Resistance and susceptibility of alfalfa (Medicago sativa L.) cultivars to

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Alfalfa (Medicago sativa L.) is an important forage crop .... using SigmaStat 2.03 (SPSS Inc., 1992J1997). ... field on different alfalfa (Medicago sativa) cultivars.
Insect Science (2006) 13, 55JSM

Resistance and susceptibility of alfalfa to T. maculata 55

Resistance and susceptibility of alfalfa (Medicago sativa L.) cultivars to the aphid Therioaphis maculata (Homoptera: Aphididae): insect biology and cultivar evaluation A L E X A N D R E D E A L M E I D A E S I LVA 1 , E L E N I C E M O U R O V A R A N D A 2 and JOSÉ RICARDO BAROSELA2 1

Laboratório de Entomologia, IPEPATRO, RO, Brasil and 2Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Abstract Biology of the aphid Therioaphis maculata was studied on alfalfa (Medicago sativa L.), including four resistant (Mesa-Sirsa, CUF101, Baker and Lahontan) and two susceptible (ARC and Caliverde) alfalfa cultivars, and one of the most cropped Brazilian cultivars, Crioula. Under controlled conditions, antibiosis (i.e., reduced longevity, fecundity and increased mortality of the aphid) was observed mainly on the resistant alfalfa cultivars, except on Lahontan. Crioula seemed to be tolerant to aphids. Present data support geographic limitation usage of cultivars, and we suggest Baker and Mesa-Sirsa as sources of antibiosis, and provide biological information of a tropical T. maculata biotype on alfalfa. Key words alfalfa cultivars, aphid, resistance, susceptibility, Therioaphis maculata DOI 10.1111/j.1744-7917.2006.00068.x

Introduction Alfalfa (Medicago sativa L.) is an important forage crop that has large amounts of protein, calcium, phosphorus, and vitamins A and D (Nuernberg et al., 1992). Formerly used in horse-feeding, numerous livestock species, mainly cattle, are alfalfa-fed nowadays. Alfalfas nutritional characteristics are also of great interest in human nutrition (Small, 1996). The crop is attacked by several insect groups (Kalvelage, 1992). Aphids, such as Therioaphis maculata (Buckton), are probably the most important herbivores in Brazil (Carvalho et al., 1996). Aphids decrease forage yield by feeding on phloem sap and are also virus vectors (Holman, 1974). Insect-resistant cultivars have been a good alternative to insecticides to keep herbivore population under their economical threshold damage levels (Lara, 1979). Most of

Correspondence: Alexandre de Almeida e Silva, Laboratório de Entomologia, IPEPATRO. Rua Beira, 7671 (BR 364, Km 3,5), Porto Velho, RO, Brasil. CEP 78.900-000. Tel: +55 69 32259345; fax: +55 69 32259345; e-mail: [email protected]

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their resistance is based on antibioses, antixenoses and tolerance that affect host utilization (Painter, 1958). However, resistant cultivar selection and evaluation for such resistance characteristics should be carefully performed because cultivar usage is geographically restricted, and because aphids have a great biotypic variation (Puterka et al., 1992). Thus, local studies on aphid biology on different cultivars are needed to provide information on aphid management and cultivar suitability under tropical conditions. The present study is to investigate T. maculata biology on resistant and susceptible alfalfa cultivars under controlled conditions.

Materials and methods Plant Four resistant cultivars, CUF 101 (Nielson & Lehman, 1977); Baker (Kehr et al., 1978); Mesa-Sirsa (Schonhorst et al., 1968); and Lahontan (Howe & Smith, 1957); two susceptible cultivars, ARC (Devine et al., 1975); Caliverde (Nielson & Lehman, 1977); and an important Brazilian www.blackwellpublishing.com/ins 55

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cultivar, Crioula (Nuernberg et al., 1992) were cultivated in small garden plots (7 m2) at the Biology Department of the FFCLRP-USP, Brazil (2105to 2115S and 4550 to 4755W). All cultivars were also cultivated under laboratory conditions in polystyrene trays (5 cm  5 cm  7 cm) filled with a mixed vegetal matter and expanded vermiculite substrate (Bioplant). Plants were irrigated daily either with Hoagland solution one-quarter-strength (Dunn & Ardith, 1968) or tap water. Light was supplied by six 20W common fluorescent lamps and six 20W GROLUX lamps with average light intensity of 165 nmoles photons m2/s during 12 hours. Humidity close to the trays was kept at 75%, and temperature at 25.

