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Plant waxy bloom on peas affects infection of pea aphids by Pandora neoaphidis
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 149–158 www.elsevier.com/locate/yjipa
Plant waxy bloom on peas affects infection of pea aphids by Pandora neoaphidis Patrick S. Duetting,1 Hongjian Ding, Jeffrey Neufeld, and Sanford D. Eigenbrode* Division of Entomology, Department of Plant, Soil, and Entomological Sciences, College of Agricultural and Life Sciences, University of Idaho, Moscow, ID 83844-2339, USA Received 28 April 2003; accepted 14 October 2003
Abstract This study examined the effects of the surface wax bloom of pea plants, Pisum sativum, on infection of pea aphids, Acyrthosiphon pisum, by the fungal pathogen Pandora neoaphidis. In prior field surveys, a higher proportion of P. neoaphidis-killed pea aphids (cadavers) had been observed on a pea line with reduced wax bloom, as compared with a sister line with normal surface wax bloom. Laboratory bioassays were conducted in order to examine the mechanisms. After plants of each line infested with aphids were exposed to similar densities of conidia, the rate of accumulation of cadavers on the reduced wax line was significantly greater than on the normal wax bloom line; at the end of the experiment (13 d), the proportion of aphid cadavers on the reduced wax line was approximately four times that on the normal wax bloom line. When plants were exposed to conidia first and then infested with aphids, the rate of accumulation of cadavers was slightly but significantly greater on the reduced wax line, and infection at the end of the experiment (16 d) did not differ between the lines. When aphids were exposed first and then released onto the plants, no differences in the proportion of aphid cadavers were observed between the pea lines. Greater infection of pea aphid on reduced wax peas appears to depend upon plants being exposed to inoculum while aphids are settled in typical feeding positions on the plant. Additional experiments demonstrated increased adhesion and germination by P. neoaphidis conidia to leaf surfaces of the reduced wax line as compared with normal wax line, and this could help explain the higher infection rate by P. neoaphidis on the reduced wax line. In bioassays using surface waxes extracted from the two lines, there was no effect of wax source on germination of P. neoaphidis conidia. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Pisum sativum; Pandora neoaphidis; Acyrthosiphon pisum; Conidia; Plant surface wax; Tritrophic interaction; Adhesion; Germination; Extrinsic resistance
1. Introduction Plant characteristics can influence the effectiveness of natural enemies of insect herbivores. When conducive to natural enemy effectiveness, plant characteristics can confer an ecologically dependent or ÔextrinsicÕ resistance against insect herbivores (Price, 1986). Among the plant characteristics influencing predators and parasitoids are trichomes (Obrycki, 1986; van Lenteren and de Ponti, *
Corresponding author. Fax: 1-208-885-7760. E-mail address: [email protected] (S.D. Eigenbrode). 1 Present address: University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, 2199 S. Rock Rd., Ft. Pierce, FL 34945-3138, USA. 0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.10.001
1990), gross morphological characteristics such as plant size and architecture (Coll et al., 1997; Frazer and McGregor, 1994; Grevstad and Klepetka, 1992; Kareiva and Perry, 1989; Kareiva and Sahakian, 1990), and surface waxes (Eigenbrode and Kabalo, 1999; Eigenbrode et al., 1995, 1996, 1998a, 1999; White and Eigenbrode, 2000a,b). Effects of plant characteristics on insect pathogens have received less attention than have effects on insect carnivores (Elliot et al., 2000). Surface waxes are a potentially important plant characteristic in this context. In a notable series of papers, Inyang and others showed that cuticular wax extracts with associated phytochemicals from cruciferous plants influenced the germination behavior and pathogenicity of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.)
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Sorokin (Inyang et al., 1998a,b). Plant surface topography also influenced the acquisition of the pathogen by the leaf feeding beetle, Phaedon cochleariae F. (Inyang et al., 1998c). Whereas M. anisopliae is a generalist attacking insects in many environments, fungal pathogens in the order Entomophthorales (Zygomycetes) tend to be more specialized and frequently associated with their arthropod hosts on plant surfaces (Pell et al., 2001). Entomophthoralean fungi might, therefore, be especially sensitive to plant characteristics, including leaf waxes. Germination of the conidia of the entomophthoralean fungus Pandora neoaphidis (Remaudiere and Hennebert) Humber is inhibited by green-leaf volatiles from tobacco, Nicotiana tabacum L. (Brown et al., 1995). Field data suggest that P. neoaphidis might also be affected by plant surface waxes. Higher proportions of pea aphids, Acyrthosiphon pisum Harris (Homoptera: Aphididae), are killed in field trials by P. neoaphidis on a line of peas, Pisum sativum L. (Rutales: Fabaceae), with genetically reduced surface wax bloom (406G) than on a normal wax bloom sister line (406N) (Duetting, 2002; White, 1998). The biology of P. neoaphidis is closely tied to leaf surfaces. The fungus disperses by the forcible ejection of conidia from fungal-killed aphid cadavers (Humber, 1989). Dispersed conidia adhere by mucus to surfaces on which they land (Brobyn and Wilding, 1977; Butt et al., 1990). A conidium is capable of germinating to produce either a germ tube that infects the host or a secondary infective conidium (Butt et al., 1990). Typically, the majority of primary conidia fall onto leaf surfaces in the vicinity of host aphids rather than directly onto hosts. As a result, adequate attachment to the leaf surface, the production and viability of secondary conidia, and transfer of these propagules to host cuticle are important for the infection dynamics of P. neoaphidis (reviewed in Pell et al., 2001). Here we report the results of experiments to confirm that a higher proportion of pea aphids are killed by P. neoaphidis on reduced wax bloom vs. normal wax bloom peas, and to examine possible mechanisms. Confirmation of the field results (Duetting, 2002; White, 1998) was required because an apparent higher infection rate on reduced wax peas could have been produced by the greater impact of predators on pea aphids on these plants (Rutledge et al., 2003) coupled with discrimination by predators favoring healthy aphids (Pell et al., 1997; Roy et al., 1998), differences in the levels of P. neoaphidis inoculum on these plants, or other ecological factors. Our first experiment determined whether more pea aphids are killed by P. neoaphidis on the reduced wax bloom pea line under controlled laboratory conditions with uniform levels of P. neoaphidis inoculum. A second experiment focused on the role of the plant surface by comparing infection rates on plants of each pea line
onto which conidia were deposited prior to infesting the plants with aphids. A third experiment compared infection rates after first exposing aphids to P. neoaphidis conidia and then placing these exposed aphids on plants of each pea line. Two additional experiments examined specific mechanisms whereby surface waxes of the two pea isolines could influence infection rates of P. neoaphidis. One compared adhesion and germination of P. neoaphidis primary conidia on the surfaces of the two pea isolines. The other examined the effects of extracted surface waxes, which differ between the two lines in chemical composition (Eigenbrode et al., 1998b), on germination of P. neoaphidis conidia.
