JP3512824B2 - Culture method of insect parasitic nematodes - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to the fields of agriculture, forestry,
The present invention relates to a method for culturing an insect parasitic nematode which is a biological control agent for harmful insects in the field of hygiene and the like. More specifically, it relates to a culture method for producing a nematode that maintains a strong insecticidal property in artificial culture of solid insects and liquid cultures of insect parasitic nematodes belonging to the genera Steinanema and Heterorhabditis.

[0002]

BACKGROUND ART Nematodes belong to the phylum Nematoda and are divided into many eyes and families. Some of them live freely, but some are parasitic on plants and animals. Those nematodes that parasitize insects are called "insect parasitic" or "insect pathogenic" nematodes. A nematode of commercial interest for the control of harmful insects is the Rabditideida, and the nematodes belonging to it belong to many insects. Of these, the ones that are drawing attention from the viewpoints of efficacy and commerce are insect parasitic nematodes belonging to the genus Steinanema and the genus Heterorhabditis.

[0003] As a general review of nematode classification and insect parasitic nematodes, Poiner's "Natural history of nematodes" [Prentice-Hall, Inc., NJ 323 pp.] And "For biological control of insects" Nematode "[(1979) CRC Press, FL.167pp.]
Will be helpful. Nematodes generally have a life cycle consisting of five stages. Each stage progresses through the growth process of forming new cuticles and molting old cuticles. The fifth stage lays eggs in the adult stage. First stage larvae emerge from the egg. And in the right environment for the second period,
It grows into 3rd and 4th stage larvae and becomes an adult. However, in insect parasitic nematodes, if the environment becomes unsuitable for growth, 2
Stage larvae grow into infectious stage 3 larvae. This infectious third stage larva is in a state where the cuticle of the second stage larva remains.
This is called the sheath. Infected 3rd stage larvae are infectious and grow into 4th stage larvae and adults only in an insect host. Only this infectious stage 3 larva is used for biological control.

Infection state of insect parasitic nematodes belonging to the genus Steinernema and the genus Heterorhabditis larvae move into the blood vessels (blood chambers) of insects when they enter the body of the insects. At this point, the nematode becomes ready to feed and releases the symbiotic bacteria present in the intestine of the nematode from the anus. This symbiotic bacterium is unique to the insect parasitic nematodes belonging to the genus Steinanema and the genus Heterorhabditis and has been identified as a genus Xenorhabdus. When the fungus grows in the insect body, the insect causes sepsis and dies. Therefore, it is considered that the nematode's insecticidal nature lies in this symbiotic bacterium.

Nematodes and this commensal bacterium have many interrelationships. This bacterium obviously cannot survive in soil or water. And, when insects directly ingest this fungus, it shows no pathogenicity. The nematode has a role of protecting the symbiotic bacteria outside the host insect and a role of migrating the symbiotic bacteria from the corpse to a new host. The role of bacteria is to supply nutrients for nematodes by their degrading power. The nematodes feed on the symbiotic bacteria together with nutrients during the growth process, and when the infectious stage 3 larvae appear, the symbiotic bacteria are retained again in these intestines. Aseptic nematodes cannot grow in aseptic insects and require the activity of bacteria to produce the proper nutritional conditions.

From the taxonomic study, regarding the specificity of nematode species and symbiotic bacterial species, "various kinds of insect parasitic nematodes have a symbiotic relationship with only one Xenorhabdus bacterial species. It is clear. A large number of patents and documents on artificial culture of insect parasitic nematodes belonging to the genus Steinernema and the genus Heterorhabditis have been issued. Typical bedding method using solid medium
[(1984) Ann. Appl. Biol., 104; 117-120] uses a solid material called a sponge impregnated with nematode-living animal tissue.

This method requires a material that is well aerated for nematode symbiotic bacteria and has shown that a single culture is suitable for the production of 1 billion nematodes. 100 ml for small-scale ones using liquid medium
For the following culture volumes, stall [(1961) J. Helmintho
l, Lister Sppl., pp.169-174], Jackson [(1973) Ex
p. Parasitol., 34; 111-114], Hansen et al. [(1967) 42nd.
Ann. Meeting Am. Soc. Parasitol.], Lower et al. [(1
979) Nematologia 16; 563-566], Bucher et al. [(1970)
Nematologia 16; 403-409].

