CN1115408C - Liquid culture method for pathogenic nematode of insect - Google Patents

Abstract

The invention provides a method for liquid culture of entomopathogenic nematodes used as biological insecticides. The method is to add antibacterial substances at the critical age of nematode development in the liquid single-bacteria culture system of entomopathogenic nematodes to change the cell density and induce symbiotic bacteria to produce mutants, control the formation of nematodes in the infection period, and increase the yield of nematodes in the infection period. The method can make the nematodes reach the worm state of the infection period with a proportion as high as 95% within 8 to 16 days.

Description

A liquid culture method for entomopathogenic nematodes

technical field

The present invention relates to a liquid culture method for the mass production of entomopathogenic nematodes as biopesticides.

Background technique

The entomopathogen Steinernematidae and Heterorhabditidae nematodes are new biological insecticides. This kind of nematode has a wide range of hosts; it has the ability to actively search for hosts, especially for soil-dwelling and borer pests; it is safe for humans, animals and the environment. In recent years, it has been widely used in the prevention and control of pests in agriculture, forestry, pasture and sanitation, and has been highly valued by domestic and foreign scholars and commercial departments, and is moving towards commercialization.

This kind of nematode enters the body of insects in the infectious juvenile state (infective juveniles, IJ) along with the host food or from the insect's natural openings (such as anus, stomata) and intersegmental membranes, and then releases the Xenorhabdus genus carried in the intestinal cavity (currently with symbiotic bacteria of the genus Photorhabdus (currently in symbiosis with Heterobacteriaceae nematodes). Toxins (virulence factors) secreted by nematodes as well as symbiotic bacteria cause insect death.

Large-scale field application of entomopathogenic nematodes requires commercialization of nematode production. At present, the industrialized culture system of nematodes is completed by adding single symbiotic bacteria and sterile nematodes to various artificial culture media through aseptic technique, that is, the single-bacteria in vitro culture system of nematodes. According to the culture medium, it can be divided into solid culture [Bedding, R.A. (1984) Annals of Applied Biology 104, 117-120; Wouts, W.M. (1981) Journal of Nematology 13, 467-469] and liquid culture [Pace et al. (1986 ) PCT Patent Application No.86/01074; Friedman et al. (1991) PCT Patent Application No.89/04602; Surrey & Davies (1996) Journal of Invertebrate Pathology 67, 92-99; Han, R.C. (1996) Nematologica 42, 546-553 ; Ehlers et al. (1998) Biocontrol 43:77-86; Tac hibana et al. (1997) US patent, Application No. 403879]. The solid culture method has relatively low technical content, is easy to operate, and can be adapted to a variety of nematodes. But it needs a lot of manpower, space, and a lot of waste. Liquid culture overcomes the shortcomings of solid culture and is the main method for industrial production of nematodes.

The life cycle of entomopathogenic nematodes includes egg, first to fourth instar larvae and adult stages. These nematodes all have a third instar infection stage. The nematodes at this age do not feed, can survive in the soil for a long time, and can invade possible host insects at any time. In the single-bacteria in vitro culture system of nematodes, the typical developmental pattern of nematodes is as follows. When the nematodes and symbiotic bacteria are inserted into the culture medium, the food signal produced by the symbiotic bacteria induces the development of nematodes in the infection stage, from the infection stage to the third instar (L3), and the fourth instar (L4) to adult worms. After the eggs hatch, from the first instar larvae (L1) to the second instar larvae (L2), if the nutritional conditions are good, they will continue to develop to L3, L4 and a new generation of adults; if the nutritional conditions are deteriorated, L2 can develop into infective stage nematodes. The new generation of infectious nematodes can continue to develop into L3 and carry out a new life cycle. Steiner's nematode adopts dioecious reproduction; while the first generation of adults developed from infected larvae is strictly hermaphrodite, and the second generation of adult worms may have mixed individuals of dioecious and hermaphroditic. The infective stage larvae of entomopathogenic nematodes are the stages capable of infecting insect hosts. In terms of application, only nematodes in the infection stage have a certain tolerance to environmental factors and are infectious to insects. Therefore, when nematodes are cultured in vitro, the proportion of nematodes in the infection stage to other instars and the culture period are very important.

