BIOLOGICAL AND MICROBIAL CONTROL Virulence of Entomopathogenic to Diaprepes abbreviatus (Coleoptera: Curculionidae) in the Laboratory

1 DAVID I. SHAPIRO AND CLAY W. MCCOY

Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850

J. Econ. Entomol. 93(4): 1090Ð1095 (2000) ABSTRACT The Diaprepes root weevil, Diaprepes abbreviatus (L.) is the most severe weevil pest in Florida citrus. Entomopathogenic nematodes have effectively suppressed larval populations of D. abbreviatus. Our objective was to conduct a broad laboratory comparison of entomopathogenic nematodes for virulence toward larvae of D. abbreviatus. The study was conducted at three temperatures (20, 24, and 29ЊC) and included nine entomopathogenic species and 17 strains: Heterorhabditis bacteriophora Poinar (Baine, NJ1, Hb, Hbl, HP88, and Lewiston strains), H. indica Poinar, Karunakar & David (original and Hom1 strains), H. marelatus Liu & Berry (IN and Point Reyes strains), H. megidis Poinar, Jackson & Klein (UK211 strain), H. zealandica Poinar (NZH3 strain), Steinernema riobrave Cabanillas, Poinar & Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (SN and UK76 strains), and S. glaseri (Steiner) (NJ43 strain). At 20ЊC, the greatest mortality was caused by S. riobrave although it was not signiÞcantly greater than H. bacteriophora (Baine), H. bacteriophora (Hb), H. bacteriophora (Hbl), and H. indica (original). At 24 and 29ЊC, S. riobrave caused greater larval mortality than other nematodes tested. Two strains of H. indica, H. bacteriophora (Baine), and S. glaseri were next in terms of virulence at 29ЊC. Our results suggest that S. riobrave has the greatest potential for control of D. abbreviatus.

KEY WORDS Diaprepes abbreviatus, Steinernema, Heterorhabditis, entomopathogenic, biological control, virulence

THE DIAPREPES ROOT weevil, Diaprepes abbreviatus (L.) Entomopathogenic nematodes are roundworms in is a major pest of citrus, sugarcane, ornamentals, and the genera Heterorhabditis and Steinernema. These vegetables in the West Indies and Florida (Woodruff obligate parasites kill insects with the aid of a mutal- 1964, McCoy 1999). This insect is the most severe istic bacterium that is carried in the intes- weevil pest in Florida citrus, infesting at least 18% of tine (Poinar 1990). The free-living infective juveniles the total acreage, and the potential for further spread generally enter their host through natural openings is vast (McCoy 1999). In citrus, adult weevils feed on (mouth, anus, or spiracles), release their bacteria foliage (causing a characteristic notching of leaves) (which kill the host within 48 h), and complete 2Ð3 and deposit eggs between leaves within the canopy generations before exiting the host (Poinar 1990). (Schroeder 1992). Neonates drop to the ground and Inundative releases, and endemic populations, of enter the soil where all instars feed on the roots, entomopathogenic nematodes have been docu- progressing from small to large roots as larvae mature mented to suppress larval populations of D. abbrevia- (Schroeder 1992). Pupation occurs in the soil and tus in Florida citrus (Schroeder 1987, Downing et al. adults emerge throughout the year with a substantial 1991, Adair 1994, Bullock et al. 1999b, Duncan and peak in the spring (MarchÐMay), and occasionally a McCoy 1996, Duncan et al. 1996; unpublished data). smaller peak between SeptemberÐOctober (Adair Reported levels of suppression from inundative appli- 1994, Stansly et al. 1997). Larval feeding alone can kill cations, however, have varied greatly (Duncan et al. trees, but the problem is often greatly exacerbated by 1999). Depending on species and rate of application, the fungal disease Phytophthora spp., which enters the levels of control have varied from no detectable con- citrus root at points of larval feeding (Graham and trol (Adair 1994, Duncan and McCoy 1996) to Ͼ90% Menge 1999, McCoy 1999). Although chemical insec- suppression (Bullock et al. 1999b, Duncan and McCoy ticides are used to control adult D. abbreviatus, and as 1996, Duncan et al. 1996). a barrier treatment against neonates, entomopatho- One of the primary reasons why entomopathogenic genic nematodes are currently the only recommended nematodes fail is because of the wrong choice of spe- control for all instars that have established themselves cies or strain (Georgis and Gaugler 1991). According in the grove (Bullock et al. 1999a). to Hominick et al. (1997) there are 22 recognized species in the genus Steinernema and eight in Heter- orhabditis. Furthermore, many of the species contain 1 Current address: USDAÐARS SE Fruit and Tree Nut Laboratory, numerous strains (Poinar 1990). Five species of en- 21 Dunbar Road, Byron, GA 31008. tomopathogenic nematodes have been tested for vir- August 2000 SHAPIRO AND MCCOY:ENTOMOPATHOGENIC NEMATODES VERSUS D. abbreviatus 1091