2. Materials and methods 2.1. Plants The wax eliminator (wel) mutation in pea (Marx, 1969) strongly reduces wax bloom over all aerial plant surfaces throughout development. Segregants of accession W6-15368 (Marx 406: P. sativum sativum) housed in the G.A. Marx Pea Genetic Stock Collection (USDA/ ARS Plant Station, Pullman Washington, USA) were used to generate two near-isogenic lines, reduced wax (glossy appearance) 406G (wel/wel), and normal wax 406N (Wel/Wel). The lines are also fixed for mutation tl (acacia leaf), which converts all tendrils to leaflets (Eigenbrode et al., 1998b). 406G and 406N are the isolines of peas on which differences in P. neoaphidis infection of pea aphid have been observed in the field (Duetting, 2002; White, 1998). Seeds were treated with the commercial rate (81.5 ml/ 50 kg seed) of Captan 400 fungicide (N-trichlormethylthio-4-cyclohexene-1,2-dicarboximide: Gustafson LLC; Plano, TX, USA), and refrigerated prior to planting. Seeds were planted at a depth of 5 cm within 10.2 10.2 cm plastic pots moderately packed with soil mix (9.5:1:1.7, Sunshine Potting Mix [Sun Gro Horticulture, Bellevue, Washington, USA]:sand:water, by volume). Plants to be used in experiments were planted one seed per pot and those for use in aphid rearing were planted five seeds per pot. Plants were grown in a greenhouse maintained at 20 °C with supplemental lighting (16:8, L:D) via sodium-halide lamps, and watered twice weekly. 2.2. Insects Pea aphid colonies were maintained in the insect biology laboratory (University of Idaho, Moscow). Aphids were originally collected from pea fields (Whitman County, Washington, USA) and have been reared in our laboratory for several years. Colonies are housed in 61 61 cm Plexiglas boxes ventilated through Ôno-see-
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umÕ netting (REI, Seattle Washington, USA). Inside the boxes, greenhouse-grown pea plants are used to support aphid populations. Plants are changed and watered as necessary. Before adding new plants, existing plants are carefully cut at the soil surface, and left within the box until the next plant change. This permits aphids to move onto new plants. Colony boxes are kept at room temperature, on shelves, and under fluorescent lighting (20 °C, 16:8 L:D). 2.3. Fungus Pandora neoaphidis fungal isolates (PANNEO 2754, 3240, and 3237) were obtained from the USDA/ARS Entomopathogenic Fungus Collection (Ithaca, New York, USA) and used together in a descending conidial shower (Papierok and Hajek, 1997) to expose individual pea plants infested with pea aphids, for the production of fungus-killed cadavers. Plants that contained cadavers were then placed into an established pea aphid colony (set up as described above) to perpetuate the fungus in vivo. This colony was kept at the H.C. Manis Laboratory for Entomological Research (University of Idaho, Moscow, Idaho, USA) in a growth chamber at 20 °C, with 16:8, L:D. Relative humidity (RH) inside the colony box ranged from 43 to 100% with a mean of 74.8 and a mode of 96.2 (N ¼ 1985). To enhance the spread of disease within this colony, water was occasionally applied as an aerosol to the plant surfaces. For all experiments, fresh P. neoaphidis cadavers were gently pried from the plant surface with forceps, placed into a new 60 15 mm plastic Petri plate sealed with Parafilm M (Pechiney Plastic Packaging; Neenah, Wisconsin, USA), and refrigerated until needed. Cadavers used in bioassays were refrigerated less than three weeks. 2.4. Infection rates on plants In the first of three experiments to estimate infection rates on the two pea isolines, plants from each isoline were infested with pea aphids and then exposed to P. neoaphidis inoculum. Individually potted plants of each isoline were cut down to three nodes with a razorblade to ensure similar size and plant architecture. Each plant was then infested with 30 mixed-age apterous, adult pea aphids from the colony, and covered with a 10 15 cm (d h) acetate tube with a Ôno-see-umÕ netting top for ventilation. The caged plants were incubated at 20 °C with 16:8, L:D for the remainder of the experiment. Aphids were then given 4 d to settle and reproduce. The morning of the fourth day, P. neoaphidis conidia were introduced from three cadavers using a descending conidial shower. The conidial shower took place inside the cage, from a 22 ml plastic rearing cup full of 1.5% water agar. Cadavers of equal age and size were placed equidistant across the agar surface. Wooden kabob skewers
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were placed in the soil and used to suspended rearing cups approximately 2 cm above the center of the plant. All cadavers sporulated copiously as evidenced by the presence of a thick whitish ring of conidia on the agar beneath each cadaver. The cadavers were left in the cage for 4 d. Based on previous work showing that a minimum of 72 h (Brobyn and Wilding, 1977; Butt et al., 1990) and typically 96 h (personal observations) is required for mycosis, plants were examined in the morning of the fourth day following conidial exposure, and every 2 d thereafter to 10 d, and finally on d 13 when aphid populations collapsed. For each plant, the numbers of aphids and cadavers were recorded. The experiment was performed twice with 17 and 18 replicates of 406G and 406N, respectively. In a second experiment, plants were infested with aphids after the plants had been exposed to P. neoaphidis conidia. This method eliminated infections caused by primary conidia falling directly onto the aphids, leaving only those caused by passive or active transfer of primary or secondary conidia from the plant surface to the aphids. Individually potted plants of both isolines were cut to three nodes, and exposed to P. neoaphidis conidia as in the first experiment, but for a period of 17.5 h with five sporulating cadavers, and prior to infestation with aphids. The number of cadavers used for inoculum was increased from the first experiment to compensate for the shorter exposure period. A shorter exposure period was used to maximize aphid exposure to viable conidia, as previous work has shown that 80% of conidia germinate within 6 h at optimal temperatures 18 and 21 °C (Morgan, 1994; Morgan et al., 1995). Following conidial exposure, plants were immediately infested with 30 apterous, adult pea aphids, and incubated at 20 °C with 16:8, L:D. Aphids and cadavers were counted every 2 d as in the previous experiment but for a period of 16 d. The experiment was performed twice, with 10 replicates of each isoline. A third experiment focused on indirect effects of the plant lines by first exposing aphids to P. neoaphidis primary conidia and then placing them on plants. Exposure was achieved by suspending sporulating cadavers over open containers of healthy aphids. Three hundred cadavers were placed on the surface of 1.5% agar in each of 4, 60 mm diam. Petri dishes, which were then sealed with Parafilm M to maintain high humidity and left at room temperature until the majority of cadavers were sporulating (16 h). To ensure uniform distribution of inoculum, the Petri dishes with sportulating cadavers were inverted over a slowly rotating carousel (1.3 rpm) holding four 100-mm-diam Petri dishes containing aphids to be exposed and covered with window screen to prevent aphid escape. To monitor exposure rate, two 60 mm Petri dishes with 22 44 mm glass cover-slides were also placed on the carousel. After exposure, these cover-slides were inspected with a microscope at 100 to
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estimate conidial dose. Exposure over 2–4 h resulted in estimated doses of 2.4 0.2, 13.4 1.3, and 127.3 3.6 (x SE) conidia/mm2 . Following exposure, 20 (for doses 1.4 and 13.4), or 25 (for dose 127.3) exposed aphids were transferred to an individual three-node plant, which was covered with an acetate cage and incubated in a growth chamber at 20 °C and 16:8, L:D. There were 12 replicates of each isoline for each dose. The number of cadavers on each plant was counted in the morning, 7 d after conidial exposure (156 h). Aphids succumbing to entomopathogenic fungi usually die in the late afternoon and evening (Milner and Bourne, 1983), and it takes a minimum of 72 h (Brobyn and Wilding, 1977; Butt et al., 1990) and generally 96 h (personal observations) following conidial exposure for A. pisum to die, so this interval was chosen to measure the maximum infection rate resulting from the initial exposure to conidia, and prior to the development of secondary infections from conidia having contacted the plant surface. For the first two experiments, replicates from the two experimental runs were pooled for analysis because the effect of experimental run was not significant in a preliminary analysis. All analyses were performed in SAS version 8.2 (SAS Institute, Cary, NC, USA). To normalize the data, the proportion of pea aphid cadavers per-plant was weighted by 1/r2 (Kleinbaum et al., 1998). A dual Quasi-Newton algorithm was used to fit the generalized non-linear model: Y ¼ b0 þ erðtlÞ , in which Y is the proportion of aphids infected, b0 ¼ 0:001 as the procedure requires a non-zero intercept, r is an instantaneous infection rate, t is time in days, and l is a lag term (time in days to disease initiation), on each plant isoline assuming a binomial response distribution (cadavers vs. healthy aphids) (proc NLMIXED). Estimated parameters for each line were subsequently compared using a dummy variable technique. In the third experiment, the progression of infection was not monitored, so analysis of variance (ANOVA) (proc GLM) was used to compare effects of dose and isoline on the proportion aphids killed by P. neoaphidis on d 7. To meet requirements of normality for parametric tests, data were transformed to arcsine square root (Kleinbaum et al., 1998). F ratios were calculated using the type III mean squares and residual error. At the end of each experiment, final proportions of cadavers on the two lines were compared with a t-test on arcsine-transformed data. 2.5. Adhesion and germination of conidia on the plant surface The relative effect of plant surface on P. neoaphidis conidial adhesion was determined by measuring the proportion of conidia that could be removed from leaf surfaces of the two pea isolines by rinsing in a stain solution with mechanical agitation. The effect of plant
