Abstract:
Compositions and methods are provided for attracting and subsequently trapping and/or killing crop pests from the Euschistus species. Alkyl esters of (2E,4Z)-decadienoic acid as a chemical attractant pheromone and, in combination with a trap or toxicant, provide an efficient means for controlling these insects.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to compositions and methods for attracting, trapping and/or killing crop pests from Euschistus species known as stink bugs. 
     1. Description of the Prior Art 
     The genus Euschistus is the largest of the genera in the so-called stink bug family Pentatomidae (order Hemiptera: suborder Heteroptera). Over 60 Euschistus species have been described, including 18 from North America. Several Euschistus species are among the most common members of the family in the western hemisphere and, to many people, these large brown insects are recognized as the stink bug. 
     Euschistus adults are strong fliers and often migrate to a succession of maturing crops where they feed on developing fruits or seeds. The immature bugs usually disperse from the initial point of attack, thereby spreading the infestation. As with other kinds of stink bugs, Euschistus adults and immatures suck sap or oil through hypodermic-like mouthparts, thus inconspicuously damaging the most valuable portion of the crop. Their feeding also indirectly reduces yield and seed quality by transmitting diseases. In the absence of food or with the onset of winter, adults are able to hibernate and resume attack the following season. 
     Various Euschistus species attack a wide range of fruit, field crops, vegetables, and nut crops. In the western United States the consperse stink bug, E. conspersus, is a major pest of tomatoes and also causes &#34;catfacing&#34; of apples, peaches, pears, grapes, and other fruits. During years of high E. conspersus populations, cotton and seed alfalfa yields in California have been seriously reduced. East of the Rocky Mountains three other Euschistus species are serious agricultural pests of field, vegetable, fruit, and nut crops: E. servus, the brown stink bug; E. variolarius, the onespotted stink bug; and E. tristigmus, the dusky stink bug. One or more of these Euschistus species frequently coexist with the green stink bugs, Acrosternum hilare and Nezara viridula, to form a complex that, along with the corn earworm, is the most damaging of the soybean pod feeders in the southern U.S. Although stink bug damage to soybean is less severe in more northerly regions of the U.S., the widespread use of reduced tillage crop systems has vastly increased the threat of E. servus and E. viriolarius to corn. This threat has arisen in fields under a soybean/corn or wheat/corn rotation because the brown and onespotted stink bugs overwinter in soybean and wheat stubble, then feed on emerging corn seedlings in the spring. A single stink bug can cause irrevocable damage to a corn plant in less than 24 hours. In Central and South America other species of Euschistus bugs are important pests. For example, E. heros is one of the three major stink bug pests of soybean in Brazil. 
     Outbreaks of Euschistus and other stink bugs are difficult to control because the bugs can migrate into a field and irreversibly damage a crop before they are noticed. Population build-ups that go undetected my lead to infestations of later maturing crops or outbreaks the following season. Moreover, several stink bug species usually coexist at varying abundances from region to region and year to year, further complicating monitoring and control strategies. 
     Although the use of insecticides has increased the quality and quantity of crop yields, there are serious disadvantages to the use of presently available insecticides. The toxicity to humans and animals is high. Additionally, they are not readily degraded and may thus pollute the environment. Therefore, reducing or eliminating the amount of toxic insecticides used in the management of insect pests is desirable. It is an object of the present invention to provide compositions and methods by which the amounts of pesticide may be reduced or eliminated in eradicating Euschistus species stink bugs. 
     Attractant pheromones have recently been identified for several species of predatory and plant-feeding stink bugs. F. Warnaar [Phytochemistry, Vol. 20, (1981), pp. 89-91] disclosed the mass spectra (MS) of methyl (2E,4Z)-decadienoate and methyl (2E,4E)-decadienoate, both of which closely resemble the MS of the E. conspersus male-specific compound. 
     Additionally, there has been reported a strong synergism between methyl E,Z-2,4-decadienoate and chalcogran at attracting the bark beetle, Pityogenes chalcographus [Byers, et al., Naturwissenschaften, Vol. 75, (1988), pp. 153-155]. The reference does not indicate a utility for the attraction and/or management of insects of the Euschistus species. 
