Evaluation of Vaccinium Spp. for Illinoia Pepperi (Hemiptera: Aphididae) Performance and Phenolic Content

Home , Vaccinium hirsutum, Vaccinium myrsinites

PLANT RESISTANCE Evaluation of Vaccinium spp. for Illinoia pepperi (Hemiptera: Aphididae) Performance and Phenolic Content

1 CHRISTOPHER M. RANGER, JENNIFER JOHNSON-CICALESE, SRIDHAR POLAVARAPU, AND NICHOLI VORSA

Philip E. Marucci Center for Blueberry and Cranberry Research and Extension, Rutgers, The State University of New Jersey, 125A Lake Oswego Road, Chatsworth, NJ 08019

J. Econ. Entomol. 99(4): 1474Ð1482 (2006) ABSTRACT Host acceptance and population parameters of the aphid Illinoia pepperi (MacGilli- vray) (Hemiptera: Aphididae) were measured on highbush blueberry, Vaccinium corymbosum L. ÔElliottÕ, and the wild species Vaccinium boreale Hall and Aalders, Vaccinium tenellum Aiton, Vaccinium pallidum Aiton, Vaccinium hirsutum Buckley, Vaccinium myrsinites Lamarck, and Vaccinium darrowi Camp. After 24 h of exposure, signiÞcantly fewer aphids remained in contact with V. boreale and V. hirsutum compared with V. corymbosum Elliott, V. darrowi, and V. pallidum. Length of the prereproductive period of I. pepperi was signiÞcantly longer on V. boreale and V. myrsinites, in contrast to V. corymbosum. Fecundity was also lower on V. boreale, V. hirsutum, V. myrsinites, and V. darrowi. Survivorship of I. pepperi 42 d after birth was signiÞcantly lower on V. hirsutum compared with the remaining Vaccinium spp. Reduced I. pepperi performance resulted in signiÞcantly lower intrinsic rate

of increase (rm) values being associated with V. myrsinites, V. boreale, V. hirsutum, and V. darrowi, compared with V. corymbosum. Net reproductive rate (R0), generation time (T), and doubling time (Td)ofI. pepperi also were affected by the Vaccinium spp. Total phenolic and ßavonol content varied between Vaccinium spp., with some high phenolic content Vaccinium spp. having reduced aphid performance. However, no signiÞcant correlation between phenolics and I. pepperi performance was detected. Results from this study identiÞed several potential sources of aphid resistance traits in wild Vaccinium spp.

KEY WORDS Illinoia pepperi, Vaccinium, blueberry, host suitability

The aphid Illinoia pepperi (MacGillivray) (Hemi- Aphid-transmitted viruses have become a major ptera: Aphididae), is the most abundant aphid infest- threat to highbush blueberry production in New Jer- ing cultivated highbush blueberry, Vaccinium corym- sey and the PaciÞc Northwest. Since Þrst being de- bosum L., in the northeastern and upper Midwest of tected in the late 1970s, blueberry scorch virus the United States (Elsner and Kriegel 1989). I. pepperi (BlScV) has become one of the most economically is monoecious, spending its entire life cycle on Vac- important diseases affecting highbush blueberries on cinium spp. (Elsner 1982, Morimoto and Ramsdell both the east and west coasts of the United States 1985). Consequently, although within-Þeld move- (Stretch 1983; Martin and Bristow 1988, 1995; Bristow ment of alate and apterae I. pepperi occurs, movement et al. 2000). BlScV belongs to the carlavirus group and outside of cultivated highbush blueberries is rare is transmitted in a nonpersistent manner (Hillman et (Elsner 1982, Morimoto and Ramsdell 1985). I. pepperi al. 1996). Because I. pepperi is the most abundant aphid also has been collected from wild V. corymbosum, infesting cultivated highbush blueberries in the north- Vaccinium angustifolium Aiton, and Vaccinium myr- eastern United States, it is recognized as the primary tilloides Michuax (Elsner and Kriegel 1989). The ho- vector of BlScV within this region (Hillman et al. 1996, locyclic life cycle of I. pepperi consists of overwinter- Bristow et al. 2000). ing eggs that hatch into wingless, parthenogenic The primary tactic currently used for reducing the fundatrices during the spring (Elsner and Kriegel spread of BlScV in conventionally grown blueberries 1989). Subsequent summer generations consist of is the use of insecticides to control aphid vectors, along alate or apterous virginoparae giving birth partheno- with rouging symptomatic plants. However, BlScV has genetically to live young. In the fall, sexual morphs of a latent period of 1Ð3 yr before symptoms manifest, apterous females and alate males are produced. and certain cultivars are asymptomatic (Bristow et al. 2000). As such, an infected plant could continue to 1 Current address: Application Technology Research Unit, USDAÐ serve as a source of inoculum. By using host plant ARS, 1680 Madison Ave., Wooster, OH 44691. resistance to reduce aphid population Þtness, trans-