surfaces on conidial germination was examined by recording the germination status of the conidia removed by rinsing and those remaining on each isoline. To apply conidia to leaflet surfaces, sporulating cadavers were affixed within a clip cage (2.9 2.5 cm, d h, Rutledge et al., 2003) attached to the first leaflet of the third node on six five-node plants of each isoline. Cotton balls moistened with distilled water were installed in the clip cages and two sporulating cadavers were positioned on the center of the cotton ball. Cages were then clipped over leaflets and positioned so that conidia produced by the cadavers would be deposited onto the surface of the leaflet. Plants and clip cages were left at room temperature (approximately 20 °C) for 24 h, after which the cadavers had sporulated copiously, producing a visible deposit of conidia on the leaflet surface. To measure adhesion by conidia, a cork borer was used to cut 6 mm disks including the patch of conidia on each leaflet. These disks were then placed in 1.5 ml microcentrifuge tubes with the conidia facing inward to prevent their abrasion by the walls of the tube. The leaf disks were covered with 450 ll of 2.5% aceto-orcein stain (Humber, 1997), and 550 ll of 95% ethyl alcohol. This was used to visualize the conidia in solution and prevent them from clumping together. Although ethanol may have denatured the adhesive mucus of the conidia, it should have done so equally on the two pea isolines. Each Eppendorf tube was then capped and vortexed at setting #6 (Vortex Genie 2: VWR Scientific; Batavia, IL) for 30 s. The leaf disks were then carefully removed and placed into new microcentrifuge tubes containing stain solution. These microcentrifuge tubes were then capped and sonicated with their contents for 5 min. Leaf disks were then removed from sonicated solutions and carefully placed onto glass slides. The leaf rinsates remaining in the microcentrifuge tubes (six vortexed and six sonicated for each pea isoline) were centrifuged for 10 min at 13,000 rpm. To concentrate the conidia in each microcentrifuge tube, 750 ll of solution was then pipetted from just under the surface and discarded. Discarded solutions were checked for conidia, but none were found. To redisperse conidia, the remaining 250 ll of solution was then vortexed for 1 min prior to sampling. Conidia on leaf disks and in solutions were counted at 100 under a compound microscope. This was possible because the brightly stained conidial nucleus was easily visible against the leaves. Each conidium counted was assigned to one of three classes: ungerminated, germinated with germ tube, or germinated with a secondary conidium. Conidia on leaf disks were counted in ten 1-mm2 quadrats along two randomly chosen transects. The mean was then used to estimate the total number of conidia on the leaf disk. Conidia removed by vortexing and sonicating were estimated by counting conidia in the concentrated solutions from each of these treatments using a haemocytometer. Each replicate was
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sampled 10 times to estimate the total number of conidia in the solution. The number in each removal treatment, or germination class was converted to a proportion of the total for each replicate. The experiment was performed twice. ANOVA was used to compare the effect of isoline on conidial adhesion and germination on the leaf surface. To meet requirements of normality for parametric tests, data were transformed to arcsine square root (Kleinbaum et al., 1998). The two experimental runs were combined and hypotheses tests were conducted using F ratios calculated with type III mean squares and replication experiment as the error term for experiment, and replication experiment isoline as the error term for isoline and experiment line. Least squares means was used to estimate parameters, which were backtransformed for presentation. 2.6. Conidial germination on extracted plant waxes Leaf waxes from 36, four- to five-node plants of each isoline were extracted with hexane. Each plant was cut at the base and dipped into hexane for 10 s. Care was taken to prevent any damaged plant portion from contacting the hexane. The hexane was evaporated under nitrogen at room temperature. Extracted waxes were weighed and redissolved in hexane (1:2, lg wax:ll hexane). Extracted wax solutions of each isoline were deposited onto 2.5-cm2 glass cover-slides at both a high and low coverages (26.6 and 1.7 lg of wax extract/cm2 ). Solutions were applied by pipette and distributed with a squeegee made from a 10-ll, glass micropipette. Hexane-rinsed cover-slides served as controls. Cover-slides were arranged in a randomized complete block design, and conidia were deposited from sporulating cadavers using the carousel as previously described. Following exposure, each cover-slide was placed within a 60 mm Petri dish lined with moistened filter paper. The dish was sealed with Parafilm M to maintain high humidity and incubated at 25 °C in the dark for 24 h. Conidia on each cover-slide were counted at 100 in ten, 1-mm2 quadrats along two randomly chosen transects and scored for germination as in the previous experiment. In each experimental run, there were five replicate blocks, and four experimental runs. ANOVA was used to compare the effect of treatment on the proportion of conidia in each germination class. To meet requirements of normality for parametric tests, data were transformed to arcsine square root (Kleinbaum et al., 1998). The four experimental runs were combined and hypotheses tests were conducted using F ratios calculated with type III mean squares, block experiment as the error term for experiment, and block (experiment treatment) as the error term for treatment. Least squares means was used to estimate
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parameters, which were back-transformed for presentation.
3. Results 3.1. Infection rates on plants In the experiment in which pea plants infested with pea aphids were exposed to P. neoaphidis conidia, the proportion of fungus-killed aphids was approximately four times greater on 406G than on 406N after 13 d (0.582 0.069 vs. 0.099 0.039, x SE, respectively) (Fig. 1). The lag term did not differ between 406G and 406N so the model was run with a common lag term (l ¼ 3:537 0:138, x SE). The rate of infection increase was significantly higher (F ¼ 698:14, df ¼ 1; 288, P < 0:0001) on 406G than on 406N (r ¼ 0:650 0:012 vs. 0.332 0.013, x SE, respectively). In the experiment in which pea plants were first exposed to P. neoaphidis conidia and then infested with pea aphids, the proportion of fungus killed-aphids did not differ between 406G and 406N after 16 d (0.756 0.108 vs. 0.621 0.095, x SE, respectively) (Fig. 2). However, the rate of infection increase and the lag term were significantly higher on 406G than on 406N
Fig. 1. The proportion of P. neoaphidis-killed pea aphid cadavers perplant from 0 to 13 d after exposing aphid-infested pea plants to P. neoaphidis conidia in a growth chamber experiment. Upper panel shows data for reduced wax pea line 406G; lower panel for normal wax pea line 406N. Plotted points are values for individual plants. The curve for the fitted model is superimposed on each plot.
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Fig. 2. The proportion of P. neoaphidis-killed pea aphid cadavers perplant from 0 to 16 d after exposing uninfested pea plants to P. neoaphidis conidia and then infesting them with aphids in a growth chamber experiment. Upper panel shows data for reduced wax pea line 406G; lower panel for normal wax pea line 406N. Plotted points are values for individual plants. The curve for the fitted model is superimposed on each plot.
(r ¼ 0:596 0:009 vs. 0.526 0.007, x SE, F ¼ 36:24, df ¼ 1; 260, P < 0:0001; l ¼ 5:111 0:142 vs. 3.920 0.148, x SE, F ¼ 33:82, df ¼ 1; 260, P < 0:0001, 406G and 406N, respectively) (Fig. 2). In the experiment in which pea aphids were first exposed to P. neoaphidis conidia and then released onto pea plants, conidial dose (F ¼ 66:90, df ¼ 2; 66, P < 0:0001), but not isoline (F ¼ 2:30, df ¼ 1; 66, P ¼ 0:1344), produced a significant effect on proportion of fungus-killed aphids after 7 d (Fig. 3).
Fig. 3. The proportion of P. neoaphidis-killed pea aphid cadavers perplant 7 d after exposing pea aphids to P. neoaphidis conidia at three doses and then placing them on pea plants. Upper panel shows data for reduced wax pea line 406G; lower panel for normal wax pea line 406N. Plotted points are values for individual plants.
3.2. Adhesion and germination of conidia on the plant surface Overall, the proportion of conidia remaining on leaf disks after vortexing and subsequent sonicating was significantly higher on 406G as compared with 406N (F ¼ 15:05, df ¼ 1; 10, P ¼ 0:0031), and although the proportions remaining on the leaves differed between the two experimental runs (F ¼ 7:66, df ¼ 1; 10, P ¼ 0:0198) there was no experimental run isoline interaction (F ¼ 0:32, df ¼ 1; 10, P ¼ 0:5826) (Fig. 4). The largest proportions of conidia were removed by vortexing (0.353 0.017 and 0.690 0.017 for run one, and 0.066 0.012 and 0.500 0.012 for run two, x SE, for 406G and 406N, respectively), with a very small proportion being removed during subsequent sonication (0.013 0.008 and 0.160 0.008 for run one, vs.
Fig. 4. The proportion of P. neoaphidis conidia remaining on upper surfaces of leaflets from the reduced wax pea line, 406G, and the normal wax pea line, 406N, following vortexing and then sonication. The hatched portion of each bar indicates the proportion of conidia removed by vortexing; the filled portion indicates the proportion of conidia removed by sonication; the open portion indicates the proportion of conidia remaining following both treatments.
0.111 0.006 and 0.023 0.006 for run two, x SE, for 406G and 406N, respectively). A significantly greater proportion of conidia inspected had germinated on 406G than on 406N
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(F ¼ 7:51, df ¼ 1; 10, P ¼ 0:0208). Overall germination rates differed significantly between experimental runs (F ¼ 15:34, df ¼ 1; 10, P ¼ 0:0029) but there was no experimental run isoline interaction (F ¼ 0:18, df ¼ 1; 10, P ¼ 0:6799) (Fig. 5). The type of germination causing the effect in each run was inconsistent. In the first experimental run, increased germination on 406G as compared with 406N was due to a greater proportion of conidia producing secondary conidia (0.241 0.009 vs. 0.075 0.009, x SE, respectively), whereas the proportion producing germ tubes was low and similar on the isolines (0.007 0.001 vs. 0.001 0.001, x SE, respectively). In the second experimental run, the increased proportion of conidia germinating on 406G as compared with 406N was due to an increased proportion of conidia producing germ tubes (0.159 0.012 vs. 0.054 0.012, x SE, respectively), whereas the proportion of conidia producing secondary conidia was the same on both isolines (0.387 0.004 vs. 0.392 0.004, x SE, respectively). 3.3. Conidial germination on extracted plant waxes The proportion of conidia having not produced a germ tube or secondary conidium after 24 h was
Fig. 5. Germination of P. neoaphidis conidia on upper leaf surfaces of two pea lines, 406G (reduced wax) and 406N (normal wax). The open portion of each bar indicates the proportion of conidia that were ungerminated, the filled portion indicates the proportion producing germ tubes, and the hatched portion indicates the proportion producing secondary conidia.