[0008] As components of the liquid medium, bull kidney grounds-yeast extract (Pace et al. [PCT patent application No. 86/01074]),
Soy peptone-yeast extract-dextrose (Bucha et al. [(1971) J. Nematol., 3; 199-200]), peptone-glucose-symbiotic cells (Datkey et al. [(1967) Nematologic]
a., 13; 140]), meat extract-yeast extract-corn oil (Wouts et al. [(1981) J. Netmatol., 13; 467-469]).

Further, in liquid mass culture, Friedman et al.
[PCT Patent Application No. 88/04124] is a 16 liter medium containing egg yolk-dried yeast cells-corn oil-soybean flour as a component,
It is reported that the symbiotic bacteria were pre-cultured and then inoculated with nematodes, and about 100,000 heads were cultured per ml at the maximum with aeration. However, in these cultures, the focus is on producing as many nematodes as possible and establishing a fermentation technique using liquid culture. It does not describe whether it has power.

Bucher and Friedman et al.
It has been shown that symbiotic bacteria are added in advance during the culturing process, the purpose of which is to decompose the media components by the metabolic capacity of the symbiotic bacteria, making them easier for the nematodes to digest. Further subculture of the nematodes on the solid medium,
It is known that the nematicidal insecticidal activity is reduced, and in particular Steinernema kushidai is markedly reduced.

On the other hand, the physiological and biochemical studies of symbiotic bacteria are basically conducted by only a few laboratories worldwide, and there are many unclear points. For the growth and secondary metabolites of this unique bacterial group, see Kaya and Goggler, “Insect parasitic nematode in biological control” [(1990) CRC Pre.
ss, FL. 365pp.]. In particular, regarding secondary metabolites, luminescent substances, antibacterial substances, pigment formation, extracellular enzymes (proteolytic enzymes (proteases), lipolytic enzymes (lipases)), and crystal inclusion bodies are described.

However, it has not been known so far what is effective for the growth of nematodes, what kills insects, and what is important for the specific symbiotic relationship between bacteria and nematodes. Moreover, it is known that the pre-introduction of bacteria in the above-mentioned culture process gives empirically good results, and the reason therefor is not known in detail.

[0013]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to correlate the properties of the bacteria to be added in advance with the insecticidal activity of nematodes in the artificial culturing method of insect parasitic nematodes in which bacteria are added to the medium during the culturing process. An object of the present invention is to provide a method for clarifying the relationship and efficiently culturing an insect parasitic nematode having a strong insecticidal activity.

[0014]

[Means for Solving the Problems] As described above, the insecticidal activity of insect parasitic nematodes is largely due to sepsis due to the growth of symbiotic bacteria in the host insect blood body cavity. Insect body cavities and internal tissues are rich in humoral proteins and fats. Therefore, it is considered that the proliferative ability of symbiotic bacteria in them is related to protein degrading ability and lipid degrading ability.

The genus Xenorhabdus, which is a symbiotic bacterium, physiologically and biochemically changes into a form that absorbs a dye (bromthymol blue) and a form that does not absorb a dye. Among them, the form that absorbs the pigment produces an antibacterial substance that suppresses the growth of many other bacteria and molds (molds) without exception. The host insect body of the nematode holds many miscellaneous bacteria, especially in the intestine, but after the nematode infection, these miscellaneous bacteria do not spoil the carcass and form an optimal environment for nematode growth. This is due to the ability of this symbiotic bacterium to produce antibacterial substances.

Therefore, by increasing the decomposing ability of insect body tissues and the ability to produce antibacterial substances by some method, a symbiotic bacterium having a proliferative ability in the insect body is prepared and incorporated into an insect parasitic nematode. It is expected that the insecticidal activity of nematodes will be improved and the culture efficiency will be improved.

[0017] Therefore, the present inventors proteolytically, antibacterially and lipolytically decompose the symbiotic bacteria belonging to the genus Xenolabdus having high insecticidal activity, which are preliminarily added to the medium of the insect parasitic nematodes belonging to the genera Steinanema and Heterorhabditis. As a result of repeated research on selection based on indicators such as sex, symbiotic bacteria are selected based on their proteolytic ability and antibacterial substance-producing ability, and the enhanced ones are inoculated into a nematode culture medium and cultured. The present invention has been completed by confirming that the above is effective.

