Multitrophic interactions and the diamondback moth: implications for pest management

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1 Multitrophic interactions and the diamondback moth: implications for pest management Robert H. J. Verkerk and Denis J. Wright Department of Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, U.K. Abstract There is a scarcity of studies concerning Plutella xylostella L. dealing with either interactions between partial plant resistance and insecticides or multitrophic interactions (with or without insecticides). Such studies are vital to allow improved understanding of possible interactions in IPM programmes and may help both to develop approaches which enhance cultural and biological controls and minimise insecticide use. Laboratory studies showing the relationship between different degrees of partial plant resistance to P. xylostella (in various Brassica oleracea var. capitata L. and B. pekinensis (Lour.) Rupr. cultivars), leaf biomass consumption, leaf surface area damage and residual toxicity of two selective insecticides are presented and reasons for consistent relationships between various factors are discussed. Insecticide toxicity to P. xylostella larvae on plants with partial plant resistance is shown to be less compared with insecticides applied to either more or less resistant plants. Tritrophic laboratory and field studies in Malaysia show that larvae of P. xylostella on different host plant species and cultivars may experience different and sometimes contrasting rates of relative survival and parasitism by the key endolarval hymenopteran parasitoids, Diadegma semiclausum Hellén and Cotesia plutellae Kurdj. Finally, the value of multitrophic studies is discussed and their potential for stimulating novel strategies is considered. Key words: host plant, parasitoid, Plutella xylostella, insecticide, integrated pest management Introduction Plutella xylostella L. (Lepidoptera: Yponomeutidae), has been intensively studied for more than forty years, reflecting its importance as a key pest of cruciferous crops in many parts of the world. During this period, most studies concerning P. xylostella have focused on its biology, methods of control or resistance to insecticides (e.g., Talekar and Griggs, 1986; Talekar, 1992). Insect/plant-based research on multitrophic interactions has recently been developing rapidly, although the focus has been predominantly on nonagricultural systems (Wright and Verkerk, 1995). A recent database search revealed that less than 5% of publications involving P. xylostella considered tritrophic (host plant-dbm-natural enemy) interactions or interactions between two or more different categories of control method (e.g., biological, cultural, physical, chemical) (Verkerk, 1995). This paper considers ways in which an improved understanding of multitrophic interactions involving P. xylostella, including interactions with selective insecticides, may potentially be used to improve levels of control with reduced input of insecticides. Such approaches are vital to help understand the multitude of interactions which may occur as a result of integrated pest management (IPM) programmes and could alleviate problems caused by insecticide resistance and resurgence which have often plagued control strategies aimed at P. xylostella. Materials and Methods All laboratory experiments were maintained in a controlled environment (20±2 o C; 65% RH, 16:8 L:D). Intrinsic host plant resistance Leaf biomass consumption. Groups of five, two-dayold second instar P. xylostella larvae (SERD 2 strain: F 18 from the field; Serdang, Malaysia) were introduced to individual Petri dishes (5 cm) containing leaf discs (5 cm dia.) cut from glasshouse-grown Brassica pekinensis (Lour.) Rupr. cv. Tip Top (84 days-old), B. oleracea var. capitata L. cv. Wheelers Imperial and cv. Red Drumhead (84 and 147 days-old for both cultivars). After 120 h, when most insects were either fourth instar or prepupae, mortality was assessed, leaf disc surface area damage estimated (see Table 1) and the leaf discs were removed, oven-dried and weighed. Control leaf discs were treated in the same way but were not infested. Biomass consumption