphids were transferred to each test plant. These were 1-month-old seedlings planted in a 28-rack tray. Aphids found either on the leaf surface or around the cage were used to inoculate the test plants. These aphids were kept on the test plants by covering with plastic cups for at least a 2 h inoculation feeding time. All plants were then sprayed with imidacloprid (Confidor® ) and transferred to a growth chamber at 26:20 ◦ C (day:night), 16:8 h, (light:dark) and a light intensity of 100 E m−2 s−1 . A 28-rack tray of lettuce (cv. Cazorla) seedlings was used as an uninoculated control in each transmission experiment. In these experiments, aphids were able to move freely around the infected and test plants. Leaf samples from all test plants were tested for LMV using double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Clark and Adams, 1977) 4–5 weeks after inoculation, using an LMV-specific monoclonal antibody (Agdia Inc., USA). Alkaline phosphatase was the enzyme used and p-nitrophenyl phosphate the substrate. Absorbance at 405 nm was measured after 1 h with an ELISA plate photometer (Lab Instruments, Austria). Test plants were recorded as infected when absorbance values were at least three times those of the mean of the healthy control. The data on transmission rate (number of infected plants divided by number of test plants) were subjected to analysis of variance (Abacus Concepts, √ 1989) after using the following transformation: X = arcsin (x + 1/100). Multiple mean comparisons were made between the transmission rate obtained for each aphid species using the Fisher’s protected LSD test (Abacus Concepts, 1989). Significant differences (F = 42.2; d.f. = 6, 14; P = 0.0001) in the transmission rate of LMV were apparent among the different aphid species tested (Table 3). M. persicae and A. gossypii were the most efficient vectors, followed by M. euphorbiae, A. fabae and H. lactucae transmitted less
M. Nebreda et al. / Virus Research 100 (2004) 83–88 Table 3 Transmission efficiencies for LMV by each of seven aphid species Aphid species
Mean ± S.E.a
M. persicae A. gossypii M. euphorbiae A. fabae H. lactucae R. padi N. ribisnigri
42.4 33.2 26.2 15.4 9.5 1.2 0
± ± ± ± ± ± ±
4.2 a 4.4 ab 5.2 b 1.2 c 1.2 c 1.2 d 0d
a Means followed by different letters are significantly different according to Fisher’s protected LSD test (n = 3).
efficiently. The transmission rate of R. padi was low (only one of 84 plants became infected), and was not significantly different from that of N. ribisnigri, which never transmitted LMV. None of the plants from the 28-rack trays of non-inoculated lettuce seedlings became infected with LMV.
4. Discussion Spread of viruses transmitted in a non-persistent manner by aphids often occurs when non-colonising species land in large numbers on the crop (Raccah et al., 1985; Perez et al., 1995). Some aphid species actively select the Moericke traps in which they can be as much as 30 times more numerous than in suction traps at the same site (Eastop, 1955). Therefore, when studying plant virus epidemics it is important to have a good approximation of the absolute landing rates from tile traps. These provide a good estimate of aphid activity (sensu Irwin and Ruesink, 1986), because they assess the aphid species that actually land on the crops and therefore are involved in transmission. In our study, the tile trap caught fewer aphid species than the Moericke trap, but its captures still represented well the most abundant aphid species landing on the crop, H. lactucae or A. fabae. Tile traps would provide even better information if they could be modified to prevent spilling of the samples when used during heavy periods of rain. LMV was the most widespread virus of lettuce in Navalcarnero during the autumn of 2001 (Moreno et al., 2003). We confirmed previous studies (Broadbent et al., 1951) showing that M. persicae is the most efficient vector of LMV. It is likely that M. persicae was involved in the spread of LMV in Navalcarnero that was detected in the last week of October 2001 because it reached a peak of 55 aphids per metre square during the 1-week of October. However, other Aphis species were also abundant at the time and probably also contributed to the spread of LMV. In Ribaforada, LMV was also present in autumn-sown broccoli crops (Moreno et al., 2003), where A. spiraecola and other Aphis spp. species were abundant (Table 2). A. spiraecola was one of the most abundant species landing on lettuce crops during the autumn season when LMV epidemics are usually most severe. It is not known if this species is a vector of LMV and transmission experiments are required to determine this. In
