JPH0734692B2 - Bacterial composition for preventing frost damage on plants - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition containing ice nucleation-deficient bacteria which antagonize ice nucleation bacteria.

[0002]

Frost sensitive crops are damaged when ice formation occurs in their tissues. Ice formation in the tissue mechanically destroys the cells, resulting in what is known as frost damage. Most frost-sensitive crops do not have a mechanism to resist ice formation in their tissues and therefore ice formation occurs in their tissues, resulting in frost damage.

Thus, these plant species are not frost resistant. However, it is known that the water in the plant tissues of these species is inert to supercooling, ie it is kept in the liquid state below 0 ° C.

Some bacteria have ice nucleation ability,
This limits the supercooling of water when such bacteria are present on the surface of plants. Strains with Pseudomonas syringae, Erwinia herbicola or Pseudomonas fluorescens catalyze ice formation and form ice below 1 ° C.

Therefore, an economical and effective means for avoiding ice nucleation by ice nucleating microorganisms and sustaining plant protection during the period of frost damage to plants without damaging the plants. Is desired to be developed.

As prior art, Am.Phytopah Soc.Annual
At the Meeting (22-28 August 1980), Gendou, S. A report made by E (Lindow, SE) `` Frost Damage To Pear Reduced By Antagonistic Bac
teria, Bactericides and Ice Nucleation Inhibitors
(Reduction of frost damage on pears by antagonistic bacteria, fungicides and ice nucleating bacteria inhibitors) ". U.S. Pat. No. 4,045,91
Nos. 0 and 4,161,084 describe the prevention of frost damage by the use of microorganisms deficient in ice nucleation ability.

[0007]

DESCRIPTION OF THE INVENTION The present invention provides a composition for preventing ice nucleation. The inventors have isolated ice nucleation-deficient microorganisms that can grow on host plants, particularly those that compete for the essential limiting nutrients provided by the host plants and antagonize the ice nucleation microorganisms. By applying the ice nucleation-deficient microorganism to a plant early in the growth cycle, the microorganism was allowed to colonize the plant, and the survival and colonization of the ice nucleating microorganism was prevented.

[0008] Microorganisms deficient in ice nucleation ability can be isolated from natural or mutagenized sources and, in addition, transformed to impart desirable properties to the microorganism. Beneficial effects other than frost protection have also been observed.

Frost damage to a host plant is prevented by donating to the host plant an ice nucleation-deficient microorganism capable of growing in the host plant, which antagonizes the host-derived ice nucleation microorganism. . The ice nucleation-deficient microorganism is selected from wild microorganisms or microorganisms that have been subjected to a mutation treatment by a method capable of confirming the ability to antagonize ice nucleation microorganisms.

Desirable properties of a microorganism deficient in ice nucleation are
Furthermore, a particular genetic capacity can be enhanced by introducing it by transformation. Microorganisms deficient in ice nucleation are applied to host plants early in the growth cycle of the plant, prior to or during the period when frost damage can occur.

The ice nucleation deficient microorganism is collected from an endophytic microorganism or a mutated microorganism of a plant or another source, and discriminates between an ice nucleation deficient bacterial species and an ice nucleation deficient bacterial species. Select according to the test method to be obtained. Mutagenesis can be performed by any conventional method including chemical methods such as treatment with ethyl methanesulfonate or nitrosoguanidine,
Alternatively, it can be performed by irradiation with ultraviolet rays or X-rays.
Wild microorganisms are isolated from the predominant microflora present in healthy leaves of frost-sensitive crops.

As microorganisms or bacteria, it is possible to use several microorganisms which can survive in plants and grow during plant growth. Since many microbial species or strains can be used as antagonistic microorganisms, it is not possible to point out the only species used as antagonistic microorganisms.

Pseudomonas, Erwinia, Corynebacterium, Xantomonas
Strains belonging to homonas and Bacillus are particularly useful. In addition, the endophyte strain A5 of these microorganisms
10-1 (ATCC # 31947), strain A506 (A
TCC # 31948), strain A501 (ATCC # 31
949), strain A505 (ATCC # 31950), strain A526 (ATCC # 31951), and strain 42B.
-10 (ATCC # 31952) has been deposited with the American Type Culture Collection (ATCC).

In separating the microorganisms, first, the microorganisms are spotted on the surface of a limiting medium containing the limiting nutrients usually found on the leaf surface of the host in respective ratios. It is expected that limiting the available nutrients will limit the total number of cells on the surface of the medium. The medium usually contains sugars and amino acids utilized by the microbiota of the host, especially dicarboxylic amino acids or their monoamides, in amounts not sufficient for the microbiota.

Usually, one or more uronic acids and inorganic salts are further included. These components are incorporated into a suitable gel, such as agar. Of the active ingredients, sugar is about 80-
95% by weight, total amino acids and total inorganic salts are each present in the range of about 2-10% by weight. The total amount of nutrients is generally about 0.01-2% by weight, based on the gel medium,
It is usually 0.1 to 1% by weight.