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approximately 0.6 (Table 1), with no effect of treatment (wax extract source or coverage, or clean glass control) on combined types of germination (F ¼ 1:02, df ¼ 4; 16, P ¼ 0:4247). A greater proportion of the conidia germinating produced secondary conidia as opposed to germ tubes (Table 1), but there was no effect of treatment on secondary conidia production (F ¼ 1:71, df ¼ 4; 16, P ¼ 0:1959). Formation of germ tubes occurred only in experimental runs two and four, creating a significant treatment run interaction for this response (F ¼ 2:83, df ¼ 12; 16, P ¼ 0:0273). Nevertheless, the proportion of conidia producing germ tubes was significantly affected by treatment (F ¼ 8:36, df ¼ 4; 16, P ¼ 0:0008), being highest on 406G wax applied at 26.6 lg/cm2 and lowest on clean glass (Table 1).
4. Discussion In a controlled laboratory experiment in which aphidinfested pea plants were exposed to a uniform level of P. neoaphidis conidia, a greater proportion of pea aphids were killed and produced cadavers on a reduced wax line (406G) than on a near isoline (406N) with normal wax bloom. Based on a fit of the data to a growth model, inception of infection was similar on the two lines, but the rate of increase in the proportion of aphid cadavers was approximately twice as great on the reduced wax line. This result appears to confirm that a physiological interaction between leaf waxes, aphids, and fungus contributes to the observed higher rates of infection of pea aphids by P. neoaphidis in the field (Duetting, 2002; White, 1998). Our experiments with conidia of P. neoaphidis suggest two possible mechanisms that could contribute to the greater infection of aphids by this fungus on reduced wax peas. First, we show that conidia of P. neoaphidis adhere more strongly to the surface of reduced wax peas. Greater retention of primary and secondary conidia on the surface of reduced wax peas should promote greater rates of contact with aphids and greater infection rates. Physical and chemical properties of the surface of the reduced-wax pea line could promote greater adhesion. Adhesion of phytopathogenic fungal spores is favored by increased hydrophilicity of the substrate (Blakeman, 1971; Heather, 1967), and contact angle
Table 1 Percent (SE) of P. neoaphidis conidia germinating to germ tubes, secondary conidia, or remaining ungerminated on extracted surface waxes of the two pea lines, 406G (reduced wax) and 406N (normal wax), deposited on glass at two levels of coverage Wax source
Coverage (lg/cm2 )
Germ tube
Secondary conidium
Ungerminated
406G 406N 406G 406N Control
26.6 26.6 1.7 1.7 0
6.3 0.01 3.5 0.01 3.2 0.01 2.0 0.01 2.0 0.01
30.4 0.02 33.6 0.02 36.2 0.02 36.1 0.02 34.5 0.02
59.4 0.02 60.7 0.02 57.1 0.02 60.7 0.02 62.1 0.02
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studies indicate that surfaces of normal wax peas are more hydrophobic than the surfaces of the reduced wax peas (Eigenbrode, unpublished data). This could be due to the greater amount of hydrophobic wax on the normal wax line (approx. 7 and 14.3 lg/cm2 on abaxial and adaxial surfaces, as compared with 1.6 and 1.8 lg/cm2 on the reduced wax line). In addition, the more strongly elaborated wax crystals on normal wax peas (Eigenbrode et al., 1998b) should further increase hydrophobic properties of the surface and interfere with conidial adhesion. Second, properties of the surfaces of the two lines also apparently influence germination of P. neoaphidis conidia. A higher proportion of conidia recovered from the surfaces of the reduced wax pea line had germinated. Higher rates of germination could indicate a greater proportion of conidia from these plants are vigorous and capable of infecting an aphid host. Our experiment with extracted waxes rules out a direct allelochemical effect of the waxes themselves, despite differences in their chemical composition (Eigenbrode et al., 1998b). Leaf wax extracts from either isoline only slightly enhanced the proportion of germ tubes produced at high wax concentrations but had no effect on overall germination rates. Although plant leaf waxes including pea leaf waxes can suppress germination of some phytopathogenic fungi (Blakeman, 1971, 1973; Carver et al., 1990; Heather, 1967), and leaf waxes of Brassica can suppress the germination of the entomopathogenic fungus M. anisopliae (Inyang et al., 1998b), pea waxes appear to have little effect on P. neoaphidis. Some hypotheses can be proposed to explain the effects of the wax mutation wel on P. neoaphidis germination, if a direct effect of wax chemistry can be ruled out. First, the RH at the phylloplane of 406G peas may be higher, and therefore more conducive to conidial germination. RH influences the viability, germination, and dispersal of several entomopathogens (Brobyn et al., 1987; Dillon and Charnely, 1990; Hajek and St. Leger, 1994), and the spread of P. neoaphidis disease can be limited by dry conditions in the field (Duetting, 2002; Feng et al., 1990; Rockwood, 1950; Wilding et al., 1986). A primary function of plant waxes is the prevention of water loss through the cuticle (Jenks and Ashworth, 1999) and the reduced amount of waxes on 406G may increase cuticular transpiration, causing higher RH at the phylloplane. It is also possible that differences in leaf wax affect the concentration or composition of plant volatiles near the leaf surface. Brown et al. (1995) have shown that green leaf volatiles of tobacco, N. tabacum, including hexanal, (E)-2-hexenal, and (E)-2-hexenol inhibit germination of P. neoaphidis conidia and reduce their infectivity to Myzus nicotianae L. RH and volatile organic compounds directly above the leaf surface of 406G and 406N have not been measured, but headspace volatiles trapped above whole plants of these isolines
and analyzed with gas chromatography–mass spectrometry do not differ (Ding and Eigenbrode, unpublished data). Thus, the evidence indicates that plant volatiles do not differently affect P. neoaphidis conidia on the two isolines. If interactions between the plant surface and conidia of P. neoaphidis can influence infection rates, we expected higher infection rates on reduced wax peas when intact pea plants were exposed to P. neoaphidis conidia immediately prior to being infested with aphids. However, in our experiment to test this, there was no difference in the proportion of aphids killed by the fungus on the two pea lines differing in surface wax. Although the rate of increase of infected aphids was significantly greater on the reduced wax line, this difference was slight (about 1.1 times that on the normal wax line). Additionally, inception of the infection was somewhat delayed in this experiment on the reduced wax line, based on a significantly greater lag term in the fitted model. We suspect, but cannot determine from our data, that much of the infection in this experiment occurred as aphids moved over the plants after being placed on them, thereby contacting the conidia that had been deposited on the upper surfaces of leaflets during the exposure. If so, probability of infection would be similar on the lines and might even initially be higher on the normal wax line, if poorer adhesion of P. neoaphidis conidia to these surfaces (Fig. 4) promoted their transfer to aphid cuticles. This effect could have caused the smaller lag term in the model for infection rate on the normal wax line. Eventually, the greater retention of conidia on the reduced wax line could have led to the greater rate of infection increase on that line and a trend towards higher proportion of infection on that line. In our experiment in which aphids were exposed to P. neoaphidis conidia prior to being placed on plants, the proportions of cadavers were not different on the two lines. This result indicates that indirect effects of the plant lines on the progression of infection and proportion of cadavers were inconsequential. In summary, greater rates of infection of pea aphid by P. neoaphidis on a reduced wax line of peas, 406G, as compared with its normal wax isoline 406G (Duetting, 2002; White, 1998) is associated with and could be explained by the greater adhesion and rates of germination of P. neoaphidis conidia on the surface of the reduced wax line. The results show that a simple genetic difference in the host plant can influence the disease-causing potential of an entomophthoralean fungus. The effects we document in the laboratory are subtle but evidently influence disease epidemiology. If sufficiently well understood, the mechanisms could guide genetic or artificial manipulations of the plant surface to promote fungal epizootics of aphids attacking peas or other crops. Our result also provides an additional mechanism whereby plant characteristics could influence insect
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pathogens to confer a form of extrinsic resistance to insect herbivory (Elliot et al., 2000).
Acknowledgments The manuscript was substantially improved by careful reviews provided by G. Knudsen, J.B. Johnson, M. Klowden, and two anonymous individuals. W. Price provided statistical advice and C.E. Rutledge provided insights throughout the execution of the experiments. The work was supported by grants to SDE from the USDA-CSREES-Cool Season Food Legume Special Grants Program.
