JP3184967B2 - Microbial herbicide and herbicidal method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microbial herbicide utilizing a microorganism exhibiting pathogenicity to S. americana and a microbial herbicide utilizing a microorganism having ice nucleation activity. The present invention also relates to herbicides containing these microbial herbicides and chemical herbicides.

[0002]

2. Description of the Related Art In Japan, the damage of weeds is enormous. If weeding is not carried out, the yield of various crops will be greatly reduced, which will directly lead to a decrease in agricultural productivity.
For example, the yield loss due to non-herbicide is 36% for paddy rice, 68% for barley, 14% for wheat, 19% for upland rice, and 37% for peanut.
% For red beans and 12% for red beans. Weeds are also severely damaged on non-cultivated lands such as turf of golf courses, railroad tracks, parks, embankments, parking lots, and slopes. Therefore, especially after World War II, large amounts of chemical herbicides have been used in agricultural lands and golf courses. Thus, chemical herbicides have played a role in increasing crop productivity and preserving landscapes.

[0003] However, new problems have emerged with the spread of chemical herbicides. That is, there has been a strong concern in recent years that the accumulation of pesticides, including chemical herbicides, in the environment adversely affects human health and the survival of species other than humans. Therefore, there is a strong demand for the development of pesticides that have a gentle effect on the environment as much as possible, especially biological pesticides. This is the same in the field of herbicides, and the development of a herbicide method utilizing microorganisms is being promoted worldwide.

[0004] Herbicidal methods using microorganisms can be divided into two methods: methods using microorganisms themselves and methods using metabolites of microorganisms. The former is called a microbial herbicide, and fungi, bacteria, viruses, or the like showing pathogenicity to plants are used. On the other hand, the latter is called a microbial herbicide and uses toxins, plant hormones, enzymes and the like, which are metabolites of pathogenic microorganisms.

At present, only about five kinds of microbial herbicides have been put to practical use, and only about 20 kinds have been put into practical use. Reasons for the extremely small number of microbial herbicides commercialized in this way include that the herbicidal effect of the microbial herbicide is not always sufficiently exhibited particularly in a large-scale test, and that the herbicidal effect is not stably exhibited.

[0006] Most of the microorganisms used as microbial herbicides are filamentous fungi showing pathogenicity to plants, and only one kind is derived from bacteria. One of them is Campellico (Agricultural Chemical Registration, Ministry of Agriculture, Forestry and Fisheries No. 19657), a microbial herbicide for Poa annua, which is a harmful weed on turf.
Symposium of the Society for the Study of Weed Kanto (1997)). This is a formulation of Xanthomonas bacteria showing pathogenicity to Poa annua, which is characterized in that Poa annua can be selectively weeded. However, because Xanthomonas bacteria grow in the tract of plants, their primary route of infection is wound infection. For this reason, there is a restriction on the utilization technology that an effective herbicidal effect cannot be obtained unless this microbial herbicide is sprayed after cutting grass or the like.

[0007] Microbial herbicides generally target specific weeds and are not available for other types of weeds.
Therefore, it is necessary to develop microbial herbicides for each weed. S. chinensis, a naturalized weed derived from North America, is the most common weed that thrives on wastelands, roadsides, etc. in Japan. For the control, physical weeding methods such as pruning and the application of chemical herbicides have been performed, but they are still prosperous. However, no research has been conducted on microbial herbicides against S. pallidum so far, and development of a microbial herbicide capable of controlling P. pallidum has been desired.

[0008] On the other hand, since the chemical herbicide and the microbial herbicide have their respective problems as described above, it has been desired to develop a herbicidal method for compensating for these problems. For example, if effective weeding can be achieved by adding a chemical herbicide to a microbial herbicide as a main component, it is considered that the number of times of application and the amount of the chemical herbicide to be applied can be reduced. Under such circumstances, the development of a novel microbial herbicide capable of controlling the S. aureus and a novel microbial herbicide that exhibits an excellent herbicidal effect on weeds and can be used in combination with a chemical herbicide has been developed. Was desired.

[0009]

DISCLOSURE OF THE INVENTION The present invention relates to a novel microbial herbicide capable of controlling the S. pallidum and a novel herbicide which exhibits an excellent herbicidal effect on weeds and can be used in combination with a chemical herbicide. It is intended to provide a microbial herbicide.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the use of microorganisms belonging to the genus Pseudomonas makes it possible to control S. pallidum. Also, the present inventors,
It has been found that weeds can be controlled using natural frost by using microorganisms having ice nucleation activity. Furthermore, the present inventors have found that weeds can be efficiently controlled by using the above microorganisms in combination with a chemical herbicide.

Based on these findings, the present invention has been completed. That is, the present invention is a herbicide characterized by containing a microorganism belonging to the genus Pseudomonas and showing pathogenicity against S. pallidum as an active ingredient. Further, the present invention is a herbicide comprising a microorganism having ice nucleation activity as an active ingredient. Further, the present invention is a herbicide comprising the above herbicide and one or more chemical herbicides.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The first (hereinafter referred to as "first invention") of the present invention is a herbicide characterized by containing, as an active ingredient, a microorganism belonging to the genus Pseudomonas and exhibiting pathogenicity against S. pallidum.

The microorganism which is an active ingredient of the herbicide of the first invention may be any microorganism as long as it belongs to the genus Pseudomonas and is a microorganism which is pathogenic to S. pallidum, and is preferably Pseudomonas syringae. (Pseudomonas syringae), most preferably Pseudomonas syringe SEI-1 strain. Since the Pseudomonas syringe SEI-1 strain was rejected from the Institute of Biotechnology, Institute of Industrial Science and Technology, the applicant has guaranteed the sale. For Pseudomonas syringae, the LOPAT method (Lelliott et al., J. Appl. Bact.,
29, pp. 470-489, 1966).

[0014] Pseudomonas syringae SEI-1 strain is a strain isolated from Solidago altissima with black-brown lesions presumed to be bacterial diseases on the leaf blades. This strain has the following mycological properties. Gram-negative bacteria,
It is a rod-shaped bacterium with extreme flagella that has motility. Produces yellow-green fluorescent dye in King B medium (Eiken Chemical). In the LOPAT method, simple identification of plant pathogenic bacteria showed levan production (-), oxidase activity (-), potato tuber rot (-), arginine hydrolysis (-), and tobacco hypersensitivity reaction (+). Belong. Based on these mycological properties, this strain was identified as a microorganism belonging to Pseudomonas syringae. The strain is selectively pathogenic to S. americana and other plants (eg, mulberry, mulberry,
Trees such as casino trees and plums, flowers such as asters, salvia, marigolds, and weeds such as mulberry, kuzu, chickweed)
Is not pathogenic to Since Pseudomonas syringae, which is selectively pathogenic to S. agaricus, was never known before, this strain was found to be a new pathogenic strain. A characteristic feature of the mycological properties is that the present strain shows a very specific response to the LOPAT method. In Pseudomonas syringae, there are pathovar types corresponding to about 50 subspecies. These pathogenic types cannot be distinguished by mycological properties, but only by the presence or absence of pathogenicity on a particular plant. For example, those showing pathogenicity to mulberry are pv.mor,
Those showing pathogenicity in tobacco are distinguished from pv.tabaci.
Among them, it is known that a specific pathogenic type (for example, pv.mori) has ice nucleus activity.