That is, the present invention provides 1) a method of culturing an insect parasitic nematode belonging to the genus Steinanema or Heterorhabditis in the presence of a symbiotic bacterium belonging to the genus Xenorhabdus. From the population of symbiotic bacteria collected from nematodes, a population having a proteolytic activity that is 1.3 times or more the average capacity of this population and an antibacterial activity that is 1.1 times or more the average capacity is selected. A method for culturing an insect parasitic nematode, which comprises culturing the insect parasitic nematode in the presence of 2), having a lipolytic property 1.2 times or more the average capacity of the symbiotic bacterial population in the selection step The method for culturing insect parasitic nematodes according to 1 above, further comprising: as an index for selection, and 3) a population in which an antibacterial property and a proteolytic property are enhanced by inducing mutation prior to selection of the population. The present invention relates to a method for culturing an insect parasitic nematode according to the above 1, which is used by selection.

[0019]

The insect-parasitic nematodes used in the present invention refer to all the insect-parasitic nematodes contained in the genus Steinanema and Heterorhabditis shown above, and also for symbiotic bacteria. It contains all Xenorhabdus bacteria that have a symbiotic relationship with insects. Further, in the present invention, the artificial culture medium of insect parasitic nematode is not particularly limited, regardless of liquid or solid medium,
It is applicable to all culture media for nematodes.

The proteolytic, antibacterial and lipolytic indexes used for selecting the symbiotic bacteria to be preliminarily added in the culture method of the present invention will be described below. [Selection of Symbiotic Bacteria Based on Strength of Protein Degradation] The protein degrading ability of a symbiotic bacterium is evaluated by how much proteolytic enzyme the symbiotic bacterium produces. Proteolytic enzymes are enzymes that hydrolyze peptide bonds in proteins and peptides. The method for selecting a symbiotic bacterium having a high enzyme activity includes a relatively simple one and a complicated one. When selecting a non-specific proteolytic enzyme-producing bacterium, a general method of determining the digestibility of a protein can be used. In the first-stage method, symbiotic bacteria are applied to an agar medium containing casein, hemoglobin, gelatin, peptone, wheat protein, etc. as the sole nutrients and cultured for several days.

In order to easily know how much the bacterial colony produces the enzyme, the protein becomes cloudy when 0.4 M trichloroacetic acid is added, and a colony having a strong enzyme-producing ability is present around the colony. A transparent band is formed. Since the gelatin-added medium solidifies at 15 ° C. or lower, in the case of colonies with strong proteolytic activity, it can be measured by the degree of liquefaction of the medium. The pH of digestive juice and blood in an insect varies depending on the species, but it is generally distributed at pH 8-9. It has been known that the optimum pH for growth of the symbiotic bacterium is 8 to 9 because it has evolved by adapting to the growth in the insect body.

Also in the above-mentioned proteolytic enzyme producing ability,
By fixing the pH of the medium to 8 to 9, it is possible to select the symbiotic bacterium having the highest enzyme-producing ability under these conditions. The temperature condition is 28 ° C, which is the optimum temperature for symbiotic bacteria.
Seems to be the best. However, in actual use, when a parasitic nematode retaining a symbiotic bacterium is sprayed as a biological control agent under natural conditions, the period in which spring or autumn pests are most frequent is effective. In these seasons, the optimum temperature for nematodes is around 25 ° C, but it is often 20 ° C or lower at night. Therefore,
Even if the temperature is 28 ° C or lower, smooth growth of the symbiotic bacteria in the insect body infected with nematodes leads to improvement of nematicidal insecticidal activity. Therefore, the optimum temperature range for the enzyme-producing ability of the symbiotic bacterium is preferably 20 to 28 ° C.

[Selection of Symbiotic Bacteria Based on Strength of Lipid Degradation] A bacterium having a lipid degrading property is characterized by growing the microorganism in a solid medium containing lipid and observing the oil and fat degrading power around the colony. Has been done. See Abstract of Microbiological Methods for these methods.
(Wiley-interscience, 1969). The lipids added to the medium include tributine, triacetin, synthetic fats and oils such as methyl laurate, butter,
Natural oils and fats such as margarine, olive oil, corn oil, lard, and tallow, and water-soluble surfactants such as Tween can be seen, but there are examples using special lipids such as fatty acid ester of propylene glycol and horse fat. These lipids are emulsified in an agar medium and then solidified, and then the target symbiotic bacteria are applied thereto, and after culturing, the lipolytic activity around the colonies is detected.

