Effects of Combining an Entomopathogenic Fungi Or Bacterium with Entomopathogenic Nematodes on Mortality of Curculio Caryae

Biological Control 30 (2004) 119–126 www.elsevier.com/locate/ybcon

Eﬀects of combining an entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of Curculio caryae (Coleoptera: Curculionidae)

David I. Shapiro-Ilan,a,* Mark Jackson,b Charles C. Reilly,a and Michael W. Hotchkissa

a USDA-ARS, SE Fruit and Tree Nut Research Lab, 21 Dunbar Road, Byron, GA 31008, USA b USDA-ARS-NCAUR 1815 N. University St. Peoria, IL 61604, USA

Received 10 July 2003; accepted 25 September 2003

Abstract

Our objective was to determine the eﬀects of combining entomopathogenic nematodes with other entomopathogens on their ability to suppress larvae of the pecan weevil, Curculio caryae. In laboratory assays, we simultaneously applied the nematodes Heterorhabditis indica or Steinernema carpocapsae with the fungus Beauveria bassiana, Metarhizium anisopliae,orPaecilomyces fumosoroseus, or the bacterium Serratia marcescens. Mortality of C. caryae was determined 14 days after application. We observed antagonism in all pathogen combinations, except H. indica combined with M. anisopliae, for which additive eﬀects were observed. The combination of H. indica and M. anisopliae may merit further study in other systems. Depending on application rate, S. carpocapsae combined with B. bassiana or S. marcescens also resulted in additivity. S. carpocapsae (applied alone) generally caused greater C. caryae mortality than other pathogens applied alone. P. fumosoroseus and S. marcescens were not pathogenic to C. caryae when applied alone. We conclude that the pathogen combinations we investigated are not likely to improve suppression of C. caryae larvae beyond what is expected from single application of the pathogen with greatest virulence. Ó 2003 Elsevier Inc. All rights reserved.

Keywords: Additivity; Antagonism; Beauveria bassiana; Heterorhabditis indica; Paecilomyces fumosoroseus; Pathogen combinations; Serratia marcescens; Steinernema carpocapsae

1. Introduction additional year in the soil as larvae and emerge as adults in the third year (Harris, 1985). The pecan weevil, Curculio caryae (Horn), is a key Control recommendations for C. caryae currently pest of pecan throughout the Southeastern US as well as consist of foliar applications of chemical insecticides in portions of Texas and Oklahoma (Payne and Dut- (e.g., carbaryl) to kill the adults (Harris, 1999). Due to cher, 1985). This insect has a 2- or 3-year life cycle the problems associated with aphid resurgence (Dutcher (Harris, 1985). Adults emerge from soil in late July- and Payne, 1985), as well as other environmental and August and feed on and oviposit in nuts (Harris, 1985). regulatory concerns, research toward developing alter- Larvae develop within the nut and fourth instars drop to native control strategies is warranted. Entomopatho- the ground where they burrow to a depth of 8–25 cm, genic nematodes, which occur naturally as pathogens of form a soil cell, and overwinter. The following year, C. caryae (Nyczepir et al., 1992), are one of the potential approximately 90% of the larvae pupate and spend the alternatives (Shapiro-Ilan, 2003). next 9 months in the soil cell as adults (Harris, 1985). Entomopathogenic nematodes are obligate parasites The remaining 10% of the larval population spend an in the families Steinernematidae and Heterorhabditidae. Entomopathogenic nematodes kill insects with the aid of a mutualistic bacterium, which is carried in their intes- * Corresponding author. Fax: 1-478-956-2929. tine (Xenorhabdus spp. and Photorhabdus spp. are as- E-mail address: [email protected] (D.I. Shapiro-Ilan). sociated with Steinernema spp. and Heterorhabditis spp.,