by surviving larvae was determined by the difference between dry weights of undamaged and damaged leaf discs for each plant group. Interactions between plant resistance and selective insecticides Residual toxicity/plant resistance interactions. Leafdip bioassays were carried out using two age-classes of two glasshouse-grown B. oleracea var. capitata cultivars (Wheelers Imperial and Red Drumhead: 84 and 147-day-old) and a field strain of P. xylostella (SERD 2: F 18 ; see above). Four concentrations of teflubenzuron (Nomolt ) (0.02, 0.07, 2.00 and 6.00 µg ai ml -1 ) and Bacillus thuringiensis (Bt) Berliner subsp. aizawai (Florbac ) (0.04, 0.11, 0.33 and 1.00 IU mg -1 ) were assayed. Treated leaf discs were removed after 5 days and replaced with fresh, untreated leaf discs ad lib. Mortality was assessed on Days 5, 9 Development and implementation of IPM 243

2 and 12. Leaf disc damage was estimated on Day 5 according to an arbitrary scale (see Leaf Biomass, above). Tritrophic interactions Laboratory studies. a) Cotesia plutellae Kurdj. (Hymenoptera: Braconidae). Single, mated three-dayold female C. plutellae (laboratory culture, IACR, Rothamsted, UK) were introduced to plastic Petri dishes (5 cm dia.), each containing a moistened filter paper (Whatmans No. 1; 4.5 cm dia.), a leaf disc (4.8 cm dia.) from the selected plant group and 10 twoday-old second instar larvae (insecticide-susceptible laboratory strain, Rothamsted Experimental Station, UK). Leaf discs were cut from outer leaves selected at random from at least 6 different plants from each group. Larvae were starved for 2 h prior to being transferred to the leaf discs. Parasitoids were removed from Petri dishes after 1 h, a duration found to allow oviposition attempts in most, if not all, ten larvae. Leaf discs were replaced after four days and subsequently ad lib. Treatments with parasitoids were replicated 8 times. Life stages were recorded after 5, 7 and 9 days and then at daily intervals until all insects had either emerged as adults or died. b) Diadegma semiclausum Hellén (Hymenoptera: Ichneumonidae). Single, mated three-day-old female D. semiclausum (laboratory culture, IACR, Rothamsted, UK) were introduced to plastic Petri dishes (9 cm dia.), each containing a moistened filter paper (Whatmans No. 1; 9 cm dia.), a leaf disc (8.8 cm dia.) from the selected cultivar and 20 two-dayold second instar host larvae. Each treatment was replicated four times. The method was otherwise identical to that used for C. plutellae. Field study (Tanah Rata, Cameron Highlands, Malaysia). Three plots (c. 10 x 5 m each) within the MARDI Cameron Highlands Field Station (Tanah Rata) were transplanted with each of the B. oleracea var. capitata cvs KY Cross, Super Dragon and B. pekinensis cv Super Queen. The plots were adjacent to one another but were separated by 3 m, the Super Queen plot being central. Except for one application of pyrethrum during the first week following transplanting, the plots were maintained insecticidefree. From four weeks after transplanting until harvest (6 weeks later), individual cabbages (n = 10) were sampled randomly at weekly intervals from each plot and fourth instar P. xylostella larvae were collected and transferred to Petri dishes for rearing through in the laboratory. Sampled cabbages were marked and not re-sampled. Results Intrinsic host plant resistance Leaf biomass consumption. The dry weights (biomass) of leaf discs (4.8 cm dia.) from mature (147-day-old) B. oleracea var. capitata cvs. Wheelers Imperial and Red Drumhead were significantly (p <0.05) greater than those from younger (84-day-old) plants of the same cultivars and B. pekinensis cv. Tip Top (also 84 days-old) (Table 1). Biomass consumption by P. xylostella larvae (SERD 2) was greater on both ages of the two B. oleracea cultivars than on B. pekinensis, and larvae consumed five-fold more dry weight on mature cv. Red Drumhead (despite slight [6.7%] mortality after five days) compared with larvae on the younger B. pekinensis. The relative damage to surface area of leaf discs appeared to be inversely related to the relative rate of leaf biomass consumption. Table 1. Relative biomass consumption compared with damage