There are many detection methods, but the method of detecting the produced fatty acid using a pH indicator has long been used. Nile blue is often used as the indicator, but all indicators having a color change range at pH 2 to 7, such as Victoria blue, methyl red, congo red, and bromocresol green, can be used. A method has also been devised in which Nile blue is used as a water-insoluble oxazine base to dissolve in oils and fats to prevent growth inhibition. There is also a report that clear distinction can be made using Victoria blue (Davis et al., J. Bact., 88, 16-19).

Although it is different from the indicator, there is also a method in which a plate on which a microorganism is grown is immersed in a saturated solution of copper sulfate, and the produced fatty acid is dyed in green to detect the fat and oil decomposing power. When fatty acids are produced by lipolytic enzymes, the emulsified state of fats and oils changes, and circular clear zones occur around colonies. There is also a method of selecting a large microorganism in this clear zone. Various fats and oils are used as lipids, but trybutine and olive oil are often used.

Since there is also a method of measuring the lipolytic enzyme activity by the method using tributine, this method is effective for separating the lipolytic enzyme-producing bacterium. There is also a method of observing the halo around the colony by pouring a thin layer of beef tallow on a petri dish and solidifying it in a refrigerator, and then pouring a medium on it to grow microorganisms and decomposing the beef tallow by the produced lipolytic enzyme. This is mainly used as a method for measuring the lipolytic activity of yeast, but it can also be applied to symbiotic bacteria. In some cases, egg yolk is used as the oil and fat, and the clear zone is observed. However, when egg yolk is used, lipoprotein lipase activity and phospholipase activity may be detected, so caution is required.

[Selection of symbiotic bacteria based on strength of antibacterial substance production] For the antibacterial properties of symbiotic bacteria, the titer assay method and susceptibility test used in the study of antibiotics can be applied. Of these, the most widely used sensitive disk method is the simplest. Place a sensitive disc impregnated with a liquid medium of an antibacterial substance-producing bacterium on a plate medium mixed with the test bacterial solution to be tested, and make a positive circle when an inhibition circle of 1 mm or more is observed from the periphery of each disc, and the inhibition circle It is possible to judge the strength of productivity by the diameter (Nobuo Tanaka, Shoshiro Nakamura, Supplement of Antibacterial Substances 3rd Edition, pages 19-23).

Thus, a symbiotic bacterium excellent in protein degrading, lipid degrading and antibacterial activity is selected and contained in the medium for infecting parasitic nematode 3rd stage larvae, and the nematode is inoculated into the medium for culturing. did. The culturing method and culturing conditions are not particularly limited, and the culturing is performed according to a conventionally known culturing method for nematodes.

[Proteolytic ability, lipolytic ability of symbiotic bacteria,
And relationship between antibacterial substance-producing ability and insecticidal activity of the bacterium against insect parasitic nematode] Belonging to the genus Xenorhabdus having high insecticidal activity selected by the above-mentioned protein degrading ability, lipid degrading ability, and antibacterial substance producing ability When the symbiotic bacteria were pre-added to the culture medium of the insect parasitic nematodes belonging to the genus Steinanema and Heterorhabditis, the symbiotic bacteria were selected with high protein degrading and antibacterial activity as indicators, preferably high protein degrading and lipid degrading. It was revealed that the parasitic nematodes cultured with the addition of the symbiotic bacteria selected based on the antibacterial activity showed excellent insecticidal activity against insects.

On the other hand, an insect which, incidentally or artificially, further enhances protein degrading ability, lipid degrading ability and antibacterial activity to obtain a symbiotic bacterium having a stronger insecticidal activity, which is preliminarily added and retained. It was confirmed that the biological control effect of insect parasitic nematodes was further enhanced by culturing the parasitic nematodes. In order to enhance certain properties of bacteria, either accidentally or artificially, generally, a method of inducing mutation and isolating mutants satisfying the condition from a population is used. In order to induce mutation, there are physically X-rays, ultraviolet rays, and the like as mutagens, and chemically, ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG), Nitric acid, acridine dye, etc. are used.