0022-0493/06/1474Ð1482$04.00/0 ᭧ 2006 Entomological Society of America August 2006 RANGER ET AL.: Vaccinium RESISTANCE TO I. pepperi 1475 mission of the nonpersistent BlScV could potentially Table 1. Vaccinium spp. used for measuring behavioral and be reduced. For example, certain selections of peach, biological responses of I. pepperi Prunus persica (L.) Batsch, are highly resistant to both Selection ID Ploidy Species Source Myzus persicae (Sulzer) and Myzus varians (David- or cultivar level son). Transmission of nonpersistent plum pox virus is V. darrowi NJ88-06-46 2x Santa Rosa Co., FL reduced on certain aphid-resistant P. persica selections V. darrowi NJ88-09-04 2x Polk Co., FL (Massonie and Maison 1980). Additional examples of V. darrowi Fla 4B 2x Marion Co., FL reduced virus transmission on aphid-resistant cultivars V. tenellum NC83-09-15 2x Bladen Co., NC exist for maize, Zea mays L. (Milinko et al. 1983); V. pallidum NJ88-18-32 2x Ocean Co., NJ V. boreale NJ88-29-35 2x Cape Brenton, NS, Canada muskmelon, Cucumis melo L. (Lecoq et al. 1979), soy- V. myrsinites NC84-06a-04 4x Lake Co., FL bean, Glycine max (L.) Merr. (Gunasinghe et al. 1988); V. myrsinites NJ88-07-43 4x Highlands Co., FL tobacco, Nicotiana sp. (Gooding and Kennedy 1985); V. hirsutum NJ90-54-07 4x Graham Co., NC and watermelon, Citrullus lanatus (Thunb.) (Gray et V. corymbosum Elliott 4x USDA Breeding Program al. 1988). Extensive information currently exists on the levels Vaccinium of pathogen resistance exhibited by spp. rooted shoots from single parent plants for V. tenellum, and blueberry cultivars (Stretch and Ehlenfeldt 2000, V. pallidum, V. boreale, V. hirsutum, V. myrsinites Ehlenfeldt and Stretch 2001, Stretch et al. 2001), yet NC84-06a-04 and NJ88-07-43, and V. darrowi NJ88- to date only one published study has examined aphid 06-46, NJ88-09-04, and Fla 4B. Thus, with the excep- resistance. In a Michigan Þeld trial, densities of adult tion of V. corymbosum Elliott, all replicates of a par- I. pepperi were recorded on 16 highbush blueberry ticular Vaccinium spp. possessed the same genetic cultivars on two dates in 1980 and three in 1981 (Han- identity. After removal from the parent plants, rooted cock et al. 1982). SigniÞcantly fewer aphids were de- cuttings were placed in a sand:peat moss (1:1) mixture. tected on four of the 16 cultivars, indicating potential One-year old V. corymbosum Elliott plants were pur- sources of resistance. Wild Vaccinium spp. represent a valuable source of chased from DeGrandchampÕs blueberry farm (South Haven, MI). All plants were maintained in a green- useful genes for insect and disease resistance. In an Ϸ attempt to identify possible sources of aphid resistance house for 6 mo before their use in experiments. for introgression into highbush blueberry cultivars, we Osmocote 14Ð14-14 (NÐPÐK) (Scotts-Sierra Horticul- characterized host acceptance, developmental, repro- tural Products, Marysville, OH) was used as a con- ductive, and life table parameters of I. pepperi in re- trolled release fertilizer. sponse to seven Vaccinium spp. Because carbon-based Colony Maintenance. A colony of I. pepperi was defense mechanisms are associated with long-lived established by collecting several apterous virginop- woody perennials adapted to nutrient poor environ- arae during June 2004 from a commercial blueberry ments (Berryman 1988), total phenolic and ßavonol planting in Atlantic Co., NJ. Because the overall ob- content also was measured in each of the Vaccinium jective was to identify sources of resistance against I. spp. to determine whether such compounds were cor- pepperi, multiple clones were used to establish the related with aphid resistance. colony. Apterous I. pepperi were occasionally added to the colony to ensure the presence of multiple genotypes. Aphids were reared on excised shoots of V. Materials and Methods corymbosum Elliott conÞned to plastic rearing con- Plant Material. Seven Vaccinium spp. were used in tainers. Cages were changed weekly and plant mate- this study: V. darrowi Camp, V. tenellum Aiton, V. rial was added as needed. The colony was maintained pallidum Aiton, V. boreale Hall and Aalders, V. hirsu- in a laboratory under ßuorescent lights with a photo- tum Buckley, V. myrsinites Lamarck, and V. corymbo- period of 16:8 (L:D) h. sum L. A single accession was tested for each species, Aphid Host Acceptance. Settling behavior of I. pep- with the exception of three accessions being tested for peri was measured on seven Vaccinium spp.: V. tenel- V. darrowi (NJ88-06-46, NJ88-09-04, and Fla 4B) and lum, V. pallidum, V. boreale, V. hirsutum, V. darrowi two accessions for V. myrsinites (NC84-06a-04 and NJ88-09-04, V. myrsinites NJ88-07-43, and V. corym- NJ88-07-43) (Table 1). For V. corymbosum, the highly bosum Elliott (Table 1). Approximately 6 to 7 cm of a susceptible highbush cultivar Elliott was used. All of single intact shoot from each of the aforementioned the Vaccinium spp., except V. corymbosum, were col- Vaccinium spp. was positioned in an individual dialy- lected from wild populations (Table 1) and partly sis-tube cage (4 cm in diameter, 13 cm in length) comprise the Vaccinium germplasm collection at the (WardÕs National Science, Rochester, NY), which was Philip E. Marucci Center for Blueberry and Cranberry sealed at both ends by using split foam plugs. Cages Research and Extension, Rutgers University, Chats- contained only leaf and stem tissue in the vegetative worth, NJ. Collection of these Vaccinium spp. were state and were positioned on the plants 24 h before described by Bruederle and Vorsa (1994). The parent their use in experiments. Five recently emerged apter- plants are potted in a (1:1) sand:peat moss mixture and ous I. pepperi adults were transferred from the labo- maintained under greenhouse conditions. ratory-reared colony and placed on the shoot in each Identical clones of a particular Vaccinium accession of the individual cages. Plants were then arranged in were prepared in May 2004 by excising multiple a completely randomized design on a single green- 1476 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4 house bench. The number of adults remaining on the A modiÞed version of the GloriesÕ method (Glories test plants was recorded 24 h after their introduction. 1979, Mazza et al. 1999) was used to estimate the total Aphid Development, Survival, and Reproduction. I. phenolic and ßavonol content of Vaccinium leaf and pepperi performance was measured on 10 Vaccinium stem tissues. Extracts were diluted 1:10 with methanol spp.: V. darrowi (NJ88-06-46, NJ88-09-04, and Fla 4B), and 0.25 ml of sample was combined in a test tube with V. myrsinites (NC84-06a-04 and NJ88-07-43), V. tenel- 0.25 ml of 0.1% HCl in 95% ethanol and 4.50 ml of 2% lum, V. pallidum, V. boreale, V. hirsutum, and V. corym- HCl (aqueous). The solution was mixed and allowed bosum L. Elliott (Table 1). Three adult apterous I. to stand for 15 min before reading absorbance levels pepperi were collected from the laboratory-reared col- at 280 nm for total phenolics and 360 nm for ßavonols ony and transferred to dialysis-tube cages containing using a spectrophotometer (Beckman DU530, Beck- a single intact shoot from the aforementioned Vac- man Coulter, Fullerton, CA). Standard curves were cinium spp. Plants were then arranged in a completely developed using gallic acid (Ն98.0% purity; Fluka Bio- randomized design on a single greenhouse bench. chemika, Buchs, Switzerland) for total phenolics and Cages were inspected daily until nymphal production myricetin (Ն95.0% purity; Fluka Biochemika) for ßa- began, at which point all individuals were removed vonols. Total phenolics are expressed as micrograms of except for Þve Þrst instars. Nymphs were allowed to gallic acid equivalent per milligrams dry weight and develop until one reached reproductive maturity, af- total ßavonols are expressed as micrograms of myr- ter which all remaining individuals were removed. icetin equivalent per milligram of dry weight. Fecundity was subsequently measured every 2 d until Statistical Analyses. For experiments measuring 42 d after birth. This procedure allowed for calculating aphid parameters and Vaccinium phenolics, plants the length of the prereproductive period (birth to were arranged in a completely randomized design on maturity), nymphal and adult survivorship over a 42-d a single bench under greenhouse conditions. The im- period, and fecundity. pact of Vaccinium spp. on aphid settling, development,