Statistics

Insect

There were significant differences in T. maculata abundance on the studied alfalfa (M. sativa) cultivars (F = 24.2; P < 0.000 1). Resistant cultivars, except for Lahontan, had the lowest abundance of aphids. The highest density of T. maculata was found on Crioula, and the lowest density was found on Baker (Table 1).

Aphids used in laboratory studies were previously collected in the field and allowed to reproduce on excised Crioula shoots dipped in Hoagland solution on plastic pots covered with fine-mesh cloth and kept on an insect-rearing chamber (25, 50% RH and constant illumination). Field investigation Five samples were collected for each cultivar, each sample comprising 20 shoots randomly collected from the field and immediately placed in plastic bags and stored in a common freezer (J20). A soap solution, 500 mL of tap water containing a few drops of common detergent, was added to the bags and shaken for a few seconds. The shoots were removed, and the solution was filtered using a finemesh cloth. Aphids, T. maculata, retained on the filter were transferred to Petri dishes and counted using a stereomicroscope (Bueno et al., 1996). Laboratory investigation Therioaphis maculata biology was studied using clip cages; basically two perpex rings opposed on opposite sides of the leaf held together with a small clip (hair-curl clip). The edges of the rings are lined with foam plastic to protect the leaf and one ring is muslin covered to contain the aphids (Adams & van Emden, 1972). Five first instars were clipped on a single leaflet of each cultivar (10 replicates/cultivar). Survivoral rates to adulthood were recorded, and after the last molt, one adult was kept inside the clip cage. The number of nymphs produced daily (fecundity rate), number of days producing nymphs (reproductive period), total number of nymphs produced (fecundity), and adult longevity were recorded, and the intrinsic rate of increase (rm) and the net reproductive rate (R0) were calculated.

Intrinsic rate of increase (rm) and the net reproductive rate (R0) were calculated using the program Two Sex Life Table Analysis (Chi, 1988). One-way ANOVA of biological data and aphid abundance, and one-way repeated measures ANOVA of survival and fecundity rate were carried out using SigmaStat 2.03 (SPSS Inc., 1992J1997). A multivariate discriminant analysis to separate cultivars based on aphid biological data was performed using STATISTICA 5.1 (StatSoft, Inc., 1998).

Results

Table 1 Abundance of Therioaphis maculata collected in the field on different alfalfa (Medicago sativa) cultivars. Cultivars Resistant

Susceptible

Mesa Sirsa CUF 101 Baker Lahontan ARC Caliverde Crioula

Number of individuals/ sample (mean  SE) 95  17 c 82  5 c 53  6 c 270  27 ab 195  23 b 242  33 b 335  22 a

One-way ANOVA, Student-Newman-Keuls (comparisons), n = 5 (20 shoots/sample). Different letters indicate significant (P < 0.05) differences in a column.

Aphid survivorship on resistant cultivars was lower than on susceptible cultivars, except for Lahontan (F = 4.77; P = 0.000 2). Significantly (F = 6.07; P < 0.000 1) shorter reproductive periods of T. maculata were observed only on Mesa-Sirsa and Baker cultivars. Fecundity rate was different only between Baker and Crioula cultivars (F = 2.4; P = 0.03). But fecundity was lower on resistant cultivars (F = 8.1; P < 0.000 1) than on susceptible cultivars. Therioaphis maculata fecundity was lowest on Mesa-Sirsa and Baker. Type of cultivar also affected aphid longevity by decreasing it on resistant plants, except on Lahontan (F = 6.1; P < 0.000 1) (Table 2). Insect Science 13, 55J60

S

R

One-way ANOVA, Student-Newman-Keuls (comparisons), n = 10. Different letters indicate significant (P < 0.05) differences in a row. R = resistant and S = susceptible.