For culturing Pseudomonas syringae SEI-1 strain, it is not necessary to use a special method, and the same culturing method as used for known strains of the genus Pseudomonas can be used. As the medium, any of a synthetic medium and a natural medium may be used as long as the medium appropriately contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth promoting substances.
Specifically, for example, King B medium (peptone 20 g, K _{2
HPO 4 1.5g, MgSO 4 · 7H} 2 O 1.5g, glycerin 10 g, agar 15
g, distilled water 1 L), sucrose-added LB medium (peptone 10 g, yeast extract 5 g, salt 10 g, agar 15 g, sucrose 50 g, distilled water 1 L) and the like can be used.
During the culture, the culture temperature is 15 to 30 ° C., preferably 25 to 30 ° C.
At 28 ° C., the pH can be 5-9, preferably 6-8.

As the herbicide of the first invention, a microorganism (live bacterium) belonging to the genus Pseudomonas and exhibiting pathogenicity for A. niger may be used as it is, or may be dissolved or dispersed in an appropriate liquid carrier. Or admixed with or adsorbed on a suitable powder carrier. At this time, add emulsifiers, dispersants, suspending agents, spreading agents, penetrants, wetting agents, stabilizers, etc. as necessary and formulate them into liquids, emulsions, oils, wettable powders, powders, etc. You can also. As a liquid carrier used for formulation, for example, water, skim milk aqueous solution, sorbitol aqueous solution,
Examples thereof include an aqueous solution of sodium glutamate and the like, and one or more of these can be used in combination. Examples of the powder carrier used for the formulation include gelatin, silk fibroin powder, zeolite, skim milk and the like.
Species or a combination of two or more can be used.
The formulation method is not particularly limited as long as the microorganism does not lose its pathogenicity against S. pallidum. For example, a microorganism belonging to the genus Pseudomonas, which exhibits pathogenicity to S. agaricus, is grown in a bacterial growth medium such as King B medium for 20 to 20 μm.
After culturing at 30 ° C. for 2 days, it can be suspended in sterile distilled water and used as the herbicide of the first invention. On this occasion,
It is preferable to add a surfactant (for example, Tween 20) to the suspension at a dilution of about 5000 times and mix it, since the adhesion between the microorganism and the leaf surface is improved.

The concentration of the microorganism, which is an active ingredient of the herbicide of the first invention, can be appropriately set according to the purpose of use. For example, when a suspension of microorganisms is used as described above, the concentration of microorganisms is preferably in the range of 10 ⁸ to 10 ⁹ cells / ml. Can be demonstrated. When the herbicide of the first invention is used, the amount of the herbicide to be applied can be appropriately set according to the degree of vegetation of the target plant. The method of spraying is not particularly limited, and examples thereof include intact inoculation and injured inoculation using methods such as application, adhesion, and spraying. At the time of spraying, it is preferable that the microorganisms be sufficiently attached to the leaf surface of the target plant using a sprayer or the like. It is effective that the wound inoculation is performed after the target plant is mechanically wounded. In the Examples, the wound was mechanically damaged by the bundle needle method. However, when the herbicide of the first invention is used on a large scale, the target plant may be damaged and inoculated after being wounded by mechanical weeding. .

Although the target plant of the herbicide of the first invention is not particularly limited, it is particularly effective against A. niger. In addition, the growth period of the plant to be controlled is not particularly limited, and the plant can be widely controlled from a plant shortly after germination to a grown plant. However, when the target plant is S. pallidum, the herbicidal effect differs depending on the growth stage and inoculation time. It is preferable that the growth stage of Aedes chinensis is as young as possible. That is, it is preferable to target the S. pallidum (for example, one having a height of 5 to 15 cm) in the growing season (5 to 6 months), and when used throughout the year, after mechanical or chemical weeding, It is preferable to use newly grown young goldenrod (e.g., one with a height of 5 to 15 cm). In addition, the time of inoculation into the S. pallidum is preferably at a time when the growth of microorganisms is active (for example, from April to September). When the temperature is low and the growth of S. pallidum is stopped, and the growth of microorganisms is suppressed (for example, from October to December), even if the microorganisms are inoculated, the infection to the s. I can't get it. On the other hand, if the inoculation time is around June, it is possible to easily infect and cause disease of S. americana. In this case, an initial lesion appears about one week after the inoculation, progresses to a gangrene spot, and expands. In addition, it can infect even the surrounding S. americana and effective herbicidal effects can be obtained.

When Pseudomonas syringae SEI-1 strain is used as an active ingredient of the herbicide of the first invention, S. pallidum can be selectively controlled. That is, Pseudomonas syringae SEI-1 strain specifically shows pathogenicity to S. pallidum and other plants (for example, trees such as mulberry, mulberry, cassia tree, plum, etc.
It does not show pathogenicity against plants such as aster, salvia, marigold, mulberry, kuzu, chickweed, etc.). Therefore, in using Pseudomonas syringae SEI-1 strain, there is no need to consider the effects on surrounding plants and the like.
This high host selectivity is an excellent herbicide condition, and its use is not limited. For example, it can be used on non-agricultural land (railway tracks, parks, embankments, parking lots, slopes) and upland. Can be used. However, since this strain also has ice nucleus activity as described in detail below, care must be taken when using it in a field when the temperature drops and natural frost occurs.

The second of the present invention (hereinafter referred to as "second invention") is a herbicide characterized by containing a microorganism having ice nucleating activity as an active ingredient. The microorganism that is the active ingredient of the second invention may be any microorganism as long as it is a microorganism having ice nucleus activity. Here, “a microorganism having an ice nucleating activity” means a microorganism having an ability to become an ice crystal nucleus and freeze or freeze water. As such a microorganism, for example, a microorganism capable of having ice nucleating activity in a temperature range of -6 ° C to 0 ° C can be exemplified. Here, “possible” means that it is not limited to the temperature range of −6 ° C. to 0 ° C., but can have ice nucleating activity even at a temperature lower than −6 ° C. Therefore, the temperature at which the microorganism having ice nucleus activity freezes or freezes water is not particularly limited.