1049-9644/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2003.09.014 120 D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126 respectively) (Poinar, 1990). The nematodes complete 2– microbial control agents for C. caryae suppression 3 generations within the host, after which free-living (Shapiro-Ilan, 2003). infective juveniles emerge to seek new hosts (Poinar, The primary objective of this study was to determine 1990). Entomopathogenic nematodes are effective at whether applications of entomopathogenic fungi or a controlling a variety of economically important pests bacterium combined with entomopathogenic nematodes including the larvae of several weevil species (Coleop- results in a synergistic, additive, or antagonistic effect on tera: Curculionidae) such as the black vine weevil, Oti- C. caryae larvae. It was also of interest to compare the orhynchus sulcatus (F.), and the Diaprepes root weevil, relative virulence of each of the entomopathogens acting Diaprepes abbreviatus (L.) (Shapiro-Ilan et al., 2002). alone and determine which treatments caused greater One approach to controlling C. caryae with ento- mortality than a non-treated control. The entomopath- mopathogenic nematodes would be to target the larvae ogens tested were B. bassiana, M. anisopliae, Paecilo- when they drop to the ground or after burrowing into myces fumosoroseus (Wize), and S. marcescens combined the soil. Prior research, however, does not indicate great with Heterorhabditis indica Poinar, Karunakar, & David potential for entomopathogenic nematodes to control or S. carpocapsae. the larval stage of C. caryae (Nyczepir et al., 1992; Shapiro-Ilan, 2001a; Smith et al., 1993). Field trials to suppress C. caryae larvae with H. bacteriophora (Po- 2. Materials and methods inar), Steinernema carpocapsae (Weiser), or Steinernema feltiae (Filipjev) resulted in less than 35% control (Ny- 2.1. Insects and pathogens czepir et al., 1992; Smith et al., 1993). Furthermore, in a laboratory study, Shapiro-Ilan (2001a) found the viru- Nematodes, H. indica (Hom1 strain) and S. carpo- lence of 15 strains from nine species of entomopatho- capsae (All strain), originally obtained from Integrated genic nematodes to be relatively poor to C. caryae larvae Biocontrol Systems (Aurora, IN) and K. Nguyen (none of the nematodes produced greater than 60% (University of Florida), respectively, were cultured in mortality when applied at ca. 40 infective juveniles/cm2). the last instar of Galleria mellonella (L.) according to Conceivably, combining entomopathogenic nema- procedures described by Kaya and Stock (1997). The todes with other entomopathogens would result in syn- two nematode species were chosen because each has ergistic interactions that would enhance the potential for shown at least some promise for C. caryae control rel- biological control of C. caryae. In studies targeting ative to other species (Shapiro-Ilan, 2001a,b; Shapiro- other insect pests, synergistic interactions have been Ilan et al., 2003). Entomopathogenic fungi, B. bassiana observed from certain combinations of entomopatho- (BbGA2) and M. anisopliae (MaLA4), were collected genic nematodes with other pathogens. For example, from soil in pecan orchards in Byron, Georgia and synergistic virulence to Cyclocephala spp. was observed Dixie, Louisiana, respectively (Shapiro-Ilan et al., 2003), in combinations of entomopathogenic nematodes with and reproduced on SabouraudÕs dextrose agar (Becton Paenibacillus popilliae (Dutky) (Thurston et al., 1993, Dickson, Sparks, MD, USA) with yeast extract (Sigma 1994) or with Bacillus thuringiensis Berliner subspecies Chemical, St. Louis, MO, USA) according to Goettel japonensis (Koppenhofer€ and Kaya, 1997; Koppenhofer€ and Inglis (1997); these strains were chosen because they et al., 1999). However, interactions between entomo- previously exhibited relatively high virulence to C. car- pathogenic nematodes and other entomopathogens can yae larvae compared with other strains in laboratory also be antagonistic (Baur et al., 1998; Brinkman and assays (Shapiro-Ilan et al., 2003). P. fumosoroseus Gardner, 2000; Koppenhofer€ and Kaya, 1997). (ARSEF 3581), was originally isolated from an infected In addition to entomopathogenic nematodes, several silverleaf whitefly (Bemisia argentifolii Bellows and other entomopathogens have been reported to occur Perring); blastospores were produced through deep-tank naturally in C. caryae including the entomopathogenic fermentation in a basal salts medium containing 2.5% fungi Beauveria bassiana (Balsamo) Vuillemin and acid hydrolyzed casein and 5% glucose and subsequent Metarhizium anisopliae (Metschnikoff) Sorokin (Sri- air-drying (Jackson et al., 2003). The S. marcescens Arunotai et al., 1975; Swingle and Seal, 1931), and isolate was obtained on potato dextrose agar from the Paecilomyces sp. (Sikorowski, 1985), and the bacterium surface of an adult pecan weevil (captured on a pecan Serratia marcescens Bizio (Sri-Arunotai et al., 1975). tree trunk in Byron, GA, identified by Bacterial Strain- Field trials with B. bassiana and M. anisopliae caused Identification and Mutant Analysis Service (BSI-MSA, significant reductions in C. caryae larval populations, Auburn University, AL) and cultured in nutrient broth but the levels of control were generally low (e.g., <35%) (Becton Dickson) (Klein, 1997). Nematodes were stored (Gottwald and Tedders, 1983; Harrison et al., 1993; at 13 °C (Kaya and Stock, 1997) and the other patho- Shapiro-Ilan, 2003). Beyond entomopathogenic nema- gens at 4 °C (Goettel and Inglis, 1997; Klein, 1997) prior todes, B. bassiana and M. anisopliae, no other pathogens to use in experiments; pathogens were not sub-cultured that naturally occur in C. caryae have been tested as more than once during the study. D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126 121

Curculio caryae cannot be cultured on artiﬁcial me- pathogen at 1, combinations of each at 0:5, and a dia. Therefore, larvae (fourth instar) were collected from non-treated (water) control (resulting in 11 treatments, infested nuts (on the USDA-ARS Research Station, see Figs. 1–3). The fourth experiment, nematodes with Byron, GA). The larvae were stored in sterile (auto- S. marcescens, contained four additional treatments claved) soil at 25 °C for 2 weeks (to remove diseased combining 0:5 rates of the nematodes with 1 S. individuals), and remaining larvae stored up to 6 marcescens, and vice versa (resulting in 15 treatments, months in sterile soil at 4–10 °C prior to experimenta- see Fig. 4). The experiments were arranged in completely tion (Shapiro-Ilan, 2001a). randomized designs with three replicates of 10 cups for each treatment. All experiments were repeated in en- 2.2. Virulence assays tirety once.