The intrinsic rate of natural increase (rm) was es- fertility, and phenolic contents were each separately timated using the equation of Wyatt and White analyzed using one-way analysis of variance ϭ (1977): rm 0.74(ln Md)/TTR, where 0.74 is a cor- (ANOVA) (PROC GLM, SAS Institute 1991). To help recting constant, Md is the number of young produced stabilize the variance, data were square root-trans- in a reproductive period equal to TTR, and TTR is the formed before analysis, but untransformed data are time from birth to reproduction (Wyatt and White presented. Means were separated using TukeyÕs stu- ϭ 1977). Doubling time (Td ln 2/rm), net reproductive dentized range (honestly signiÞcant difference ϭ͚ ϭ ␣ ϭ rate (Ro lxmx), and generation time (T ln Ro/rm) [HSD]) test at 0.05, which is suitable for both also were calculated, whereby lx is age-speciÞc survi- balanced and unbalanced data (SAS Institute 1991). vorship and mx is age-speciÞc fecundity (Andrewartha PearsonÕs correlation coefÞcient between Vaccinium and Birch 1954, DeLoach 1974). Because Td and T are phenolics and I. pepperi performance parameters were ␣ ϭ functions of rm, and R0 represents the populationÕs analyzed at 0.05 by using the PROC CORR pro- response, statistical analyses were only performed on cedure (SAS Institute 1991). rm; however, values for each are provided for com- The bootstrap technique was used to estimate 95% parative purposes. conÞdence intervals (CIs) for rm values associated Determination of Total Phenolics and Flavonols in with each Vaccinium spp. (Meyer et al. 1986, Pettit et Vaccinium Tissue. For each Vaccinium spp. (Table 1), al. 1994, R Project for Statistical Computing 2005). Ϸ15 actively growing shoots were harvested on Þve Nonoverlapping 95% CI corresponds to the rejection separate occasions from greenhouse grown plants dur- of no treatment effect hypothesis at ␣ ϭ 0.05 (Maia et ing JuneÐJuly 2005. To prepare for extractions, excised al. 2000). stems were placed in paper bags in a drying oven set The percentage of aphids surviving on each Vac- to 60 Ϯ 5ЊC for 24 h and stored at Ϫ20ЊC until analysis. cinium spp. 42 d after birth were compared using a R ϫ After drying, leaves were separated from stems, and C contingency table using the PROC FREQ proce- each was ground into a homogenous powder. Ground dure (SAS Institute 1991), and Fisher exact test (two- leaf and stem tissue were extracted for phenolics and tail) was used to test for signiÞcant differences be- ßavonols according to a modiÞed protocol of Witzell tween observed and expected frequencies (␣ ϭ 0.05). et al. (2003), whereby 30 mg of leaf or stem tissue were placed in an Eppendorf tube with 1000 ␮l of chilled Results methanol:acetone:water:acetic acid (40:40:20:0.1). Samples were placed on a shaker (Labnet Shaker 20, Aphid Host Acceptance. After 24 h of exposure, Labnet Edison, NJ) at 1000 rpm for 2 min and then signiÞcant differences were detected in the settling centrifuged at 12,000 ϫ g for 5 min (Eppendorf cen- behavior of I. pepperi in response to the seven Vac- trifuge 5417C, Eppendorf, Hamburg, Germany). The cinium spp. (Fig. 1; F ϭ 4.79; df ϭ 6, 45; P ϭ 0.0007). supernatant was collected, and the samples were re- SigniÞcantly fewer adults were in contact with V. extracted as described above with 500 ␮l of solvent. hirsutum compared with V. corymbosum Elliott, V. Both supernatants were pooled, Þltered using a sy- darrowi NJ88-09-04, and V. pallidum. SigniÞcant dif- ringe Þlter (pore size 0.2 ␮m; Agilent Technologies, ferences were not detected between V. corymbosum, Palo Alto, CA), and concentrated to dryness in a fume V. myrsinites NJ88-07-43, V. darrowi NJ88-09-04, V. hood. Residues were redissolved in 1 ml of methanol. tenellum, V. pallidum, and V. boreale. All of the aphids August 2006 RANGER ET AL.: Vaccinium RESISTANCE TO I. pepperi 1477