The medium employed supports the limited growth of almost randomly selected foliar bacteria. The bacteria grown on this nutrient medium selectively deplete the nutrients in the medium around the growth region of the bacteria. Antagonist microorganisms that deplete the nutrients essential for the survival and growth of ice nucleating bacteria spotted randomly are separated by allowing the microorganisms to grow on the surface of the gel nutrient medium to form colonies and to deplete the nutrient source. can do.

Then, a suspension of ice nucleating cells such as Pseudomonas syringer or Erwinia herbicola is sprayed onto the surface of the medium containing the colonies. Ice nucleating bacteria sprayed onto the medium plate grow between spot areas containing antagonistic bacteria.

Antagonistic bacteria deplete the nutrients necessary for the growth of ice nucleating bacteria and are used to prevent the growth of ice nucleating bacteria. Antagonistic bacteria are detected by the non-growth of spray-applied ice nucleating bacteria around a small area of the nutrient medium surface spotted with antagonistic bacteria.

With respect to the antagonistic bacteria which showed a positive reaction in the above-mentioned test, it is confirmed by a radial diffusion test or the like that an anti-substance which inhibits the growth of ice nucleating bacteria is not produced. This confirmation is performed by spotting the selected antagonistic bacteria on a rich medium such as the above-mentioned restriction medium and King's medium B.

This method is performed as described above, and
Record the presence or absence of growth in the immediate vicinity of the spotted colony. Antagonistic bacteria that inhibit the growth of ice nucleating bacteria on limiting medium but not on King's B medium are isolated bacteria that consume the nutrients essential for ice nucleating bacteria and prevent the growth of the bacteria. It indicates that there is.

Antagonists are further selected by greenhouse and laboratory methods. Host plants in the early stages of growth are sprayed with the selected antagonist and allowed to grow for a short period of time. The plants inoculated with the antagonist and the plants not inoculated as a control are then inoculated with ice nucleating bacteria capable of growing on the host plants. After culturing for a relatively short time in a moist chamber, the plants are dried and placed in an environmental chamber regulated at about -5 ° C.

After exposure to a temperature of -5 ° C. for about 1 hour, all plants were left under growth conditions, for example 20 ° C., for 1 day, at which point the leaves having the symptoms of frost damage, ie wetted and weakened. Count leaves. A significant reduction in the number of wet leaves indicates that it is an effective antagonistic bacterium.

When selecting an ice nucleation-deficient strain from among cells treated with mutations of an ice nucleation strain, a method different from the above method can be used. The mutagenized cell mixture was plated on a suitable nutrient gel medium surface and after culturing for a limited period of time, the cultured cells were replicated on the surface of a paraffin-coated aluminum foil, and the aluminum foil was cooled to the ice formation temperature, For example, it is maintained at -5 ° C or -9 ° C.

Wild-type colonies possessing ice nucleation ability are immediately frozen and can be distinguished from mutant strains lacking ice nucleation ability. These mutants remain liquid when droplets of water are sprayed onto the sheet. Furthermore, it is confirmed by the above-mentioned method that these mutant strains lack ice nucleation ability and that they maintain the host range of the strain and the effective antagonistic ability to the wild-type strain.

Microorganisms deficient in ice nucleation ability can be changed in their properties by introducing new genetic ability by a conventional method. These techniques often involve transformation, transduction and conjugation. Various genetic abilities to confer on antagonistic microorganisms include antibiotic resistance, bacteriocin production, host range, growth characteristics such as colicin resistance, nitrogen fixation, or the like.

Then, a desired microorganism is selected by using a label capable of selecting a transformant or a zygote. This label includes antibiotic resistance, colicin resistance, heavy metal resistance,
It includes conversion of prototrophic strains to auxotrophic strains, or the like.

The target bacteria can be used in a wide range of crops, especially vegetables, fruit trees, grains and nut trees. Host plants include lemons, oranges, such as citrus plants such as navel, grapefruit, tomatoes, potatoes, cereals, legumes such as soybeans, and the like.

Depending on the nature of the plant, the plant part to which the antagonistic microorganism is to be applied, various methods and compositions for applying the microorganism to the plant can be employed. Furthermore, it is desirable to use a mixture of two or more microorganisms, rather than one microorganism.

The number of cells per unit formulation depends on whether the formulation is a dry formulation or a wet formulation.
In the case of wet formulations such as foliar sprays, suspensions, aerosols, sprays etc., the cell number is generally about 10 ⁵ to 10 ¹⁰ cells / ml. Generally, about 10 ⁴ per 1 g of fresh leaf weight in the application period
It is desirable that the number is 10 to ¹⁰ . For dry formulations, the cell number is generally about 10 ⁴ to 10 ⁹ cells per gram of formulation.

The number of cells in the formulation is necessary to compete with the microorganisms originally present in the environment and to settle on the host plant, taking into account the relatively high killing rate that occurs when the spray is used. Must be a number. The cell number should be sufficient to allow colonies to form within about a week or less after application.

Aqueous formulations include biological or chemical agents such as surfactants such as nonionic surfactants, pigments, nutrients, buffers, penetrants for introducing cells into the leaves, herbicides, insecticides. A small amount of specific agricultural chemicals can be contained.