Virulence assays were based on methods described by 2.3. Statistical analyses Shapiro-Ilan (2001a) and Shapiro-Ilan et al. (2003). Experiments were conducted in plastic cups (Bioserv, To evaluate whether pathogen interactions were ad- Frenchtown, NJ) (3–4 cm in diam., 3.5 cm deep) filled ditive, antagonistic, or synergistic we applied the anal- with 27 g of (oven-dried) soil from the USDA-ARS pe- yses used by Nishimatsu and Jackson (1998). The nature can orchard (Byron, GA), and contained one larva each. The soil was a loamy sand with the percentage sand:silt:clay ¼ 84:10:6, pH 6.1, and organic matter ¼ 2.8% by weight. Pathogens were each pipetted (at approximately the same time) onto the soil surface of each cup in 0.5 ml of water so that the final moisture was standardized at field capacity (14%). After inoculation, cups were incubated at 25 °C for 14 days at which time C. caryae larval mortality was recorded. Each of the pathogens was applied in combination or alone at two rates (one twice the rate of the other), which will hereafter be referred to as 1 and 0:5. The 1 rate for nematodes was approximately 40 infective juveniles/cm2, which is a typical rate for field applications (Shapiro-Ilan et al., 2002). Based on prior viru- Fig. 1. Mean percentage mortality (SEM) of Curculio caryae larvae lence assays using the same strains (Shapiro-Ilan et al., 14 days after treatment with various entomopathogens applied alone 2003), the 1 rate for B. bassiana and M. anisopliae was or in combination at 1 (1) and 0:5 (.5) rates (see text for specific approximately 8 104 viable conidia/cm2. The 1 rate rates used). Different letters above bars indicate statistical differences 6 2 (based on LSD tests, a ¼ 0:05). Hi, Heterorhabditis indica; Bb, Beau- for P. fumosoroseus was 8 10 viable blastospores/cm ; veria bassiana; Ma, Metarhizium anisopliae; and C, untreated control. a reported effective field rate for P. fumosoroseus was in the range of 1.25–5 105 conidia/cm2 (Wraight et al., 2000), but we selected a higher rate based on low pathogenicity observed in preliminary tests (unpublished data). The 1 rate for S. marcescens was approximately 8 106 cells/cm2; field applications of another Serratia species, Serratia entomophila (Grimont, Jackson, Ageron, and Noonan), have been reported in the range of 105/cm2 (Klein, 1997) but, again, we chose a higher rate based on a lack of pathogenicity observed in preliminary tests (unpublished data). Four separate experiments were conducted: (1) H. indica combined with B. bassiana or M. anisopliae, (2) S. carpocapsae with B. bassiana or M. anisopliae, (3) H. indica or S. carpocapsae with P. fumosoroseus, and (4) H. indica or S. carpocapsae with S. marcescens. Nema- Fig. 2. Mean percentage mortality (SEM) of Curculio caryae larvae todes were combined with other pathogens (fungi or 14 days after treatment with various entomopathogens applied alone bacteria) but not with each other, and fungal pathogens or in combination at 1 (1) and 0:5 (.5) rates (see text for specific rates used). Different letters above bars indicate statistical differences and bacteria were not combined (not the focus of this (based on LSD tests, a ¼ 0:05). Sc, Steinernema carpocapsae; Bb, study). All experiments included 0:5 and 1 rates of Beauveria bassiana; Ma, Metarhizium anisopliae; and C, untreated each pathogen applied alone, combinations of each control. 122 D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126

the arcsine-transformed means (square root of arcsine). If the ANOVA was signiﬁcant, the above-mentioned pair-wise comparisons were made using LSD (SAS, 1999). An a level of 0.05 was used in all analyses.