Fig. 1. Mean Ϯ SE number of adult I. pepperi in contact with terminal shoots of the following Vaccinium spp. after 24 h of no-choice conÞnement: Vc, V. corymbosum Elliott; Vb, V. boreale; Vh, V. hirsutum; Vm, V. myrsinites NJ88-07-43; Vd, V. darrowi NJ88-09-04; Vt, V. tenellum; Vp, V. pallidum (n ϭ 8 for all, except n ϭ 6 for V. tenellum and V. pallidum). Five Fig. 2. Age-speciÞc survivorship (lx)ofI. pepperi con- apterous adults were conÞned to each dialysis-tube cage (see Þned to terminal shoots of wild Vaccinium spp. V. corymbo- Materials and Methods for details). Bars headed by the same sum Elliott (f)(n ϭ 11), V. boreale (⌬)(n ϭ 11), V. hirsutum letter are not signiÞcantly different (␣ ϭ 0.05; TukeyÕs stu- (Ⅺ)(n ϭ 9), V. myrsinites NC84-06a-04 (Œ)(n ϭ 9), V. dentized range HSD test). myrsinites NJ88-07-43 (ϫ)(n ϭ 11), V. darrowi NJ88-06-46 (छ)(n ϭ 13), V. darrowi NJ88-09-04 (E)(n ϭ 10), V. darrowi Fla 4B ()(n ϭ 7), V. tenellum (F)(n ϭ 12), and conÞned to V. corymbosum Elliott and V. darrowi V. pallidum (ࡗ)(n ϭ 8). Survivorship values at the 42-d time NJ88-09-04 were in contact with the plant after 24 h. point followed by the same letter are not signiÞcantly dif- Aphid Development and Survival. Length of the ferent (Fisher exact test; ␣ ϭ 0.05). prereproductive period of I. pepperi was signiÞcantly affected by the Vaccinium host (Table 2; F ϭ 8.14; df ϭ Ͻ 9, 92; P 0.0001). The longest mean prereproductive Survivorship (lx) of reproductive adults, reared on period of 19.3 d on V. myrsinites NC84-06a-04 was the 10 Vaccinium selections, differed 42 d after birth signiÞcantly longer than all of the other Vaccinium (Fig. 2). The 30% survival associated with V. hirsutum spp., except V. boreale. The shortest mean prerepro- differed signiÞcantly from the remaining Vaccinium ductive period of 10.2 d occurred on V. corymbosum spp. (Fisher exact test, P Ͻ 0.0001). No mortality was Elliott, which was signiÞcantly different from V. myr- associated with I. pepperi nymphs or reproductive sinites NC84-06a-04 and V. boreale. SigniÞcant differ- adults on V. corymbosum Elliott, V. pallidum, V. tenel- ences in the length of the prereproductive period of lum, V. darrowi Fla 4B, and V. myrsinites NC84-06a-04. I. pepperi were not detected between V. corymbosum Aphid Fecundity. Reproductive capacity of I. pep- Elliott, V. pallidum, V. myrsinites NJ88-07-43, V. tenel- peri was variable on the Vaccinium hosts, resulting in lum, V. hirsutum, and the V. darrowi accessions Fla 4B, signiÞcant differences in cumulative nymphal produc- NJ88-06-46, and NJ88-09-04. SigniÞcant differences in tion up to 42 d after birth (Table 2; F ϭ 10.58; df ϭ 9, the length of the prereproductive period were de- 91; P Ͻ 0.0001). The highest mean total fecundity of tected between the two allopatric V. myrsinites ac- 59.9 nymphs was associated with V. corymbosum El- cessions NC84-06a-04 and NJ88-07-43, but differences liott and was 3 times higher than the lowest mean were not detected between the three allopatric V. fecundity of 18.6 nymphs from V. boreale. Compared darrowi accessions NJ88-06-46, NJ88-09-04, and Fla 4B with V. corymbosum Elliott, signiÞcantly fewer (Table 2). nymphs were produced on V. boreale, V. hirsutum, V.