0.158 0.249 0.122 0.260 0.295 0.286 0.288 6.6 a 20.6 b 5.7 a 24.9 b 30.0 b 33.7 b 44.1 b 13.1  2.7 b 13.8  2.5 b 9.7  2.7 c 21.5  1.0 a 19.5  2.1 a 23.0  1.3 a 21.7  2.8 a 6.7  2.3 c 19.3  4.3 b 5.0  1.9 c 23.0  5.8 b 34.2  4.6 a 34.2  8.8 a 44.7  3.0 d 2.5  0.8 b 6.0  1.0 a 2.0  0.6 b 6.3  1.2 a 8.5  0.9 a 7.8  2.0 a 9.6  1.0 a

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Mesa-Sirsa CUF 101 Baker Lahontan ARC Caliverde Crioula

44.7  7.2 b 57.7  7.5 b 38.0  7.2 c 67.5  7.3 a 80.0  7.3 a 78.0  7.2 a 55.4  7.8 b

1.1  0.4 ab 2.0  0.6 ab 0.6  0.2 a 1.3  0.3 ab 2.1  0.5 ab 2.02  0.6 ab 2.8  0.4 b

Intrinsic rate of increase (rm) (individuals/individual/day) Net reproductive rate (R0) (∑lxmx) Longevity (days) Fecundity (progeny/female) Fecundity rate (progeny/female/day) Reproductive period Survival (%) (days) Cultivar

Table 2 Biology of Therioaphis maculata on different alfalfa (Medicago sativa) cultivars under controlled conditions (RH = 75%; T = 25 and photoperiod = 12 h) (mean  SE).

Resistance and susceptibility of alfalfa to T. maculata 57

Discriminant analysis of biological data of T. maculata indicated significant differences among cultivars (Wilks lambda = 0.27; F = 3.9; P < 0.001). On the scatter plot, data points on susceptible cultivars were clustered, whereas on resistant cultivars, data points were scattered. A distinct separation between resistant group, mostly Mesa-Sirsa and Baker, and susceptible group was observed, except for Lahontan. Variables contributing significantly for group separation were fecundity and longevity (Fig. 1).

Fig. 1 Discriminant analysis scatter plot of biological data from Therioaphis maculata on different alfalfa (Medicago sativa) cultivars. White background symbols indicate the resistant cultivars. Different letters indicate significant (P < 0.05) differences among cultivars.

Therioaphis maculata life table analysis indicated a significant decrease in the survivorship (lx) and on fecundity (mx) by the beginning of the adult stage on the resistant cultivars (5th day), except on Lahontan (F = 89.3; P < 0.000 1 and F = 11.1; P < 0.000 1, respectively). Net reproductive rate (R0) of T. maculata was significantly lower on Mesa-Sirsa and Baker only (F = 11.9; P < 0.000 1). Intrinsic rate of increase was higher on the susceptible cultivars (Fig. 2, Table 2).

Discussion Low abundance of herbivorous insects on resistant cultivars in field collections, such as in the present work, is not a new issue because resistant cultivars usually have a drastic impact on insect herbivore populations (Lara, 1979).

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Fig. 2 Survival (lx) and fecundity (mx) rates of Therioaphis maculata on different alfalfa (Medicago sativa) cultivars under controlled conditions (RH = 75%; T = 25 and photoperiod = 12 h) during 25 days.

In a previous work, Silva and Varanda (2005) reported a low T. maculata density on the resistant cultivar CUF 101 in the field. Despite that, aphid biological data on resistant and susceptible alfalfa cultivars are important for cultivar selection and evaluation, and can be incorporated into population dynamics models. Present data suggest that antibiosis, mostly in the cultivars Baker and Mesa-Sirsa, character-

ized by reduced fecundity and longevity and increased mortality (Painter, 1958), plays a role in alfalfa resistance to tropical T. maculata biotypes, corroborating previous studies (Hackerott & Harvey, 1959; Hughes & Hughes, 1988; Nielson & Curie, 1959; Ruggle & Gutierrez, 1995) and also suggesting both cultivars as sources of antibiosis. Differently, Crioula, a Brazilian cultivar developed in the southern region (Nuernberg, 1992), is possibly tolerant Insect Science 13, 55J60

