BIOLOGICAL AND MICROBIAL CONTROL
Toxicity of Chromobacterium subtsugae to Southern Green Stink Bug (Heteroptera: Pentatomidae) and Corn Rootworm (Coleoptera: Chrysomelidae) PHYLLIS A. W. MARTIN,1 EDSON HIROSE,2,3
AND
JEFFREY R. ALDRICH2
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 80 Ð100% 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, although certain species of diabroticite beetles also feed on soybean, Glycine max (L.)Merr.; melons (Cucumis spp.), cucumbers, Cucumis sativus L., and peanuts, Arachis hypogea L. In addition to damaging the crop, the adult beetles transmit bacterial wilt and viral diseases (York 1992). The southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae), is a pest of solanaceous crops, but it also has more general tastes that extend to soybean, other legumes, cotton (Gossypium spp.), and various vegetables, including tomatoes (Lycopersicum spp.) (McKinlay et al. 1992). These insects are presently controlled by the use of insecticides, either prophylactically or in an integrated pest management program. The propensity of insects to develop resistance to insecticides has encouraged the use of other means of control. An alternative to traditional chemical control is the use of pathogens. Fungi, such as Beauveria bassiana Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 1 Corresponding author: USDAÐARS Insect Biocontrol Laboratory, Beltsville, MD 20705-2350 (e-mail:
[email protected]). 2 Chemicals Affecting Insect Behavior Laboratory, Beltsville, MD 20705-2350. 3 Current address: Universidade Federal do Parana ´, Departamento de Zoologia, Caixa Postal 19020, Curitiba, PR 81531-990, Brazil.
(Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff), have been suggested for control of these pests (Krueger and Roberts 1997, Pereira and Roberts 1991, Sosa-Gomez and Moscardi 1998). Bacteria, other than Bacillus thuringiensis Berliner strains for corn rootworm (Diabrotica spp.) (Baum et al. 2004), are not used for control of these insects. Other bacteria such as pigmented Serratia marcescens Bizo have been isolated from dead insects and implicated as the causative disease agent (Grimont and Grimont 1978). Pathogens have traditionally been identiÞed using KochÕs postulates (as per Black 1996) in which the Þrst step in identifying the causal agent is isolation from diseased insects. Violet-pigmented bacteria such as Chromobacterium violaceum Bergonzini have infrequently been isolated from insects, and they have not been previously considered harmful to insects (Bucher 1981). In the larger grain borer, Prostephanus truncatus (Horn), these bacteria may be involved in cellulose digestion (Vazquez-Artista et al. 1997), forming a symbiotic rather than a pathogenic association. The genome of C. violaceum has been sequenced. In addition to the violet pigment violacein, which has antimicrobial activity against gram-positive and gram-negative bacteria (Duran et al. 1983) and Trypanosoma cruzi Chagas (Duran et al. 1994), it contains a gene similar to an insecticidal gene found in Photorhabdus luminescens
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Thomas and Poinar and in Xenorhabdus nematophilia Poinar and Thomas (Brazilian National Genome Project Consortium 2003). A new species of Chromobacterium, Chromobacterium subtsugae Martin et al., was found to be toxic to Colorado potato beetle, Leptinotarsa decemlineata (Say) Martin et al. 2004). Here, we describe the toxicity of these bacteria in laboratory assays to other pest insects: two corn rootworm species and southern green stink bug. Materials and Methods Bacterial Strains and Media. Violet colonies of bacterial strain PRAA4-1 were isolated in summer 2000, from Maryland forest soil plated on L-agar, by S. Stone (Martin et al. 2004). The violet bacteria were Gramrods, preliminarily identiÞed as C. violaceum by comparison with descriptions in BergeyÕs manual (Sneath 1984). When the Þrst 500 bp of the 16S ribosomal DNA were sequenced, the identiÞcation was only to the genus Chromobacterium (Accugenix, Newark, DE). This bacterial strain has been subcultured weekly at room temperature on L-agar (Atlas 1997). For bacterial counts, we used RM-agar (Martin et al. 1998), containing half the nutrients of L-agar. Because viable bacterial counts were not correlated with toxicity, we used optical density as a measure of cell density. The correlation between optical density and toxicity for Colorado potato beetle was 0.77 (P.A.W.M., unpublished data). Preliminary Toxin Characterization. Bacteria were grown on L-agar plates at 25⬚C for 48 h to test whether bacteria could be recovered from dead insects, thus fulÞlling KochÕs postulates to identify