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other areas where open-field lettuce is widely grown such as Murcia, LMV has been detected recently in some samples collected at three different sites in October, 2002 (Moreno et al., 2003). Moreover, there is a high risk of LMV epidemics in this region because M. persicae and other vector aphid species are abundant during the autumn (Table 2). It is unlikely that aphids found feeding and reproducing in lettuce are involved in the secondary spread of LMV within plantings because the only aphid species that formed large colonies was N. ribisnigri, which is not a vector. LMV epidemics tend to be much more severe in autumn than in spring-sown lettuce crops. The reason for this is unknown. Nevertheless, the fact that aphid species trapped in autumn differ from those present in spring probably does not account for such differences because high numbers of potential vectors of LMV land on the crop during both seasons. However, the most efficient vector of LMV, M. persicae, was not trapped on spring lettuce in Navalcarnero until late May 2001, by which time most crops had been harvested. Also, the lack of LMV disease outbreaks in spring lettuce may be related to the absence of virus reservoirs (lettuce and weeds) during the winter months. In central Spain, where severe LMV epidemics have occurred in each of the years 2000–2002, lettuce is grown from March to November. LMV is likely to over-winter in the seeds of several weed species such as S. vulgaris that were found infected at the beginning of March in Navalcarnero before any aphid flight activity was detected. Also, weeds are commonly infected with LMV during summer and the beginning of autumn in this region. Therefore, both summer lettuce crops and weeds may act as a primary source of inoculum for LMV epidemics during early autumn when transient aphid vectors are searching for new hosts. Control of LMV is difficult because it is seed-borne in lettuce and also in certain common weed species. Although production of virus-tested lettuce seed is the best option to control the disease (Grogan, 1980), weeds that act as important reservoirs for the virus should be also removed, especially S. vulgaris and other weed species in which LMV is seed-borne (Tomlinson, 1970). The seed bank of such weed species may maintain an important source of virus inoculum during the periods where no susceptible crops are available and is probably important in initiating LMV epidemics. Acknowledgements We thank R. Biurrun, C. Ortuño, and J.L. Nieves for their help in aphid sampling. This work was supported by the Comisión Interministerial de Ciencia y Tecnolog´ıa (Research Grant CICYT No. AGL-2000-2006). References Abacus Concepts, 1989. SuperANOVA. Abacus Concepts. Berkeley, CA, p. 322.
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Moreno, A., De Blas, C., Palacios, I., Duque, M., Fereres, A., 2003. The incidence and distribution of viruses infecting lettuce, cultivated Brassica and associated natural vegetation in Spain. Ann. Appl. Biol. (in press). Perez, P., Collar, J.L., Avilla, C., Duque, M., Fereres, A., 1995. Estimation of vector propensity of potato virus Y (PVY) in open-field pepper crops of central Spain. J. Econ. Entomol. 88, 986–991. Plumb, R.T., Lennon, E.A., Gutteridge, R.A., 1986. Forecasting barley yellow dwarf virus by monitoring vector populations and infectivity. In: McLean, G.D., Garrett, R.G., Ruesink, W.G. (Eds.), Plant Virus Epidemics: Monitoring, Modelling and Predicting Outbreaks. Academic Press, Sydney, pp. 387–398. Raccah, B., Gal-On, A., Eastop, V.F., 1985. The role of flying aphid vectors in the transmission of cucumber mosaic virus and potato virus Y to peppers in Israel. Ann. Appl. Biol. 106, 451–460. Ruesink, W.G., Irwin, M.E., 1986. Soybean mosaic virus epidemiology: a model and some implications. In: McLean, G.D., Garrett, R.G., Ruesink, W.G. (Eds.), Plant Virus Epidemics: Monitoring, Modelling and Predicting Outbreaks. Academic Press, Sydney, pp. 295– 313. Thackray, D.J., Diggle, A.J., Berlandier, F.A., Jones, R.A.C., 2003. Forecasting aphid outbreaks and epidemics of cucumber mosaic virus in lupin crops in a mediterranean-type environment. Virus Res. (this volume). Thresh, J.M., 1986. Plant virus disease forecasting. In: McLean, G.D., Garrett, R.G. Ruesink, W.G. (Eds.), Plant Virus Epidemics: Monitoring, Modelling and Predicting Outbreaks. Academic Press, Sydney, pp. 359–386. Tomlinson, J.A., 1970. Lettuce mosaic virus. Descriptions of Plant Viruses No. 9. Commonw. Mycol. Inst. Assoc. Appl. Biol., Kew, England. Zerbini, F.M., Koike, S.T., Gilbertson, R.L., 1997. Gazania spp.: a new host of lettuce mosaic potyvirus, and a potential inoculum source for recent lettuce mosaic outbreaks in the Salinas valley of California. Plant Dis. 81, 641–646.