Examples of the microorganism having ice nucleus activity include microorganisms belonging to the genus Pseudomonas, Erwinia or Zanthomonas. Examples of ice-nucleating bacteria belonging to the genus Pseudomonas include Pseudomonas fluorescens.
ens), Pseudomonas syrin
gae) and Pseudomonas v.
iridiflava), and examples of the ice-nucleating bacteria belonging to the genus Erwinia include, for example, Erwinia ananas, Erwinia herbicola, Erwinia stewartii, and the like. Can be
Examples of ice-nucleating bacteria belonging to the genus Zanthomonas include, for example, Xanthomonas campestris (Xanthomonas camp)
estris) etc. (Lindow, S. Annual
Review of Plant Pathology, 21, 363-384, 1982, Mamoru Sato, Genetics 42, 30-34. 1988). More specifically, Pseudomonas sp. SEI-1 strain, Pseudomonas sp.
23 strains and Pseudomonas syringae Mei40 strain can be exemplified, and as the ice nucleating bacteria belonging to the genus Erwinia, Erwinia ananas TM2 strain and Erwinia ananas Mei7 strain can be exemplified. In addition, since these strains were rejected by the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, the applicants guarantee the distribution.

The mycological properties of Pseudomonas syringae SEI-1 strain are as described above. The supercooling point of this strain (SCP
(Super cooling point), ice nucleation temperature) is -1.8 ° C, and the minimum concentration of ice nucleation is 10 ⁴ cells / ml. In general,
It can be determined that the higher the supercooling point and the lower the minimum concentration of ice nucleation, the higher the ice nucleus activity, and that this strain has the best ice nucleus activity among conventional ice nucleation active bacteria. found. Such excellent ice nucleus activity can be specifically enhanced by culturing under specific culture conditions (medium, culture temperature, etc.). That is, the ice nucleus activity of the ice nucleation active bacteria cultured under the optimal culture conditions is 10% when cultured in a normal medium.
It can be increased more than twice. A convenient medium for enhancing the activity of ice-nucleating bacteria is King B medium (peptone 20
_{g, K 2 HPO 4 1.5g,} MgSO 4 · 7H 2 O 1.5g, glycerin 10 g,
Agar 15g, distilled water 1L), sucrose-supplemented LB medium (peptone 10g, yeast extract 5g, salt 10g, agar 15
g, sucrose 50 g, distilled water 1 L) and the like. Culture temperature is also important for enhancing ice nucleus activity, and the culture temperature is preferably 18 to 22 ° C. If the culture temperature is lower than this temperature range, there is a problem that it takes a long time to obtain the target amount of bacteria, and if it is too high, the ice nucleation active protein on the bacterial surface is denatured and the ice nucleus ability is reduced. There is a point. Note that such conditions for enhancing ice nucleus activity can be applied to any microorganism having ice nucleus activity.

The Pseudomonas syringae Ni23 strain is an ice-nucleation-active bacterium isolated from mulberry leaves. The mycological properties of this strain are as follows. Gram-negative bacterium with polar flagellated bacilli that is motile. Produces yellow-green fluorescent dye in King B medium. It has properties of levan production (-), oxidase activity (-), potato tuber rot (-), arginine hydrolysis (-), and tobacco hypersensitive reaction (+). Based on these mycological properties, this strain was identified as a microorganism belonging to Pseudomonas syringae. The supercooling point of this strain is -3.0 ° C, and the minimum concentration of ice nucleation is 10 ⁴ to 10 ⁶ c
ells / ml.

The strain Pseudomonas syringae Mei40 is an ice-nucleation-active bacterium isolated from the body of Kuwanomeiga. The mycological properties of this strain are as follows. Gram-negative bacterium with polar flagellated bacilli that is motile. Produces yellow-green fluorescent dye in King B medium. Levan production (-),
Oxidase activity (-), potato tuber rot (-),
It has the properties of arginine hydrolysis (-) and tobacco hypersensitive reaction (+). Based on these mycological properties, this strain was identified as a microorganism belonging to Pseudomonas syringae. The supercooling point of this strain is -3.0 ° C, and the minimum concentration of ice nucleation is 1
It is 0 ⁴ to 10 ⁶ cells / ml.

The Erwinia ananas TM2 strain is an ice-nucleated active bacterium isolated from mulberry leaves. The mycological properties of this strain are as follows. A gram-negative bacterium that has motile bacilli. Form yellow colonies in many growth media. Does not form endospores or aerial hyphae.
Does not reduce nitrate. Utilizes inositol, raffinose, cellobiose, melibiose, and glycerol. Based on these mycological properties, this strain was identified as a microorganism belonging to Erwinia ananas. The supercooling point of this strain is -2.7 ° C, and the minimum concentration of ice nucleation is 10 ⁴ to 10 ⁶ cel.
ls / ml.

[0027] Erwinia ananas Mei7 strain is an ice-nucleation-active bacterium isolated from the body of Kuwanomeiga. The mycological properties of this strain are as follows. A gram-negative bacterium that has motile bacilli. Form yellow colonies in many growth media. Does not produce yellow-green fluorescent dye in King B medium. Aerobic and anaerobic growth.
Does not form endospores or aerial hyphae. Does not reduce nitrate. Inositol, raffinose, cellobiose,
Use melibiose and glycerol. Based on these mycological properties, this strain was identified as a microorganism belonging to Erwinia ananas. The supercooling point of this strain is -2.7 ° C, and the minimum concentration of ice nucleation is 10 ⁶ cells / ml.

As for Erwinia ananas, intestinal bacteria identification kit API20E (manufactured by BioMerieux) and major saccharide resolution assay (Dye, DW (1983): Erwinia: Th)
e “Amylovora” and “Herbicola” groups. Plant Bacter
ial Diseases (Fahy, PC and Persley, GJ Ed.) A
cademic Press, pp. 67-86).

For culturing a microorganism having ice nucleus activity, it is not necessary to use a special method, and a culture method similar to that of a known microorganism having ice nucleus activity can be used. As the medium, any of a synthetic medium and a natural medium may be used as long as the medium appropriately contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth promoting substances. Specifically, for example, a King B medium, an LB medium with sucrose, and the like can be used. When culturing, adjust the culture temperature to 15-25.
C., preferably 18-22.degree. C., pH 5-9, preferably 6-7.

As the active ingredient of the second invention, it is preferable to use a microorganism having ice nucleus activity and showing pathogenicity to plants. If such a microorganism is used, the microorganism can be transmitted from the opening of the plant by the activity of the ice nucleus, whereby the pathogenicity of the microorganism can be exhibited without causing wound infection of the microorganism. Examples of microorganisms having ice nucleation activity and showing pathogenicity to plants include, for example, microorganisms belonging to Pseudomonas syringae, and more specifically, Pseudomonas syringae SEI-1 strain. Can be.