By these methods, the symbiotic bacteria selected by using each excellent physiological activity as an index were mutated, and nematodes were cultured in the medium containing the mutant symbiotic bacteria in the same manner as above. When the mutant symbiotic bacterium thus obtained was compared with the non-mutated symbiotic bacterium, this mutation had no effect on the growth of the host nematode, but retained it in the nematode. By doing so, it was confirmed that the insecticidal activity against insects was further improved.

Therefore, a wild-type symbiotic bacterium having high protein degrading ability and strong antibacterial activity was selected from symbiotic bacteria showing insecticidal activity and mutated to enhance various physiological activities. By the culture method of the present invention in which a mutant-type symbiotic bacterium is contained in a medium and the insect-parasitic nematode retaining the symbiotic bacterium in the medium is cultivated, an insect-parasitic nematode superior in insecticidal activity can be obtained. .

[0034]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

Example 1 Isolation Method of Symbiotic Bacteria Steinenema carpocapsae ol larvae of the third stage of infectious state were washed 5 times with 0.1% formalin solution, and then 0.1
% Merthiolate aqueous solution for surface sterilization for 2 hours.
Furthermore, it was washed 3 times with sterilized water, and the infectious third stage larva was inoculated on a normal agar nutrient medium using a micropipette. After standing at 25 ° C for 48 hours, the symbiotic bacterium Xenorhabdus nematophyllus that has grown on the medium is separated, again applied to the ordinary agar nutrient medium using a platinum loop, streak-cultured, and 40 or more colonies are selected. The test was carried out.

1-1: Ranking of Symbiotic Bacteria by Strength of Protein Decomposition The proteolytic enzyme-producing ability was investigated using the following methods and conditions. Meat extract 0.3%, peptone 0.5%, gelatin 1
Sterilize 2.0% medium (pH 8.0) (120 ℃, 20 minutes)
After that, each colony that had been pre-cultured (normal agar nutrient medium at 28 ° C. for 48 hours) was subjected to stab culture. Then, culture at 28 ° C and gelatin (protein) on the 3rd, 11th, 18th and 25th days.
The height (mm) of the liquefied part by decomposing was measured and each colony was ranked. The results are shown in Table 1.

[0037]

[Table 1]

1-2: Ranking of Symbiotic Bacteria by Strength of Lipid Degradability The lipid degrading ability of symbiotic bacteria was measured by the following method. (1) Using a 1% peptone medium (pH 8.0) containing 0.5% of lipid, each precultured colony was plated at 28 ° C. The following three lipids were used. Tween 20 polyoxyethylene sorbitan monolaurate Tween 60 polyoxyethylene sorbitan monostearate Tween 85 polyoxyethylene sorbitan trioleate (2) The degree of growth (colony diameter) of each colony was measured 4 days after the start of culture.

(3) The ratio between the size of each colony grown in a lipid-free medium and the size of a colony grown in each lipid-containing medium was calculated, compared and evaluated. (4) The amount of calcium precipitate formed by lipolysis around each colony grown on the lipid-containing medium was investigated. Using the results of (2) to (4) as an index, the strength of the lipid decomposing ability of each selected colony was ranked. The results are shown in Table 2.

[0040]

[Table 2] In the table, the lipolytic activity distinguishes the amount of calcium formed around each colony on a medium containing each of three lipids (Tween 20, Tween 60, and Tween 85) into 5 levels (-to +++). Then, + was set to 1 and the total was taken as the lipid decomposing ability. For example, when Tween 20 → ++++, Tween 60 → ++, Tween 85 → +, ++++, ++, +
The total of 6 is defined as the lipolytic power.

1-3: Ranking of Symbiotic Bacteria by Strength of Antibacterial Substance-Producing Presence or Absence of 40 or more Antibacterial Activities of Xenolabdus nematophyllus, a Symbiotic Bacterium from Steinernema carpocasae All strains, Obtained The antibacterial activity of the colonies was investigated by the following method. (1) 2 each precultured colony in normal broth liquid medium
After shaking culture at 8 ° C for 48 hours, the culture solution was centrifuged (10
(000 rpm, 10 minutes) to separate the supernatant and the bacterial cell residue. The obtained supernatant was sterilized by filtration to be completely sterile. (2) Bacillus subtilis, which is the target bacterium whose antibacterial property is to be investigated, at about 5 ml in a normal broth liquid medium at 28 ° C.
After shaking culture for 24 hours, sterilize 1.5 ml (120 ml).
(20 ° C., 20 minutes), and mixed in ordinary agar medium cooled to about 35 ° C., dispensed into a petri dish having a diameter of 9 cm and solidified.