a pathogen; or for 120 h for increased stability upon storage. Bacteria were harvested into 15 ml of sterile distilled water. Ten-milliliter volumes were autoclaved at 121⬚C for 10 min to test for stability. Cells were removed from suspension by centrifugation and Þltered through a 0.45-m Millipore Þlter (Millipore Corporation, Billerica, MA) to yield a cell-free supernatant. The pellet fraction was formed by resuspending the pellet to the original volume. Insects. The southern green stink bug colony originated from specimens collected near Stoneville, MS, and insects were reared on sunßower, Helianthus annuus L., seeds and green beans, Phaseolus vulgaris L., at 28⬚C, 65% RH, and a photoperiod of 16:8 (L:D) h (Aldrich et al. 1993). Initially, southern corn rootworm adults, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), were obtained from French Agricultural Research, Inc. (Lamberton, MN). Adults were held for 3 wk before testing at 24⬚C, 30% RH, and a photoperiod of 16:8 (L:D) h. Beetles were fed artiÞcial diet (Branson and Jackson 1988). Later, a southern corn rootworm colony was established from Þeldcollected insects from Beltsville, MD, in 2003. The colony has been maintained on corn roots for larvae, and on squash and water for the adults. Field-collected insects are introduced yearly to maintain genetic di-
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versity. Western corn rootworms, Diabrotica virgifera virgifera LeConte, were obtained from C. Neilson (USDAÐARSÐNGIRL, Brookings, SD) and maintained under the same conditions as described for southern corn rootworms. Bioassays. Southern green stink bugs were incubated 48 h in plastic cages without water. Twenty adults were used per treatment with Þve stink bugs per cage. Males and females were tested separately. The 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 microcentrifuge tubes containing the test material or water as a control were plugged with cotton and replaced every 3 d. Mortality was recorded daily for 6 d. Survival times were determined with LIFEREG procedure by using the Weibull distribution with 95% conÞdence intervals (SAS Institute 2004), because of limitation of numbers of insects (Preisler and Robertson 1989). We attempted feeding C. subtsugae to nymphs, but the nymphs did not feed consistently in a liquid delivery system. The toxicity of C. subtsugae to green stick bug adults also was tested by injection of killed cultures (autoclaved at 121⬚C for 20 min). One microliter of undiluted culture was injected ventrally into the abdomen of 10 males and 10 females. Controls of 10 males and 10 females also were injected with sterile distilled water. Mortality was recorded daily for 5 d. Corn root worm assays were performed with adult beetles of either southern corn rootworm or western corn rootworm of mixed sexes as described by Schroder et al. (2001). C. subtsugae was harvested at 48 h into 20 ml of watermelon juice containing 0.08% cucurbitacin-E glycoside (Matsuo et al. 1999) mixed with 3% Mirasperse starch (A.E. Staley Manufacturing Co., Decatur, IL). Five 25-l droplets were added to each 100-mm petri dish. Five beetles were added to each dish, and controls (without the bacteria) and treatments were repeated Þve times. At 48 h, additional moisture was introduced by a premoistened dental wick and additional food was added (either western corn rootworm diet or squash). Mortality was recorded 5 d after treatment was initiated. For corn rootworm larval assays, eggs were hatched and larvae were fed on corn roots germinated in sand at room temperature. Roots were kept moist by daily watering. Larvae (16) were removed after 3 wk days and transferred to freeze-dried diet pellets (Martin 2004a) made with corn rootworm diet (BioServ, Frenchtown, NJ), which had been rehydrated with water or C. subtsugae suspension. The larvae in sealed trays were incubated at 25⬚C and 50% RH in the dark. Mortality was recorded every 24 h. Surviving larvae were weighed after 4 d. Differences in weights were compared using PROC MIXED (SAS Institute 2004). Recovery of C. subtsugae from Dead Insects. Because viability of C. subtsugae declines after 72 h of growth on media, recovery from insects was done using cultures that were grown on solid media for 48 h. Insects that had died after ingesting C. subtsugae cells were ground in 0.5 ml of sterile water by using a
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Fig. 1. Survival times of 48- and 120-h cultures of C. subtsugae against the southern green stink bug. Dark bars, males; light bars, females. Error bars represent 95% CL. Control mortality for females was 0% (0/20) and for males was 15% (3/20).