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 149–158 www.elsevier.com/locate/yjipa
Plant waxy bloom on peas affects infection of pea aphids by Pandora neoaphidis Patrick S. Duetting,1 Hongjian Ding, Jeffrey Neufeld, and Sanford D. Eigenbrode* Division of Entomology, Department of Plant, Soil, and Entomological Sciences, College of Agricultural and Life Sciences, University of Idaho, Moscow, ID 83844-2339, USA Received 28 April 2003; accepted 14 October 2003
Abstract This study examined the effects of the surface wax bloom of pea plants, Pisum sativum, on infection of pea aphids, Acyrthosiphon pisum, by the fungal pathogen Pandora neoaphidis. In prior field surveys, a higher proportion of P. neoaphidis-killed pea aphids (cadavers) had been observed on a pea line with reduced wax bloom, as compared with a sister line with normal surface wax bloom. Laboratory bioassays were conducted in order to examine the mechanisms. After plants of each line infested with aphids were exposed to similar densities of conidia, the rate of accumulation of cadavers on the reduced wax line was significantly greater than on the normal wax bloom line; at the end of the experiment (13 d), the proportion of aphid cadavers on the reduced wax line was approximately four times that on the normal wax bloom line. When plants were exposed to conidia first and then infested with aphids, the rate of accumulation of cadavers was slightly but significantly greater on the reduced wax line, and infection at the end of the experiment (16 d) did not differ between the lines. When aphids were exposed first and then released onto the plants, no differences in the proportion of aphid cadavers were observed between the pea lines. Greater infection of pea aphid on reduced wax peas appears to depend upon plants being exposed to inoculum while aphids are settled in typical feeding positions on the plant. Additional experiments demonstrated increased adhesion and germination by P. neoaphidis conidia to leaf surfaces of the reduced wax line as compared with normal wax line, and this could help explain the higher infection rate by P. neoaphidis on the reduced wax line. In bioassays using surface waxes extracted from the two lines, there was no effect of wax source on germination of P. neoaphidis conidia. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Pisum sativum; Pandora neoaphidis; Acyrthosiphon pisum; Conidia; Plant surface wax; Tritrophic interaction; Adhesion; Germination; Extrinsic resistance
1. Introduction Plant characteristics can influence the effectiveness of natural enemies of insect herbivores. When conducive to natural enemy effectiveness, plant characteristics can confer an ecologically dependent or ÔextrinsicÕ resistance against insect herbivores (Price, 1986). Among the plant characteristics influencing predators and parasitoids are trichomes (Obrycki, 1986; van Lenteren and de Ponti, *
Corresponding author. Fax: 1-208-885-7760. E-mail address: [email protected] (S.D. Eigenbrode). 1 Present address: University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, 2199 S. Rock Rd., Ft. Pierce, FL 34945-3138, USA. 0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.10.001
1990), gross morphological characteristics such as plant size and architecture (Coll et al., 1997; Frazer and McGregor, 1994; Grevstad and Klepetka, 1992; Kareiva and Perry, 1989; Kareiva and Sahakian, 1990), and surface waxes (Eigenbrode and Kabalo, 1999; Eigenbrode et al., 1995, 1996, 1998a, 1999; White and Eigenbrode, 2000a,b). Effects of plant characteristics on insect pathogens have received less attention than have effects on insect carnivores (Elliot et al., 2000). Surface waxes are a potentially important plant characteristic in this context. In a notable series of papers, Inyang and others showed that cuticular wax extracts with associated phytochemicals from cruciferous plants influenced the germination behavior and pathogenicity of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.)
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Sorokin (Inyang et al., 1998a,b). Plant surface topography also influenced the acquisition of the pathogen by the leaf feeding beetle, Phaedon cochleariae F. (Inyang et al., 1998c). Whereas M. anisopliae is a generalist attacking insects in many environments, fungal pathogens in the order Entomophthorales (Zygomycetes) tend to be more specialized and frequently associated with their arthropod hosts on plant surfaces (Pell et al., 2001). Entomophthoralean fungi might, therefore, be especially sensitive to plant characteristics, including leaf waxes. Germination of the conidia of the entomophthoralean fungus Pandora neoaphidis (Remaudiere and Hennebert) Humber is inhibited by green-leaf volatiles from tobacco, Nicotiana tabacum L. (Brown et al., 1995). Field data suggest that P. neoaphidis might also be affected by plant surface waxes. Higher proportions of pea aphids, Acyrthosiphon pisum Harris (Homoptera: Aphididae), are killed in field trials by P. neoaphidis on a line of peas, Pisum sativum L. (Rutales: Fabaceae), with genetically reduced surface wax bloom (406G) than on a normal wax bloom sister line (406N) (Duetting, 2002; White, 1998). The biology of P. neoaphidis is closely tied to leaf surfaces. The fungus disperses by the forcible ejection of conidia from fungal-killed aphid cadavers (Humber, 1989). Dispersed conidia adhere by mucus to surfaces on which they land (Brobyn and Wilding, 1977; Butt et al., 1990). A conidium is capable of germinating to produce either a germ tube that infects the host or a secondary infective conidium (Butt et al., 1990). Typically, the majority of primary conidia fall onto leaf surfaces in the vicinity of host aphids rather than directly onto hosts. As a result, adequate attachment to the leaf surface, the production and viability of secondary conidia, and transfer of these propagules to host cuticle are important for the infection dynamics of P. neoaphidis (reviewed in Pell et al., 2001). Here we report the results of experiments to confirm that a higher proportion of pea aphids are killed by P. neoaphidis on reduced wax bloom vs. normal wax bloom peas, and to examine possible mechanisms. Confirmation of the field results (Duetting, 2002; White, 1998) was required because an apparent higher infection rate on reduced wax peas could have been produced by the greater impact of predators on pea aphids on these plants (Rutledge et al., 2003) coupled with discrimination by predators favoring healthy aphids (Pell et al., 1997; Roy et al., 1998), differences in the levels of P. neoaphidis inoculum on these plants, or other ecological factors. Our first experiment determined whether more pea aphids are killed by P. neoaphidis on the reduced wax bloom pea line under controlled laboratory conditions with uniform levels of P. neoaphidis inoculum. A second experiment focused on the role of the plant surface by comparing infection rates on plants of each pea line
onto which conidia were deposited prior to infesting the plants with aphids. A third experiment compared infection rates after first exposing aphids to P. neoaphidis conidia and then placing these exposed aphids on plants of each pea line. Two additional experiments examined specific mechanisms whereby surface waxes of the two pea isolines could influence infection rates of P. neoaphidis. One compared adhesion and germination of P. neoaphidis primary conidia on the surfaces of the two pea isolines. The other examined the effects of extracted surface waxes, which differ between the two lines in chemical composition (Eigenbrode et al., 1998b), on germination of P. neoaphidis conidia.