Table 1. Entomopathogenic nematodes used in this study

Species Strain Source Abbreviation Heterorhabditis bacteriophora Poinar Baine K. Nguyen, University of Florida, Gainesville HbBai H. bacteriophora NJ1 Bio Integrated Technology S. R. L. Perugia, Italy HbNj1 H. bacteriophora Hb VPI ϩ SU, Blacksburg, VA HbHb H. bacteriophora Hb1 Integrated BioControl Systems Inc., Lawrenceburg, IN HbHb1 H. bacteriophora HP88 MicroBio, L. T. D., Cambridge, UK HbHP8 H. bacteriophora Lewiston Integrated BioControl Systems, Lawrenceburg, IN HbLew H. indica Poinar, Karanukar & David Original isolate K. Nguyen, University of Florida, Gainesville HiOrg H. indica Hom1 Integrated BioControl Systems, Lawrenceburg, IN HiHom H. marelatus Liu & Berry IN Integrated BioControl Systems, Lawrenceburg, IN HmIn H. marelatus Point Reyes P. Stock, University of California, Davis HmPoi H. megidis Poinar, Jackson & Klein UK211 MicroBio, L. T. D., Cambridge, UK HmUk2 H. zealandica Poinar NZH3 P. Stock, University of California, Davis HzNzh Steinernema riobrave Cabanillas, 355 Thermo Trilogy, Columbia, MD Sr355 Poinar & Raulston S. carpocapsae (Weiser) All K. Nguyen, University of Florida, Gainesville ScAll S. feltiae (Filipjev) SN K. Nguyen, University of Florida, Gainesville SfSn S. feltiae UK76 MicroBio, L. T. D., Cambridge, UK SfUk7 S. glaseri (Steiner) NJ43 K. Nguyen, University of Florida, Gainesville SgNj4 ulence against D. abbreviatus: H. bacteriophora Poinar, in tap water at 10ЊC (Kaya and Stock 1997) except H. H. indica Poinar, Karunakar & David, S. carpocapsae indica, which was stored at 15ЊC (D.I.S., unpublished (Weiser), S. glaseri (Steiner), and S. riobrave Cabanil- data). In all experiments, nematodes were used within las, Poinar & Raulston (Schroeder 1987, 1994; Down- 2 wk of emergence from G. mellonella. ing et al. 1991; Duncan et al. 1999; Shapiro et al. 1999). Experimental units consisted of 25-dram lidded These Þve species represent only a small portion of the plastic containers (3.5 cm i.d., 8.5 cm deep) Þlled with variety of nematodes that are known to infect insects. Candler sand (estimated soil characteristics are the Additionally, not more than three of these species percentage of sand:silt:clay ϭ 96.6:2.0:1.4%, pH ϭ 6.3, have been compared for virulence against D. abbre- organic matter ϭ 0.3%). Soil moisture was standard- viatus in any one study, and some of the comparisons ized within the containers at Ϸ8% by weight (approx- were made using nematodes produced or formulated imately Ϫ0.03 bars). A single eighth- or ninth-instar D. under different conditions (Schroeder 1987, 1994; abbreviatus was placed on the bottom of each con- Downing et al. 1991; Shapiro et al. 1999; Duncan et al. tainer before Þlling with soil, and 500 infective juve- 1996, 1999; Shapiro and