     SUMMARY OF THE INVENTION 
     A chemical attractant pheromone for Euschistus species as well as methods for concentrating and destroying these pests is provided. In one alternative embodiment the attractant could be used to monitor area levels of these pests. In another embodiment the attractant would be useful for concentrating these pests in early maturing &#34;trap-crops&#34; where they could be economically destroyed by limited insecticide application. Analogously, this chemical attractant could be applied to artificially create overwintering aggregations of adults that would then be destroyed, thus preventing spring outbreaks. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows the peak and elution time of the attractant pheromone obtained from aeration experiments. 
     FIG. 2 is the mass spectrum and chemical structure of methyl (2E,4Z) -decadienoate. 
     FIG. 3 shows the structure of some minor male-specific volatile components from Euschistus spp. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLE 1 
     Isolation of Pheromones. 
     Euschistus conspersus adults were collected near Davis, California and reared in the laboratory at the UC-Davis Entomology Department on sunflower seeds, green beans, and water. Volatiles were trapped from groups of 10-30 virgin adult males or females by coaxing the bugs into a ca. 1 liter glass column and drawing air by vacuum (100 ml/min) over the bugs through 30 mg of activated charcoal for 24 hr. Trapped volatiles were washed from the filter in 200-300 μl of CH 2  Cl 2  and stored under nitrogen at -20° C. 
     In four months, four species of Euschistus stink bugs were caught in traps deployed at the Agricultural Research Center, Beltsville, Md.: E. servus, E. tristigmus, E. variolarius, and E. politus. Field-collected adults and/or laboratory-reared adults of E. servus, E. tristigmus, and E. politus were used for aeration experiments as for E. conspersus. In addition, specimens of E. obscurus and E. ictericus were obtained from Florida and aerated for pheromone isolation. 
     Samples were analyzed by gas chromatography (GC) on a bonded methyl silicone column (0.25 μm film, 14-mm ID; DB-1™, J&amp;W Scientific, Folsom, Calif.) using a Varian 3700 GC with helium as carrier (40 cm/sec), and a temperature program from 45° C. for 2 min to 230° C. at 15°/min. When the odor of the stink gland secretion was detected at the start of a male or female aeration, or when one or mere adults died during the aeration, usually only the stink gland secretion components were detected by GC. However, in uncontaminated aeration samples of mature male Euschistus species a single major component eluting at 7.4 min was observed (FIG. 1). This component was never present in samples from female Euschistus species. Other minor components (&lt;5%) were detected in some male Euschistus samples. 
     EXAMPLE 2 
     Identification of Pheromone Components. 
     Mass spectra (MS) were obtained in the electron impact mode at 70 eV using a Finnigan 4510 GC-MS. The MS of the major male-specific compound detected in E. conspersus aeration samples gave a prominent molecular ion a m/z 182 (ca. 20%), but a matching spectrum was not found in the GC-MS computer library of spectra (&gt;31,000 MS) (FIG. 2). The presence of an M-31 peak at m/z 151 suggested a methyl ester structure for this component, as did ions at m/z 59 ([O═C--O--CH 3  ] + ) and m/z 74 ([CH 2  ═C(OH)--OCH 3  ] + ). Thus, the molecular formula was deduced to be C 11  H 18  O 2  and a di-unsaturated, unbranched acidic moiety was thought most likely. A 20 g sample of the related compound, ethyl (2E,4Z)-decadienoate (called the pear ester), was obtained from International Flavors &amp; Fragrances Inc. (IFF, Union Beach, N.J.). The pear ester standard was saponified and re-esterified with methanol by normal procedures to give methyl (2E,4Z)-decadienoate and a small percentage of the corresponding (2Z,4E)- and (2E,4E)-isomers. This standard of methyl (2E,4Z)-decadienoate gave an identical MS to that of the E. conspersus male-specific compound and the standard coeluted with the unknown by GC. 
     Certain minor components were identified from Euschistus male aeration samples (FIG. 3). Methyl (2E,4E)-decadienoate (2-7%) was present in extracts from all species examined except E. tristigmus. Methyl (2Z,4E)-decadienoate (0.2%) was also detected in an extract of E. politus. Decanoic acid (ca. 10%) was detected in a sample from E. tristigmus males. Geranyl acetone (6,10-dimethyl-5E,9-undecadien-2-one) (ca. 5%) occurred in extracts from E. conspersus, E. servus, and E. tristigmus. 