2. Materials and methods 2.1. Plants The wax eliminator (wel) mutation in pea (Marx, 1969) strongly reduces wax bloom over all aerial plant surfaces throughout development. Segregants of accession W6-15368 (Marx 406: P. sativum sativum) housed in the G.A. Marx Pea Genetic Stock Collection (USDA/ ARS Plant Station, Pullman Washington, USA) were used to generate two near-isogenic lines, reduced wax (glossy appearance) 406G (wel/wel), and normal wax 406N (Wel/Wel). The lines are also fixed for mutation tl (acacia leaf), which converts all tendrils to leaflets (Eigenbrode et al., 1998b). 406G and 406N are the isolines of peas on which differences in P. neoaphidis infection of pea aphid have been observed in the field (Duetting, 2002; White, 1998). Seeds were treated with the commercial rate (81.5 ml/ 50 kg seed) of Captan 400 fungicide (N-trichlormethylthio-4-cyclohexene-1,2-dicarboximide: Gustafson LLC; Plano, TX, USA), and refrigerated prior to planting. Seeds were planted at a depth of 5 cm within 10.2 10.2 cm plastic pots moderately packed with soil mix (9.5:1:1.7, Sunshine Potting Mix [Sun Gro Horticulture, Bellevue, Washington, USA]:sand:water, by volume). Plants to be used in experiments were planted one seed per pot and those for use in aphid rearing were planted five seeds per pot. Plants were grown in a greenhouse maintained at 20 °C with supplemental lighting (16:8, L:D) via sodium-halide lamps, and watered twice weekly. 2.2. Insects Pea aphid colonies were maintained in the insect biology laboratory (University of Idaho, Moscow). Aphids were originally collected from pea fields (Whitman County, Washington, USA) and have been reared in our laboratory for several years. Colonies are housed in 61 61 cm Plexiglas boxes ventilated through Ôno-see-
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umÕ netting (REI, Seattle Washington, USA). Inside the boxes, greenhouse-grown pea plants are used to support aphid populations. Plants are changed and watered as necessary. Before adding new plants, existing plants are carefully cut at the soil surface, and left within the box until the next plant change. This permits aphids to move onto new plants. Colony boxes are kept at room temperature, on shelves, and under fluorescent lighting (20 °C, 16:8 L:D). 2.3. Fungus Pandora neoaphidis fungal isolates (PANNEO 2754, 3240, and 3237) were obtained from the USDA/ARS Entomopathogenic Fungus Collection (Ithaca, New York, USA) and used together in a descending conidial shower (Papierok and Hajek, 1997) to expose individual pea plants infested with pea aphids, for the production of fungus-killed cadavers. Plants that contained cadavers were then placed into an established pea aphid colony (set up as described above) to perpetuate the fungus in vivo. This colony was kept at the H.C. Manis Laboratory for Entomological Research (University of Idaho, Moscow, Idaho, USA) in a growth chamber at 20 °C, with 16:8, L:D. Relative humidity (RH) inside the colony box ranged from 43 to 100% with a mean of 74.8 and a mode of 96.2 (N ¼ 1985). To enhance the spread of disease within this colony, water was occasionally applied as an aerosol to the plant surfaces. For all experiments, fresh P. neoaphidis cadavers were gently pried from the plant surface with forceps, placed into a new 60 15 mm plastic Petri plate sealed with Parafilm M (Pechiney Plastic Packaging; Neenah, Wisconsin, USA), and refrigerated until needed. Cadavers used in bioassays were refrigerated less than three weeks. 2.4. Infection rates on plants In the first of three experiments to estimate infection rates on the two pea isolines, plants from each isoline were infested with pea aphids and then exposed to P. neoaphidis inoculum. Individually potted plants of each isoline were cut down to three nodes with a razorblade to ensure similar size and plant architecture. Each plant was then infested with 30 mixed-age apterous, adult pea aphids from the colony, and covered with a 10 15 cm (d h) acetate tube with a Ôno-see-umÕ netting top for ventilation. The caged plants were incubated at 20 °C with 16:8, L:D for the remainder of the experiment. Aphids were then given 4 d to settle and reproduce. The morning of the fourth day, P. neoaphidis conidia were introduced from three cadavers using a descending conidial shower. The conidial shower took place inside the cage, from a 22 ml plastic rearing cup full of 1.5% water agar. Cadavers of equal age and size were placed equidistant across the agar surface. Wooden kabob skewers
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were placed in the soil and used to suspended rearing cups approximately 2 cm above the center of the plant. All cadavers sporulated copiously as evidenced by the presence of a thick whitish ring of conidia on the agar beneath each cadaver. The cadavers were left in the cage for 4 d. Based on previous work showing that a minimum of 72 h (Brobyn and Wilding, 1977; Butt et al., 1990) and typically 96 h (personal observations) is required for mycosis, plants were examined in the morning of the fourth day following conidial exposure, and every 2 d thereafter to 10 d, and finally on d 13 when aphid populations collapsed. For each plant, the numbers of aphids and cadavers were recorded. The experiment was performed twice with 17 and 18 replicates of 406G and 406N, respectively. In a second experiment, plants were infested with aphids after the plants had been exposed to P. neoaphidis conidia. This method eliminated infections caused by primary conidia falling directly onto the aphids, leaving only those caused by passive or active transfer of primary or secondary conidia from the plant surface to the aphids. Individually potted plants of both isolines were cut to three nodes, and exposed to P. neoaphidis conidia as in the first experiment, but for a period of 17.5 h with five sporulating cadavers, and prior to infestation with aphids. The number of cadavers used for inoculum was increased from the first experiment to compensate for the shorter exposure period. A shorter exposure period was used to maximize aphid exposure to viable conidia, as previous work has shown that 80% of conidia germinate within 6 h at optimal temperatures 18 and 21 °C (Morgan, 1994; Morgan et al., 1995). Following conidial exposure, plants were immediately infested with 30 apterous, adult pea aphids, and incubated at 20 °C with 16:8, L:D. Aphids and cadavers were counted every 2 d as in the previous experiment but for a period of 16 d. The experiment was performed twice, with 10 replicates of each isoline. A third experiment focused on indirect effects of the plant lines by first exposing aphids to P. neoaphidis primary conidia and then placing them on plants. Exposure was achieved by suspending sporulating cadavers over open containers of healthy aphids. Three hundred cadavers were placed on the surface of 1.5% agar in each of 4, 60 mm diam. Petri dishes, which were then sealed with Parafilm M to maintain high humidity and left at room temperature until the majority of cadavers were sporulating (16 h). To ensure uniform distribution of inoculum, the Petri dishes with sportulating cadavers were inverted over a slowly rotating carousel (1.3 rpm) holding four 100-mm-diam Petri dishes containing aphids to be exposed and covered with window screen to prevent aphid escape. To monitor exposure rate, two 60 mm Petri dishes with 22 44 mm glass cover-slides were also placed on the carousel. After exposure, these cover-slides were inspected with a microscope at 100 to
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estimate conidial dose. Exposure over 2–4 h resulted in estimated doses of 2.4 0.2, 13.4 1.3, and 127.3 3.6 (x SE) conidia/mm2 . Following exposure, 20 (for doses 1.4 and 13.4), or 25 (for dose 127.3) exposed aphids were transferred to an individual three-node plant, which was covered with an acetate cage and incubated in a growth chamber at 20 °C and 16:8, L:D. There were 12 replicates of each isoline for each dose. The number of cadavers on each plant was counted in the morning, 7 d after conidial exposure (156 h). Aphids succumbing to entomopathogenic fungi usually die in the late afternoon and evening (Milner and Bourne, 1983), and it takes a minimum of 72 h (Brobyn and Wilding, 1977; Butt et al., 1990) and generally 96 h (personal observations) following conidial exposure for A. pisum to die, so this interval was chosen to measure the maximum infection rate resulting from the initial exposure to conidia, and prior to the development of secondary infections from conidia having contacted the plant surface. For the first two experiments, replicates from the two experimental runs were pooled for analysis because the effect of experimental run was not significant in a preliminary analysis. All analyses were performed in SAS version 8.2 (SAS Institute, Cary, NC, USA). To normalize the data, the proportion of pea aphid cadavers per-plant was weighted by 1/r2 (Kleinbaum et al., 1998). A dual Quasi-Newton algorithm was used to fit the generalized non-linear model: Y ¼ b0 þ erðtlÞ , in which Y is the proportion of aphids infected, b0 ¼ 0:001 as the procedure requires a non-zero intercept, r is an instantaneous infection rate, t is time in days, and l is a lag term (time in days to disease initiation), on each plant isoline assuming a binomial response distribution (cadavers vs. healthy aphids) (proc NLMIXED). Estimated parameters for each line were subsequently compared using a dummy variable technique. In the third experiment, the progression of infection was not monitored, so analysis of variance (ANOVA) (proc GLM) was used to compare effects of dose and isoline on the proportion aphids killed by P. neoaphidis on d 7. To meet requirements of normality for parametric tests, data were transformed to arcsine square root (Kleinbaum et al., 1998). F ratios were calculated using the type III mean squares and residual error. At the end of each experiment, final proportions of cadavers on the two lines were compared with a t-test on arcsine-transformed data. 2.5. Adhesion and germination of conidia on the plant surface The relative effect of plant surface on P. neoaphidis conidial adhesion was determined by measuring the proportion of conidia that could be removed from leaf surfaces of the two pea isolines by rinsing in a stain solution with mechanical agitation. The effect of plant