The microorganism having ice nucleus activity, which is the active ingredient of the second invention, may be a viable cell, or may be a processed cell having ice nucleus activity. The treated cells include any treated cells as long as they have ice nucleating activity. Examples of such treated cells include dead cells obtained by a sterilization treatment (for example, an ultraviolet irradiation treatment, a γ-ray irradiation treatment, or the like) that does not destroy the ice nucleus protein on the cell surface layer. As long as it has ice nucleation activity, it can be used as an active ingredient of herbicides whether it is a viable cell or a processed bacterial cell.This means that ice nucleation active proteins derived from microorganisms having ice nucleation activity are mixed in chemical herbicides. Means that promising biological herbicides are obtained.

As the herbicide of the second invention, a microorganism having ice nucleus activity or a treated product thereof may be used as it is,
It may be dissolved or dispersed in a suitable liquid carrier, or mixed with a suitable powder carrier or adsorbed on the powdered carrier to be formulated and used. In this case, examples of the liquid carrier used in the formulation include water, an aqueous solution of skim milk, an aqueous solution of sodium glutamate, an aqueous solution of sorbitol, and the like, and one or more of these can be used in combination. . Examples of the powder carrier used for formulation include gelatin, silk fibroin, zeolite, skim milk and the like, and one or more of these can be used in combination. Further, if necessary, an emulsifier,
A dispersing agent, a suspending agent, a spreading agent, a penetrating agent, a wetting agent, a stabilizer and the like can be added, and the composition can be formulated into a solution, emulsion, oil, wettable powder, powder and the like. For example, a microorganism having ice nucleus activity can be cultured in a medium for growing bacteria such as King B medium at 22 ° C. for 2 days, then suspended in sterile distilled water and used as the herbicide of the second invention. At this time, it is preferable to add a surfactant (for example, Tween 20 (manufactured by Sigma)) to the suspension at a dilution of about 5,000 times and mix it, since the adhesion between the leaf surface and the microorganisms becomes good.

The practical use of a microbial herbicide depends on the possibility of formulation. That is, it is very important to maintain a stable and high level of the characteristics of the target microorganism (ice nucleus activity in the second invention),
It is essential to develop protective agents to maintain stable and high levels of microbial properties. The medium used as such a protective agent must have a high ability to adsorb and retain microorganisms, maintain the characteristics of microorganisms, and be relatively inexpensive and easy to formulate. Microorganism protectants that can be used when formulating the herbicide of the second invention include gelatin, silk protein, silk fibroin and the like. When the preparation is formulated using these protective agents, the preparation can be performed while maintaining the characteristics (ice nucleus activity) of the microorganism having ice nucleation activity at a stable and high level. For example, after the above-mentioned protective agent is added to a suspension of microorganisms having ice nucleation activity, the microorganisms are adsorbed on the protective agent and dried to maintain the ice nucleus activity of the microorganisms at a stable and high level by drying. Can be formulated. At this time, the microorganism concentration of the suspension is preferably about 2 to 10%.
In addition, it is important that microorganisms are sufficiently adsorbed to the protective agent before the drying treatment. For this reason, after adding the protective agent to the suspension, the suspension is allowed to stand at 20 to 25 ° C. for about 1 hour. It is preferable to keep it.

The concentration of the microorganism, which is the active ingredient of the herbicide of the second invention, can be appropriately set according to the dosage form, the purpose of use, and the like. For example, when a microorganism having ice nucleus activity is used by suspending it as described above, the concentration of the microorganism in the suspension is preferably in the range of 10 ³ to 10 ⁹ / ml, preferably 10 ⁶ to 10 ⁹ / ml. can do. At this time, when using Pseudomonas syringae SEI-1 strain as a microorganism having ice nucleus activity, the concentration of Pseudomonas syringe SEI-1 strain in the suspension is 10 ³ /
If it is at least ml, a herbicidal effect can be exhibited. Under the natural environment, the bacteria are sterilized under the ultraviolet irradiation environment of the sun, but even if the bacterial concentration is reduced to 10 ³ / ml, they still have ice nucleating activity. The ice nucleus activity of the SEI-1 strain is maintained for a long time. For example, 10 ⁸ /
When the plant is sufficiently sprayed with mL of Pseudomonas syringae SEI-1 strain, the herbicidal effect can be maintained for 2 weeks or more after spraying.

When the herbicide of the second invention is used, the amount of application can be appropriately set according to the degree of weed growth. The method of spraying is not particularly limited, and examples thereof include intact inoculation and injured inoculation using methods such as application, adhesion, and spraying. The spraying is preferably performed by a sprayer or the like so that microorganisms having ice nucleation activity sufficiently adhere to the leaf surface of the target plant. The wounded inoculation can be performed by, for example, spraying the second invention after damaging the plant by mechanical weeding. By spraying the herbicide of the second invention after mechanical weeding, it is possible to suppress secondary occurrence of the target plant.

The target plant for the herbicide of the second invention is not particularly limited. The target plants for the herbicide of the second invention include, for example, autumn-winter weeds, more specifically, grass weeds (e.g., barley, mugwort, barley, sparrowgrass, spruce blossom, camphor japonicus), legume weeds (E.g., pomegranate, rape), weeds of the family Papilionidae (e.g., minamixa, nominophora), weeds of the lamiaceae family (e.g., scrophulariaceae), weeds of the family Scrophulariaceae (e.g., Daphnia japonicus), weeds of the Brassicaceae (e.g., nazuna) , Rubiaceae weeds (for example, yamgra), and Himeodorikosou.

Since a microorganism having ice nucleus activity has an ice nucleus protein on its surface, it becomes an ice crystal nucleus and freezes or freezes surrounding water droplets. If this property is utilized, it is possible to induce frost damage on the target plant by lowering the temperature or natural frost, and thereby the target plant can be frozen to death and controlled. At this time, for example, natural frost of about −5 ° C.
When the time is from 2 minutes to 2 hours, frost damage by microorganisms having ice nucleation activity can be sufficiently induced, and the target plant can be efficiently controlled. In addition, even if spontaneous frost of about −2 to 3 ° C. is present for 30 minutes or more, preferably for a long time, it is possible to sufficiently induce frost frost damage caused by microorganisms having ice nucleation activity, Control becomes possible.

In using the herbicide of the second invention, it is desirable to sufficiently consider the place of use. That is, since the herbicidal effect of the microorganism having the ice nucleus activity can be exerted non-specifically, freezing and frost damage caused by the microorganism having the ice nucleus activity is also exerted on useful plants other than the target plant (for example, crops such as vegetables). May be induced. Therefore,
In using the herbicide of the second invention, it is preferable to consider that the place of use is limited. For example, places where there are no useful plants, non-agricultural lands (railroad tracks, parks,
It is preferably used in places where useful plants are not affected by frost damage induced by microorganisms having ice nucleation activity. In addition, as shown in the following examples, turfgrass such as Koryo turf is not affected by frost damage induced by microorganisms having ice nucleation activity and exhibits strong frost resistance, so it is used in turfgrass cultivation fields and golf courses. It is also preferable to do so.

