CN1180375A - Heterorhabditis bacteriophora insecticide compositions and related processes - Google Patents

Abstract

The present invention provides an insecticide composition comprising an effective dose of an entomopathogenic Heterorhabditisbacteriophora hybrid strain Hb (NJ) capable of killing insects or a mutant or variant thereof in a juvenile infective stage, and an organism therefor. Compatible carrier. The pesticidal composition provides improved pesticidal activity compared to known pesticidal compositions comprising the known H. bateriophora nematode HBHP88. The invention also discloses a method for reducing the population of soil habitual insects in the soil, which is carried out by applying the insecticidal composition to the soil infected with the insects. In addition, the Hb(NJ) hybrid nematode strain is also provided.

Description

Heterorhabditis bacteriophora insecticide compositions and related methods

Field of the invention

The present invention relates to novel insecticide compositions containing heterorhabditid hybrid nematodes with enhanced insecticidal effect; novel nematodes having this characteristic; The pesticidal composition is used to reduce the population of soil habitual pests.

Background of the invention

Insecticide compositions containing biopesticides or entomopathogenic nematode ingredients open up an interesting field of biocontrol agents against soil-inhabiting pests. They have a number of important advantages over commercially available chemical insecticides. The advantages of nematode insecticides include high toxicity, broad host range, safety, government registration exemption and ease of mass production.

Nematodes of the Heteroblastoid family form a unique symbiotic association with the Photorhabdus bacterium. They are pathogenic to many groups of insects. The heterotypic rods are hermaphroditic. Typically, infectious larvae penetrate the hemocoel of their host insects, such as white grubs, usually through the gut or stigma. The shaped rods have a dorsal tooth to facilitate penetration into other areas (eg, intersegmental membranes). Bacteria from the nematode's digestive system are released in the host, and the host typically dies of sepsis within 48 hours. The nematode feeds, develops, matures, and reproduces within the host's cadaver, and typically after many generations, in turn produces infective larvae, emerges, and seeks out new host insects.

It would be desirable to provide insecticidal compositions containing those insecticidal nematodes having improved effects against soil-inhabiting pests; new nematodes having such improved effects; methods; and processes and equipment for producing the improved insecticide nematodes.

Summary of the invention

Provided by the present invention are novel insecticide compositions and the control of soil-inhabited pests by applying to existing infestations a new insecticide composition comprising an effective dose of the entomopathogenic nematodes of the present invention in the infestation stage. Improved methods for insects. The insecticidal composition of the present invention comprises an effective dose of an insecticidal entomopathogen of the juvenile infective stage of Heterorhabditis bacteriophora hybrid strain HB (NJ) and effective variants thereof, mutants or hybrids. Insecticidal entomopathogenic nematodes and bioadaptive vectors for the juvenile infective stage of the insecticide properties of sexual nematodes and living hybrids therefor. Also provided by the present invention are novel entomopathogenic nematodes in the pesticidal compositions of the present invention that are highly effective in controlling soil-dwelling insects, including increased pathogenicity and enhanced reproduction in a variety of insect hosts force. Detailed Description of the Invention

The Heterorhabditis bacteriophora HB(NJ) hybrid was obtained by crossing a large series of wild-type isolates of H. bacteriophora from soil samples collected from four different habitats. The habitat was grassland, grassland with scattered trees, weedy field, and weedy field with scattered trees, and the habitat represented a number of collection points.

Enhanced properties of the hybrid include enhanced pathogenicity or infectivity to a variety of insect hosts and increased fecundity.

Infectivity was determined by the assay described below. The infectivity standardized test used was to place 50 infective larvae on top of a sand column (5 cm diameter x 5 cm height) with a target insect (wax moth, Galleria mellonella) at the bottom. The test is generally carried out at 25°C and the sand contains 10% by weight of moisture. The nematodes were allowed 24 hours to find the target insect, at which point the insects were removed, rinsed in distilled water and incubated at 25°C for an additional 24 hours to allow the nematodes time to develop in the host. Then, the host was dissected and the number of those nematodes that successfully colonized the host were counted. In two groups of experiments repeated 20 times in each group, the average infection rate of HB(NJ) was 10.45 and 11.1 nematodes per host (standard deviation (SE) were 1.053 and 1.095, respectively), all hosts were infected and in died during the experiment. In comparable tests, this HB(NJ) strain performed much better than the two known H. bacteriophora strains (HBNC and HBHP88). They killed only 50% and 70% of the hosts, respectively, and the average infection rates were only 0.08 and 1.20 nematodes per host, respectively. The infection rate of this HB(NJ) strain to other insects also showed its similar advantages.