Table 2. Life history parameters of I. pepperi conﬁned to Vaccinium spp.

Selection ID Prereproductive perioda Total fecundityb No. nymphs/reproductive adult/2 dc Vaccinium spp. n or cultivar [mean (Ϯ SE)] [mean (Ϯ SE)] [mean (Ϯ SE)] V. myrsinites NC84-06a-04 9 19.3 Ϯ 1.1a 19.0 Ϯ 1.9a 1.5 Ϯ 0.1a V. boreale NJ88-29Ð35 11 16.4 Ϯ 2.0ab 18.6 Ϯ 3.9a 1.6 Ϯ 0.2a V. hirsutum NJ90-54-07 9 13.3 Ϯ 1.0bc 23.7 Ϯ 5.7ab 2.1 Ϯ 0.3ab V. myrsinites NJ88-07-43 11 12.4 Ϯ 0.9bc 31.5 Ϯ 3.5ab 2.3 Ϯ 0.1bc V. pallidum NJ88-18-32 8 12.3 Ϯ 1.0bc 42.5 Ϯ 6.1bcd 2.6 Ϯ 0.3bcd V. darrowi NJ88-06-46 13 12.0 Ϯ 0.5c 40.6 Ϯ 4.5bc 2.9 Ϯ 0.3cde V. darrowi NJ88-09-04 10 11.8 Ϯ 0.4c 40.1 Ϯ 2.7bc 2.5 Ϯ 0.2bcd V. darrowi Fla 4B 7 11.4 Ϯ 0.6c 51.3 Ϯ 4.4cd 3.3 Ϯ 0.3de V. tenellum NC83-09-15 12 11.3 Ϯ 0.5c 50.6 Ϯ 4.9cd 3.0 Ϯ 0.3cde V. corymbosum Elliott 11 10.2 Ϯ 0.4c 59.9 Ϯ 4.4d 3.6 Ϯ 0.3e

Means within columns followed by the same letter are not signiÞcantly different (TukeyÕs studentized range ͓HSD͔ test; (␣ ϭ 0.05). a Number of days from birth to reproductive maturity. b Total offspring produced over a period of 42 d after birth. c Represents the mean number of offspring produced every 2 d from reproductive maturity until death or 42 d after birth. 1478 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4

Table 3. Demographic statistics for I. pepperi conﬁned to Vaccinium spp

Selection ID Vaccinium spp. nra 95% CIb R c Td T e or cultivar m o d V. myrsinites NC84-06a-04 9 0.121 0.099Ð0.145a 30.2 27.9 5.7 V. boreale NJ88-29-35 11 0.129 0.083Ð0.173ab 67.7 32.9 5.4 V. hirsutum NJ90-54-07 9 0.160 0.129Ð0.188abc 26.9 20.7 4.4 V. myrsinites NJ88-07-43 11 0.167 0.151Ð0.181b 39.1 21.9 4.2 V. pallidum NJ88-18-32 8 0.192 0.169Ð0.223bcd 68.0 22.1 3.6 V. darrowi NJ88-09-04 10 0.193 0.184Ð0.201c 65.7 21.7 3.6 V. darrowi NJ88-06-46 13 0.202 0.177Ð0.222bcd 53.8 19.7 3.4 V. darrowi Fla 4B 7 0.207 0.194Ð0.236cd 51.3 19.0 3.4 V. tenellum NC83-09-15 12 0.214 0.199Ð0.229cd 78.9 20.4 3.2 V. corymbosum Elliott 11 0.237 0.222Ð0.254d 94.1 19.2 2.9

a ϭ rm 0.74(ln Md)/TTR, where Md is the number of young produced in a reproductive period equal to TTR, and TTR is the prereproductive period. Bootstrap estimate of rm. b Nonoverlapping 95% CI corresponds to the rejection of the no treatment effect hypothesis (␣ ϭ 0.05). 95% CI values followed by the same letter are not signiÞcantly different (TukeyÕs studentized range ͓HSD͔ test; ␣ ϭ 0.05). c ϭ͚ Ro lxmx. d ϭ T lnRo/rm. e ϭ Td ln(2)/rm

myrsinites NC84-06a-04 and NJ88-07-43, and V. dar- Net reproductive rates (Ro) were considerably rowi NJ88-06-46 and NJ88-09-04. SigniÞcant differ- lower for I. pepperi reared on V. hirsutum and V. ences in total fecundity of I. pepperi were not detected myrsinites NC84-06a-04 than for aphids conÞned to V. between V. corymbosum Elliott, V. tenellum, and V. corymbosum Elliott (Table 3). The longest generation darrowi Fla 4B. time of T ϭ 32.9 was associated with V. boreale, The number of nymphs produced per reproductive whereas the shortest value of T ϭ 19.0 occurred on V. ϭ adult per 2 d also was signiÞcantly affected by the darrowi Fla 4B. The shortest doubling time of Td 2.9 Vaccinium hosts (Table 2; F ϭ 7.24; df ϭ 9, 86; P Ͻ was associated with V. corymbosum, whereas the long-