surfaces on conidial germination was examined by recording the germination status of the conidia removed by rinsing and those remaining on each isoline. To apply conidia to leaflet surfaces, sporulating cadavers were affixed within a clip cage (2.9 2.5 cm, d h, Rutledge et al., 2003) attached to the first leaflet of the third node on six five-node plants of each isoline. Cotton balls moistened with distilled water were installed in the clip cages and two sporulating cadavers were positioned on the center of the cotton ball. Cages were then clipped over leaflets and positioned so that conidia produced by the cadavers would be deposited onto the surface of the leaflet. Plants and clip cages were left at room temperature (approximately 20 °C) for 24 h, after which the cadavers had sporulated copiously, producing a visible deposit of conidia on the leaflet surface. To measure adhesion by conidia, a cork borer was used to cut 6 mm disks including the patch of conidia on each leaflet. These disks were then placed in 1.5 ml microcentrifuge tubes with the conidia facing inward to prevent their abrasion by the walls of the tube. The leaf disks were covered with 450 ll of 2.5% aceto-orcein stain (Humber, 1997), and 550 ll of 95% ethyl alcohol. This was used to visualize the conidia in solution and prevent them from clumping together. Although ethanol may have denatured the adhesive mucus of the conidia, it should have done so equally on the two pea isolines. Each Eppendorf tube was then capped and vortexed at setting #6 (Vortex Genie 2: VWR Scientific; Batavia, IL) for 30 s. The leaf disks were then carefully removed and placed into new microcentrifuge tubes containing stain solution. These microcentrifuge tubes were then capped and sonicated with their contents for 5 min. Leaf disks were then removed from sonicated solutions and carefully placed onto glass slides. The leaf rinsates remaining in the microcentrifuge tubes (six vortexed and six sonicated for each pea isoline) were centrifuged for 10 min at 13,000 rpm. To concentrate the conidia in each microcentrifuge tube, 750 ll of solution was then pipetted from just under the surface and discarded. Discarded solutions were checked for conidia, but none were found. To redisperse conidia, the remaining 250 ll of solution was then vortexed for 1 min prior to sampling. Conidia on leaf disks and in solutions were counted at 100 under a compound microscope. This was possible because the brightly stained conidial nucleus was easily visible against the leaves. Each conidium counted was assigned to one of three classes: ungerminated, germinated with germ tube, or germinated with a secondary conidium. Conidia on leaf disks were counted in ten 1-mm2 quadrats along two randomly chosen transects. The mean was then used to estimate the total number of conidia on the leaf disk. Conidia removed by vortexing and sonicating were estimated by counting conidia in the concentrated solutions from each of these treatments using a haemocytometer. Each replicate was
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sampled 10 times to estimate the total number of conidia in the solution. The number in each removal treatment, or germination class was converted to a proportion of the total for each replicate. The experiment was performed twice. ANOVA was used to compare the effect of isoline on conidial adhesion and germination on the leaf surface. To meet requirements of normality for parametric tests, data were transformed to arcsine square root (Kleinbaum et al., 1998). The two experimental runs were combined and hypotheses tests were conducted using F ratios calculated with type III mean squares and replication experiment as the error term for experiment, and replication experiment isoline as the error term for isoline and experiment line. Least squares means was used to estimate parameters, which were backtransformed for presentation. 2.6. Conidial germination on extracted plant waxes Leaf waxes from 36, four- to five-node plants of each isoline were extracted with hexane. Each plant was cut at the base and dipped into hexane for 10 s. Care was taken to prevent any damaged plant portion from contacting the hexane. The hexane was evaporated under nitrogen at room temperature. Extracted waxes were weighed and redissolved in hexane (1:2, lg wax:ll hexane). Extracted wax solutions of each isoline were deposited onto 2.5-cm2 glass cover-slides at both a high and low coverages (26.6 and 1.7 lg of wax extract/cm2 ). Solutions were applied by pipette and distributed with a squeegee made from a 10-ll, glass micropipette. Hexane-rinsed cover-slides served as controls. Cover-slides were arranged in a randomized complete block design, and conidia were deposited from sporulating cadavers using the carousel as previously described. Following exposure, each cover-slide was placed within a 60 mm Petri dish lined with moistened filter paper. The dish was sealed with Parafilm M to maintain high humidity and incubated at 25 °C in the dark for 24 h. Conidia on each cover-slide were counted at 100 in ten, 1-mm2 quadrats along two randomly chosen transects and scored for germination as in the previous experiment. In each experimental run, there were five replicate blocks, and four experimental runs. ANOVA was used to compare the effect of treatment on the proportion of conidia in each germination class. To meet requirements of normality for parametric tests, data were transformed to arcsine square root (Kleinbaum et al., 1998). The four experimental runs were combined and hypotheses tests were conducted using F ratios calculated with type III mean squares, block experiment as the error term for experiment, and block (experiment treatment) as the error term for treatment. Least squares means was used to estimate
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parameters, which were back-transformed for presentation.
3. Results 3.1. Infection rates on plants In the experiment in which pea plants infested with pea aphids were exposed to P. neoaphidis conidia, the proportion of fungus-killed aphids was approximately four times greater on 406G than on 406N after 13 d (0.582 0.069 vs. 0.099 0.039, x SE, respectively) (Fig. 1). The lag term did not differ between 406G and 406N so the model was run with a common lag term (l ¼ 3:537 0:138, x SE). The rate of infection increase was significantly higher (F ¼ 698:14, df ¼ 1; 288, P < 0:0001) on 406G than on 406N (r ¼ 0:650 0:012 vs. 0.332 0.013, x SE, respectively). In the experiment in which pea plants were first exposed to P. neoaphidis conidia and then infested with pea aphids, the proportion of fungus killed-aphids did not differ between 406G and 406N after 16 d (0.756 0.108 vs. 0.621 0.095, x SE, respectively) (Fig. 2). However, the rate of infection increase and the lag term were significantly higher on 406G than on 406N
Fig. 1. The proportion of P. neoaphidis-killed pea aphid cadavers perplant from 0 to 13 d after exposing aphid-infested pea plants to P. neoaphidis conidia in a growth chamber experiment. Upper panel shows data for reduced wax pea line 406G; lower panel for normal wax pea line 406N. Plotted points are values for individual plants. The curve for the fitted model is superimposed on each plot.
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Fig. 2. The proportion of P. neoaphidis-killed pea aphid cadavers perplant from 0 to 16 d after exposing uninfested pea plants to P. neoaphidis conidia and then infesting them with aphids in a growth chamber experiment. Upper panel shows data for reduced wax pea line 406G; lower panel for normal wax pea line 406N. Plotted points are values for individual plants. The curve for the fitted model is superimposed on each plot.
(r ¼ 0:596 0:009 vs. 0.526 0.007, x SE, F ¼ 36:24, df ¼ 1; 260, P < 0:0001; l ¼ 5:111 0:142 vs. 3.920 0.148, x SE, F ¼ 33:82, df ¼ 1; 260, P < 0:0001, 406G and 406N, respectively) (Fig. 2). In the experiment in which pea aphids were first exposed to P. neoaphidis conidia and then released onto pea plants, conidial dose (F ¼ 66:90, df ¼ 2; 66, P < 0:0001), but not isoline (F ¼ 2:30, df ¼ 1; 66, P ¼ 0:1344), produced a significant effect on proportion of fungus-killed aphids after 7 d (Fig. 3).
Fig. 3. The proportion of P. neoaphidis-killed pea aphid cadavers perplant 7 d after exposing pea aphids to P. neoaphidis conidia at three doses and then placing them on pea plants. Upper panel shows data for reduced wax pea line 406G; lower panel for normal wax pea line 406N. Plotted points are values for individual plants.
3.2. Adhesion and germination of conidia on the plant surface Overall, the proportion of conidia remaining on leaf disks after vortexing and subsequent sonicating was significantly higher on 406G as compared with 406N (F ¼ 15:05, df ¼ 1; 10, P ¼ 0:0031), and although the proportions remaining on the leaves differed between the two experimental runs (F ¼ 7:66, df ¼ 1; 10, P ¼ 0:0198) there was no experimental run isoline interaction (F ¼ 0:32, df ¼ 1; 10, P ¼ 0:5826) (Fig. 4). The largest proportions of conidia were removed by vortexing (0.353 0.017 and 0.690 0.017 for run one, and 0.066 0.012 and 0.500 0.012 for run two, x SE, for 406G and 406N, respectively), with a very small proportion being removed during subsequent sonication (0.013 0.008 and 0.160 0.008 for run one, vs.
Fig. 4. The proportion of P. neoaphidis conidia remaining on upper surfaces of leaflets from the reduced wax pea line, 406G, and the normal wax pea line, 406N, following vortexing and then sonication. The hatched portion of each bar indicates the proportion of conidia removed by vortexing; the filled portion indicates the proportion of conidia removed by sonication; the open portion indicates the proportion of conidia remaining following both treatments.
0.111 0.006 and 0.023 0.006 for run two, x SE, for 406G and 406N, respectively). A significantly greater proportion of conidia inspected had germinated on 406G than on 406N
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(F ¼ 7:51, df ¼ 1; 10, P ¼ 0:0208). Overall germination rates differed significantly between experimental runs (F ¼ 15:34, df ¼ 1; 10, P ¼ 0:0029) but there was no experimental run isoline interaction (F ¼ 0:18, df ¼ 1; 10, P ¼ 0:6799) (Fig. 5). The type of germination causing the effect in each run was inconsistent. In the first experimental run, increased germination on 406G as compared with 406N was due to a greater proportion of conidia producing secondary conidia (0.241 0.009 vs. 0.075 0.009, x SE, respectively), whereas the proportion producing germ tubes was low and similar on the isolines (0.007 0.001 vs. 0.001 0.001, x SE, respectively). In the second experimental run, the increased proportion of conidia germinating on 406G as compared with 406N was due to an increased proportion of conidia producing germ tubes (0.159 0.012 vs. 0.054 0.012, x SE, respectively), whereas the proportion of conidia producing secondary conidia was the same on both isolines (0.387 0.004 vs. 0.392 0.004, x SE, respectively). 3.3. Conidial germination on extracted plant waxes The proportion of conidia having not produced a germ tube or secondary conidium after 24 h was
Fig. 5. Germination of P. neoaphidis conidia on upper leaf surfaces of two pea lines, 406G (reduced wax) and 406N (normal wax). The open portion of each bar indicates the proportion of conidia that were ungerminated, the filled portion indicates the proportion producing germ tubes, and the hatched portion indicates the proportion producing secondary conidia.
