BIOLOGICAL AND MICROBIAL CONTROL Toxicity of Chromobacterium subtsugae to Southern Green Stink Bug (Heteroptera: Pentatomidae) and Corn Rootworm (Coleoptera: Chrysomelidae) 1 2,3 2 PHYLLIS A. W. MARTIN, EDSON HIROSE, AND JEFFREY R. ALDRICH USDAÐARS, Insect Biocontrol Laboratory and Chemicals Affecting Insect Behavior Laboratory, Beltsville, MD 20705-2350 J. Econ. Entomol. 100(3): 680Ð684 (2007) ABSTRACT Diabrotica spp. (Coleoptera: Chrysomelidae) beetles and southern green stink bugs, Nezara viridula (L.) (Heteroptera: Pentatomidae), are pests on corn, Zea mays L., and soybean, Glycine max (L.) Merr., as well as on cucurbits. Control of these insects has depended on chemicals. An alternative to chemical control is the use of biologicals. Use of bacteria, fungi, viruses, pheromones, and metabolites to control these insects can potentially improve resistance management and reduce pesticide use. Other than Bacillus thuringiensis Berliner, few bacteria have been discovered that are lethal to either of these pests. Chromobacterium subtsugae Martin et al., a newly described bacterium that is known to be toxic to Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae, was found to be toxic to both diabroticite adult beetles and southern green stink bug adults. In laboratory assays, toxins produced by these bacteria kill 80Ð100% of the adults of two species of diabroticite beetles, Diabrotica undecimpunctata howardi Barber and Diabrotica virgifera virgifera LeConte, and 100% of southern green stink bug adults within 6 d. For green stink bug, live bacteria were not needed for toxicity. KEY WORDS Diabrotica, Nezara viridula, biocontrol Corn rootworm beetles are principal pests of corn, (Balsamo) Vuillemin and Metarhizium anisopliae although certain species of diabroticite beetles also (Metschnikoff), have been suggested for control of feed on soybean, Glycine max (L.)Merr.; melons (Cu- these pests (Krueger and Roberts 1997, Pereira and cumis spp.), cucumbers, Cucumis sativus L., and pea- Roberts 1991, Sosa-Gomez and Moscardi 1998). Bac- nuts, Arachis hypogea L. In addition to damaging the teria, other than Bacillus thuringiensis Berliner strains crop, the adult beetles transmit bacterial wilt and viral for corn rootworm (Diabrotica spp.) (Baum et al. diseases (York 1992). The southern green stink bug, 2004), are not used for control of these insects. Other Nezara viridula (L.) (Heteroptera: Pentatomidae), is bacteria such as pigmented Serratia marcescens Bizo a pest of solanaceous crops, but it also has more gen- have been isolated from dead insects and implicated as eral tastes that extend to soybean, other legumes, the causative disease agent (Grimont and Grimont cotton (Gossypium spp.), and various vegetables, in- 1978). Pathogens have traditionally been identiÞed cluding tomatoes (Lycopersicum spp.) (McKinlay et using KochÕs postulates (as per Black 1996) in which al. 1992). These insects are presently controlled by the the Þrst step in identifying the causal agent is isolation use of insecticides, either prophylactically or in an from diseased insects. integrated pest management program. The propensity Violet-pigmented bacteria such as Chromobacte- of insects to develop resistance to insecticides has rium violaceum Bergonzini have infrequently been encouraged the use of other means of control. isolated from insects, and they have not been previ- An alternative to traditional chemical control is the ously considered harmful to insects (Bucher 1981). In use of pathogens. Fungi, such as Beauveria bassiana the larger grain borer, Prostephanus truncatus (Horn), these bacteria may be involved in cellulose digestion Mention of a trademark or proprietary product does not constitute (Vazquez-Artista et al. 1997), forming a symbiotic a guarantee or warranty of the product by the United States Depart- rather than a pathogenic association. The genome of ment of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. C. violaceum has been sequenced. In addition to the 1 Corresponding author: USDAÐARS Insect Biocontrol Laboratory, violet pigment violacein, which has antimicrobial ac- Beltsville, MD 20705-2350 (e-mail:[email protected]). tivity against gram-positive and gram-negative bacte- 2 Chemicals Affecting Insect Behavior Laboratory, Beltsville, MD ria (Duran et al. 1983) and Trypanosoma cruzi Chagas 20705-2350. 