In general, adding high densities of nematodes to a nutrient-rich medium will facilitate obtaining the greatest number of infective nematodes in the shortest time. However, adding high-density nematodes will reduce the reproduction ratio of nematodes and is not economically viable. If the nematodes can reach the maximum number of infectious stage worms in the first generation, it will shorten the culture time and reduce the culture cost. Pace et al. (1986) (PCT Patent Application No.86/01074) reported that by diluting the culture solution with sterile distilled water and lowering the temperature of the culture solution to 15°C, 60-90% of the nematodes in the infection period could be obtained. But when this method is operated, it is both inconvenient and increases expenses. Friedman et al. (1989) Tachibana et al. (1997) patents describe the culture medium of nematodes and the method of controlling the stirring rate according to the different stages of nematodes, but do not mention the method of controlling the formation of nematodes during the infection period.

Contents of the invention

The object of the present invention is to propose a liquid culture method for entomopathogenic nematodes capable of controlling and increasing the output of nematodes in the infection period.

The method for liquid culture of entomopathogenic nematodes provided by the present invention comprises: in a commonly used artificial bacterial culture medium, insert the symbiotic bacteria and nematodes of the nascent nematodes under aseptic conditions; , For example, nematode liquid culture is carried out in shaker shaking culture or oxygen fermenter, which is characterized by the developmental period of nematodes, mainly adding antibacterial substances (ie antibiotics) during the key developmental stages of nematodes. Antibiotics used include all species that have inhibitory effects on nematode symbiotic strains, such as Chlorotetracycline, Streptomycin, Chloramphenicol, Ampicillin, Nalidixic acid, etc., preferred It's streptomycin. The dose of antibiotics ranges from 0.01-1 μg/ml, which varies according to different commensal strains and types of antibiotics. Antibiotics in this dose range can cause some strains (<20% CFU; CFU=colony forming unit) to undergo phenotypic changes. The critical developmental stage of the nematode is generally selected after the emergence of the first instar nematode L1 of the new generation.

The nematodes inserted in the commonly used artificial bacterial culture medium in the method of the present invention can be nematodes of various ages, and the nematodes in the infection stage are the best.

The present invention uses antibacterial substances to reduce the bacterial density and induce the mutant type of symbiotic bacteria in the culture medium at the developmental stage of the nematode liquid culture, reduces the nutritional information of the bacterium liquid, changes the physiological state of some symbiotic bacteria, and controls and improves infection. The formation of stage nematodes makes the nematode larvae develop in the direction of infecting stage nematodes. In addition, it also controls the further development of already formed infecting stage nematodes. The invention can greatly increase the proportion of nematodes in the infection stage in the culture liquid, shorten the culture time, and improve the liquid culture effect of the entomopathogenic nematodes. The method can make the nematodes reach a high proportion of infection stage worms (>95%) within 8-16 days (varies according to the nematode species).

specific implementation plan

The following experiments and operational examples are further illustrations of the present invention. It should be noted that these examples are illustrative and should not be taken as limiting the invention. Only S.carpocapsae A24 nematode and H.bacteriophora H06 nematode are described in the examples, but the method of this patent is applicable to all nematode species and strains.

Embodiment one

Add 100ml liquid culture medium (2% soybean powder, 1% flour, 0.5% yeast extract, 1.2% egg powder, 3% corn oil and 92.3% water) in 500ml Erlenmeyer flask, autoclave 30 minutes under 121 ℃. Then insert the X.nematophilus primary symbiotic bacteria (5×10 ⁸ cells) isolated from the S.carpocapsae A24 nematode, place it on a shaking table at 25°C and 150rpm for 48 hours, and then insert 5000 bacteria/ml S. carpocapsae infection stage nematodes, the next day after the nematodes were inoculated, samples were taken to check the development of the nematodes under a dissecting microscope, and 0.05 μg/ml streptomycin sulfate was added to the culture bottle after 4 days of culture. After 48 hours, samples were taken to check the development of nematodes under an anatomical microscope, and the nematode output and the proportion of nematodes in the infection period were counted on the 8th, 10th, 12th and 16th days after inoculation.