     EXAMPLE3 
     Behavioral Testing. 
     Two field tests for E. conspersus attraction were conducted in Yolo County, California, in the following manner. Traps consisted of one gallon transparent plastic cylindrical containers with wire mesh funnels inserted in opposite sides of the container. A rubber septum (Thomas Scientific) was used as a receptacle for the pheromone. Each septum was washed with CH 2  Cl 2 , clipped inside a piece of PVC pipe (2.5 cm diameter×3.0 cm length), and suspended 7.0 cm from the top of the trap. Traps were baited by applying either 1 μl or 10 μl of methyl (2E,4Z)-decadienoate in the septum, followed by 100 μl of CH 2  Cl 2  ; control traps lacked the ester. The first field tests used material prepared from the pear ester obtained from IFF Inc. as described above consisting of 88% methyl (2E,4Z)-decadienoate, 9% of the corresponding (2Z,4E)-isomer, and 3% of the (2E,4E)-isomer. The second field tests used material purchased from Bedoukian Research Inc. (Danbury, Conn.) determined by GC and GC-MS to contain 92.1% methyl (2E,4Z)-decadienoate, 1.4% of the (2Z,4E)-isomer, and 6.5% of the (2E,4E)-isomer. Traps were placed on the ground ca. 20 m apart in a randomized block design, checked and rotated daily, and rebaited at 3-5 day intervals. 
     Field tests for the attraction of Euschistus species native to the eastern U.S. were conducted at the Beltsville Agricultural Research Center, Prince George&#39;s County, Md., as follows. Transparent plastic traps were constructed as for the California field tests except that the rubber septum was fastened in the bottom of the trap and was unprotected by PVC pipe. One test also used traps constructed of 15×25 cm transparent plastic sheets coated on each side with Tack Trap™. A rubber septum was fastened in a 4 cm diameter hole in the center of the sheet. The bottom and sides of the trap had a 1.5 cm perpendicular rim that prevented insects from falling off the trap. Traps were hung from tree branches at least 20 m apart along powerline clearings through forest and around the borders of grassland, or from stakes at least 10 m apart in a weedy field. The following lures were tested: 1) methyl (2E,4Z)-decadienoate (Bedoukian Research Inc.), 2) ethyl (2E,4Z)-decadienoate (IFF Inc.), 3) 60% methyl (2E, 4Z)-decadienoate/40% decanoic acid (Aldrich Chemical), and 4) 98.5% methyl (2E,4Z) -decadienoate/1.5% geranylacetone (Bedoukian Research Inc.). One part of each of these lures was blended with 2  parts of heptane. Traps were monitored daily and rebaited at 2-3 day intervals with 10 μl of a solution; control traps were unbaited. 
     Euschistus conspersus females and males were significantly attracted to the 10 μl dosage of methyl (2E,4Z)-decadienoate in the California field tests (Tables 1 and 2). Small nymphs of E. conspersus were also significantly attracted to this ester, and many more medium-and large-sized nymphs were caught in baited traps than control traps but not at a significant level because of high variability between traps (Table 2). 
     In a later field test conducted in and around forest at the Agricultural Research Center, Beltsville, Md., 201 E. tristigmus, 67 E. politus, 8 E. servus, and 76 tachinid fly parasites of Euschistus bugs were attracted to traps baited with methyl (2E,4Z)-decadienoate; whereas, only one female E. tristigmus and five tachinids were attracted to unbaited controls (Tables 3 and 4). Euschistus tristigmus adults and tachinid parasites were significantly more attracted to methyl (2E,4Z) -decadienoate than ethyl (2E,4Z)-decadienoate (Chi 2  =9.81 and 22.62, respectively; Table 3). The inclusion of decanoic acid with methyl (2E,4Z)-decadienoate did not significantly improve attraction of any Euschistus species or parasites (Table 4). Similarly, inclusion of geranyl acetone with methyl (2E,4Z)-decadienoate did not improve attraction (Table 5), although small samples may have precluded detection of this effect. 