McCoy 2000). It is conceivable niles were applied to the soil surface. Larval mortality that nematode species or strains exist that are more was determined 12 d after inoculation. virulent than the relatively few that have been tested. Experiments were arranged in completely random- Our objective was to expand comparative testing of ized designs and were conducted at 20, 24, and 29 entomopathogenic nematodes for virulence toward D. (Ϯ0.5)ЊC. These temperatures represent the approx- abbreviatus using constant conditions. Our study, imate range of soil temperatures beneath the tree which included nine entomopathogenic nematode canopy in a Florida citrus grove at times of nematode species and 17 strains, was intended to serve as a application (springÐfall) (DuCharme 1971). Because preliminary foundation on which further comparisons of incubator availability, we could not conduct exper- can be built. Because entomopathogenic nematode iments at all temperatures simultaneously. The Þrst virulence against D. abbreviatus can be affected by Њ temperature (Shapiro et al. 1999) our experiments experiment compared all nematodes at 20 and 24 C and was repeated twice (i.e., three trials). The second were conducted at three temperatures that are rep- Њ resentative of soil temperatures in a citrus grove at experiment tested nematode virulence at 29 C and times of nematode application. was repeated once (i.e., two trials). All nematodes listed in Table 1 were used in experiment 1. Because of extremely low virulence observed in the Þrst ex- Materials and Methods periment, S. carpocapsae (All) and S. feltiae (SN) were Larvae of D. abbreviatus reared on artiÞcial diet omitted in the second experiment (all other nema- were obtained from the USDAÐARS Horticultural todes in Table 1 were included). A control (tap water) Laboratory, Orlando, FL. Nematode species and was included in all trials. Each treatment contained strains and their sources are listed in Table 1. All three replicates of 10 U (containers) in all trials. nematodes were reared in parallel at Ϸ24ЊC in last Mean percentage D. abbreviatus mortality was com- instar greater waxmoth, (L.), lar- pared among nematodes at each temperature using vae according to procedures described in Woodring arcsine transformation and analysis of variance (SAS and Kaya (1988). G. mellonella were obtained from Institute 1985). Treatment differences were further Northern Bait (Chetek, WI). Before experimentation, elucidated by StudentÐNewmanÐKeulsÕ test (SAS In- culture of nematodes did not exceed Þve passages in stitute 1985). Temperature effects (20 versus 24ЊC the host. After harvesting, all nematodes were stored only) were compared for each nematode strain with 1092 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 93, no. 4