A third aspect of the present invention is a herbicide comprising the herbicide of the first invention or the herbicide of the second invention and one or more chemical herbicides. As the chemical herbicide, any known chemical herbicide may be used. For example, commercially available chemical herbicides such as those for paddy fields, fields, and non-agricultural lands can be used. More specifically, railway tracks,
Glyphosate herbicides used as chemical herbicides for non-cultivated lands such as parks, embankments, parking lots, slopes, etc. (for example,
"Round Up" (manufactured by Monsanto Japan Limited))
A glyphosinate herbicide (manufactured by Hoechst) and a bipyridylium herbicide (for example, paraquat and diquat (manufactured by Zeneca)) can be exemplified. As a herbicide for paddy fields, a sulfonylurea herbicide can be exemplified. .

When the herbicide of the first invention or the herbicide of the second invention and the chemical herbicide are mixed and used, the frequency or amount of application of the chemical herbicide can be reduced. Chemical herbicides generally have an excellent feature of having an immediate and powerful herbicidal effect, but have the problem of environmental pollution.Therefore, it is extremely difficult to reduce the frequency or amount of application of chemical herbicides. Useful.

When weeds (for example, S. pallidum) are used in combination with the herbicide of the first invention and the chemical herbicide, the period of use of the weeds is such that the growth of the microorganism, which is the active ingredient of the first invention, is active. Time (for example, from April to September) is preferred,
If used at this time, the frequency or amount of application of the chemical herbicide can be greatly reduced. In addition, since the herbicidal effect of microorganisms having ice nucleation activity is induced by a decrease in temperature and frost, by using microorganisms having ice nucleation activity together with a chemical herbicide, especially when the temperature is low (for example, in October) To March), the number of times or amount of application of the chemical herbicide can be greatly reduced.

[0041]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. [Example 1] Isolation of ice-nucleated bacteria from mulberry and mulberry mulberry Leaf or insect mulberry plant of mulberry plant was put in a mortar, and an appropriate amount of sterilized water was added thereto and triturated sufficiently. (Peptone 10g, yeast extract 5g, salt 10
g, agar 15 g, distilled water 1 L) at 28 ° C. A single colony obtained by culturing for 3 days was further purified to obtain an isolated strain.

The presence or absence of ice nucleus activity of these isolates was assayed by the following method. That is, after culturing the isolated strain at 22 ° C. for 1 to 2 days using the LB plate medium method, the cells are placed in sterile water.
The test solution was suspended at about 10 ⁸ / mL. Approximately 1 mL of the test solution was placed in an Eiken test tube (Sterile F Spitz, 10 mL
Volume) and immersed in an antifreeze solution at -5 ° C. for about 30 minutes, and the presence or absence of ice nucleus activity was visually determined by the presence or absence of freezing of the bacterial suspension. The strain that showed ice nucleus activity was skim milk medium (10 g skim milk, 1.5 g sodium glutamate, 100 mL)
And stored at -30 ° C. When used again, it was dissolved and cultured in LB medium.

According to the above method, two ice-nucleating bacteria were isolated from the mulberry leaf surface (Ni23 strain and TM2 strain), and two strains (Mei40 strain and Mei7 strain) were isolated from the body of Kuwanomeiga. Therefore, the bacteriological properties of these strains were examined, and identification of each strain was attempted. As a result, the Ni23 strain had the following mycological properties. Gram-negative bacteria Motile bacilli with polar flagella produce yellow-green fluorescent pigment in King B medium Levan production (-), oxidase activity (-), potato tuber rot (-), arginine hydrolysis (-), Based on the above microbiological properties having the characteristic of tobacco hypersensitivity reaction (+), Ni23 strain was identified as a microorganism belonging to Pseudomonas syringae,
It was named Pseudomonas syringae Ni23 strain.

The TM2 strain had the following mycological properties. Gram-negative bacterium Motile with perihaired bacilli Forms yellow colonies in many growth media Endospores, does not form aerial hyphae Does not reduce nitrate Uses inositol, raffinose, cellobiose, melibiose, glycerol Based on the above bacteriological properties, the TM2 strain was identified as a microorganism belonging to Erwinia ananas and named as Erwinia ananas TM2 strain.

The Mei40 strain had the following mycological properties. Gram-negative bacteria Motile bacilli with polar flagella produce yellow-green fluorescent pigment in King B medium Levan production (-), oxidase activity (-), potato tuber rot (-), arginine hydrolysis (-), The Mei40 strain was identified as a microorganism belonging to Pseudomonas syringae and named as Pseudomonas syringae Mei40 strain based on the above microbiological properties having the characteristic of tobacco hypersensitivity reaction (+).

The Mei7 strain had the following mycological properties. Gram-negative bacterium Motile bacillus with perihairs Forms yellow colonies in many growth media Does not produce yellow-green fluorescent dye in King B medium Aerobic and anaerobic growth Endospores, ki Does not form medium hyphae Does not reduce nitrate Utilizes inositol, raffinose, cellobiose, melibiose, and glycerol

From the above mycological properties, the Mei7 strain was identified as a microorganism belonging to Erwinia ananas and named as Erwinia ananas Mei7 strain. For the Pseudomonas syringae Ni23 strain, Erwinia ananas TM2 strain, Pseudomonas syringae Mei40 strain, and Erwinia ananas Mei7 strain, the recruitment of these strains to the Institute of Biotechnology, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology was rejected. Guaranteed by applicant.

Example 2 Isolation of Ice-Nucleating Active Bacteria from Solidago altissima Ice-nucleating Active Bacteria were isolated from Solidago altissima in the following manner. In May 1998, a black-brown lesion was identified on the leaf blade of A. niger, which grows on a farm road in Tsukuba, Ibaraki Prefecture, as a bacterial disease. There is no known bacterial disease of S. americana, and there is a possibility of a new strain, so we decided to isolate the bacteria. As a result, it was confirmed that the isolated bacteria showed a clear pathogenicity in both healthy and intact inoculation methods, and was a new pathogenic bacterium.