3. Results

Interactions among pathogens consisted mostly of antagonism and some additive effects; no synergy was observed (Table 1). All combinations of nematodes with either S. marcescens or P. fumosoroseus resulted in an- Fig. 3. Mean percentage mortality ( SEM) of Curculio caryae larvae tagonism, except the 1 S. carpocapsae and 0:5 S. 14 days after treatment with various entomopathogens applied alone marcescens which were additive. Interactions between or in combination at 1 (1) and 0:5 (.5) rates (see text for specific H. indica and B. bassiana were antagonistic, whereas rates used). Different letters above bars indicate statistical differences interactions between H. indica and M. anisopliae were (based on LSD tests, a ¼ 0:05). Hi, Heterorhabditis indica; Sc, Stein- ernema carpocapsae; Pf, Paecilomyces fumosoroseus; and C, untreated additive. Interactions between S. carpocapsae and B. control. bassiana or M. anisopliae differed depending on application rate. Antagonism was observed when S. carpocapsae and B. bassiana were applied at the 0:5 rate, but additivity was observed when these pathogens were applied at 1 rates. Interactions were antagonistic when M. anisopliae and S. carpocapsae were applied at 1 rates, but additive when the pathogens were applied at 0:5 rates. In all ANOVAs, interaction between trial (the repeats of experiments) and treatment was not significant (P > 0:05). Thus, data from both trials were combined in each analysis. The high level of control mortality, Fig. 4. Mean percentage mortality (SEM) of Curculio caryae larvae particularly in experiments including S. marcescens or P. 14 days after treatment with various entomopathogens applied alone or in combination at 1 (1) and 0:5 (.5) rates (see text for specific fumosoroseus (Figs. 1–4), was likely related to the length rates used). Different letters above bars indicate statistical differences of time the insects were stored. However, the range of C. (based on LSD tests, a ¼ 0:05). Hi, Heterorhabditis indica; Sc, Stein- caryae control mortality observed in this study was not ernema carpocapsae; Sm, Serratia marcescens; and C, untreated con- unusual in comparison with similar studies (Shapiro- trol. Ilan, 2001a; Shapiro-Ilan et al., 2003). In experiment 1, including H. indica, B. bassiana,and of the interaction was determined through a comparison M. anisopliae, only combination treatments of H. indica of expected and observed percentage C. caryae mortal- and M. anisopliae caused greater C. caryae mortality ity. Expected mortality was based on the formula than the control (F ¼ 2:80; df ¼ 10:44; P ¼ 0:009) PE ¼ P0 þð1 P0ÞðP1Þþð1 P0Þð1 P1ÞðP2Þ, where PE (Fig. 1). No differences were observed among pathogens is the expected mortality of the combination, P0 is the applied alone (Fig. 1). In experiment 2 that included S. control mortality, P1 is the mortality from one pathogen carpocapsae, B. bassiana, and M. anisopliae, all treat- treatment applied alone, and P2 is the mortality from the ments caused greater mortality than the control except other pathogen applied alone. A v2 test was applied to the fungal applications applied alone (F ¼ 8:22; 2 2 the observed and expected results: v ¼ðL0 LEÞ =LEþ df ¼ 10:44; P < 0:0001) (Fig. 2). S. carpocapsae applied 2 ðD0 DEÞ =DE, where L0 is the number of living larvae at the 0:5 or 1 rate caused greater mortality than the observed, LE the number of living larvae expected, D0 fungi at corresponding rates (Fig. 2). Only treatments of the number of dead larvae observed, and DE the number S. carpocapsae alone produced greater mortality than of dead larvae expected. Interactions were additive if the control in experiment 3 (with H. indica, P. fumos- 2 2 v < 3:84, antagonistic if v > 3:84 and PC < PE, and oroseus, and S. carpocapsae), and the S. carpocapsae 2 synergistic if v > 3:84 and PC > PE, where PC is the treatments at 0:5 and 1 caused greater mortality than observed mortality from the combination and PE is the the other pathogens applied alone at 0:5 and 1, re- expected mortality from the combination. spectively (F ¼ 5:94; df ¼ 10:44; P < 0:0001) (Fig. 3). To compare mortality from the treatments to the In experiment 4 (with S. marcescens and nematodes), control and to compare the relative virulence of only three treatments caused greater mortality than the the pathogens acting alone, we conducted ANOVA on control (0:5 S. carpocapsae,1 S. carpocapsae with 1 D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126 123

Table 1 Interactions observed when combining entomopathogenic nematodes with other entomopathogensa for suppression of Curculio caryae larvae Experiment Nematode Application Other Application Observed Expected v2 Interactionf rateb pathogen ratec mortalityd mortalitye 1Hi0:5 Bb 0:5 16.7 43.3 17.7 Antagonism 1Hi1 Bb 1 31.7 45.7 4.7 Antagonism 1Hi0:5 Ma 0:5 41.7 47.8 0.9 Additive 1Hi1 Ma 1 43.3 54.3 2.9 Additive 2Sc1 Bb 1 75.0 76.0 0.04 Additive 2Sc0:5 Bb 0:5 46.7 71.6 17.8 Antagonism 2Sc1 Ma 1 61.7 80.0 11.4 Antagonism 2Sc0:5 Ma 0:5 60.0 70.0 3.3 Additive 3Hi0:5 Pf 0:5 28.3 65 35.5 Antagonism 3Hi1 Pf 1 28.3 73.3 12.9 Antagonism 3Sc0:5 Pf 0:5 48.3 78.3 31.8 Antagonism 3Sc1 Pf 1 53.3 83.3 38.9 Antagonism