Fig. 1. Nematode induced mortality of D. abbreviatus larvae at 20ЊC. Different letters above bars indicate statistical signiÞcance (P Ͻ 0.05). C, control (water). a t-test (i.e., least signiÞcant difference) (SAS Institute in D. abbreviatus than the other steinernematids and 1985). some of the heterorhabditids (Fig. 1). Larval mortality caused by S. feltiae, S. glaseri, and S. carpocapsae was not different from the mortality observed in the con- Results trol (Fig. 1). No differences in virulence were de- Differences in virulence were detected among tected among heterorhabditids at 20ЊC (Fig. 1). nematode species at 20, 24, and 29ЊC(F ϭ 13.57; df ϭ At 24ЊC, S. riobrave caused greater mortality than all 17, 143; P ϭ 0.0001 for 20ЊC, F ϭ 17.96; df ϭ 17, 149; P ϭ other nematode species and strains tested (Fig. 2). 0.0001 for 24ЊC, and at 29ЊC F ϭ 19.09; df ϭ 15, 95; P ϭ Larval mortality caused by S. feltiae and S. carpocapsae 0.0001). At 20ЊC, S. riobrave caused greater mortality was not different from the mortality observed in the

Fig. 2. Nematode induced mortality of D. abbreviatus larvae at 24ЊC. Different letters above bars indicate statistical signiÞcance (P Ͻ 0.05). C, control (water). August 2000 SHAPIRO AND MCCOY:ENTOMOPATHOGENIC NEMATODES VERSUS D. abbreviatus 1093

Fig. 3. Nematode induced mortality of D. abbreviatus larvae at 29ЊC. Different letters above bars indicate statistical signiÞcance (P Ͻ 0.05). C, control (water). control (Fig. 2). No differences in virulence were be equal to or greater than the virulence of S. riobrave. detected among heterorhabditids at 24ЊC (Fig. 2). This discrepancy may have been caused by host age, At 29ЊC, S. riobrave caused greater mortality in D. experimental conditions, or attenuation. Host age can abbreviatus than all other nematode species and affect the virulence of entomopathogenic nematodes strains (Fig. 3). Larval mortality caused by H. indica, against D. abbreviatus (Shapiro et al. 1999). Shapiro et H. bacteriophora (Baine), and S. glaseri was relatively al. (1999) observed greater virulence in H. indica rel- high compared with all other nematodes tested except ative to S. riobrave only in fourth- and Þfth-instar D. S. riobrave (Fig. 3). Larval mortality caused by S. abbreviatus, whereas the current study was conducted feltiae was not different from the mortality observed with older larvae. Thus, it is possible that superiority in the control (Fig. 3). in virulence (between H. indica and S. riobrave) may Mortality of D. abbreviatus was compared at 20 and be dependent on host age. Alternatively, the discrep- 24ЊC for each nematode strain. Mortality caused by S. ancy in results may have been caused by differing riobrave and H. indica (Hom1) was signiÞcantly experimental conditions. The containers used by Sha- greater at 24ЊC relative to 20ЊC(P Յ 0.05). Larval piro et al. (1999) were substantially smaller (3Ð4 cm mortality compared at 20 and 24ЊC was not signiÞ- i.d., 3.5 cm deep) than those used in this study. There- cantly different for all other nematodes tested (P Ͼ fore, the containers in the previous study may have 0.05). been too small to adequately compare virulence of species because nematode dispersal and host-search- ing behavior may have been removed as a factor. Discussion Finally, it is possible that the virulence of H. indica Our study shows that S. riobrave has the greatest (Hom1) may have attenuated in the period between virulence toward mature larvae of D. abbreviatus the two studies. BeneÞcial traits in entomopathogenic among nematodes tested in this study. The virulence nematodes can be reduced during normal laboratory of entomopathogenic nematodes can be affected by culture techniques (e.g., in G. mellonella) after only production and formulation methodology (Baur et al. eight passages (Shapiro et al. 1996). Attenuation is also 1997). The nematodes tested in this study, however, a widespread occurrence in other insect pathogen were cultured under parallel conditions and therefore groups (Tanada and Fuxa 1987, Tanada and Kaya our results represent a comparison of the nematodeÕs 1993). ability to kill D. abbreviatus. Our Þndings are consis- Heterorhabditis marelatus and H. megidis are known tent with laboratory, greenhouse, and Þeld studies that to be highly effective biocontrol agents against cur- indicate that S. riobrave has a higher virulence than S. culionids in the genus Otiorhynchus (Berry et al. 1997, carpocapsae, H. bacteriophora, and H. indica (Schroe- Kakouli-Duarte et al. 1997) and are relatively active at der 1994, Duncan and McCoy 1996, Duncan et al. 1996, lower temperatures (Grewal et al. 1994, Berry et al. Shapiro and McCoy 2000). 1997). Therefore, we hypothesized that these nema- Contrary to the results of this study, Shapiro et al. todes might also be highly virulent toward D. abbre- (1999) reported the virulence of H. indica (Hom1) to viatus, particularly at 20ЊC. The observed mortality in 1094 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 93, no. 4