miniature mortar and pestle. Large particles were allowed to settle and liquid was plated directly on L-agar and incubated for 48 h at 25⬚C and scored for the violet colonies typical of the bacteria. Results Southern Green Stink Bug. Male and female southern green stink bugs are known to differ in their response to insecticides (Panizzi and Hirose 1995), so they were tested against C. subtsugae separately. For treatment with undiluted cultures, mortality for both sexes usually reached 100% by 6 d, and the main difference between males and females was how quickly they died. In most assays, female stink bugs did not begin to die until day 4, whereas mortality often occurred in the treated male stink bugs by the second day. This difference is best expressed by differences in survival times with cultures that were 48 h old. For undiluted cultures at the same optical density (OD600 ⫽ 2.3), the males died 29% faster than the females for cultures that were harvested at 48 h but only 15% faster for cultures harvested at 5 d (Fig. 1). Final mortality for females was 90% and for the males it was 95%. However, the males died quicker only when the cell-associated fraction was used. When the cell-free supernatant was used for treatment, the survival time was essentially identical for both males and females, although there was more variability for females (Fig. 2). In these assays, all the males were dead at 6 d after treatment with the whole culture, the supernatant or the resuspended pellet. However, the female mortality was 100% only in the whole culture and the supernatant treatments. The Þnal mortality for the females fed the resuspended pellet was only 70%. To test for stability, the autoclaved whole cultures were tested. There were little differences in the survival times between the whole cultures and the autoclaved suspension for either males or females (Fig. 3). In treatments with the autoclaved suspension, the difference in survival times between males and females also was absent, as determined by overlapping 95% conÞdence intervals. Final mortality for both males and females in the autoclaved cultures was 95%. A 1:3 dilution of supernatants killed 50% of the females, and a 1:6 dilution killed 50% of the males.
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Fig. 2. Survival times for whole cultures and separated cultures of C. subtsugae against the southern green stink bug. Dark bars, males; light bars, females. Error bars represent 95% CL. Control mortality for females was 5% (1/20) and for males was 10% (2/20).
Upon injection, only the males that received the C. subtsugae injection died. All mortality (70%) occurred within 2 d. No females receiving C. subtsugae injections and no controls died. Corn Rootworm. Approximately 80% of both southern and western corn rootworm adults died when fed C. subtsugae in a bait formulation (Fig. 4). The beetles consumed the entire control bait droplets, whereas the violet droplets containing C. subtsugae were still visible at the end of the assay. In additional assays, 100% of southern corn rootworm adults died within 120 h, whereas control mortality was 13.3%. For larvae the mortality reached only 40% when fed whole cultures at 4 d (OD600 ⫽ 2.31). However, there was signiÞcant a signiÞcant difference (df ⫽ 24, P ⫽ 0.0057) in weight between surviving controls (15.02 ⫾ 1.24 mg) and those treated with C. subtsugae (9.81 ⫾ 0.86 mg). The control larvae burrowed into the diet pellets and produced frass. For the C. subtsugaetreated larvae, there was no evidence of feeding and no frass. Recovery of C. subtsugae from Dead Insects. Eleven southern green stink bug adults that died were ground after being fed C. subtsugae. Although living bacteria were recovered from all of them, with titers ranging from 103 to 105 cells per insect, no violet colonies were
Fig. 3. Survival times for autoclaved versus nonautoclaved cultures of C. subtsugae against the southern green stink bug. Dark bars, males; light bars, females. Error bars represent 95% CL. Control mortality for females was 15% (3/20) and for males 20% (4/20).
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Fig. 4. Percentage of mortality of southern (dark bars) and western (light bars) corn rootworm adults when fed whole cultures of C. subtsugae. Error bars represent standard error. Control mortality for western corn rootworm is shown by thick line, and control mortality for southern corn rootworm is shown by thin line.