4Hi0:5 Sm 0:5 50.0 56.4 4.9 Antagonism 4Hi1 Sm 1 36.7 63.2 17.4 Antagonism 4Sc0:5 Sm 0:5 48.3 59.3 8.8 Antagonism 4Sc1 Sm 1 55.0 63.2 4.2 Antagonism 4Hi0:5 Sm 1 36.3 58.7 13.5 Antagonism 4Hi1 Sm 0:5 25.5 56.6 21.8 Antagonism 4Sc0:5 Sm 1 50.0 61.7 7.8 Antagonism 4Sc1 Sm 0:5 56.7 56.6 3.3 Additive a Hi, Heterorhabditis indica; Sc, Steinernema carpocapsae; Bb, Beauveria bassiana; Ma, Metarhizium anisopliae; Pf, Paecilomyces fumosoroseus; and Sm, Serratia marcescens. b Application rates for nematodes were 40 and 20 infective juveniles/cm2 for 1 and 0:5, respectively. c 1 application rates were 8 104 conidia/cm2 for Bb and Ma, and 8 106 blastospores and cells for Pf and Sm, respectively; 0:5 rates were 50% of the 1 rates. d Observed mortality (%) in average of three replicates of 10 C. caryae larvae in two trials (60 total). e Expected mortality (%) ¼ P0 þð1 P0ÞðP1Þþð1 P0Þð1 P1ÞðP2Þ, where P1 is mortality from nematodes alone, P2 is mortality from other pathogen alone, and P0 is control mortality. f Interaction was based on v2 ratio of expected:observed mortality.

S. marcescens, and 1 S. carpocapsae with 0:5 S. Koppenhofer€ et al., 2000; Nishimatsu and Jackson, marcescens), and S. carpocapsae applied at the 0:5 rate 1998). Our results, however, did not indicate any syn- caused greater mortality than the other pathogens ap- ergism; for the most part, we observed antagonism with plied alone (F ¼ 2:64; df ¼ 14:60; P ¼ 0:005) (Fig. 4). a few exceptions of additivity. In all experiments that included S. carpocapsae, one or Similar to our results, various studies have also in- both of the nematodeÕs application rates produced dicated less than synergistic interactions between ento- greater C. caryae mortality than the other pathogen(s) mopathogenic nematodes and chemical or other applied alone (Figs. 2–4). Application of H. indica and entomopathogenic agents (Brinkman and Gardner, M. anisopliae together at 0:5 was the only combination 2000; Koppenhofer€ and Kaya, 1997; Mannion et al., that caused greater mortality than either pathogen ap- 2000; Nishimatsu and Jackson, 1998). We observed plied alone at the same rates (Figs. 1–4). antagonism in all the experiments that included entomopathogenic nematodes and P. fumosoroseus,and most that included S. marcescens; to our knowledge 4. Discussion these entomopathogens had not been applied together previously. Synergistic eﬀects resulting from combination of en- The antagonistic interactions we observed may have tomopathogenic nematodes with other entomopatho- been due to pathogen interactions prior to or during gens have been reported in a number of studies infection. In the case of S. marcescens and P. fumos- (Koppenhofer€ and Kaya, 1997; Koppenhofer€ et al., oroseus, it is possible that these organisms are directly 1999; Thurston et al., 1993, 1994). Additionally, synergy pathogenic to entomopathogenic nematodes, and has been observed in combination of entomopathogenic therefore the nematodes may have been killed or their nematodes with certain chemical insecticides (Kermar- ﬁtness reduced prior to infection. Indeed, S. marcescens rec and Mauleon, 1989; Koppenhofer€ and Kaya, 1998; has been reported to kill Caenorhabditis elegans 124 D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126