(3) A paper disc (diameter of about 1) immersed in the sterile culture supernatant obtained in (1) on the medium of (2) and air-dried.
cm) was placed and cultured at 28 ° C. for 48 hours. Then, the diameter of the growth inhibition circle against Bacillus subtilis formed around the paper disk was measured and used as an index of antibacterial activity. Also,
Use 4 paper disks per colony,
Four average values were obtained as the diameter of the growth inhibition circle. (4) The above experiment was performed twice, and the strength of the antibacterial activity was ranked by the average value of the diameters of the two inhibition circles. The results are shown in Table 3.

[0043]

[Table 3]

1-4: Insecticidal activity of the selected colonies against the 6th instar larva of Spodoptera litura, among the colonies ranked from 1-1 to 1-3,
All three physiological properties are strong (No.44), all weak (No.8), all moderate (No.28), lipid degrading is strong but other two properties are weak (No. .27) was selected, and these bacteria were cultured by shaking in about 5 ml of a normal broth liquid medium at 28 ° C. for 48 hours, and then diluted so that the number of bacteria was about 1000 cells per 5 μl. Further, 5 μl of the obtained bacterial suspension was injected into the blood body cavity of the 6th instar larva of Spodoptera litura using a 10 μl microinjector. Then, the strength of insecticidal activity of the four selected colonies was ranked using the time required for the test insects to die as an index.
The results are shown in Table 4.

[0045]

[Table 4] In the table, the concentration was adjusted by culturing each selected colony, diluting it with physiological saline, and adjusting the concentration of symbiotic bacteria to 1000 cells per 5 μl. The insecticidal rate was calculated by the following formula. Insect kill rate (%) = (number of dead insects + number of agony insects x 0.5) / number of test insects x 100

From these results, the symbiotic bacterium Xenorhabdus nematophyllus selected from all of these, which have excellent protein degrading ability, lipid degrading ability and antibacterial activity, has a strong insecticidal activity, and particularly excellent in protein degrading ability and antibacterial activity. It was revealed that selection based on the above is important for high insecticidal activity and proliferative ability.

Based on the above-mentioned research results of Examples 1-1 to 1-4, the present inventors found that Xenolabdus nematophilus, which is a symbiotic bacterium from the insect parasitic nematode Steinanema carpocapsae all strain, is in the insect blood cavity. It was confirmed that the root cause of the death of insects after being propagated in Escherichia coli is the growth of symbiotic bacteria in the body of insects, and the strength of this growth power is related to the protein degrading ability and antibacterial substance-producing ability of symbiotic bacteria. did. Then, it was judged to be effective for the expression of high insecticidal activity of symbiotic bacteria to be selected by using the fact that they possess high protein degrading ability and strong antibacterial activity as indicators. The specified value of proteolytic property of 1.3 was obtained by dividing the lowest value 4.5 of the top-performing colony group (group of gelatin liquefaction amount: 4.5 to 7.0 mm in Table 1) with an average capacity of 3.5 mm in the gelatin liquefaction amount of Table 1. It is a value (4.5 ÷ 3.5 = 1.3). Antibacterial regulation value 1.1
Is a top-performing colony group with an average capacity of 12.0 mm in the diameter of the growth inhibition circle in Table 3 (the diameter of the growth inhibition circle in Table 3: 1
The value obtained by dividing the minimum value 13.1 of the group of 3.1 to 19.0 mm) (13.
1 ÷ 12.0 = 1.1). In addition, the specified value of lipolysis is 1.2
Is a value (6/5 = 1.2) obtained by dividing the lowest value 6 of the top-performing colony group (lipolytic power of the table: 6 to 8 group) with the average capacity of 5 in the lipolytic power of Table 2. is there.

Example 2: Enhancement of proteolytic ability and antibacterial activity of symbiotic bacteria The present inventors irradiate the symbiotic bacteria No. 28 and No. 44 having strong insecticidal activity selected in Example 1 with ultraviolet rays. Mutants were induced by using a general microbiological experiment method by S., and colonies with more excellent protein degrading and antibacterial activity were selected. Table 5 shows the results of measuring various physiological activities.