to leaf surface area after larval feeding by a field strain of Plutella xylostella on leaf discs for five days in the laboratory a Host plant / Plant age Initial Day 5 Mean wt. Relative rate Relative cultivar (days) leaf disc leaf disc difference of leaf damage to dry wt. ±SE dry wt. ±SE after biomass surface area (mg) (mg) 5 days consumption of leaf disc b Brassica pekinensis Tip Top ± 2.9a 41.6 ± 17a a (n = 12) (n = 12) (n = 6) Brassica oleracea var. capitata Wheelers ± 5.3a 33.5 ± 8.3a a Imperial (n = 12) (n = 6) (n = 6) ± 6.4b 68.4 ± 7.2b a (n = 8) (n = 12) (n = 6) RD ± 2.5a 26.0 ± 7.5a b (n = 6) (n = 6) (n = 6) ± 10b 74.8 ± 3.9b b (n = 8) (n = 6) (n = 6) a Within columns, means followed by a common letter are not significantly (p >0.05) different (ANOVA and pairwise t-tests or 2 tests). TT = cv.tip Top (mortality after 120 h = 0%); WI = cv. Wheelers Imperial (mortality after 120 h = 0%); RD = cv. Red Drumhead (mortality after 120 h = 6.7%). All plants glasshouse-grown. b Surface area damage based on visual assessment according to leaf surface area damage rating scale (0 = <5%; 1 = 5 25%; 2 = 26 50%; 3 = 51 80%; 4 = %): each rating determined as a proportion of total damage (sum of six replicates: maximum = 24). Significance testing using 2 test with binomial errors. 244 Proceedings: The Management of Diamondback Moth and Other Crucifer Pests

3 Interactions between plant resistance and selective insecticides The different plant groups caused more than eight-fold and five-fold variations in residual toxicity of teflubenzuron and Bt subsp. aizawai respectively when exposed to larvae of the P. xylostella field strain (Table 2). With both insecticides, plant groups of intermediate ditrophic status (based on survival of control insects) contributed to the lowest insecticide toxicity, whilst apparent toxicities were significantly (p <0.05) greater with both the most and the least intrinsically susceptible plant groups (84-day-old cv. Wheelers Imperial and 147- day-old cv. Red Drumhead, respectively). Tritrophic interactions Laboratory studies. Percentage parasitism by C. plutellae was significantly (p <0.05) greater on P. xylostella maintained on mature B. oleracea cvs Wheelers Imperial and Red Drumhead than on young B. pekinensis cv Tip Top (Table 3). In contrast, parasitism by D. semiclausum was greatest (p <0.05) on cv Wheelers Imperial, intermediate (p <0.05) on cv Tip Top and least (p <0.05) on cv Red Drumhead. Larval mortality was caused directly by parasitism and by resistance factors in the (partially resistant) plants; the net effect of parasitism and plant resistance factors was assessed using survival analysis (Table 3). Table 2. Logit analysis of mortality data (Day 12) and leaf damage consumption rates (Day 5) for a field strain (SERD 2) of Plutella xylostella with teflubenzuron (TFB) and Bt ssp. aizawai (Bta) in leaf-dip bioassays on two glasshouse-grown Brassica oleracea var. capitata cultivars at two ages a Insecticide Control data Cultivar b Plant age % Mean leaf LC e,f 50 95% FL Slope f ± SE RT g (days) mortality c damage (Day 12) rating d,f (Day 5) TFB WI a 3.8 ± 0.2a 0.10ab ± 0.22a ab 3.7 ± 0.2a 0.44bc ± 0.22a 1 RD ab 3.7 ± 0.2a 0.20bc ± 0.23a b 2.5 ± 0.2b 0.05a ± 0.22a 8.8 Bta WI 84 as above h 0.11a ± 0.39a b ± 0.39a 1 RD i 84 > 1 < a ± 0.42a 5.2 a Source: Verkerk & Wright (1996a). b WI and RD = B.oleracea var. capitata cvs. Wheelers Imperial and Red Drumhead, respectively. c Control mortality not used for correction in logit analysis in order to include plant-induced effects. Significance test: 2 with binomial errors ( 2 = 26.7, 3 df, p <0.001). d Based on rating scale: 0 = <5%, 1 = 5 25%, 2 = 26 50%, 3 = 51 80%, 4 = % damage to leaf disc area. e Units: mg ai ml -1 for teflubenzuron; iu mg -1 for Bt ssp. aizawai. f For each toxicant (or controls), values within columns followed by a common letter are not significantly (p > 0.05) different (5 insects / replicate; 6 replicates / treatment; n = 30). g Relative Toxicity factor for each insecticide: toxicity (measured by LC 50 ) of specific plant/insecticide combination in relation to least toxic plant/insecticide combination. h Controls shared between treatments. i Inadequate