The isolated bacterium was Gram-negative, and exhibited motility in rods having extremely flagella. In addition, since a yellow-green fluorescent dye was produced in King B medium (manufactured by Eiken Chemical Co., Ltd.), it was confirmed that the medium belongs to the genus Pseudomonas. Furthermore, the LOPAT method for simple identification of plant pathogenic bacteria showed levan production (-), oxidase activity (-), potato tuber rot (-), arginine hydrolysis (-), and tobacco hypersensitive reaction (+).
The strain was identified as belonging to the LOPAT; Ib group, and thus the strain was identified as a microorganism belonging to Pseudomonas syringae. Although this strain has pathogenicity in Aspergillus niger as described below, since microorganisms belonging to Pseudomonas syringae showing pathogenicity to the plant are not known at all,
The strain was identified as a new pathogen, ie, a new strain, and named Pseudomonas syringae SEI-1 strain. This strain is
Since the contract with the Institute of Biotechnology and Industrial Technology is denied, the applicant guarantees the sale.

[Example 3] Ice nucleus activity of a novel ice nucleus active bacterium Pseudomonas syringae S isolated in Examples 1 and 2
The ice nucleus activity of EI-1, Strain Ni23, Mei40 strain, Erwinia ananas TM2 strain, and Mei7 strain was compared with known non-ice nucleus active bacteria. The comparison of ice nucleation activity should be evaluated with respect to two factors: the supercooling temperature (temperature at which ice nucleation activity starts) in a bacterial suspension of about 10 ⁸ / mL and the minimum concentration of ice nucleation at -5 to -6 ° C Was performed. By this evaluation, it can be determined that the higher the supercooling temperature and the lower the minimum concentration of ice nucleation, the higher the ice nucleus ability. The supercooling temperature was measured by a supercooling temperature measuring device designed as a prototype device. At that time, the sample volume was 20 μL, and the cooling rate was 1 ° C./min.

The minimum concentration of ice nucleation is measured by preparing a stepwise bacterial concentration test solution in a test tube in a volume of 1 mL by usually serially diluting the bacterial suspension to be measured 10-fold. This was carried out by keeping the mixture in the air-cooled or antifreeze solution for about 30 minutes at the above-mentioned temperature, and examining to what concentration the suspension was frozen. Here, the method using air cooling refers to a method in which a test tube containing ice-nucleated bacteria is placed in a constant temperature chamber at a constant temperature (-6 ° C to 0 ° C), and the state of ice nucleation of the sample is examined after 30 minutes. means. Also, the method in antifreeze is
This means a method in which a test tube containing ice-nucleated bacteria is placed in a refrigerant kept at a constant temperature using ethylene glycol as a refrigerant and the change over time is examined. At the same time, the number of bacteria (the number of viable bacteria) in each dilution was examined, and the bacterial concentration was expressed as the number of viable bacteria per mL. Ice nucleation temperature of various ice-nucleating bacteria (supercooling point)
Table 1 shows the minimum ice nucleation concentration (CFU / mL).

[0052]

[Table 1]

As is clear from Table 1, all five strains of Pseudomonas syringae SEI-1, Ni23 and Mei40, and Erwinia ananas TM2 and Mei7 showed high ice nucleus activity. In particular, the Pseudomonas syringae SEI-1 strain exhibited the best ice nucleating ability with an ice nucleation temperature of -1.8 ° C and a minimum ice nucleation concentration of 10 ⁴ / mL, and was stable. Meanwhile, Pseudomonas syringe Ni23
The strain and the Mei40 strain, and the Erwinia ananas TM2 strain and the Mei7 strain had ice nucleation minimum concentrations of 10 ⁴ to 10 ⁶ / mL, and had somewhat lower ice nucleus activity than Pseudomonas syringae SEI-1 strain.

Example 4 Pathogenicity of SEI-1 Strain to Solidago altissima Inoculation of a suspension of SEI-1 strain (concentration of 10 ⁹ / ml) into Solidago serrata (young and mature seedlings) having different growth stages. , SEI-
We evaluated the herbicidal effect of Aedes aegypti using one pathogenic function. At that time, the inoculation time was set to three wards on June 12, June 5, and November 1 to examine the appropriateness of the spraying time. Inoculation was performed in two zones: intact vaccination and injured vaccination. Sprays and smears may be included in intact inoculations. Spray inoculation is 10 ⁹ / mL
(Tween 20, 5000-fold diluted solution) is sprayed by a sprayer so that the bacterial solution sufficiently covers both sides of the leaves. For wound inoculation, about 10 sewing needles are bundled together, and the tip is stuck to the surface of the leaf with 10 ⁹ / mL of bacterial solution, and about 10 spots per leaf are slightly damaged. (20 to 25 ° C.) and observe for about 2 weeks. Furthermore, as young seedlings and mature seedlings inoculated at the time of June, S. aureus with a height of about 10 cm and 60 cm, respectively, and as young seedlings at an inoculation time of November, S. aureus with a height of about 10 cm were used. Table 2 shows the obtained results.

[0055]

[Table 2]

As is apparent from Table 2, when a suspension of the SEI-1 strain was sprayed and inoculated on young seedlings of S. serrata with a height of about 10 cm, initial lesions appeared about one week after the inoculation. It became gangrene spots and spread, which made it easier to cause A. niger. In addition, the disease spread to the surrounding young seedlings. The same was true for wound inoculation, but the lesions developed quickly. In addition, comparing the effects of spray inoculation and wound inoculation on the pathogenic expression of plants, wound inoculation showed a wider range of application.
For example, as shown in Table 2, even mature seedlings showed moderate progression of lesions.

However, in November, when the temperature dropped, both the spray inoculation and the injured inoculation showed almost no infection and no symptoms. Therefore, it has been found that it is particularly preferable to spray younger plants during the high temperature period from April to September in order to herbicide the S. pallidum with the SEI-1 strain. In addition, since the SEI-1 strain easily penetrates the plant through the wound, it has been found that secondary emergence can be suppressed, for example, by spraying after mechanical weeding.

Example 5 Pathogenicity of SEI-1 Strain to Various Weeds and Useful Plants A suspension of the SEI-1 strain (concentration: 10 ⁹ / m
l) was inoculated by spraying or wounding, and the pathogenicity of the SEI-1 strain against various weeds and useful plants was examined. The inoculation time and the experimental time were set in early June. After the inoculation, they were placed in a greenhouse at 20-25 ° C and observed for about 2 weeks. Table 3 shows the obtained results.

[0059]

[Table 3]

As is apparent from Table 3, in the case of spray inoculation, the SEI-1 strain is pathogenic only to the S. pallidum and has no pathogenicity to other various weeds and useful plants. It showed extremely high host selectivity. In the case of the wounded inoculation, gangrene was formed on some plants, but the spray inoculation showed no symptom. As described above, it was found that the SEI-1 strain has an excellent advantage as a microbial herbicide in that it does not harm plants other than the target weed, S. pallidum.