Fertility is determined by the following test. The standard fecundity assay quantifies those newly infectious from single hosts (Galleria mellonella) of standard weight (0.24-0.26 g) that have been routinely inoculated (n=20) with infectious larvae at 25°C. number of larvae. For 30 replicate experiments with HB (NJ), an average fecundity level of 383,046.7 (standard deviation (SE) of 15212.9) was obtained. It shows that the fecundity of HB(NJ) is superior.

The Heterorhabditis bacteriophora hybrid strain HB (NJ) provided by the present invention has the preservation number ATCC_.

The insecticide composition may be in the form of an aqueous spray composition; in the form of calcium alginate or in the form thereof.

Carriers used to immobilize the nematodes or to partially dry them can slow their metabolism and improve their tolerance to extreme temperatures. Nematodes are commercially available on moist substrates such as foam, vermiculite, and peat, although persistent storage at room temperature is generally less effective due to the rapid decline in nematode viability. The Heterorhabditis composition of the present invention is suitable for storage at 10°C to 15°C and the organism should be kept in an aerobic environment.

Nematodes can be applied using common agricultural chemical equipment including small pressure sprayers, mist blowers, electrostatic sprayers, fan sprayers, and aerial sprayers. Entomopathogenic nematodes can withstand a pressure of 300 ^psi and can be sprayed with all conventional orifice-type sprayers (such as "01" nozzles) with opening diameters as small as 50 microns. However, some types of extrusion equipment generate a lot of heat, which has a negative effect on the nematode once the temperature in the nebulizer line rises above 32°C. Luckily it happens very rarely and is usually only a problem when the sprayer tank is almost empty and there is little heat sinking water.

Field concentrations in excess of 2.5 billion nematodes per hectare (1 billion per acre) are commonly used to ensure that a sufficient population of nematodes contacts the target insects for control. High concentrations are required to overcome the negative effects of both animate and animate soil environments. High nematode concentrations are also needed against certain insects that stay in the soil for only a few days before burrowing into roots, such as cabbage maggots (D. radicum), and against insects that are less susceptible to nematode infestation due to their small size and mobility. For example, high concentrations of nematodes are required against early immature stages of mole crickets (Scapteriscus spp.), root maggots (Delia spp.) and corn root worms (Diabroticas pp.).

An extensive discussion of formulations and applications suitable for providing the pesticidal compositions of the present invention, as well as processes related to the present invention, can be found in the collection of papers edited by Dr. Randy Gaugler and Dr. Harry K. kaya, "Use of Entomopathogenic Nematodes in Biocontrol ", Chapter 9, "Ramon Georgis Formulation and Application Techniques", CRC Press (Boca Raton, Ann Arbor, Boston), 1990.

The Galleria mellonella recognition test used to evaluate the insecticidal activity of nematodes was carried out as follows. Whatman No. 1 filter paper was placed in a 35mm petri dish. Then put the Galleria mellonella host on the filter paper. 100 juvenile nematodes were suspended in 500 microliters of water. The aqueous suspension was placed on the paper by wetting the filter paper in several places. If the Galleria mellonella turned red at the end of the experiment, it indicated that the Galleria mellonella host was dead.

In the Galleria mellonella bait method used to isolate nematodes, Galleria mellonella infected with Heterorhabditisbacteriophora usually dies and turns red. The soil samples taken of the bacteriophora Galleria mellonella usually die and turn red. The soil samples taken were 2 cm in diameter and 15 cm in length. To provide the Heterorhabditis bacteriophora hybrid strain HB(NJ) of the present invention, about 150 Heterorhabditis bacteriophora isolates were used.

The cross-breeding method for providing the HB(NJ) strain involves conventional steps.

Variations and modifications to the present invention are provided which will be obvious to those skilled in the art and are within the scope of the invention which are within the spirit of the invention.

Claims (6)

1. A pesticidal composition for reducing the population of one or more soil-dwelling insects, comprising an effective dose of a Heterorhabditis bacteriophora hybrid strain Hb (NJ) or an effective variant or mutant thereof Entomopathogenic nematodes that kill insects during the larval infectivity phase of insect characteristics and bioadaptable vectors therefor. 2. The composition of claim 1, wherein the nematode is Heterorhabditis bacteriophora hybrid strain Hb (NJ). 3. A method for reducing soil habitual white grubs or other insect populations, comprising applying an effective dose of Heterorhabditis bacteriophora hybrid strain Hb (NJ) or its variant or mutant at the soil infested by the insects Insecticidal properties of an insecticidal composition against an entomopathogenic nematode in the infective stage. 4. The method of claim 3, wherein the nematode is an infectious stage Heterorhabditisbacteriophora hybrid strain Hb(NJ) nematode. 5. A nematode of Heterorhabditis bacteriophora hybrid bacterial strain HB (NJ), which can reduce the population of one or more soil-dwelling insects when it is applied to the soil where the insects have been infested during the juvenile infection period. 6. The nematode of a juvenile infective stage according to claim 5.