(Maupas), a nematode in the same order as Steinernema among experiments. Alternatively, the C. caryae larvae and Heterorhabditis (Kurz et al., 2003), and P. fumos- used in this study were generally stored longer before oroseus is pathogenic to the root knot nematode, use than those in the previous study (Shapiro-Ilan, Meloidogyne javanica (Treub) (Tigano-Milani et al., 2001a) (up to 6 months vs less than 3 months), and 1995). The negative interactions observed may also have Shapiro-Ilan (2001a) indicated that longer storage may been due to antagonistic toxins produced by the ento- decrease C. caryae susceptibility to nematodes. Indeed, mopathogens after infection was initiated. Thus, the when C. caryae larvae were stored 125–135 days in the overall pathogen titer in the insect would be reduced previous study, H. indica did not cause greater mortality thereby decreasing the potential to kill the host. The than the control. Finally, although the nematode and bacteria associated with entomopathogenic nematodes fungal strains were only sub-cultured a few times (less produce various antibiotics capable of suppressing a than three) since the previous tests, it is possible that variety of microbes (Akhurst, 1982; Webster et al., some attenuation occurred during that period (Shapiro 2002). Likewise, various toxins are produced by ento- et al., 1996; Wang and Grewal, 2002). mopathogenic fungi (Lysenko and Kucera, 1971; Mc- Despite application at rates greater or equivalent to Coy et al., 1988) and bacteria (such as S. marcescens) those used in the field for P. fumosoroseus and S. ento- (Lysenko and Kucera, 1971; Tanada and Kaya, 1993) mophila, the strains we tested of P. fumosoroseus and S. and may have inhibited the nematodes or their bacteria. marcescens did not show pathogenicity to C. caryae. The Martin (2002) observed that P. luminescens (a symbiont lack of pathogenicity in P. fumosoroseus was somewhat of Heterorhabditis spp.) and S. marcescens inhibited unexpected because it was pathogenic to other Coleop- each other in vitro, and exhibited antagonistic toxicity tera (James and Lighthart, 1994; Tigano-Milani et al., to the Colorado potato beetle, Leptinotarsa decemline- 1995), and there was some indication of pathogenicity in ata (Say). Similar interactions in our study may have our preliminary tests (unpublished data). A lack of caused the observed antagonism. In contrast to these pathogenicity in S. marcescens was not surprising be- direct effects, antagonism between entomopathogenic cause the bacterium is opportunistic and generally in- nematodes and other pathogens has also been attributed capable of penetrating into the hostÕs hemocoel (though to indirect interactions related to competition for the once inside the hemocoel they can be quite virulent). We same host resources (Kaya, 2002). Yet such indirect hypothesized that the nematodes entry into the hemo- interactions are generally associated with inhibition of coel would facilitate S. marcescens to enter and enhance nematode development and reproduction within the mortality, but overall this hypothesis did not hold up. host (Kaya, 2002) and not the ability to kill the insect, We conclude that the pathogen combinations we which was the focus of this study. investigated are not likely to improve suppression of C. Conceivably we may have observed more additivity caryae larvae beyond what is expected from single or synergism had we applied the pathogens sequentially application of the pathogen with greatest virulence. Of (Koppenhofer€ and Kaya, 1997). However, in some the combinations we investigated, H. indica applied studies synergy or best results were observed when with M. anisopliae might have the most potential for nematodes were applied with another control agent si- being beneficial to a pest management strategy because multaneously (Barbercheck and Kaya, 1990; Kermarrec it is the only combination that was additive at all rates and Mauleon, 1989). In addition to timing of applica- tested, and at the 0:5 rate caused greater C. caryae tion, the nature of the pathogen interactions (antago- mortality than when the pathogens were applied alone nism, additivity, or synergism) can depend on nematode at that rate. To our knowledge, the combination of M. species (Koppenhofer€ and Kaya, 1997; Thurston et al., anisopliae with entomopathogenic nematodes has not 1994), host species (see James and Elzen, 2001; Quintela been studied previously and may merit further study and McCoy, 1998), or application rate (Koppenhofer€ with other insects, nematodes, rates, and timing of and Kaya, 1997; Nishimatsu and Jackson, 1998). For application. example, in our study we observed varying effects of application rate in interactions between S. carpocapsae and B. bassiana, M. anisopliae,orS. marcescens). Acknowledgments Substantial differences in virulence were observed among the pathogen treatments applied alone. Overall, We thank Erika Brewer, Chevona Clark, Wanda S. carpocapsae exhibited the greatest virulence, and Evans, Kathy Halat, Grace Lathrop, and Angela R. produced mortality similar to previous studies (Shapiro- Payne for technical assistance, Dr. Khuong Nguyen and Ilan, 2001a). The B. bassiana, H. indica, and M. ani- Integrated BioControl Systems, for supplying nematode sopliae strains caused lower mortality than expected strains, William Joyner and James Campbell for advice based on previous experiments (Shapiro-Ilan, 2001a; and assistance on statistical methods, and the Georgia Shapiro-Ilan et al., 2003). Possibly, the lower virulence Agricultural Commodity Commission for Pecans for observed in this study was due to natural variation funding a portion of this research. D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126 125