responses at concentrations tested prevented logit analysis. Table 3. Survival analysis a of Plutella xylostella (laboratory strain) maintained in the laboratory on three host plant groups following exposure to two species of endolarval parasitoid Parasitoid species Cultivar b Hazard Mean time to % censored % parasitism c (age in days) function death (days) C. plutellae [shape parameter = 2.667] Tip Top (42) a 34 29a Wheelers Imperial (121) b 8 52b Red Drumhead (121) c 10 44b D. semiclausum [shape parameter = 1.946] Tip Top (63) a 13 32a Wheelers Imperial (132) a 2 52b Red Drumhead (133) b 5 14c a Within species, means followed by a common letter are not significantly (p > 0.05) different, based on estimated standard errors derived from GLIM best-fit analysis with Weibull error distribution and censoring. b Tip Top = B. pekinensis; the remaining two cvs are B. oleracea var. capitata. c Significance tested (p = 0.05) by ANOVA and LSD following angular transformation. Development and implementation of IPM 245

4 Table 4. Fate of fourth instar Plutella xylostella collected at weekly intervals over a six week period ( ) from three field-grown host plant groups in Tanah Rata (Cameron Highlands, Malaysia) Fate a of field collected L4 Plutella xylostella (mean proportion ± SE ) Host plant / Parasitised by Parasitised by Adult Dead n cultivar Cotesia spp. Diadegma spp. Brassica pekinensis cv Super Queen 0.40 ± 0.24a 0a 0.60 ± 0.25ab 0a 5 Brassica oleracea cv KY Cross 0.06 ± 0.06b 0.17 ± 0.08b 0.69 ± 0.17a 0.07 ± 0.03a 45 Brassica oleracea cv Super Dragon 0.04 ± 0.04b 0.33 ± 0.11b 0.29 ± 0.08b 0.33 ± 0.08b 24 a Following rearing through in the laboratory. Including censored individuals (those alive at the experimental end-point), mean time to death of P. xylostella exposed to C. plutellae was significantly ( p <0.05) less on mature B. oleracea cv Wheelers Imperial than on similar-aged cv Red Drumhead, which was in turn significantly ( p <0.05) less than on young B. pekinensis cv Tip Top (Table 3). Mean time to death for D. semiclausum-exposed P. xylostella was significantly ( p <0.05) less on cv Red Drumhead compared with cvs Wheelers Imperial and Tip Top (Table 3). Field study (Tanah Rata, Cameron Highlands, Malaysia). The mean (± SE) number of immature (L2 to pupae) P. xylostella per five plants (on a single assessment, over the six week period) was 3.04 (± 1.56), 1.96 (± 1.12) and 0.75 (± 0.37) for cvs KY Cross, Super Dragon and Super Queen, respectively. Although the number of fourth instar larvae recovered on B. pekinensis cv Super Queen was very low (n = 5), a pattern of parasitoid-host plant preference was noted, with C. plutellae emerging predominantly from larvae recovered from B. pekinensis, rather than from either of the two B. oleracea cultivars (Table 4). This preference appeared to be reversed for D. semiclausum. Mortality of larvae (presumably parasitised and unparasitised) increased through the host plant sequence cvs Super Queen, KY Cross and Super Dragon and the latter cultivar contributed to significantly ( p <0.05) greater mortality of larvae (unsuccessfully or not parasitised) than the other two plant groups. Discussion A reciprocal relationship was apparent in laboratory studies between the relative rate of biomass consumption and the relative damage to the surface area of leaf discs (Table 1). On the most resistant cultivar/age group tested (147-day-old B. oleracea var. capitata cv Red Drumhead) five times more biomass was consumed by larvae of P. xylostella, with only two-thirds of damage to leaf disc surface area, compared with on the most susceptible plant group (84-day-old B. pekinensis cv Tip Top) (Table 1). This was caused by the tendency for herbivores to consume greater amounts of lower status food plants to compensate for their low nutritional content and has been reported previously in other lepidopterous larvae (e.g., Feeny, 1970; Slansky and Feeny, 1977; Price et al., 1980; Clancy and Price, 1987). When insecticide toxicity was assessed in relation to these interacting processes mediated by differing degrees of partial plant resistance, the toxicity of both teflubenzuron and Bt subsp. aizawai to larvae of P. xylostella was