Activity of aphids associated with lettuce and broccoli in Spain and their efficiency as vectors of Lettuce mosaic virus M. Nebreda, A. Moreno, N. Pérez, I. Palacios, V. Seco-Fernández, A. Fereres∗ Departamento de Protección Vegetal, Centro de Ciencias Medioambientales (CSIC), c/Serrano 115 Dpdo, Madrid 28006, Spain
Abstract This research sought to identify the aphid virus vector species associated with lettuce and broccoli crops in Spain, and to determine their population dynamics and ability to transmit Lettuce mosaic virus (LMV). Green tile traps and Moericke yellow water-pan traps were used to monitor aphid flights during the spring and autumn growing seasons of 2001. Aphid species feeding on lettuce were counted weekly. The transmission efficiencies of LMV were determined for the aphid species caught most frequently. The Moericke traps generally caught more aphid species than the tile trap, but the latter was the most suitable to estimate flight activity of species involved in virus spread. Spring aphid catches indicated that the main aphid species landing on lettuce in the regions of Madrid and Murcia was Hyperomyzus lactucae, but Brachycaudus helichrysi was also abundant in both regions. In broccoli in the Navarra region, the most abundant species in spring were Aphis fabae, B. helichrysi and H. lactucae. In autumn-sown crops, the main species landing on lettuce in the Madrid region were Hyadaphis coriandri and Aphis spiraecola. In Murcia, A. spiraecola and Myzus persicae were the most abundant, while in Navarra, Therioaphis trifolii, and various Aphis spp. were the most numerous landing on broccoli. The main aphid species colonising lettuce was Nasonovia ribisnigri, but other less abundant colonising species were Aulacorthum solani and Macrosiphum euphorbiae. The most efficient vectors of LMV were M. persicae, Aphis gossypii and M. euphorbiae, while A. fabae and H. lactucae transmitted with low efficiency, and Rhopalosiphum padi and N. ribisnigri did not transmit. Occurrence of LMV epidemics in central Spain in relation to aphid flights and the role of weeds as virus reservoirs is discussed. © 2003 Elsevier B.V. All rights reserved. Keywords: Lettuce mosaic virus; Virus transmission; Nasonovia ribisnigri; Hyperomyzus lactucae; Myzus persicae
1. Introduction Lettuce and Brassica crops (cauliflower, broccoli and cabbage) are important in the Spanish economy because they are exported, mainly to central and northern Europe. Cauliflower and broccoli exports have more than doubled in the past 5 years (from 90,922 mt in 1995 to 213,476 mt in 2000). A similar situation has occurred with lettuce and exports increased from 295,545 mt in 1995 to 459,568 mt in 2000 (Anonymous, 2002). Lettuce and Brassica spp. require less irrigation than other vegetables because they are grown during rainy seasons of the year (spring and autumn), which is particularly important in countries with limited water resources. The increase in yield of these crops achieved in Spain is due to intensification of crop production and increasing use of pesticides. However, there have been losses
∗
Corresponding author. Tel.: +34-91-5627620; fax: +34-91-5640800. E-mail address: [email protected] (A. Fereres).