recovered on RM agar. Five southern corn rootworms were ground, and no violet colonies were recovered on RM agar. Discussion The deaths of both southern green stink bug and corn rootworm adults after consuming C. subtsugae in water or in a bait formulation were similar to results with Colorado potato beetle (Martin et al. 2004). Reduction in weight or feeding inhibition with corn rootworm larvae was similar to that seen with Lymantria dispar (L.) (Martin 2004b). Although whole bacterial cultures were used in most experiments, live bacteria do not seem to be necessary for lethality. Several lines of evidence support this hypothesis. First, violet pigment-producing bacteria could not be isolated from either stink bugs or corn rootworms that had died after feeding on C. subtsugae cells. Thus, the Þrst step in identifying a pathogen could not be fulÞlled. Second, for stink bugs, cell-free Þltrates alone caused mortality. Third, cells killed by autoclaving also killed stink bugs in a manner similar to viable whole cultures. Fourth, cells killed by autoclaving killed 70% of male stink bugs in 48 h by injection. The injection data showed the most obvious difference in susceptibility between males and females (Panizzi and Hirose 1995). This suggests a heat-stable toxin(s) is responsible for the mortality. Heat-stable oral insecticidal toxins, such as the -exotoxin, are known to be produced by strains of B. thuringiensis (Sˇ ebesta et al. 1981). Attempts to localize the toxicity in the soluble (supernatant) or particulate (pellet) fractions were unsuccessful, because signiÞcant toxicity was noted with both fractions. Although undiluted bacterial cultures were used to kill these insects, bait formulations with attractants for N. viridula (Aldrich et al. 1993) or feeding stimulants for diabroticite beetles (Schroder et al. 2001) could increase the effective concentration of the toxins in the Þeld. For corn rootworm adults, the compulsive feeding on formulations containing cucurbitacin ensures the ingestion of a lethal dose (Schroder et al. 1998). Southern corn rootworm larval mortality was
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low (⬍40%), but feeding was inhibited as evidenced by lack of weight gain. What remains is to determine whether attractant or bait combinations can be effective under in the Þeld and whether the nymphs of N. viridula will feed on and be killed by C. subtsugae. It is unusual that an orally active compound produced by bacteria is Þrst discovered as lethal to adults. The ␦-endotoxin of B. thuringiensis kills the larval stage of most insects (Schnepf et al. 1998). Even the toxin of C. subtsugae did not kill adult Colorado potato beetles, but it killed in all instars of feeding larvae (Martin et al. 2004). Acknowledgments We thank the following Eleanor Roosevelt High School students; S. Stone for isolation of PRAA from soil, and H. Romani for some bioassays; and USDA employees L. Liska for rearing insects, and A. Shropshire for coordinating bioassays and general technical assistance.
References Cited Aldrich, J. E., H. Numata, M. Borges, F. Bin, G. K. Waite, and W. R. Lusby. 1993. Artifacts and pheromone blends from Nezara spp. and other stink bugs (Heteroptera: Pentatomidae) A. Naturforsh. C. 48: 73Ð79. Atlas, R. M. 1997. Handbook of microbiological media. CRC, Boca Raton, FL. Baum, J. A., C.-R. Chu, M. Rupar, G. R. Brown, W. P. Donovan, J. E. Huesing, O. Ilagan, T. M. Malvar, M. Pleau, M. Walters, and T. Vaughn. 2004. Binary toxins from Bacillus thuringiensis are active against the western corn rootworm, Diabrotica virgifera virgifera LeConte. Appl. Environ. Microbiol. 70: 4889 Ð 4898. Black, J. G. 1996. Microbiology: principles and applications, 3rd ed. Prentice Hall, Upper Saddle River, NJ. Branson, T. F., and J. J. Jackson. 1988. An improved diet for adult Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). J. Kans. Entomol. Soc. 61: 353Ð355. Brazilian National Genome Project Consortium. 2003. The complete sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. U.S.A. 100: 11660 Ð11665. Bucher, G. E. 1981. IdentiÞcation of bacteria found in insects, pp. 7Ð33. In H. D. Burges [ed.], Microbial control of pests and plant diseases 1970 Ð1980. Academic, New York. Duran, N., R. V. Antonio, M. Haun, and R. A. Pilli. 1994. Biosynthesis of a trypanocide by Chromobacterium violaceum. World J. Microbiol. Biotechnol. 10: 685Ð 690. Duran, N., S. Erazo, and V. Campos. 1983. Bacterial chemistry II. Antimicrobial photoproduct from pigment of Chromobacterium violaceum. An. Acad. Bras. Cie´ n. 55: 231Ð234. Grimont, A. D., and F. Grimont. 1978. The genus Serratia. Annu. Rev. Microbiol. 32: 221Ð248. Krueger, S. R., and D. W. Roberts. 1997. Soil treatment with entomopathic fungi for corn rootworm (Diabrotica spp.) larval control. Biol. Control Theor. Appl. Pest Manag. 9: 67Ð74. Martin, P.A.W., S. Mischke, and R.F.W. Schroder. 1998. Compatibility of photoactive dyes with insect biocontrol agents. Biocontrol Sci. Technol. 8: 501Ð508. Martin, P.A.W. 2004a. A freeze-dried diet to test pathogens of Colorado potato beetle. Biol. Control 29: 109 Ð114.