[Example 6] Induction of frost damage in barley by the ice nucleus activation function of SEI-1 strain (relation with low-temperature treatment time) An experiment was carried out to cause frost damage in canine using ice nucleus-active bacteria SEI-1 strain. Was. The implementation was October 1998, and young seedlings of barley growing in the field were transplanted into pots. It was sprayed with a bacterial solution at a concentration of 10 ⁸ / mL, contacted at −5 ° C. for a certain time, returned to the greenhouse, and observed for about 1-2 weeks. As a control, a P. syringae SM-2 strain having no ice nucleus activity was used. The time of low temperature treatment of dog wheat at -5 ° C was changed, and the frost damage rate at each treatment time was evaluated. Note that “Cont (DW)” used in the following tables means a control group to which distilled water was applied. “M” represents minutes, and “h” represents hours.

The frost damage rate of the target plants after Experimental Example 6 was evaluated from the ratio of useful plant leaves that caused frost damage according to the following evaluation criteria. -: 0-10% +: 11-50% ++: 51-80% +++: 81-100% The obtained results are shown in Table 4.

[0063]

[Table 4]

As is apparent from Table 4, the control group was -5 ° C.
The contact within 2 hours of the above did not cause any damage at all or was slight. On the other hand, in the section to which the ice-nucleated bacterium SEI-1 strain (10 ⁸ / ml) was sprayed, contact for more than 10 minutes caused severe damage (frost damage) and most of the cells died. As a result, it was found that if there was natural frost for about 30 minutes at -5 ° C (or about 2 hours at -5 ° C, which was lightly damaged in the control plot), it was possible to control winter weeds with ice-nucleating bacteria. did.

[Example 7] Induction of frost damage in barley by the ice nucleus activity function of SEI-1 strain (difference in ice nucleus activity function by live and dead SEI strains) by live and dead SEI-1 strains We investigated the differences in the activity of ice nuclei and the relationship between the bacterial concentration and the damage rate of frost damage on wheat. The dead bacteria were obtained by irradiating the bacterial solution concentration with an ultraviolet lamp directly under 30 cm for about 15 minutes. This was diluted in the same manner as in the case of the viable bacteria of Example 6 and tested. As a control, only distilled water was sprayed. These were subjected to low-temperature treatment at -5 ° C for 30 minutes, and the frost damage rate was evaluated. Table 5 shows the obtained results.

[0066]

[Table 5]

As is apparent from Table 5, the optimal bacterial concentration for inoculating ice nucleated bacteria on dog wheat is 10 ⁹ -10 ⁸ / ml.
And was found to be particularly effective during frosting at -5 ° C for 30 minutes to 2 hours. When weeding canine, the concentration of viable bacteria is preferably 10 ⁷ / ml or more, but low concentration (for example, 10 ² / m
In l), a slight herbicidal effect was observed. On the other hand, even the dead bacteria of the SEI-1 strain irradiated with ultraviolet light showed a sufficient ice nucleus activation function. This result indicates that the herbicidal effect is not significantly reduced even if the bacterial count on the leaf surface is reduced by external factors such as ultraviolet rays. It was found that the ice nucleus activation function was maintained for a long period of time (this analogy was experimentally confirmed in Example 8 below). In addition, it has been found that even a UV-killed bacterium has an ice nucleating activity, so that it is not always necessary to use a living bacterium when formulating a microbial herbicide, and the application range of the formulation method can be expanded.

[Example 8] Persistence of the effect of the SEI-1 strain on ice nucleus activity inducing frost damage to barley To what extent the effect of preventing the damage to frost damage on barley by the SEI-1 strain was examined. The 10 ⁸ / ml strain was sufficiently sprayed on the plants with a sprayer, kept in a greenhouse for a predetermined period, and then subjected to a low-temperature treatment. As a control, distilled water was sprayed and the same treatment was performed. In addition, the frost damage experiment of the dog wheat was treated at -5 ° C for 30 minutes to examine the persistence of the effect, and the frost damage damage rate was evaluated. Table 6 shows the obtained results.

[0069]

[Table 6]

As is clear from Table 6, the efficacy was maintained for 2 weeks or more after application of the SEI-1 strain. As described above, persistence of two weeks or more was confirmed even when the viable cells were used. In addition, long-term maintenance can be expected by adding protection operations for ice core proteins. Thus, a useful perspective for formulation was obtained.

[Example 9] Induction of frost damage in barley by the ice nucleating activity of SEI-1 strain (effect of combined use of herbicide) Herbicide (Round-up (manufactured by Nippon Monsanto)) alone, SEI-1 strain alone or both The mixed solution of (1) and (2) was dispersed in young barley seedlings with a sprayer and treated at -5 ° C for 10 minutes or 30 minutes, and then returned to a greenhouse (20-25 ° C), and the degree of frost damage was observed. At this time, 10 ⁸ / ml viable bacteria were used as the SEI-1 strain. The mixture ratio of the herbicide and the bacterial suspension was 1: 1 and distilled water was sprayed as a control. Although damage from frost damage is seen relatively early, immediately after the treatment, to 2 days, the appearance of damage by the herbicide usually takes 1-2 weeks, so observation was made for about 3 weeks. In this way, the rate of frost damage of dog wheat by the combined use of chemical herbicides was evaluated. Table 7 shows the obtained results.

[0072]

[Table 7]

The damage rate was evaluated in the following four stages. +++ Extremely significant ++ Significant + Normal-N / A The damage rate marked (*) in the table means that both frost damage and herbicide damage are included. In the table, "R
`` A '' means herbicide round-up, `` X '' means dilution factor, `` X 100 RA '' means 100-fold round-up herbicide, `` X 100 RA + SEI-1 '' Dilution ratio
It means a mixture of 100 times herbicide and viable SEI-1 strain at a mixing ratio of 1: 1.

As is clear from Table 7, frost damage appeared early after the low-temperature treatment, and damage caused by the chemical herbicide developed after a relatively long time. Therefore, almost all died early (about 80%) due to frost damage, and the remaining healthy leaves died further with chemical herbicides, resulting in a damage rate of almost 100%. In the case of chemical herbicides alone (X 100, X 500), 100% died similarly.

As is clear from Table 7, when a 100-fold and 500-fold diluted solution of the chemical herbicide was sprayed alone, a remarkable herbicidal effect was exhibited at any dilution ratio.
No herbicidal effect was observed when the dilution ratio was 1000 times. On the other hand, when ice nucleation active bacteria were mixed with the chemical herbicide, the degree of damage was similar to that when the chemical herbicide was applied alone.However, the enhancement of the herbicidal effect by mixing the ice nucleation active bacteria was not improved. I could not confirm. In addition, the effects of ice nucleation active bacteria on frost damage were not reduced by the addition of chemical herbicides.