D. abbreviatus caused by H. marelatus and H. megidis, can persist in the soil for relatively long periods, could however, was not greater than mortality caused by the be an attractive biocontrol agent for D. abbreviatus. other heterorhabditids. Nematodes that have been applied commercially to Our results indicated no effect of S. carpocapsae on control D. abbreviatus (H. bacteriophora, H. indica, S. D. abbreviatus larvae. S. carpocapsaeÕs host-searching carpocapsae, and S. riobrave) persist poorly in a Florida behavior is that of an ambusher, i.e., the nematode citrus ecosystem; nematode populations levels decline does not actively seek hosts but rather sits and waits drastically within a 2-wk period (Duncan et al. 1996; for a target to come into close range (Lewis et al. unpublished data). S. glaseri has high dispersal 1992). When S. carpocapsae is applied to the soil sur- (Schroeder and Beavers 1987) and host Þnding (Lewis face the majority will remain there (Moyle and Kaya et al. 1992) abilities, and because of high biochemical 1981, Schroeder and Beavers 1987). In our study, it is energy reserves it has a good potential for persistence likely that an insufÞcient number of S. carpocapsae (Selvan et al. 1993). It is not clear whether a nematode reached the larvae for infection. Studies (e.g., such as S. glaseri would, in the long run, be a more Schroeder 1987, 1994) demonstrated signiÞcant con- effective biocontrol agent than S. riobrave despite its trol of D. abbreviatus by S. carpocapsae in laboratory lower virulence. Although laboratory screening of en- and greenhouse tests, but in most cases this low dis- tomopathogenic nematodes for virulence can be an persal behavior may have been overcome by applica- important component of developing a biological con- tion of excessive numbers of nematodes or the size of trol program for a particular pest (Mannion and Jans- the container. The success of certain Þeld trials with son 1992, Ricci et al. 1996, Patterson Stark and Lacey S. carpocapsae (Schroeder 1987, 1992) may also have 1999), relative virulence among nematodes in the lab- been the result of high rates of applied nematodes or oratory may not be consistent with what is observed of the nematodes being passively dispersed toward the in the Þeld (Grewal and Georgis 1998). Once the hosts (perhaps by water) (Kaya 1990). Various other biocontrol candidates are deÞned based on characters studies indicated no treatment effects after Þeld ap- tested in the laboratory, the ultimate test of efÞcacy plication of S. carpocapsae (Adair 1994, Bullock et al. must be conducted under Þeld conditions. 1999b, Duncan et al. 1996). The virulence of H. indica (Hom1) and S. riobrave were reduced at 20ЊC relative to 24ЊC, but no tem- Acknowledgments perature effect was detected in other nematodes. The The authors thank I. Jackson and A. Hoyte for technical temperature dependent differences observed in H. assistance, G. Smart (University of Florida, Gainesville) and indica (Hom1) and S. riobrave in D. abbreviatus mor- K. Smith (University of Arizona, Tucson) for reviewing an tality are consistent with prior research conducted at earlier draft of the manuscript, Ed Lewis (VPI and SU, Blacks- 21 and 24ЊC (Shapiro et al. 1999). Our results are also burg), K. Nguyen (University of Florida, Gainesville), P. consistent with research indicating that a temperature Stock (University of California, Davis), Integrated BioCon- range of 21Ð24ЊC will not affect the virulence of H. trol System, Incorporated (Lawrenceburg, IN), Bio Inte- grated Technology, S.R.L. (Perugia, Italy), MicroBio, L.T.D., megidis, S. carpocapsae, S. feltiae, and S. glaseri (Grewal and Thermo Triology, Incorporated, for providing nema- et al. 1994). Contrary to our results, other studies todes, and K. Crosby (USDAÐARS, Orlando, FL) for rearing reported that certain strains of H. bacteriophora (i.e., D. abbreviatus. This work was supported in part by FCPRAC HP88, and Hbl) showed a reduction in virulence grant No. 942-18E. Florida Agricultural Experiment Station within a temperature range similar to ours (Grewal et Journal Series No. R-07293. al. 1994, Shapiro et al. 1999). Grewal et al. 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