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Martin, P.A.W. 2004b. A stilbene optical brightener can enhance bacterial pathogenicity to gypsy moth (Lepidoptera: Lymantriidae) and Colorado potato beetle (Coleoptera: Chrysomelidae). Biocontrol Sci. Technol. 14: 275Ð383. Martin, P.A.W., M. Blackburn, and A. S. Shropshire. 2004. Two new bacterial pathogens of Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 97: 774 Ð 780. Matsuo, K., A. B. DeMilo, R.F.W. Schroder, and P.A.W. Martin. 1999. Rapid high performance liquid chromatography method to quantitate elaterinide in juice and reconstituted residues from a bitter mutant of Hawkesbury watermelon. J. Agric. Food Chem. 47: 2755Ð2759. McKinlay, R. G., A. M Spaull, and R. W. Staub. 1992. Pest of Solaneceous crops, pp. 263Ð326. In R. G. McKinlay [ed.], Vegetable crop pests. CRC, Boca Raton, FL. Panizzi, A. R., and E. Hirose. 1995. Survival, reproduction, and starvation resistance of adults southern green stink bug (Heteroptera: Pentatomidae) reared on sesame or soybean. Ann. Entomol. Soc. Am. 88: 661Ð 665. Pereira, R. M., and D. W. Roberts. 1991. Alginate and constarch mycelial formulations of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae. J. Econ. Entomol. 84: 1657Ð1661. Preisler, H. K., and J. L. Robertson. 1989. Analysis of timedose mortality data. J. Econ. Entomol. 82: 1534 Ð1542. SAS Institute. 2004. SAS OnlineDoc7, version 9.1. SAS Institute, Cary, NC. Schnepf, E. N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler and D. H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 775Ð 806.
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Schroder, R.F.W., A. B. DeMilo, C.-J. Lee, and P.A.W. Martin. 1998. Evaluation of a water-soluble bait for corn rootworm (Coleoptera: Chrysomelidae) control. J. Entomol. Sci. 33: 355Ð364. Schroder, R.F.W., P.A.W. Martin, and M. M. Athanas. 2001. Effect of a phloxine B-cucurbitacin bait on diabroticite beetles (Coleoptera: Chrysomelidae). J. Econ. Entomol. 94: 892Ð 897. Sˇ ebesta, K. J. Farkasˇ, K. Horska´ , and J. Van˜ ova´ . 1981. Thuringiensis, the beta exotoxin of Bacillus thuringiensis. pp. 249 Ð282. In H. D. Burges [ed.], Microbial control of pests and plant diseases 1970 Ð1980. Academic, London, United Kingdom. Sneath, P.H.A. 1984. Genus Chromobacterium Bergonzini, pp. 580 Ð582. In N. R. Krieg and J. G. Holt [eds.], BergeyÕs manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore, MD. Sosa-Gomez, D. R., and F. Moscardi. 1998. Laboratory and Þeld studies on the infection of stink bugs, Nezara viridula, Piezodorus guildinii, and Euschistus heros (Hemiptera: Pentatomidae) with Metarhizium anisopliae and Beauveria bassiana in Brazil. J. Invertebr. Pathol. 71: 115Ð120. Vazquez-Arista, M., R. H. Smith, V. Olalde-Portugal, R. E. Hinojosa, R. Hernandez-Delgadillo, and A. Blanco-Labra. 1997. Cellulolytic bacteria in the digestive system of Prostephanus truncatus (Coleoptera: Bostrichidae). J. Econ. Entomol. 90: 1371Ð1376. York, A. 1992. Pests of cucurbit crops: marrow, pumpkins, squash melon, and cucumber, pp. 144 Ð147. In R. G. McKinlay [ed.], Vegetable crop pests. CRC, Boca Raton, FL. Received 30 December 2004; accepted 19 January 2007.