lowest on plants with intermediate levels of partial plant resistance and significantly greater on both more resistant and susceptible plant groups (Table 2). This U-shaped response between insecticide toxicity and increasing degree of plant resistance is likely to be attributed to the tendency for insects on highly susceptible plants to consume a greater surface area of leaf (and so ingest more insecticide) whereas on the most resistant plant groups they succumb to the combined effects of the insecticide and plant resistant factors (Verkerk and Wright, 1996a). There are many factors which could complicate this relationship in the field, such as the bioavailability of insecticides on different plant surfaces, the inherent susceptibility and vigour of the herbivore (including activity/burden of detoxification enzymes), rate of foliar consumption, ratio of insecticide treated vs untreated material ingested, insecticide dose in relation to insect body weight and pressure from natural enemies. Field studies to allow better understanding of such interactions are much needed. Tritrophic laboratory studies with three different plant groups which could be categorised as being of low, intermediate and high host plant status (121-dayold B. oleracea var. capitata cvs Red Drumhead, Wheelers Imperial and 42-day-old B. pekinensis cv Tip Top, respectively) according to a range of ditrophic bionomic criteria (Verkerk and Wright, 1994a) showed significant differences in the rate of parasitism on the different plant groups (Table 3). Tip Top, the plant group with best ditrophic status to P. xylostella, contributed to significantly (p <0.05) lower rates of parasitism by C. plutellae and D. semiclausum than the other two plant groups (Table 3). The mechanism giving rise to this effect is currently being investigated in the laboratory and may be related to differential host defence and occurs even when parasitism occurs in the absence of the host plant (R. H. J. Verkerk, unpublished data). Mortality of parasitoid-exposed larvae was likely to be caused not only by parasitism of a proportion of the test insects, but also by plant 246 Proceedings: The Management of Diamondback Moth and Other Crucifer Pests

5 resistance factors and the combined interactions between parasitoid-induced stress and plant resistance factors. Survival analysis (Crawley, 1993) provides a way of assessing the combined impact of such complex interacting processes and showed that the intermediate plant group (cv Wheelers Imperial) gave rise to the shortest survival time in the presence of C. plutellae. However, when larvae were exposed to D. semiclausum, the most resistant plant group (cv Red Drumhead) contributed to the shortest survival time. This suggests that partial plant resistance is likely to be of benefit in integrated control programmes involving these endolarval parasitoids. When parasitism by the same two species of endolarval parasitoid was observed in the field (Tanah Rata, Cameron Highlands, Malaysia) over almost an entire cropping period on three host plant groups (B. oleracea var. capitata cvs KY Cross and Super Dragon, and B. pekinensis cv Super Queen respectively) parasitism by D. semiclausum was absent on B. pekinensis and almost double (33% vs 17%) that on the more resistant of the two B. oleracea var. capitata cultivars (cv Super Dragon) (Table 4). However, this latter difference was not significant (p >0.05). In contrast, parasitism by C. plutellae was very low (c. 5%) on the two B. oleracea var. capitata cultivars, but with a significantly (p <0.05) greater proportion of larvae being parasitised on B. pekinensis. The low abundance of P. xylostella on B. pekinensis found in Tanah Rata was also noted in two other sites in the Cameron Highlands (Mensum Valley and Boh Road) during the same year (1995) (R. H. J. Verkerk, unpublished data) and may have been associated with host plant conditioning/antixenosis (to B. oleracea var. capitata) by certain field strains of P. xylostella; based on these and previous studies (e.g., Verkerk and Wright, 1994a), it is unlikely to be a result of antibiosis. The contrasting trends in C. plutellae and D. semiclausum parasitism of P. xylostella