References grub (Coleoptera: Scarabaeidae) control in turfgrass. J. Econ. Entomol. 91, 618–623. Akhurst, R.J., 1982. Antibiotic activity of Xenorhabdus spp., bacteria Koppenhofer,€ A.M., Choo, H.Y., Kaya, H.K., Lee, D.W., Gelernter, symbiotically associated with insect pathogenic nematodes of the W.D., 1999. Increased field and greenhouse efficacy against scarab families Heterorhabditidae and Steinernematidae. J. Gen. Micro- grubs with a combination of an entomopathogenic nematode and biol. 128, 3061–3065. Bacillus thuringiensis. Biol. Control 14, 37–44. Barbercheck, M.E., Kaya, H.K., 1990. Interactions between Beauveria Koppenhofer,€ A.M., Grewal, P.S., Kaya, H.K., 2000. Synergism of bassiana and the entomogenous nematodes Steinernema feltiae and imidacloprid and entomopathogenic nematodes against white Heterorhabditis heliothidis. J. Invertebr. Pathol. 55, 225–234. grubs: the mechanism. Entomol. Exp. Appl. 94, 283–293. Baur, M.E., Kaya, H.K., Tabashnik, B.E., Chilcutt, C.E., 1998. Kurz, C.L., Chauvet, S., Andres, E., Aurouze, M., Vallet, I., Michel, Suppression of diamondback moth (Lepidoptera: Plutellidae) with G.P.F., Uh, M., Celli, J., Filloux, A., de Bentzmann, S., Steinmetz, an entomopathogenic nematode (Rhabditida: Steinernematidae) I., Hoffman, J.A., Finlay, B.B., Gorval, J.P., Ferrandon, D., and Bacillus thuringinensis Berliner. J. Econ. Entomol. 91, 1089– Ewbank, J.J., 2003. Virulence factors of the human opportunistic 1095. pathogen Serratia marcescens identified by in vivo screening. Brinkman, M.A., Gardner, W.A., 2000. Possible antagonistic activity EMBO J. 22, 1451–1460. of two entomopathogens infecting workers of the red imported fire Lysenko, O., Kucera, M., 1971. Micro-organisms as sources of new ant (Hymenoptera: Formicidae). J. Entomol. Sci. 35, 205–207. insecticidal chemicals: toxins. In: Burges, H.D., Hussey, N.W. Dutcher, J.D., Payne, J.A., 1985. The impact of pecan weevil control (Eds.), Microbial Control of Insects and Mites. Academic Press, strategies on non-target arthropods. In: Neel, W.W. (Ed.), Pecan London, pp. 205–228. Weevil: Research Perspective. Quail Ridge Press, Brandon, MS, Martin, P.A.W., 2002. An in vitro inhibition test that predicts toxicity pp. 39–50. of bacterial pathogen combinations in the Colorado potato beetle. Goettel, M.S., Inglis, G.D., 1997. Fungi: Hyphomycetes. In: Lacey, Biocontrol Sci. Technol. 12, 643–647. L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Mannion, C., Winkler, H.E., Shapiro, D.I., Gibb, T., 2000. Interac- Press, San Diego, pp. 213–249. tions between halofenozide and Heterorhabditis marelatus for Gottwald, T.R., Tedders, W.L., 1983. Suppression of pecan weevil control of the Japanese beetle, Popillia japonica. J. Econ. Entomol. (Coleoptera: Curculionidae) populations with entomopathogenic 93, 48–53. fungi. Environ. Entomol. 12, 471–474. McCoy, C.W., Samson, R.A., Boucias, D.G. 1988. Entomogenous Harris, M.K., 1985. Pecan phenology and pecan weevil biology and fungi. In: Ignoffo, C.M. (Ed.), CRC Handbook of Natural management. In: Neel, W.W. (Ed.), Pecan Weevil: Research Pesticides. Microbial Insecticides, Part A. Entomogenous Protozoa Perspective. Quail Ridge Press, Brandon, MS, pp. 51–58. and Fungi, vol. 5. CRC Press, Boca Raton, pp. 151–236. Harris, M.K., 1999. Pecan weevil management considerations. In: Nishimatsu, T., Jackson, J.J., 1998. Interaction of insecticides, McCraw, B.E., Dean, E.H., Wood, B.W. (Eds.), Pecan Industry: entomopathogenic nematodes, and larvae of the western corn Current Situation and Future Challenges, Third National Pecan rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 91, Workshop Proceedings. USDA, Agricultural Research Service, 410–418. 1998–2004, pp. 66–73. Nyczepir, A.P., Payne, J.A., Hamm, J.J., 1992. Heterorhabditis Harrison, R.D., Gardner, W.A., Kinard, D.J., 1993. Relative suscep- bacteriophora: a new parasite of pecan weevil Curculio caryae.J. tibility of pecan weevil fourth instars and adults to selected isolates Invertebr. Pathol. 60, 104–106. of Beauveria bassiana. Biol. Control 3, 34–38. Payne, J.A., Dutcher, J.D., 1985. Pesticide efficacies for the pecan Jackson, M.A., Cliquet, S., Iten, L., 2003. Media and fermentation weevil past present and future. In: Neel, W.W. (Ed.), Pecan Weevil: process for the rapid production of high concentration of stable Research Perspective. Quail Ridge Press, Brandon, MS, pp. 103– blastospores of the bioinsecticidal fungus Paecilomyces fumosoro- 116. seus. Biocontrol Sci. Technol. 13, 23–33. Poinar Jr., G.O., 1990. Biology and taxonomy of Steinernematidae James, R.R., Elzen, G.W., 2001. Antagonism between Beauveria and Heterorhabditidae. In: Gaugler, R., Kaya, H.K. (Eds.), bassiana and imidacloprid when combined for Bemisia argentifolii Entomopathogenic Nematodes in Biological Control. CRC Press, (Homoptera: Aleyrodidae) control. J. Econ. Entomol. 94, 357–361. Boca Raton, FL, pp. 23–62. James, R.R., Lighthart, B., 1994. Susceptibility of the convergent lady Quintela, E.D., McCoy, C.W., 1998. Synergistic effect of imidacloprid beetle (Coleoptera: Coccinellidae) to four entomogenous fungi. and two entomopathogenic fungi on the behavior and survival of Environ. Entomol. 23, 190–192. larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae) in soil. Kaya, H.K., 2002. Natural enemies and other antagonists. In: Gaugler, J. Econ. Entomol. 91, 110–122. R. (Ed.), Entomopathogenic Nematology. CABI, New York, pp. SAS, 1999. SAS Software: Version 8.2. SAS Institute, Cary, NC. 189–204. Shapiro, D.I., Glazer, I., Segal, D., 1996. Trait stability and fitness of Kaya, H.K., Stock, S.P., 1997. Techniques in insect nematology. In: the heat tolerant entomopathogenic nematode Heterorhabditis Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. bacteriophora IS5 strain. Biol. Control 6, 238–244. Academic Press, San Diego, CA, pp. 281–324. Shapiro-Ilan, D.I., 2001a. Virulence of entomopathogenic nematodes Kermarrec, A., Mauleon, H., 1989. Synergy between chlordecone and to pecan weevil larvae Curculio caryae (Coleoptera: Curculionidae) Neoaplectana carpocapsae Weiser (Nematoda: Steinernematidae) in in the laboratory. J. Econ. Entomol. 94, 7–13. the control of Cosmopolites sordidus (Coleoptera: Curculionidae). Shapiro-Ilan, D.I., 2001b. Virulence of entomopathogenic nematodes Rev. Nematol. 12, 324–325. to pecan weevil adults (Coleoptera: Curculionidae). J. Entomol. Klein, M.G., 1997. Bacteria of soil-inhabiting insects. In: Lacey, L.A. Sci. 36, 325–328. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, Shapiro-Ilan, D.I., 2003. Microbial control of the pecan weevil, San Diego, CA, pp. 101–116. Curculio caryae. In: Dutcher, J.D., Harris, M.K., Dean, D.A. Koppenhofer,€ A.M., Kaya, H.K., 1997. Additive and Synergistic (Eds.), Integration of Chemical and Biological Insect Control in interactions between entomopathogenic nematodes and Bacillus Native, Seedling, and Improved Pecan Production. Southwest. thuringiensis for scarab grub control. Biol. Control 8, 131–137. Entomol. Supplement 27, 100–114. Koppenhofer,€ A.M., Kaya, H.K., 1998. Synergism of imidacloprid Shapiro-Ilan, D.I., Gouge, D.H., Koppenhofer,€ A.M., 2002. Factors and an entomopathogenic nematode: a novel approach to white affecting commercial success: case studies in cotton, turf and citrus. 126 D.I. Shapiro-Ilan et al. / Biological Control 30 (2004) 119–126