0.0001). The highest mean number of nymphs pro- est Td of 5.7 occurred on V. myrsinites NC84-06a-04. duced per adult per 2 d was 3.6 on V. corymbosum Total Phenolics and Flavonols. Concentrations of Elliott, whereas the lowest mean number of 1.5 total leaf phenolics varied signiÞcantly between the nymphs per 2 d occurred on V. myrsinites NC84-06a- Vaccinium spp. (Table 4; F ϭ 24.42; df ϭ 9, 40; P Ͻ 04. Compared with V. corymbosum Elliott, nymphal 0.0001). Total leaf phenolics ranged from 17.4 ␮gof production per reproductive adult per 2 d was signif- gallic acid equivalent (GAE)/mg of dried V. hirsutum icantly lower on V. boreale, V. hirsutum, V. pallidum, tissue to 62.8 ␮g GAE/mg of dried V. myrsinites NJ88- V. myrsinites NC84-06a-04 and NJ88-07-43, and V. dar- 07-43 tissue. Compared with V. corymbosum Elliott, rowi NJ88-09-04. SigniÞcant differences were not de- total leaf phenolics were signiÞcantly higher for V. tected between V. corymbosum Elliott, V. tenellum, myrsinites NJ88-07-43 and V. darrowi NJ88-09-04. As and V. darrowi NJ88-06-46 and Fla 4B. previously noted, I. pepperi exhibited reduced perfor- Aphid Population Fitness. The intrinsic rate of in- mance on these V. myrsinites and V. darrowi acces- crease (rm), net reproductive rate (R0), generation sions. However, the population Þtness of I. pepperi was time (T), and doubling time (Td) were calculated for also reduced on V. boreale and V. hirsutum, yet total I. pepperi on the 10 Vaccinium hosts (Table 3). De- leaf phenolics for V. boreale were not signiÞcantly layed developmental rates and reduced offspring pro- different from V. corymbosum. Indeed, V. hirsutum duction resulted in I. pepperi reared on V. myrsinites contained signiÞcantly fewer total leaf phenolics than

NC84-06a-04 exhibiting the lowest mean rm value of V. corymbosum. 0.121, whereas comparatively rapid developmental For each Vaccinium spp., total stem phenolics were rates and high fecundity resulted in V. corymbosum lower compared with leaves. SigniÞcant differences

Elliott being associated with the highest mean rm value were detected in total stem phenolics between the ϭ ϭ ϭ of 0.237. Compared with V. corymbosum Elliott, rm Vaccinium spp. (Table 4; F 4.41; df 9, 40; P values were signiÞcantly lower for I. pepperi reared on 0.0005). Total stem phenolics ranged from 3.0 ␮g V. boreale, V. hirsutum, V. darrowi NJ88-09-04, and the GAE/mg for V. darrowi Fla 4B to 15.3 ␮g GAE/mg for V. myrsinites accessions NC84-06a-04 and NJ88-07-43. V. myrsinites NJ88-07-43. Compared with V. corym-

However, signiÞcant differences in rm values were not bosum Elliott, stem phenolics were signiÞcantly higher detected between V. corymbosum, V. pallidum, V. in V. myrsinites NJ88-07-43. As with leaves, stem phe- tenellum, and V. darrowi accessions Fla 4B and NJ88- nolics also were comparatively low for V. hirsutum,

06-46. The rm value of 0.121 associated with V. myr- which was identiÞed as a poor host for I. pepperi. sinites NC84-06a-04 was signiÞcantly lower than the Total leaf ßavonols ranged from 9.6 ␮g ME/mg for allopatric V. myrsinites accession NJ88-07-43, but sig- V. tenellum to 31.2 ␮g ME/mg for V. darrowi NJ88- niÞcant differences were not detected in rm values 09-04. SigniÞcant differences in total ßavonols were among the three allopatric V. darrowi accessions. detected between the Vaccinium spp. (Table 4; F ϭ August 2006 RANGER ET AL.: Vaccinium RESISTANCE TO I. pepperi 1479

Table 4. Total phenolic and ﬂavonol content in leaf and stem tissue of Vaccinium spp

Mean (Ϯ SE) Selection ID Vaccinium spp. or cultivar Total leaf phenolics Total stem phenolics Total leaf ßavonols Total stem ßavonols (␮g of GAE/mg)a (␮g of GAE/mg)a (␮g of ME/mg)b (␮g of ME/mg)b V. myrsinites NJ88-07-43 62.8 Ϯ 4.5a 15.3 Ϯ 1.5a 22.0 Ϯ 4.5ab 5.2 Ϯ 1.2a V. darrowi NJ88-09-04 55.5 Ϯ 4.0ab 12.9 Ϯ 1.2ab 31.2 Ϯ 6.2a 9.2 Ϯ 1.7a V. myrsinites NC84-06a-04 43.2 Ϯ 4.2bc 5.1 Ϯ 1.7ab 26.5 Ϯ 3.9ab 4.0 Ϯ 2.6a V. darrowi NJ88-06-46 41.8 Ϯ 3.6bcd 8.7 Ϯ 0.5ab 23.7 Ϯ 4.8ab 6.0 Ϯ 1.9a V. corymbosum Elliott 33.2 Ϯ 3.0cde 3.8 Ϯ 0.9b 15.1 Ϯ 2.1ab 4.1 Ϯ 1.9a V. boreale NJ88-29-35 27.9 Ϯ 3.6def 13.0 Ϯ 5.8ab 16.1 Ϯ 1.7ab 12.2 Ϯ 3.9a V. darrowi Fla 4B 26.9 Ϯ 2.0def 3.0 Ϯ 0.8b 14.7 Ϯ 1.5ab 2.7 Ϯ 2.4a V. pallidum NJ88-18-32 19.9 Ϯ 0.9ef 6.5 Ϯ 1.3ab 16.3 Ϯ 1.5ab 6.6 Ϯ 1.9a V. tenellum NC83-09-15 18.5 Ϯ 2.2ef 4.7 Ϯ 1.4b 9.6 Ϯ 3.2b 4.8 Ϯ 1.8a V. hirsutum NJ90-54-07 17.4 Ϯ 2.1f 3.4 Ϯ 1.4b 11.6 Ϯ 2.3b 4.6 Ϯ 2.6a