on B. oleracea var. capitata and B. pekinensis were also found in field studies in the Cameron Highlands in 1994 (Verkerk and Wright, 1996b) and 1996 (Arthurs, 1996). Possible mechanisms giving rise to such effects may be related to olfactory (synomone) attraction to host plants, host plant adaptation by parasitoids or co-evolution (Verkerk & Wright, 1996b). One or more of these mechanisms may occur in addition to those giving rise to plant-induced variation in parasitism and host survival in the laboratory (Table 3). Since all field-based agricultural systems are inevitably multitrophic, it is essential to evaluate and research them within a multitrophic context. Improved understanding of the many complex and inter-related processes allows informed decisions to be made about the compatibility of different tactics within control strategies. Multitrophic research within a real-world context may also open up channels for novel approaches, such as enhancing biological control through the use of partial plant resistance or low doses of specific insecticides (see Verkerk and Wright, 1994b). Acknowledgements We thank M.A.R.D.I. for cooperating in our field studies in Malaysia ( ). The work was conducted under MAFF licences PHF 909A/1447/45 and PHF 909A/1031/54 and was supported by the Leverhulme Trust. References Arthurs, S. (1996). Personal communication. Dept. Biology, Imperial College of Science, Technology & Medicine, University of London, UK. Clancy, K. M. and Price, P. W. (1987). Rapid herbivore growth enhances enemy attack: Sublethal plant defenses remain a paradox. Ecology 68: Crawley, M. J. (1993) GLIM for Ecologists, Blackwell Scientific Publications, Oxford, 379 pp. Feeny, P. (1970). Seasonal changes in oak leaf tannins as a cause of spring feeding by winter moth caterpillars. Ecology 51: Price, P. W., Bouton, C. E., Gross, P., McPheron, B. A., Thompson, J. N. and Weis, A. J. (1980). Interactions among three trophic levels: influence of plants on interactions between insect herbivores and natural enemies. Annual Review of Ecology and Systemmatics 11: Price, P. W., Westoby, M., Rice, B., Atsatt, P. R., Fritz, R. S., Thompson, J. M. and Mobley, K. (1986). Parasite mediation in ecological interactions. Annual Review of Ecology and Systemmatics 17: Slansky, F. and Feeny, P. (1977). Stabilisation of the rate of nitrogen accumulation by larvae of the cabbage butterfly on wild and cultivated food plants. Ecological Monographs 47: Talekar, N. S. (Ed.) (1986). Diamondback Moth Management: Proc. First Inter. Workshop, March 1985, Tainan, Taiwan, Shanhua, Taiwan: Asian Vegetable Research & Development Center, 471 pp. Talekar, N. S. and Griggs, T. D. (1992). Diamondback Moth and Other Crucifer Pests: Proc. Second Inter. Workshop, Dec. 1990, Tainan, Taiwan, Taipei, Taiwan: Asian Vegetable Research & Development Center, 603 pp. Verkerk, R. H. J. (1995). Studies on interactions between diamondback moth, host plants, endolarval parasitoids and selective toxicants, PhD Thesis, University of London, pp Verkerk, R. H. J. and Wright, D. J. (1994a). Interactions between the diamondback moth, Plutella xylostella L. and glasshouse and outdoor-grown cabbages. Annals of Applied Biology 125: Verkerk, R. H. J. and Wright, D. J. (1994b). The potential for induced extrinsic host plant resistance in IRM strategies targeting the diamondback moth. In: Brighton Crop Protection Conference: Pests and Diseases , p Farnham, UK: British Crop Protection Council. Verkerk, R. H. J. and Wright, D. J. (1996a). Effects of host plant-selective insecticide interactions on larvae of Plutella xylostella (Lepidoptera: Yponomeutidae) in the laboratory. Pesticide Science 44: Development and implementation of IPM 247

6 Verkerk, R. H. J. and Wright, D. J. (1996b). Field-based studies with the diamondback moth tritrophic system in Cameron Highlands of Malaysia: implications for pest management. International Journal of Pest Management (in press). Wright, D. J. and Verkerk, R. H. J. (1995). Integration of chemical and biological control systems for arthropods: evaluation in a multitrophic context. Pesticide Science 44: Proceedings: The Management of Diamondback Moth and Other Crucifer Pests