Resistance and susceptibility of alfalfa to T. maculata 59

to T. maculata and also to other aphid species. This cultivar was heavily infested under laboratory conditions but had lesser visible damage (chlorosis) than the susceptible cultivars ARC and Caliverde. Silva and Varanda (2005) also found heavy infestation by several alfalfa aphid species on this cultivar in the field. Despite that, Crioula had a higher dry matter production than the resistant CUF 101 (Rassini, 1998). Alfalfa tolerance is interesting because it might reduce selective pressure for development of resistant T. maculata biotypes (Jimenez et al., 1989). Studies including aphid biological response to plant genetic variation should not be neglected. Despite good separation between data points of aphid biological data from resistant and susceptible cultivars, there was a great variation in T. maculata biological response, as observed on the scatter plot, mainly for resistant cultivars. Hughes and Hughes (1987) related great variation in alfalfa (CUF 101) resistance to this aphid species. Most plants were highly resistant, few were susceptible, and the other fraction was partially resistant. Howe and Smith (1957) also related a wide variation in the reproductive rate of T. maculata on individual seedlings, suggesting a considerable segregation for antibiosis occurring within a resistant cultivar. Interestingly, some resistant cultivars, such as Baker and Mesa-Sirsa, but not CUF 101, had a high degree of antibiosis to T. maculata despite any genetic variation on resistance features. Howe and Smith (1957) and Nielson and Curie (1959) previously related Lahontan resistance to T. maculata. Present results, however, suggest that the utilization of resistant cultivars is geographically limited, possibly due to biotypic variation of aphids (Puterka et al., 1992). Aphid biotypes able to overcome alfalfa resistance were related by Zarrabi et al. (1995), and a high genetic variation of aphid populations reproducing asexually, such as those in tropical regions, was related by Bournoville et al. (2000). Weather and/or edaphic conditions also affect biology and resistance expression (Risebrow & Dixon, 1987). Lahontan was developed to withstand severe winter climates (Howe & Smith, 1957). Poor development of this cultivar, also of ARC and Baker, in field conditions in the present work supports this hypothesis. Despite that, Baker resistance was not disrupted. Although temperature effects on the aphid were not investigated, biological characteristics of T. maculata, such as reproductive period, fecundity rate and fecundity, were usually lower than previously reported, even at the same temperature and on the same cultivar (Graham, 1959; Messenger, 1964; Hackerott & Harvey, 1959; Isaak et al., 1963). These results suggest that tropical biotypes of this aphid species have better development rates at higher temperatures, even better than other alfalfa aphid species. Insect Science 13, 55J60

Silva et al. (unpublished data) argued that the higher T. maculata abundance than other aphid species was related to the maximum temperatures found in alfalfa fields. Thus, in situ biological data of aphids should be considered before using population dynamics models developed elsewhere.

Acknowledgments The authors thank CAPES for financial support, Dr Allan K. Stoner (USDA) for providing the seeds of most cultivars and also Dr J. B. Rassini (CPPSE-EMBRAPA) for the Crioula seeds, and Dr Carlos Alberto Martinez Y Huaman (Dept. Biology-FFCLRP-USP) for helping us to cultivate alfalfa under laboratory conditions.