Means within columns followed by the same letter are not signiÞcantly different (n ϭ 5 for phenolics and n ϭ 4 for ßavonols; TukeyÕs studentized range ͓HSD͔ test). a Total phenolics measured at 280 nm and quantiÞed as GAE. b Total ßavonols measured at 360 nm and quantiÞed as ME.

3.64; df ϭ 9, 39; P ϭ 0.0036). SigniÞcantly higher total ples exist of ßavonoids being used as host recognition leaf ßavonols were associated with V. darrowi NJ88- cues by monophagous and oligophagous insects (Har- 09-04 compared with V. tenellum and V. hirsutum. No borne and Grayer 1993). The performance of I. pepperi signiÞcant differences were detected in concentra- was reduced on several Vaccinium spp. that exhibited tions of total stem ßavonols between the Vaccinium comparatively high total phenolic and/or ßavonol spp. (Table 4). content, such as the V. myrsinites accessions NC84- Overall, the population Þtness of I. pepperi was 06a-04 and NJ8807-43, and the V. darrowi accessions reduced on several Vaccinium spp. with comparatively NJ88-09-04 and NJ88-06-46. However, total phenolic high phenolics and/or ßavonols concentrations, such or ßavonol content did not provide a strong diagnostic as V. myrsinites NJ88-07-43, V. darrowi NJ88-09-04 and tool for Vaccinium resistance or susceptibility. Further NJ88-06-46. However, I. pepperi performance also was characterization of Vaccinium chemotypes might pro- reduced on V. boreale and V. hirsutum; yet, these vide useful information regarding pathways involved Vaccinium spp. were not associated with particularly in aphid resistance. high concentrations of phenolics or ßavonols, except The population Þtness of I. pepperi was poorest for V. boreale stem phenolics. Analysis of correlation when reared on V. myrsinites NC84-06a-04, as evident coefÞcients failed to indicate any signiÞcant correla- by low fecundity and rm, and delayed development. tion (P Ͼ 0.05) between leaf and stem phenolics and The tetraploid V. myrsinites is thought to be derived I. pepperi development (r ϭ 0.160 and Ϫ0.125), total from the diploid sympatric species V. darrowi and V. ϭϪ Ϫ ϭϪ fecundity (r 0.305 and 0.350), or rm (r 0.300 tenellum (Camp 1945), all of which are found in the and Ϫ0.151), respectively. Similarly, no signiÞcant southeastern United States and outside of the known correlation (P Ͼ 0.05) was detected between leaf and range of I. pepperi (Elsner and Kriegel 1989). Being of stem ßavonols and I. pepperi development (r ϭ 0.366 the same ploidy as cultivated V. corymbosum, favor- and Ϫ0.087), total fecundity (r ϭϪ0.362 and Ϫ0.383), able alleles from the tetraploid V. myrsinites could be ϭϪ Ϫ or rm (r 0.390 and 0.016), respectively. introgressed into V. corymbosum, thereby providing an opportunity to improve the aphid resistance prop- erties of highbush blueberry cultivars. Although Discussion Lyrene (1997) considered V. myrsinites disadvanta- This study represents the Þrst screening of wild geous for improving V. corymbosum because of short,

Vaccinium spp. for resistance against I. pepperi. The rm spreading growth exhibited by F1 plants and small, values suggest the least suitable hosts for I. pepperi are dark berries, backcrossing could be used to introgress V. myrsinites, V. darrowi, V. hirsutum, and V. boreale. the traits responsible for reduced aphid performance.

Comparing rm values provides more insight into an Based on rm values, I. pepperi Þtness also was re- organismÕs population Þtness than independently duced on V. darrowi NJ88-09-04, although it was not comparing speciÞc life table parameters (Petitt et al. characterized as highly resistant. However, instead of