[Example 10] Induction of frost damage on barley by various ice-nucleating bacteria Ice-nucleating bacteria other than strain SEI-1 (Pseudomonas syringae Me)
i40 strain and Ni23 strain, Erwinia ananas Mei40 strain and TM2 strain)
In order to confirm whether or not the same effect was also observed, live germs of various ice-nucleating bacteria (concentration: 10 ⁸ / mL) were sprayed on the wheat with a sprayer and contacted at -5 ° C for 10 or 30 minutes. After that, it was transferred to a greenhouse and the frost damage rate was observed. Table 8 shows the obtained results.

[0077]

[Table 8]

As is evident from Table 8, all the ice-nucleating bacteria were as effective as the SEI-1 strain. From these results, it was found that all the strains can be used for herbicidal use similarly to the SEI-1 strain. [Example 11] Induction of frost damage of various winter weeds by the function of ice nucleus activity of SEI-1 strain In order to know the frost damage induction effect of SEI-1 strain on winter weeds other than dog wheat, bacterial fluid (10 ⁸ / ml) was applied to various plants. Spray at -5 ℃
And then placed outdoors (natural temperature) to evaluate the frost damage rate. In addition, what sprayed distilled water was used as a control. Table 9 shows the obtained results.

[0079]

[Table 9]

As is clear from Table 9, spray inoculation of the SEI-1 strain showed an effect on most winter weeds. That is, in the case of no treatment, there was almost no effect even at a low-temperature contact (−5 ° C.) for 3 hours, but severe damage (frost damage) was induced even with a contact time of about 30 minutes by inoculation of the SEI-1 strain.
Some of the weeds were originally sensitive to low temperatures. [Example 12] Induction of frost damage on useful plants by the ice nucleation activity function of SEI-1 strain To know the degree of damage to useful plants when SEI-1 strain was sprayed, frost damage on turf and Komatsuna, a typical winter crop The damage rate was evaluated. Table 10 shows the obtained results.

[0081]

[Table 10]

As is evident from Table 10, turf (Koroshiba)
Was extremely low temperature resistant and showed little effect of the SEI-1 strain. On the other hand, Komatsuna was also more resistant than winter weeds and the like, and caused minor damage. As described above, some types of useful crops were found to have a small effect. [Example 13] Evaluation assay of various adsorption protective agents for the preparation of ice nucleation active bacteria (SEI-1 strain) Suspension of ice nucleation active bacteria (SEI-1 strain) (3.3 × 10 ⁸ / ml) ) Was added with 6 to 10% of the following adsorption protective agents, and the mixture was stirred well. The ice nucleation active bacteria were adsorbed to the adsorption protective agent by allowing to stand for about 1 hour, and then the supernatant was removed by centrifugation (5000 rpm, 15 min). Next, the sample solution was kept in a thermostat at 22 ° C. for one week, the sample aqueous solution was gradually evaporated by natural drying, and naturally dried for about one day to obtain a dried and solidified state.

The gelatin used as the adsorption protective agent was
DIFCO company, silica gel is MERCK product name Kieselgel 60 (7
0-230 mesh ASTM), skin milk is from DIFCO. Fibroin powder was prepared as follows. The silkworm silk was refined with a 0.2% aqueous sodium carbonate solution at 95 ° C. to obtain a silk fibroin fiber, which was then placed in an aqueous lithium bromide solution and dissolved at 55 ° C. for 30 minutes. This was put into a cellulose dialysis membrane, replaced with pure water to prepare an aqueous solution of silk fibroin, and once frozen at −30 ° C., dried under reduced pressure to prepare powdered silk fibroin. Sericin powder was prepared by placing silkworm cocoons in distilled water and boiling to remove sericin covering the surface of the cocoons, and then freeze-drying the aqueous solution.

The ice nucleating activity of the protective agent adsorbing the ice nucleating bacteria was assayed as follows. The same amount of sterilized distilled water as before drying was added to the dried sample, and the mixture was stirred well. Using this as a stock solution, a 10-fold serial dilution was prepared. These at −5 ° C.
It was contacted for 0 minutes and the strength of the ice nucleus activity was determined by judging to what dilution concentration the ice was frozen. Table 11 shows the obtained results.

[0085]

[Table 11]

As is clear from Table 11, the maximum dilution ratio for maintaining the ice nucleus activity in the distilled water group used as the control group was 10, whereas the ability of silk fibroin powder and gelatin to maintain the ice nucleus activity was higher than that in the control group. 1000 times the capacity (1000 dilution)
0). On the other hand, sericin, silica gel, and skim milk, which has been conventionally used as a medium for freezing or freeze-drying bacteria, hardly exhibited an adsorption effect, a retention effect, or a high quality property maintaining effect. In addition, in a freezing treatment experiment performed separately, skim milk showed a high protective ability. From these facts, it was revealed that silk fibroin powder and gelatin have excellent ability as an adsorption protectant for preparing a dry powder of ice-nucleating bacteria.

[0087]

Industrial Applicability According to the present invention, there is provided a microbial herbicide capable of controlling Aedes aegypti. Further, in particular, when Pseudomonas syringae SEI-1 strain is used, it is possible to selectively control S. pallidum. Further, the present invention provides a microbial herbicide utilizing a microorganism having ice nucleus activity. In particular, if a microorganism having ice nucleus activity and showing pathogenicity to plants is used, a more efficient herbicidal effect can be obtained. A herbicide using a microorganism having ice nucleation activity uses the action in the natural world as it is, and therefore has a small burden on the environment and does not cause harm to other organisms. The herbicide using the microorganism having the ice nucleus activity is characterized by a low environmental load, high safety, and is economically advantageous. For preparation of a microorganism having ice nucleus activity, gelatin or silk fibroin can be used as a protective agent for maintaining a stable and high level of ice nucleus activity. By using such a protective agent, it becomes possible to formulate a preparation while maintaining the characteristics of the target microorganism (ice nucleus activity in the present invention) at a stable and high level. Further, the present invention provides a herbicide containing the microbial herbicide and the chemical herbicide. As a result, the number of times and the amount of chemical herbicides used can be reduced, which contributes to environmental conservation.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-58-122020 (JP, A) JP-A-59-98627 (JP, A) JP-A-60-102186 (JP, A) 115223 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 63/00 A01N 25/00 A01N 25/04 A01N 25/06 A01N 25/10 A01N 25/22 A01N 25/24 C12N 1/20

Claims (4)

(57) [Claims] 1. A herbicide comprising, as an active ingredient, a microorganism belonging to Pseudomonas syringae and exhibiting pathogenicity against S. americana. 2. A herbicide comprising the herbicide according to claim 1 and one or more chemical herbicides. 3. A method for controlling Solidago altissima using the herbicide according to claim 1 or 2. 4. The method according to claim 1, wherein the herbicide is intactly inoculated or inoculated into S. pallidum by one or more methods selected from the group consisting of application, adhesion and spraying.
The control method according to claim 3.