In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New scarabaeid larvae to an entomopathogenic nematode. J. Invertebr. York, pp. 333–356. Pathol. 61, 167–172. Shapiro-Ilan, D.I., Gardner, W.A., Fuxa, J.R., Wood, B.W., Nguyen, Thurston, G.S., Kaya, H.K., Gaugler, R., 1994. Characterizing the K., Adams, B., Humber, R.A., Hall, M.J., 2003. Survey of enhanced susceptibility of milky disease-infected scarabaeid grubs entomopathogenic nematodes and fungi endemic to pecan orchards to entomopathogenic nematodes. Biol. Control 4, 67–73. of the southeastern US and their virulence to the pecan weevil Tigano-Milani, M.S., Carneiro, R.G., de Faria, M.R., Frazao, H.S., (Coleoptera: Curculionidae). Environ. Entomol. 32, 187–195. McCoy, C.W., 1995. Isozyme characterization and pathogenicity Sikorowski, P.P., 1985. Pecan weevil pathology. In: Neel, W.W. (Ed.), of Paecilomyces fumosoroseus and P. lilacinus to Diabrotica Pecan Weevil: Research Perspective. Quail Ridge Press, Brandon, speciosa (Coleoptera: Chrysomelidae) and Meloidogyne javanica MS, pp. 87–101. (Nematoda: Tylenchidae). Biol. Control 5, 378–382. Smith, M.T., Georgis, R., Nyczepir, A.P., Miller, R.W., 1993. Wang, X., Grewal, P.S., 2002. Rapid genetic deterioration of Biological control of the pecan weevil, Curculio caryae (Coleoptera: environmental tolerance and reproductive potential of an entomo- Curculionidae), with entomopathogenic nematodes. J. Nematol. pathogenic nematode during laboratory maintenance. Biol. Con- 25, 78–82. trol 23, 71–78. Sri-Arunotai, S., Sikorowski, P.P., Neel, W.W., 1975. Study of the Webster, J.M., Chen, G., Hu, K., Jianxiong, L., 2002. Bacterial pathogens of the pecan weevil larvae. Environ. Entomol. 4, 790–792. metabolites. In: Gaugler, R. (Ed.), Entomopathogenic Nematol- Swingle, H.S., Seal, J.L., 1931. Some fungus and bacterial diseases of ogy. CABI, New York, pp. 99–114. pecan weevil larvae. J. Econ. Entomol. 24, 917. Wraight, S.P., Carruthers, R.I., Jaronski, S.T., Bradley, C.A., Garza, Tanada, Y., Kaya, H.K., 1993. Insect Pathology. Academic Press, San C.J., Galaini-Wraight, S., 2000. Evaluation of the entomopatho- Diego, CA. genic fungi Beauveria bassiana and Paecilomyces fumosoroseus for Thurston, G.S., Kaya, H.K., Burlando, T.M., Harrison, R.E., 1993. microbial control of the silverleaf whiteﬂy, Bemisia argentifolii. Milky disease bacterium as a stressor to increase susceptibility of Biol. Control, 203–217.