0168-1702/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2003.12.016
caused by aphid-borne viruses, of which Lettuce mosaic virus (LMV) is the most important. Aphids transmit most of the viruses causing disease problems in lettuce and vegetable Brassica crops. Essentially, there are few aphid species that colonise these crops, despite the many species able to transmit viruses to them (Kennedy et al., 1962). Precise information on the population dynamics of aphid vectors alighting on crops is essential to develop appropriate forecasting methods that can predict the timing and extent of virus epidemics (Thresh, 1986). Also, it is essential to develop effective and practical aphid sampling methods to forecast the occurrence of aphid populations. This is especially important for viruses transmitted in a persistent manner. By obtaining sufficient information on aphid abundance and relative transmission efficiencies for a given virus it has been possible to calculate specific indexes that forecast virus epidemics. Examples are the one developed by Plumb et al. (1986) for estimating the incidence of Barley yellow dwarf virus in cereal crops, the vector intensity index used by Ruesink and Irwin (1986) to develop
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a simulation model for epidemics of Soybean mosaic virus in soybean crops, and the one developed by Thackray et al. (2003) for use in a simulation model for epidemics of Cucumber mosaic virus in lupin (this volume). Also, the proportion of viruliferous aphids trapped, as revealed by squash capture-PCR of individual aphids, has been used to forecast the incidence of Citrus tristeza virus (CTV) (Marroquin et al., 2002). Broadbent et al. (1951) studied the temporal and spatial patterns of spread of LMV in lettuce crops in England and identified two major vectors of the virus, Myzus persicae and Macrosiphum euphorbiae. These authors also showed that the use of virus-tested seed is the most effective way of reducing the incidence of LMV. In recent years, outbreaks of LMV have occurred in large areas of central Spain, causing serious yield losses, especially in autumn-grown lettuce (Moreno et al., 2003). The reasons for these outbreaks are unknown, but the recent introduction of ornamentals that act as alternative hosts of LMV has been associated with outbreaks of the virus in the Salinas valley, CA, USA (Zerbini et al., 1997). Also, several weed and ornamental species of the Compositae are potential reservoirs of LMV, which is seed-borne in common weeds including Lactuca serriola and Senecio vulgaris (Tomlinson, 1970). This paper reports, the relative abundance of aphid species alighting on lettuce and broccoli crops in three regions of Spain during the spring and autumn growing seasons in 2001. In addition, aphid species able to colonise lettuce and the transmission efficiencies of LMV by species that alight frequently on lettuce were determined. Finally, the key factors that drive LMV epidemics in lettuce in central Spain during the autumn growing season are discussed.
2. Aphid species associated with lettuce and broccoli crops Aphid species alighting on the crop were monitored in fields of lettuce at two different sites in Navalcarnero (Madrid) and Roldan (Murcia), and in fields of broccoli at one site in Ribaforada (Navarra) during the spring and autumn growing seasons of 2001. Two types of traps were used at each sampling site: a horizontal mosaic-green tile (15.5 cm × 15.5 cm) trap (Irwin, 1980) and a Moericke yellow pan (60 cm × 60 cm surface) water trap. In addition, the aphid species colonising lettuce were sampled directly by visual counting at Navalcarnero. 2.1. Aphid flights in lettuce and broccoli crops The two trapping periods were from date of transplanting until harvest: March–May for spring crops and September–November for autumn crops. Insect samples were collected weekly from the traps and aphids were separated in the laboratory using a stereomicroscope (Leica Microsystems). All aphid specimens were preserved in 70%