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approximately 0.6 (Table 1), with no effect of treatment (wax extract source or coverage, or clean glass control) on combined types of germination (F ¼ 1:02, df ¼ 4; 16, P ¼ 0:4247). A greater proportion of the conidia germinating produced secondary conidia as opposed to germ tubes (Table 1), but there was no effect of treatment on secondary conidia production (F ¼ 1:71, df ¼ 4; 16, P ¼ 0:1959). Formation of germ tubes occurred only in experimental runs two and four, creating a significant treatment run interaction for this response (F ¼ 2:83, df ¼ 12; 16, P ¼ 0:0273). Nevertheless, the proportion of conidia producing germ tubes was significantly affected by treatment (F ¼ 8:36, df ¼ 4; 16, P ¼ 0:0008), being highest on 406G wax applied at 26.6 lg/cm2 and lowest on clean glass (Table 1).
4. Discussion In a controlled laboratory experiment in which aphidinfested pea plants were exposed to a uniform level of P. neoaphidis conidia, a greater proportion of pea aphids were killed and produced cadavers on a reduced wax line (406G) than on a near isoline (406N) with normal wax bloom. Based on a fit of the data to a growth model, inception of infection was similar on the two lines, but the rate of increase in the proportion of aphid cadavers was approximately twice as great on the reduced wax line. This result appears to confirm that a physiological interaction between leaf waxes, aphids, and fungus contributes to the observed higher rates of infection of pea aphids by P. neoaphidis in the field (Duetting, 2002; White, 1998). Our experiments with conidia of P. neoaphidis suggest two possible mechanisms that could contribute to the greater infection of aphids by this fungus on reduced wax peas. First, we show that conidia of P. neoaphidis adhere more strongly to the surface of reduced wax peas. Greater retention of primary and secondary conidia on the surface of reduced wax peas should promote greater rates of contact with aphids and greater infection rates. Physical and chemical properties of the surface of the reduced-wax pea line could promote greater adhesion. Adhesion of phytopathogenic fungal spores is favored by increased hydrophilicity of the substrate (Blakeman, 1971; Heather, 1967), and contact angle
Table 1 Percent (SE) of P. neoaphidis conidia germinating to germ tubes, secondary conidia, or remaining ungerminated on extracted surface waxes of the two pea lines, 406G (reduced wax) and 406N (normal wax), deposited on glass at two levels of coverage Wax source
Coverage (lg/cm2 )
Germ tube
Secondary conidium
Ungerminated
406G 406N 406G 406N Control
26.6 26.6 1.7 1.7 0
6.3 0.01 3.5 0.01 3.2 0.01 2.0 0.01 2.0 0.01
30.4 0.02 33.6 0.02 36.2 0.02 36.1 0.02 34.5 0.02
59.4 0.02 60.7 0.02 57.1 0.02 60.7 0.02 62.1 0.02
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studies indicate that surfaces of normal wax peas are more hydrophobic than the surfaces of the reduced wax peas (Eigenbrode, unpublished data). This could be due to the greater amount of hydrophobic wax on the normal wax line (approx. 7 and 14.3 lg/cm2 on abaxial and adaxial surfaces, as compared with 1.6 and 1.8 lg/cm2 on the reduced wax line). In addition, the more strongly elaborated wax crystals on normal wax peas (Eigenbrode et al., 1998b) should further increase hydrophobic properties of the surface and interfere with conidial adhesion. Second, properties of the surfaces of the two lines also apparently influence germination of P. neoaphidis conidia. A higher proportion of conidia recovered from the surfaces of the reduced wax pea line had germinated. Higher rates of germination could indicate a greater proportion of conidia from these plants are vigorous and capable of infecting an aphid host. Our experiment with extracted waxes rules out a direct allelochemical effect of the waxes themselves, despite differences in their chemical composition (Eigenbrode et al., 1998b). Leaf wax extracts from either isoline only slightly enhanced the proportion of germ tubes produced at high wax concentrations but had no effect on overall germination rates. Although plant leaf waxes including pea leaf waxes can suppress germination of some phytopathogenic fungi (Blakeman, 1971, 1973; Carver et al., 1990; Heather, 1967), and leaf waxes of Brassica can suppress the germination of the entomopathogenic fungus M. anisopliae (Inyang et al., 1998b), pea waxes appear to have little effect on P. neoaphidis. Some hypotheses can be proposed to explain the effects of the wax mutation wel on P. neoaphidis germination, if a direct effect of wax chemistry can be ruled out. First, the RH at the phylloplane of 406G peas may be higher, and therefore more conducive to conidial germination. RH influences the viability, germination, and dispersal of several entomopathogens (Brobyn et al., 1987; Dillon and Charnely, 1990; Hajek and St. Leger, 1994), and the spread of P. neoaphidis disease can be limited by dry conditions in the field (Duetting, 2002; Feng et al., 1990; Rockwood, 1950; Wilding et al., 1986). A primary function of plant waxes is the prevention of water loss through the cuticle (Jenks and Ashworth, 1999) and the reduced amount of waxes on 406G may increase cuticular transpiration, causing higher RH at the phylloplane. It is also possible that differences in leaf wax affect the concentration or composition of plant volatiles near the leaf surface. Brown et al. (1995) have shown that green leaf volatiles of tobacco, N. tabacum, including hexanal, (E)-2-hexenal, and (E)-2-hexenol inhibit germination of P. neoaphidis conidia and reduce their infectivity to Myzus nicotianae L. RH and volatile organic compounds directly above the leaf surface of 406G and 406N have not been measured, but headspace volatiles trapped above whole plants of these isolines
and analyzed with gas chromatography–mass spectrometry do not differ (Ding and Eigenbrode, unpublished data). Thus, the evidence indicates that plant volatiles do not differently affect P. neoaphidis conidia on the two isolines. If interactions between the plant surface and conidia of P. neoaphidis can influence infection rates, we expected higher infection rates on reduced wax peas when intact pea plants were exposed to P. neoaphidis conidia immediately prior to being infested with aphids. However, in our experiment to test this, there was no difference in the proportion of aphids killed by the fungus on the two pea lines differing in surface wax. Although the rate of increase of infected aphids was significantly greater on the reduced wax line, this difference was slight (about 1.1 times that on the normal wax line). Additionally, inception of the infection was somewhat delayed in this experiment on the reduced wax line, based on a significantly greater lag term in the fitted model. We suspect, but cannot determine from our data, that much of the infection in this experiment occurred as aphids moved over the plants after being placed on them, thereby contacting the conidia that had been deposited on the upper surfaces of leaflets during the exposure. If so, probability of infection would be similar on the lines and might even initially be higher on the normal wax line, if poorer adhesion of P. neoaphidis conidia to these surfaces (Fig. 4) promoted their transfer to aphid cuticles. This effect could have caused the smaller lag term in the model for infection rate on the normal wax line. Eventually, the greater retention of conidia on the reduced wax line could have led to the greater rate of infection increase on that line and a trend towards higher proportion of infection on that line. In our experiment in which aphids were exposed to P. neoaphidis conidia prior to being placed on plants, the proportions of cadavers were not different on the two lines. This result indicates that indirect effects of the plant lines on the progression of infection and proportion of cadavers were inconsequential. In summary, greater rates of infection of pea aphid by P. neoaphidis on a reduced wax line of peas, 406G, as compared with its normal wax isoline 406G (Duetting, 2002; White, 1998) is associated with and could be explained by the greater adhesion and rates of germination of P. neoaphidis conidia on the surface of the reduced wax line. The results show that a simple genetic difference in the host plant can influence the disease-causing potential of an entomophthoralean fungus. The effects we document in the laboratory are subtle but evidently influence disease epidemiology. If sufficiently well understood, the mechanisms could guide genetic or artificial manipulations of the plant surface to promote fungal epizootics of aphids attacking peas or other crops. Our result also provides an additional mechanism whereby plant characteristics could influence insect
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pathogens to confer a form of extrinsic resistance to insect herbivory (Elliot et al., 2000).
Acknowledgments The manuscript was substantially improved by careful reviews provided by G. Knudsen, J.B. Johnson, M. Klowden, and two anonymous individuals. W. Price provided statistical advice and C.E. Rutledge provided insights throughout the execution of the experiments. The work was supported by grants to SDE from the USDA-CSREES-Cool Season Food Legume Special Grants Program.
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