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Hackerott, H.L. and Harvey, T.L. (1959) Effect of temperature on the spotted alfalfa aphid reaction to resistance in alfalfa. Journal of Economic Entomology, 52, 949J953. Holman, J. (1974) Los áfidos de Cuba. Editorial Organismos, Cuba. Howe, W.L. and Smith, O.L. (1957) Resistance to the spotted alfalfa aphid in Lohantan alfalfa. Journal of Economic Entomology, 50, 320J324. Hughes, R.D. and Hughes, M.A. (1987) The responses of the spotted alfalfa aphid to variation between plants of an aphidresistant Lucerne cultivar. Entomologia Experimentalis et Applicata, 44, 177J185. Hughes, R.D. and Hughes, M.A. (1988) Temporary loss of antibiosis in plants of a Lucerne cultivar selected for resistance to spotted alfalfa aphid. Entomologia Experimentalis et Applicata, 49, 75J82. Isaak, A., Sorensen, E.L. and Ortman, E.E. (1963) Influence of temperature and humidity on the resistance in alfalfa to the spotted alfalfa aphid and pea aphid. Journal of Economic Entomology, 56, 53J57. Jimenez, H.O., Caddel, J.L., Berberet, R.C. and McNew, R.W. (1989) Indices of plant damage and heritability of tolerance to spotted alfalfa aphid in alfalfa. Crop Science, 29, 1337J1340. Kalvelage, H. (1992) Principais insetos que atacam a cultura de alfafa no Brasil. Manual de Produção de Alfalfa (ed. N.J. Nuernberg), pp. 63J83. EPAGRI, Florianópolis. Kehr, W.R. Manglitz, G.G. and Ogden, R.L. (1978) Registration of Baker alfalfa. Crop Science, 18, 692. Lara, F.M. (1979) Princípios de Resistência de Plantas a Insetos. Ed. Livroceres, Piracicaba. Messenger, P.S. (1964) The influence of rhythmically fluctuating temperatures on the development and reproduction of the spotted alfalfa aphid, Therioaphis maculata. Journal of Economic Entomology, 57, 71J76. Nielson M.W. and Lehman, W.F. (1977) Multiple aphid resistance in CUF 101 alfalfa. Journal of Economic Entomology, 70, 13J14. Nielson, N.W. and Curie, W.E. (1959) Effect of alfalfa variety on

the biology of the spotted alfalfa aphid in Arizona. Journal of Economic Entomology, 52, 1023J1024. Nuernberg, N.J., Milan, P.A. and Silveira, C.A.M. (1992) Cultivo, Manejo e Utilização da alfafa. Manual de Produção de Alfalfa (ed. N.J. Nuernberg), pp. 15J21. EPAGRI, Florianópolis. Painter, R.H. (1958) Resistance of plants to insects. Annual Review of Entomology, 3, 267J290. Puterka, G.J., Burd, J.D. and Burton, R.L. (1992) Biotypic variation in a worldwide collection of Russian wheat aphid (Homoptera: Aphididae). Journal of Economic Entomology, 85, 1497J1506. Rassini, J.B. (1998) Alfalfa (Medicago sativa L.): estabelecimento e cultivo no estado de São Paulo. Circular Técnica-CPPSE. Risebrow, A. and Dixon, A.F.G. (1987) Nutritional ecology of phloem feeding insects. Nutritional Ecology of Insects, Mites, Spiders and Related Invertebrates (eds. F. Slansky Jr. & Rodriguez), pp. 421J448. John Wiley and Sons, New York. Ruggle, P. and Gutierrez, A.P. (1995) Use of life tables to assess host plant resistance in alfalfa to Therioaphis trifolii f. maculata (Homoptera: Aphididae): Hypothesis for maintenance of resistance. Environmental Entomology, 24, 313J325. Schonhorst, M.H., Nielson, M.W., Thompson, R.K., Liberman, F.V., Keener, P.D. and Night, E.L.Jr. (1968) Registration of Mesa-Sirsa alfalfa. Crop Science, 8, 396. Silva, A.A.E. and Varanda, E.M. (2005) Relationship between amino acid concentration and aphid (Hemiptera: Aphididae) abundance on alfalfa (Medicago sativa L.). Acta Biologica Leopoldensia, 27, 33J42. Small, E. (1996) Adaptations to herbivory in alfalfa (Medicago sativa L.). Canadian Journal of Botany, 74, 807J822. Zarrabi, A.A., Berberet, R.C. and Caddel, J.L. (1995) New biotype of Acyrthosiphon kondoi (Homoptera: Aphididae) on alfalfa in Oklahoma. Journal of Economic Entomology, 85, 1462J1465.

Accepted November 7, 2005

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