Embodiment two

Add 100ml liquid culture medium (2% soybean powder, 1% flour, 0.5% yeast extract, 1.2% egg powder, 3% corn oil and 92.3% water) in 500ml Erlenmeyer flask, autoclave 30 minutes under 121 ℃. Then insert P.luminescens primary symbiotic bacteria (5×10 ⁸ bacterium) isolated from H.bacteriophora nematodes, place them on a shaking table at 25°C and 150 rpm for 48 hours, and then insert 5000/ml H.bacteriophora For nematodes in the infection stage, samples were taken from the second day after the nematodes were inoculated to check the development of the nematodes under a dissecting microscope. After 6 days of culture, 0.05 μg/ml streptomycin sulfate was added to the culture bottle. After 48 hours, samples were taken to check the development of nematodes under an anatomical microscope, and the nematode output and the proportion of nematodes in the infection period were counted on the 8th, 10th, 12th and 16th days after inoculation.

The method for the separation of Xenorhabdus and Photorhabdus primary symbiotic bacteria described in the above-mentioned embodiment one and embodiment two is as follows: from the last instar (seventh instar) larvae of Galleria mellonella infected by entomopathogenic nematodes, place 25 DEG C, insect After death (generally 2 to 4 days), sterilize the dead worm with 70% alcohol, then cut the worm body with sterile scissors, take hemolymph and draw NBTA, NA and MacConkey plates. Nematode symbiotic bacteria were identified according to the method described by Akhurst (1980) (Journal of General Microbiology 121, 303-309). The X.nematophilus symbiont was isolated from S.carpocapsae A24 nematode; the P.luminescens symbiont was isolated from H.bacteriophora nematode.

The entomopathogenic nematode liquid culture method described in embodiment one and embodiment two and conventional method (that is not adding antibacterial substance) comparative structure shows: after adding streptomycin sulfate, S.carpocapsae nematode infection stage nematode ratio in 8 days Reach 95%, the nematode output of infection stage is 267 * 1000/ml; And the nematode ratio of infection stage of the culture bottle that does not add streptomycin sulfate is 6%, the nematode output of infection stage is 18 * 1000/ml, although the total output of nematode is 304×1000/ml. After 12 days, the proportion of infective nematodes in the culture bottle added with streptomycin sulfate reached 97%, while the proportion of infective nematodes in the culture bottle without streptomycin sulfate was 32%. It can be seen that after the addition of streptomycin sulfate, the nematodes developed to the stage of infection and the culture time was shortened.

After streptomycin sulfate was added to the H.bacteriophora nematode culture solution, the proportion of nematodes in the infection period reached 96% in 10 days, and the output of nematodes in the infection period was 197×1000/ml; while the infection period of the culture bottle without streptomycin sulfate The proportion of nematodes was 84%, and the output of nematodes during the infection period was 178×1000/ml. After 16 days, the proportion of infective nematodes in the culture bottle added with streptomycin sulfate reached 98%, while the proportion of infective nematodes in the culture bottle without streptomycin sulfate was 89%. It can be seen that after adding streptomycin sulfate, the nematodes also developed to the stage of infection, and the culture time was shortened.

Claims (4)

1. A liquid culture method for entomopathogenic nematodes, comprising in an artificial culture medium, inserting primary nematode symbiotic bacteria and nematodes under aseptic conditions, and carrying out nematode liquid culture under aeration and stirring culture environment, characterized in that Streptomycin sulfate was added as an antibacterial substance during the developmental stage of nematodes. 2. The method for liquid culture of entomopathogenic nematodes according to claim 1, characterized in that the time for adding the antibacterial substances is the critical age of development after the appearance of the first-instar larvae L1 of the new generation of nematodes. 3. The method for liquid culture of entomopathogenic nematodes according to claim 1, characterized in that the amount of the antibacterial substance added is 0.01-1 μg per milliliter of culture medium. 4. The liquid culture method of entomopathogenic nematodes according to claim 1, characterized in that the nematodes inserted in the artificial culture medium during the culture are infective stage nematodes.