ethanol and they were prepared and identified as described previously (Perez et al., 1995). A permanent collection of voucher specimens provided by J.M. Nieto (Universidad de León, Spain) was used to verify identification. The Moericke traps generally caught more aphid species than the tile traps at all sampling locations. That fewer species were caught by the tile trap is probably because of its smaller size (Boiteau, 1990). However, the tile trap is the most suitable to determine which aphids land on the crop because its colour closely resembles that of row crops, based on data from reflectance spectrophotometry (Irwin and Ruesink, 1986). Moreover, the Moericke trap tends to overestimate numbers of certain aphid species, such as M. persicae, which alight preferentially on yellow targets (Boiteau, 1990; Fereres et al., 1999). Fig. 1 shows a 10-fold increase in the numbers of M. persicae trapped in the Moericke trap compared with those caught in the tile trap at Navalcarnero in spring 2001. In our study, although the tile trap was more selective than the Moericke trap, its catches represented well the most abundant aphid species present, including Hyperomyzus lactucae and Aphis fabae. H. lactucae was the most abundant aphid species caught in lettuce during spring, 2001. Cumulative catches of H. lactucae at Navalcarnero were 750 and 600 individuals per metre square in the tile and Moericke trap, respectively (Table 1). Cumulative catches of H. lactucae at Roldan were 1292 and 1381 individuals per metre square in the tile and Moericke traps, respectively. Other abundant species landing on lettuce were A. fabae and Brachycaudus helichrysi at Navalcarnero, and B. helichrysi and Aploneura lentisci at Roldan. In broccoli, A. fabae was most abundant. Cumulative catches of A. fabae at Ribaforada were 8083 and 506 individuals per metre square in the tile and Moericke traps, respectively (Table 1). Other abundant aphid species landing in broccoli plantings were B. helichrysi and H. lactucae. During the spring growing season at Navalcarnero, there was a peak of M. persicae landing in the week of 22–29 May (125 aphids per metre square in the tile trap and 958 aphids per metre square in the Moericke trap). At Roldan, H. lactucae and Hayhurstia atriplicis were most numerous during the week of 10–17 April, when catches in the tile trap were 625 and 208 aphids per metre square, respectively. At Ribaforada, the peak density occurred during the week of 10–17 May when there were many A. fabae (3083 aphids per metre square in the tile trap). The most abundant aphid species present in autumn-sown lettuce or broccoli differed from those found in spring of the same year. H. lactucae was most numerous in spring and was almost absent in the autumn season. During the autumn heavy rains were frequent and an unknown number of aphids landing on the tile traps were washed out of the trap and lost before the collection date. Therefore, Moericke trap captures were considered more reliable in the autumn because they had four drainage holes protected by nets that retain aphids inside the trap. The most abundant species captured in Moericke traps in Navalcarnero during autumn
M. Nebreda et al. / Virus Research 100 (2004) 83–88
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Fig. 1. Comparison between the cumulative number of aphids caught in each type of trap located in a lettuce field at Navalcarnero, Madrid during spring, 2001.
were Hyadaphis coriandri and Aphis spiraecola (Table 2). Peak flights were in the 1-week of October for H. coriandri (97 aphids per metre square) and mid-September for A. spiraecola (83 aphids per metre square). Other Aphis spp. were
Table 1 Cumulative numbers of aphids per metre square and percentage (in parentheses) of total catches of the most abundant aphid species landing in two lettuce fields located at Navalcarnero (Madrid) and Roldan (Murcia), and one broccoli field located at Ribaforada (Navarra) during spring, 2001 Location
Aphid speciesa
Navalcarnero (Madrid)
H. lactucae
750 (24)
600 (17)
A. fabae B. helichrysi Brachycaudus rumexicolens M. persicae
375 (12) 292 (9) 167 (5)
253 (7) 178 (5) 228 (7)
Roldan (Murcia)
Ribaforada (Navarra)
125 (4)
Moericke trap
Table 2 Cumulative numbers of aphids per metre square and percentage (in parentheses) of total catches of the most abundant aphid species landing in two lettuce fields located at Navalcarnero (Madrid) and Roldan (Murcia), and one broccoli field located at Ribaforada (Navarra) during autumn, 2001 Location
Aphid speciesa
Navalcarnero (Madrid)