     There has been provided in accordance with the present invention, compositions and methods for controlling insects of the Euschistus species that do not suffer the disadvantages described by the prior art. It can be seen from the specification and specific embodiments therein that there are many alternatives and variations apparent to those skilled in the art. It is understood that all such alternatives and variations are included within the spirit and scope of the invention. 
     
                                           TABLE 1__________________________________________________________________________Chemical Attraction of Euschistus conspersus, Yolo County, CA.           Number Trapped           Test I*        Test II**Treatment       nymph               male                  female                      total                          nymph                              male                                 female                                     total__________________________________________________________________________blank           0   0  0   0a  0   0  1   1a1 μl methyl 2E,4Z-decadienoate           1   5  6   12b 3   0  1   4a10 μl methyl 2E,4Z-decadienoate           0   5  2   7b  2   6  14  22b__________________________________________________________________________ *Test I: 4 replications per treatment in weedy dwarf peach orchard. **Test II: 5 replications per treatment in sugar beet field.  Data subjected to ANOVA after log (x + 1) transformation. Total means in a column not followed by a common letter are significantly different at the 5% level by Duncan&#39;s Multiple Range Test. 
    
     
                       TABLE 2______________________________________Chemical attraction of Euschistus conspersus;, Yolo County.*Mean Catch/Trap**Nymphs              AdultsTreat-ment   small   medium   large male  female Total______________________________________blank  2.6a    2.4a     1.6a  0.8a  0.4a   7.8a10 μlmethyl2E,4Z-deca-  16.8b   10.4a    11.4a 7.8b  9.0b   55.4bdienoate______________________________________ *Five replications per treatment in a weedy field or blackberry patch. **Data subjected to ANOVA after log (x + 1) transformation. Means in a column not followed by a common letter are significantly different at the 5% level by Duncan&#39;s Multiple Range Test.  Small nymphs = 2nd instars, medium nymphs = 3-4th instars, large nymphs 4-5th instars. 
    
     
                       TABLE 3______________________________________Chemical attraction of Euschistusspecies and parasitic tachinids,Prince George&#39;s County, Maryland.*     Treatment [No. Attracted]**       methyl 2E,4Z)-                    ethyl 2E,4Z-Insect      decadienoate decadienoate                               Blank______________________________________E. tristigmusmale        50           24         0female      22           15         1E. politusmale        17           13         0female      30           18         0E. servusmale         0            0         0female       3            0         0Tachinid Parasites       60           18         5______________________________________ *Six stickytrap replications and 6 transparent cylindricaltrap replications per treatment deployed in and around deciduous forest. **Number attracted includes insects within 1 m of a trap.  The following tachinid species were captured: Gymnosoma par, Euthera tentatrix, Gymoclytia occidua, and Euclytia flava. 
    
     
                       TABLE 4______________________________________Chemical attraction of Euschistusspecies and parasitic tachinids,Prince George&#39;s County, Maryland.*     Treatment [No. Attracted]**                   methyl 2E,4Z-       methyl 2E,4Z-                   decadienoate +Insect      decadienoate                   decanoic acid                                Blank______________________________________E. tristigmusmale        54          52           0female      71          74           0nymph       4           13           0E. politusmale        6           10           0female      9           15           0nymph       5            3           0E. servusmale        1            5           0female      2            4           0nymph       1            2           0Tachinid Parasites       16          10           0______________________________________ *Six transparent cylindricaltrap replications per treatment deployed in same location as test of Table 3. **Number attracted includes within 1 m of a trap.  The following tachinid species were captured: Gymnosoma par, Euthera tentatrix, and Euclytia flava. 
    
     
                       TABLE 5______________________________________Chemical attraction of Euschistus species andparasitic tachinids, Prince George&#39;s County, Maryland.*         Treatment [No. Attracted]**                        methyl 2E,4Z-           methyl 2E,4Z-                        decadienoate +Insect          decadienoate geranylacetone______________________________________E. servusmale            9            6female          4            6E. variolariusmale            1            0female          0            0E. servus/variolarius nymphs           0            3Tachinid Parasites           12           9______________________________________ *Three replicates per treatment deployed in weedy field. **Number attracted includes insects within 1 m of a trap.  Nymphs of E. servos and E. variolarius could not be distinguished.    The following tachinid species were captured: Gymnosoma fuliginosum, Euthera tentatrix, and Gymnoclytia occidua.