[0049]

[Table 5] In the table, the lipid degradability was evaluated by comparing the amount of calcium precipitate formed around the colonies grown on each medium. +++: Very large ++: Slightly large +: Large

That is, the No. 28PS strain having a stronger protein degrading power, the No. 28AS strain having a stronger antibacterial activity, and the parent strain of the symbiotic bacterium Xenorhabdus nematophilus No. 28 (designated as No. 28N), and No.28P with enhanced both activities
AS strain was obtained, and from the parent strain of No.44 (designated as No.44N), No.44PS strain, No.44AS strain and No.44PAS strain were similarly obtained.

Example 3: Improvement of nematode insecticidal activity by addition of a medium of a mutant symbiotic bacterium having high insecticidal activity 3-1: Effect on host nematode growth Induced by mutation in Example 2 and isolated After culturing symbiotic bacteria with excellent protein degrading and antibacterial activity, they were cultivated in a normal broth liquid medium at 28 ° C for 48 hours, and about 10 ⁹ cells were added to the dog food medium for culturing insect parasitic nematodes. It was pre-added and left at 28 ° C. for 48 hours.

Thereafter, surface-sterilized insect-parasitic nematode Steinanema carpocapsae-all strain infectious stage 3 larvae were inoculated into the medium and cultured at 25 ° C. for about 20 days,
Next-generation infectious 3 larvae were collected. The results are shown in Table 6, and the total number of the infectious 3rd stage larvae obtained per test tube was as follows. There was some difference in the number of proliferation, but it did not substantially change.

[0053]

[Table 6] In the table, colony No. represents the type of colony preliminarily added to the dog food medium, and the meaning of each abbreviation is as shown below. PS: those with enhanced protein degrading ability, AS: those with enhanced antibacterial activity, PAS: those with enhanced both protein degrading ability and antibacterial activity, N: those without any selection. In addition, the number of infectious larvae multiplied is 2 when the nematodes are inoculated into the medium.
1 test tube collected on day 0 (caliber 18 mm, length 18
Number of infectious larvae per 0 mm (average value of 5 tests)
Represents

3-2: Insecticidal activity of nematodes carrying a mutant symbiotic bacterium having high insecticidal activity On the other hand, using the thus obtained larvae of the insect-parasitic nematode in the infectious stage 3 When the insecticidal activity against instar larvae was compared with the infectious 3rd stage larvae obtained by ordinary culture, surprisingly, the nematode Hasmonyoto 6 cultivated by pre-adding symbiotic bacteria with enhanced proteolytic activity and antibacterial activity 6 It was found that the insecticidal activity against instar larvae is significantly improved. Below, the insecticidal activity-evaluating method and results for the 6th instar larva of Spodoptera litura are shown.

(1) Each of the insect-parasitic nematodes Steinernema carpocapsae-all strains was infected with 0.4 larvae of the third stage of infection.
It was suspended and adjusted in distilled water so that the number of nematodes was 100 per ml. (2) 0.4 ml of the nematode suspension of 1 was dropped on a 5.5-cm plastic petri dish covered with filter paper. (3) (2) One 6th-instar larva of Hehasmon Yoto was released for 20
Repeated. (4) The number of deaths of the tested insects was investigated up to 48 hours after the treatment.

When the nematode was proliferated by preliminarily adding the symbiotic bacterium No. 8 which was originally weak in proteolytic activity and antibacterial activity, the nematicid obtained had an insecticidal activity against the 6th instar larva of Spodoptera litura after 48 hours. However, although it killed only 40%, the insecticidal activity of the infectious stage 3 larvae obtained by culturing nematodes using the above-mentioned parent strains of No. 28 and 44 with high resolution is
After 30 hours, it showed an insecticidal rate of 85% to 100%. Furthermore, when a mutant strain having enhanced proteolytic activity and antibacterial activity is used from this parent strain, the infectious condition 3 obtained is
The insecticidal activity against the 6th instar larva of Spodoptera litura was higher than that of the parent strain. The results of this test are shown in Table 7.
As is clear from this result, as compared with the infectious 3rd stage larvae obtained by ordinary culture, the infectious 3rd stage larvae having a symbiotic bacterium having a stronger insecticidal activity can be killed. It was confirmed that the time was short and the insecticidal activity was improved.