1994). Furthermore, rm is the only statistic that ade- breeding a highly resistant cultivar, Van Emden quately represents the physiological qualities of an (1991) argued for developing varieties with relatively organism relative to its capacity of increase (Andre- low or medium levels of resistance, but which are wartha and Birch 1954). compatible with other components of an integrated Phenolics and ßavonoids are well documented to pest management program. Because V. darrowi pro- play a role in aphid deterrence and reduced perfor- duces an effective frequency of 2n gametes, it can be mance (Joerdens-Roettger 1979, Dreyer and Jones successfully crossed with V. corymbosum (Ortiz et al. 1981, Hedin and Waage 1986, Peng and Miles 1988, 1999, Qu and Vorsa 1999). V. darrowi exhibits high Lattanzio et al. 2000). Furthermore, numerous exam- fruit quality and drought and temperature tolerance 1480 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4 and has been used as an important source of favorable method of Birch (1948) or the revised method of traits for improving numerous southern highbush cul- Wyatt and White (1977). tivars (Chandler et al. 1985, Erb et al. 1990, Hancock In addition to monitoring each individual until et al. 1992). death, the rm method of Birch (1948) also requires that The V. darrowi accession Fla 4B is actually consid- survival, prereproductive period, and fecundity be ered a hybrid, which developed from introgression assessed daily. However, daily assessments of biolog- between V. darrowi and diploid highbush V. corym- ical parameters are often unsuitable for large, long- bosum (Lyrene 1997). Fla 4B has been used exten- term screening programs. By evaluating biological pa- sively to improve highbush blueberry, mainly south- rameters of M. persicae on potato genotypes at ern highbush cultivars (Draper 1995, Qu and Hancock different frequencies, Le Roux et al. (2004) found no 1997). Indeed, all Florida highbush cultivars and ad- signiÞcant differences in rm values estimated for vanced breeding lines contain Fla 4B lineage (Lyrene aphids with a sampling frequency of biological param- 1997). This study did not Þnd any signiÞcant differ- eters at 1, 2, or 3 d. Screening efforts were optimized ences in I. pepperi biological parameters between V. by evaluating adult aphids only during a period equiv- darrowi Fla 4B and V. corymbosum. As such, V. dar- alent to their prereproductive period and assessing rowi accessions other than Fla 4B offer more promise fecundity every 3 d (Le Roux et al. 2004). A 2-d as a source of aphid resistance traits. sampling frequency of biological parameters was used V. hirsutum and V. boreale also were identiÞed in in our study. our study as sources of aphid resistance. Longer pre- Lewontin (1965) argued that prereproductive de- reproductive periods, reduced offspring production, velopmental rate is essentially the single most impor- and decreased host acceptance were all exhibited by tant parameter inßuencing rm values, such that a 10% I. pepperi toward these Vaccinium spp. Decreased set- increase in developmental rate is equivalent to a 100% tling by I. pepperi on V. hirsutum may have been due increase in fertility. Compared with V. corymbosum to a dense covering of simple, nonglandular trichomes Elliott, the length of time required for I. pepperi to on stems and leaves (Camp 1945). Because only a few develop from birth to reproductive maturity was sig- minutes of probing by aphids is necessary for acqui- niÞcantly longer on V. myrsinites and V. boreale (Table sition and transmission of nonpersistent viruses (Har- 2). Therefore, reduced fecundity mainly accounted for the r values associated with V. myrsinites NJ88- ris 1990), such as BlScV, a resistance mechanism based m 07-43 and V. darrowi NJ88-09-04 being signiÞcantly on antixenosis could theoretically result in increased lower than V. corymbosum. However, despite differ- transmission. Alternatively, reducing population Þt- ences in offspring production between V. darrowi ness of the vector by targeting parameters such as NJ88-06-46 and V. corymbosum, signiÞcant differences developmental rates, fecundity, and longevity is a in r values were not detected. more suitable approach for attempting to use host m In summary, accessions of V. myrsinites and V. dar- plant resistance in a multifaceted approach to man- rowi represent poor hosts for I. pepperi and both spp. aging transmission of nonpersistent viruses. In addi- can be effectively crossed with highbush V. corymbo- tion, little movement of I. pepperi occurs outside of sum. Studies are currently underway to determine the cultivated highbush blueberries (Elsner 1982, Mori- variability in aphid resistance within and between moto and Ramsdell 1985) and transmission of BlScV populations of each of these Vaccinium spp., along occurs mainly within a row (Bristow et al. 2000). with characterizing the inheritance of resistance. Consequently, reducing I. pepperi population Þtness through plant resistance could be a useful management tactic. Acknowledgments In our study, rm values were calculated using the method of Wyatt and White (1977), which considers We thank Karen Geogehan for technical assistance and only the amount of offspring produced over a period colony maintenance and James Polashock (USDAÐARS, equivalent to the developmental time from birth to Chatsworth, NJ) for assistance with phenolic analyses. We also thank Patrick C. Tobin (USDAÐForest Service, Morgan- reproduction. Because calculating rm values using de- tailed life tables requires that each individual be mon- town, WV) for assistance with statistical analyses. This paper is dedicated to Professor Sridhar Polavarapu (1961Ð2004), itored until death (Birch 1948), the revised method of who served as Extension Entomologist for the Philip E. Ma- Wyatt and White (1977) is considerably less time- rucci Center for Blueberry and Cranberry Research and consuming and more appropriate for long-term Extension, Rutgers-The State University of New Jersey, for 10 screening programs. Hutchinson and Hogg (1984) yr. This work was supported in part by USDAÐCSREES later conÞrmed that the Þrst few days of aphid repro- 2005-34155Ð15749. duction contribute most to the rate of population increase. Furthermore, DeLoach (1974) and Le Roux et al. (2004) determined that 95 and Ͼ98%, respec- References Cited tively, of Myzus persicae (Sulzer) r values were ex- m Andrewartha, H. G., and L. C. Birch. 1954. 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