H. coriandri A. spiraecola M. persicae A. fabae Aphis sp.
1,028 (30)
Total
3,125
H. lactucae B. helichrysi Aploneura lentisci H. atriplicis Hyalopterus pruni
1,292 875 500 42 0
Total
3,833
A. fabae
8,083 (30)
506 (11)
B. helichrysi H. lactucae Aphis sp. Brachycaudus cardui
4,000 1,958 1,750 1,500
225 273 202 297
3,447 (34) (23) (13) (1) (0)
1,381 750 411 517 583
Total (27) (15) (8) (10) (12)
Roldan (Murcia)
5,061 Ribaforada (Navarra)
Total a
Tile trap
also very common at the end of September along with M. persicae that reached a peak of 55 aphids per metre square during the 1-week of October. At Roldan, the most abundant species landing on autumn-sown lettuce were A. spiraecola and M. persicae (Table 2). In Ribaforada, the most abundant aphid species landing in broccoli fields were Therioaphis
26,541
(15) (7) (7) (6)
(5) (6) (5) (7)
4,412
The species shown were those most abundant in the tile traps.
a
A. spiraecola M. persicae Aphis sp Pemphigus sp. Sitobion avenae
Tile trap 42 0 42 0 84
(17) (0) (17) (0) (33)
252 42 42 84 84 84
Total
417
Therioaphis trifolli A. craccivora Aphis sp A. spiraecola A. fabae
208 84 84 125 125
Total
917
Moericke trap 278 183 156 128 125
(21) (14) (12) (10) (10)
1,300 (10) (10) (20) (20) (20)
492 200 22 5 0
(60) (24) (3) (1)
822 (23) (9) (9) (14) (14)
61 56 19 14 8
(15) (14) (5) (3) (2)
411
The species shown were those most abundant in the Moericke traps.
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trifolii and Aphis craccivora, probably flying from fields of alfalfa located near the sampling area (Table 2). 2.2. Aphid species colonising lettuce fields Aphid species feeding on lettuce were also sampled weekly in 2001 during spring and autumn at Navalcarnero. Spring crops were sampled from 24 April to 29 May and autumn crops from 21 September to 25 October. Each lettuce field was divided into four quadrants and 10 plants per quadrant were selected randomly following a zig-zag sampling pattern. No pesticides were used in the selected quadrants of the lettuce field during the entire crop growth cycle. All leaves of each plant were sampled by counting the number of aphids and morphs of each species. Aphid species and morphs were identified directly in the field using a magnifying hand lens. The most abundant aphid species in both spring and autumn crops was Nasonovia ribisnigri. Other species that formed small colonies on lettuce were M. euphorbiae in spring and Aulacorthum solani in autumn. Peak populations of N. ribisnigri occurred at the end of each crop growth cycle: on 29 May (80 individuals per plant) for the spring crop and on 25 October (50 individuals per plant) for the autumn crop. Most of the morphs recorded were nymphs followed by adult apterae and alatae, which were far fewer. Population densities of the two other species were much lower. In spring, M. euphorbiae reached a peak of two individuals per plant just after transplanting on 21 April, but was displaced by N. ribisnigri 2–3 weeks later. In autumn, A. solani reached a maximum density of 11 individuals per plant on 18 October, but its numbers were always much smaller than those of N. ribisnigri.