3 Current address: Universidade Federal do Parana´, Departamento (Duran et al. 1994), it contains a gene similar to an de Zoologia, Caixa Postal 19020, Curitiba, PR 81531-990, Brazil. insecticidal gene found in Photorhabdus luminescens June 2007 MARTIN ET AL.: TOXICITY OF C. subtsugae TO PEST INSECTS 681 Thomas and Poinar and in Xenorhabdus nematophilia versity. Western corn rootworms, Diabrotica virgifera Poinar and Thomas (Brazilian National Genome virgifera LeConte, were obtained from C. Neilson Project Consortium 2003). (USDAÐARSÐNGIRL, Brookings, SD) and maintained A new species of Chromobacterium, Chromobacte- under the same conditions as described for southern rium subtsugae Martin et al., was found to be toxic to corn rootworms. Colorado potato beetle, Leptinotarsa decemlineata Bioassays. Southern green stink bugs were incu- (Say) Martin et al. 2004). Here, we describe the tox- bated 48 h in plastic cages without water. Twenty icity of these bacteria in laboratory assays to other pest adults were used per treatment with Þve stink bugs per insects: two corn rootworm species and southern cage. Males and females were tested separately. The green stink bug. suspensions of C. subtsugae were delivered to the adults in 1 ml of liquid composed of 0.5 ml of distilled water and 0.5 ml of bacterial suspension. The micro- Materials and Methods centrifuge tubes containing the test material or water Bacterial Strains and Media. Violet colonies of bac- as a control were plugged with cotton and replaced terial strain PRAA4-1 were isolated in summer 2000, every 3 d. Mortality was recorded daily for 6 d. Survival from Maryland forest soil plated on L-agar, by S. Stone times were determined with LIFEREG procedure by (Martin et al. 2004). The violet bacteria were Gram- using the Weibull distribution with 95% conÞdence rods, preliminarily identiÞed as C. violaceum by com- intervals (SAS Institute 2004), because of limitation of parison with descriptions in BergeyÕs manual (Sneath numbers of insects (Preisler and Robertson 1989). We 1984). When the Þrst 500 bp of the 16S ribosomal DNA attempted feeding C. subtsugae to nymphs, but the were sequenced, the identiÞcation was only to the nymphs did not feed consistently in a liquid delivery genus Chromobacterium (Accugenix, Newark, DE). system. This bacterial strain has been subcultured weekly at The toxicity of C. subtsugae to green stick bug adults room temperature on L-agar (Atlas 1997). For bacte- also was tested by injection of killed cultures (auto- rial counts, we used RM-agar (Martin et al. 1998), claved at 121ЊC for 20 min). One microliter of undi- containing half the nutrients of L-agar. Because viable luted culture was injected ventrally into the abdomen bacterial counts were not correlated with toxicity, we of 10 males and 10 females. Controls of 10 males and used optical density as a measure of cell density. The 10 females also were injected with sterile distilled correlation between optical density and toxicity for water. Mortality was recorded daily for 5 d. Colorado potato beetle was 0.77 (P.A.W.M., unpub- Corn root worm assays were performed with adult lished data). beetles of either southern corn rootworm or western Preliminary Toxin Characterization. Bacteria were corn rootworm of mixed sexes as described by Schro- grown on L-agar plates at 25ЊC for 48 h to test whether der et al. (2001). C. subtsugae was harvested at 48 h bacteria could be recovered from dead insects, thus into 20 ml of watermelon juice containing 0.08% cu- fulÞlling KochÕs postulates to identify a pathogen; or curbitacin-E glycoside (Matsuo et al. 1999) mixed for 120 h for increased stability upon storage. Bacteria with 3% Mirasperse starch (A.E. Staley Manufacturing were harvested into 15 ml of sterile distilled water. Co., Decatur, IL). Five 25-␮l droplets were added to Ten-milliliter volumes were autoclaved at 121ЊC for each 100-mm petri dish. Five beetles were added to 10 min to test for stability. Cells were removed from each dish, and controls (without the bacteria) and suspension by centrifugation and Þltered through a treatments were repeated Þve times. At 48 h, addi- 0.45-␮m Millipore Þlter (Millipore Corporation, Bil- tional moisture was introduced by a premoistened lerica, MA) to yield a cell-free supernatant. The pellet dental wick and additional food was added (either fraction was formed by resuspending the pellet to the western corn rootworm diet or squash). Mortality was original volume. recorded 5 d after treatment was initiated. Insects. The southern green stink bug colony orig- For corn rootworm larval assays, eggs were hatched inated from specimens collected near Stoneville, MS, and larvae were fed on corn roots germinated in sand and insects were reared on sunßower, Helianthus an- at room temperature. Roots were kept moist by daily nuus L., seeds and green beans, Phaseolus vulgaris L., watering. Larvae (16) were removed after 3 wk days at 28ЊC, 65% RH, and a photoperiod of 16:8 (L:D) h and transferred to freeze-dried diet pellets (Martin (Aldrich et al. 1993). 2004a) made with corn rootworm diet (BioServ, Initially, southern corn rootworm adults, Diabrotica Frenchtown, NJ), which had been rehydrated with undecimpunctata howardi Barber (Coleoptera: Chry- water or C. subtsugae suspension. The larvae in sealed somelidae), were obtained from French Agricultural trays were incubated at 25ЊC and 50% RH in the dark. Research, Inc. (Lamberton, MN). Adults were held Mortality was recorded every 24 h. Surviving larvae for 3 wk before testing at 24ЊC, 30% RH, and a pho- were weighed after 4 d. Differences in weights were toperiod of 16:8 (L:D) h. Beetles were fed artiÞcial compared using PROC MIXED (SAS Institute 2004). diet (Branson and Jackson 1988). Later, a southern Recovery of C.