[0057]

[Table 7] In the table, colony No. represents the type of colony preliminarily added to the dog food medium (the meaning of each symbol is the same as above). Regarding the killing rate, the infectious larvae obtained from each medium were adjusted to a concentration of 100 heads / 0.4 ml, dropped on a 5.5 cm Petri dish with a filter paper bed, and then one lotus larva of the last year was released. Death of insects was confirmed over time and calculated by the above-mentioned calculation formula.

[0058]

According to the above-mentioned series of studies, the present inventors have found that in order to enhance the insecticidal activity of insect parasitic nematodes, which is effective for biological control of pests, the metabolic ability of the symbiotic bacterium is high. Specifically, it was found that it is effective to enhance proteolytic activity and antibacterial activity, and in order to culture and propagate insect parasitic nematodes with strong insecticidal activity, enhance the metabolic ability of the symbiotic bacterium, It was confirmed that it is effective to preliminarily add the nematodes before culturing the nematodes to grow the nematodes.

It goes without saying that the present invention is applicable to all species of the insect parasitic nematodes Steinanema and Heterorhabditis nematodes having the symbiotic bacterium Xenolabdus. In addition, the method of the present invention is highly practical because it does not utilize taxonomically different insect pathogenic bacteria or the like, but selects and selects unique symbiotic bacteria. According to the invention,
In addition to being able to prevent the decrease in insecticidal activity of insect parasitic nematodes by artificial culture, it is possible to produce insect parasitic nematodes having higher insecticidal activity, and the insect parasitic nematodes are used as biological control agents. Improves practicality when used outdoors.

Front Page Continuation (56) References Tokuhyo 5-506149 (JP, A) Tokusho Sho 61-503001 (JP, A) Kondo E. , Dependen cy of Three Steinne rnnematid Nematodes on Their Symbiotic Bacteria for Grow and and Propagion, Nihonbushi Kenkyukai, 1991, V. 1991. 21, pp. 11-17 Satoshi Yamanaka, Current Status and Future Prospects of Biopesticides-Microbiocides, BIO INDUSTRY, 1988, Vol. 5, No. 1, pp. 38-43 Ogura N. , Artificial culture of an entomogenous nemato de, Appl. Entomol. Zool. , 1989, Vol. 24, No. 1, pp. 112-116 Nobuyoshi Ishibashi, Protecting Plants with Microorganisms-Pest Control Using Nematodes, Science of Organisms-Genetics, 1988, Vol. 42, No. 6, pp. 20-24 Ogura Nobuo, Possibility of control of scarab beetle larvae by the insect parasitic nematode Stein ernema kushidai, Plant protection, 1992, Vol. 46, No. 9, pp. 334-337 Boemare N.C. E. , Bioc chemical and physiologic charasterization of colony forms variants in Xe norhabdus spp. J. Gen. Microbiol. , 1988, Vol. 134, No. 3, pp. 751-761 Akhurst R. J. , Anti biotic activity of Xenorhabdus spp. J. Gen. Microbio l. , 1982, Vol. 128, No. 12, pp. 3061-3065 (58) Fields surveyed (Int.Cl. ⁷ , DB name) A01N 63/00 A01K 67/033 BIOSIS (STN) CAPLUS (STN)

Claims (3)

(57) [Claims] 1. A method for cultivating an insect parasitic nematode belonging to the genus Steinanema or Heterorhabditis in the presence of a symbiotic bacterium belonging to the genus Xenorhabdus. From the population of symbiotic bacteria, a population having a proteolytic activity that is 1.3 times or more the average capacity of this population and an antibacterial activity that is 1.1 times or more the average capacity is selected, and insect parasitism is performed in the presence of the obtained population. A method for culturing an insect parasitic nematode, which comprises culturing a sex nematode. 2. The method for cultivating an insect parasitic nematode according to claim 1, wherein in the selection step, having a lipolytic property 1.2 times or more the average capacity of the symbiotic bacterial population is further used as an index for selection. 3. The method for culturing an insect parasitic nematode according to claim 1, wherein mutation is induced prior to selection of a population, and a population having enhanced antibacterial properties and proteolytic properties is selected and used.