3. Estimation of the transmission efficiency of LMV by different aphid species LMV was the virus most frequently detected in lettuce and vegetable Brassica crops surveyed in Spain in 2001 and 2002 (Moreno et al., 2003). The virus was particularly abundant in lettuce crops and weeds in the Madrid region. LMV occurred frequently in lettuce at Navalcarnero during October of 2001 and 2002. LMV was detected in Sonchus tenerrimus and S. vulgaris collected at Navalcarnero in early March 2002 and in several weed species (Sonchus oleraceus, Malva sp., S. vulgaris, Gallinsoga parviflora and Chenopodium quinoa) collected at Navalcarnero and Villa del Prado (Madrid) during August–October, 2002. Therefore, the aphid species found landing frequently on lettuce in Navalcarnero were tested to assess their potential as vectors of LMV. Also, the aphid species found colonising lettuce, together with Aphis gossypii, which is another vector of LMV (Kennedy et al., 1962) were included in the tests. All transmission experiments were repeated three times for each aphid species. Aphid clones used for the
study were from different Spanish localities and maintained in the following host plants: M. persicae (from El Encin, Madrid) on sweet pepper, A. gossypii (El Ejido, Almeria) on melon, M. euphorbiae and N. ribisnigri (Villa del Prado, Madrid) on lettuce, A. fabae (Murcia) on faba bean, H. lactucae (Matalpino, Madrid) on sowthistle (S. oleraceus) and Rhopalosiphum padi (El Encin, Madrid) on barley. Transmission experiments were conducted under laboratory conditions (21 ± 2 ◦ C) using a procedure that provides a good estimate of aphid propensity (sensu Irwin and Ruesink, 1986). Accordingly, groups of 25–30 aphids were placed inside empty plastic cages for a 1 h pre-acquisition starvation period. The youngest expanded leaf of a young lettuce (cv. Cazorla) plant aphid-inoculated 3–4 weeks earlier was used as a source for virus acquisition by the aphids. The leaf was detached from the plant after completion of the aphid starvation period. The petiole of the infected leaf was then placed in an Eppendorf tube previously filled with water to keep the leaf turgid during the test and the whole set-up was placed in a plastic cage. Twenty young apterous adult aphids were released at a time on the upper surface of the infected leaf. After a 5 min acquisition access period, groups of five aphids were transferred to each test plant. These were 1-month-old seedlings planted in a 28-rack tray. Aphids found either on the leaf surface or around the cage were used to inoculate the test plants. These aphids were kept on the test plants by covering with plastic cups for at least a 2 h inoculation feeding time. All plants were then sprayed with imidacloprid (Confidor® ) and transferred to a growth chamber at 26:20 ◦ C (day:night), 16:8 h, (light:dark) and a light intensity of 100 E m−2 s−1 . A 28-rack tray of lettuce (cv. Cazorla) seedlings was used as an uninoculated control in each transmission experiment. In these experiments, aphids were able to move freely around the infected and test plants. Leaf samples from all test plants were tested for LMV using double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Clark and Adams, 1977) 4–5 weeks after inoculation, using an LMV-specific monoclonal antibody (Agdia Inc., USA). Alkaline phosphatase was the enzyme used and p-nitrophenyl phosphate the substrate. Absorbance at 405 nm was measured after 1 h with an ELISA plate photometer (Lab Instruments, Austria). Test plants were recorded as infected when absorbance values were at least three times those of the mean of the healthy control. The data on transmission rate (number of infected plants divided by number of test plants) were subjected to analysis of variance (Abacus Concepts, √ 1989) after using the following transformation: X = arcsin (x + 1/100). Multiple mean comparisons were made between the transmission rate obtained for each aphid species using the Fisher’s protected LSD test (Abacus Concepts, 1989). Significant differences (F = 42.2; d.f. = 6, 14; P = 0.0001) in the transmission rate of LMV were apparent among the different aphid species tested (Table 3). M. persicae and A. gossypii were the most efficient vectors, followed by M. euphorbiae, A. fabae and H. lactucae transmitted less
M. Nebreda et al. / Virus Research 100 (2004) 83–88 Table 3 Transmission efficiencies for LMV by each of seven aphid species Aphid species
Mean ± S.E.a
M. persicae A. gossypii M. euphorbiae A. fabae H. lactucae R. padi N. ribisnigri
42.4 33.2 26.2 15.4 9.5 1.2 0
± ± ± ± ± ± ±
4.2 a 4.4 ab 5.2 b 1.2 c 1.2 c 1.2 d 0d
a Means followed by different letters are significantly different according to Fisher’s protected LSD test (n = 3).
efficiently. The transmission rate of R. padi was low (only one of 84 plants became infected), and was not significantly different from that of N. ribisnigri, which never transmitted LMV. None of the plants from the 28-rack trays of non-inoculated lettuce seedlings became infected with LMV.
4. Discussion Spread of viruses transmitted in a non-persistent manner by aphids often occurs when non-colonising species land in large numbers on the crop (Raccah et al., 1985; Perez et al., 1995). Some aphid species actively select the Moericke traps in which they can be as much as 30 times more numerous than in suction traps at the same site (Eastop, 1955). Therefore, when studying plant virus epidemics it is important to have a good approximation of the absolute landing rates from tile traps. These provide a good estimate of aphid activity (sensu Irwin and Ruesink, 1986), because they assess the aphid species that actually land on the crops and therefore are involved in transmission. In our study, the tile trap caught fewer aphid species than the Moericke trap, but its captures still represented well the most abundant aphid species landing on the crop, H. lactucae or A. fabae. Tile traps would provide even better information if they could be modified to prevent spilling of the samples when used during heavy periods of rain. LMV was the most widespread virus of lettuce in Navalcarnero during the autumn of 2001 (Moreno et al., 2003). We confirmed previous studies (Broadbent et al., 1951) showing that M. persicae is the most efficient vector of LMV. It is likely that M. persicae was involved in the spread of LMV in Navalcarnero that was detected in the last week of October 2001 because it reached a peak of 55 aphids per metre square during the 1-week of October. However, other Aphis species were also abundant at the time and probably also contributed to the spread of LMV. In Ribaforada, LMV was also present in autumn-sown broccoli crops (Moreno et al., 2003), where A. spiraecola and other Aphis spp. species were abundant (Table 2). A. spiraecola was one of the most abundant species landing on lettuce crops during the autumn season when LMV epidemics are usually most severe. It is not known if this species is a vector of LMV and transmission experiments are required to determine this. In
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other areas where open-field lettuce is widely grown such as Murcia, LMV has been detected recently in some samples collected at three different sites in October, 2002 (Moreno et al., 2003). Moreover, there is a high risk of LMV epidemics in this region because M. persicae and other vector aphid species are abundant during the autumn (Table 2). It is unlikely that aphids found feeding and reproducing in lettuce are involved in the secondary spread of LMV within plantings because the only aphid species that formed large colonies was N. ribisnigri, which is not a vector. LMV epidemics tend to be much more severe in autumn than in spring-sown lettuce crops. The reason for this is unknown. Nevertheless, the fact that aphid species trapped in autumn differ from those present in spring probably does not account for such differences because high numbers of potential vectors of LMV land on the crop during both seasons. However, the most efficient vector of LMV, M. persicae, was not trapped on spring lettuce in Navalcarnero until late May 2001, by which time most crops had been harvested. Also, the lack of LMV disease outbreaks in spring lettuce may be related to the absence of virus reservoirs (lettuce and weeds) during the winter months. In central Spain, where severe LMV epidemics have occurred in each of the years 2000–2002, lettuce is grown from March to November. LMV is likely to over-winter in the seeds of several weed species such as S. vulgaris that were found infected at the beginning of March in Navalcarnero before any aphid flight activity was detected. Also, weeds are commonly infected with LMV during summer and the beginning of autumn in this region. Therefore, both summer lettuce crops and weeds may act as a primary source of inoculum for LMV epidemics during early autumn when transient aphid vectors are searching for new hosts. Control of LMV is difficult because it is seed-borne in lettuce and also in certain common weed species. Although production of virus-tested lettuce seed is the best option to control the disease (Grogan, 1980), weeds that act as important reservoirs for the virus should be also removed, especially S. vulgaris and other weed species in which LMV is seed-borne (Tomlinson, 1970). The seed bank of such weed species may maintain an important source of virus inoculum during the periods where no susceptible crops are available and is probably important in initiating LMV epidemics. Acknowledgements We thank R. Biurrun, C. Ortuño, and J.L. Nieves for their help in aphid sampling. This work was supported by the Comisión Interministerial de Ciencia y Tecnolog´ıa (Research Grant CICYT No. AGL-2000-2006). References Abacus Concepts, 1989. SuperANOVA. Abacus Concepts. Berkeley, CA, p. 322.
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