Dry formulations include inert powders, antimicrobial stabilizers, salts, anti-caking agents, nutrients, buffers, film formers,
Various additives including biological or chemical pesticides such as herbicides and insecticides can be contained. Various additives are added in the range of about 1 × 10 ^-4 to 1% by weight.

Further additives may be added under special conditions as described later. One of the methods of applying the antagonistic bacteria is a method of applying the fungi to plant seeds or bulbs or tubers (seed inoculation method). Bacteria are blended as a dry powder according to a conventional method.

A cell-containing powder formulation prepared by mixing about 1 part by volume of a cell-containing gum suspension with an inert powder carrier, for example 4 parts by volume of talc, is particularly advantageous. This gum suspension is 1
Volume of dense cell suspension (about 10 ⁹ -10 ¹¹ cells / m
l) and about 10 volumes of a dilute magnesium salt solution, and then this mixture is added to a concentrated aqueous suspension of natural gum 10
Prepare by mixing with the volume. Gum is about 90-9
9% by weight of the mixture, which is first used as a 10-30% by weight suspension.

The mixture is dried and ground to a uniform fine powder. A slightly moistened seed or bulb or tuber is contacted with the powder. When seeded, the cells settle on germinated stems and leaves while the plants emerge from the soil.

The powder formulation can also be used by the spray method. In this case, the bacteria are conveniently applied to the leaves so that they are inoculated at a ratio of about 10 ⁷ to 10 ⁸ bacteria per 1 g of fresh leaf. Dust inoculation is particularly preferred on sunny days in hot climates, with relatively low humidity, from mid-afternoon to late afternoon.

A typical method other than the above for fixing the antagonistic bacteria is foliar application. In this case, the antagonistic bacteria may simply be applied as an aqueous suspension substantially free of nutrients and other additives such as surfactants. The applied amount is generally about 10 ⁶ to 10 ⁸ vegetative cells / ml
Aqueous suspension of about 10 ⁴ to 10 ⁸ cells / g fresh leaf weight after application.

If the antagonistic microorganism is resistant to the biocide, either by nature of the wild strain or by transformation, especially when it is applied during the period when the ice nucleating bacteria are already established in the growth stage of the plant. , A biocide can be included in the formulation. By using a biocide, the ice nucleating bacteria are killed, the place where the ice nucleation deficient bacteria are colonized is secured, and the bacteria are colonized, and the ice nucleating bacteria are recolonized. Can be prevented.

Examples of biocides include antibiotics, toxins and the like. Specific antibiotics include streptomycin, oxytetracycline, tetracycline, kanamycin and similar substances. The amount of antibiotic can vary widely depending on its type, but is generally about 5 for the formulation.
Used in an amount of 0-100 ppm. The following example is provided to illustrate the invention. However, the examples are for the purpose of illustration and do not limit the scope of the invention.

Example The predominant microflora present in healthy leaves of selected frost-sensitive crops was used as a source of isolation for ice nucleation-deficient bacteria. The following media were used to ensure the amount of carbon and nitrogen sources normally dissolved and harvested from the plant surface. The amount of the following substance may be an amount per 1 liter of the medium or a higher concentration, and the medium may be diluted up to 10 times.

[0041]

[0042]

The above plate medium was prepared and allowed to form colonies at 24 ° C. for 2 days. Next, a suspension containing approximately 10 ⁸ cells / ml of Pseudomonas syringe or Erwinia herbicola was sprayed onto the plate. Around a small area on the agar plate surface spotted with antagonistic bacteria,
When the sprayed ice nucleating bacteria did not grow and a transparent region was generated, it was regarded as a positive reaction of the antagonistic bacteria.

The following tests were carried out in order to confirm that the above-mentioned positive reaction was not based on antibiotics secreted by antagonistic bacteria. Selected antagonists were spotted on the medium and on King's medium B enriched with nutrients, and the method described above was applied to both plates to show the growth of the sprayed bacteria in the area surrounding the spotted colonies. The presence or absence was recorded.

The antagonistic bacteria which inhibit the growth of the sprayed bacteria on the limiting medium and do not inhibit the growth on the enriched medium are isolates that have consumed the essential nutrients that limit the growth of ice nucleating bacteria. Means These isolates were further selected by greenhouse and laboratory methods.

100 three-leaf corn seedlings
In addition, about 10 cells of the selected antagonistic bacteria ⁸Suspension containing cells / ml
And was placed in a moist chamber in a greenhouse at about 24 ° C. for 2 days. Next
Pre-treatment with these plants and antagonistic bacteria
Control plants that did not have about 10 ^FivePseudomonas per piece / ml
・ Inoculation by spraying the cells of the syringe on the leaf surface
did.

The plants were left in a moist chamber for 2 days, then dried and placed in a controlled environment room at about -5 ° C. −
All plants were exposed to 20 ° C for 1 hour after exposure to 5 ° C for 1 hour.
I left it. The resulting dark leaves that had become wet and weak in water indicating frost damage were counted. The number of wet leaves in plants pretreated with antagonistic bacteria was reduced compared to that in plants not pretreated with antagonistic bacteria, indicating that effective antagonistic microorganisms reduced frost damage. It was

Mutant bacteria were selected by the following experiment.
Respectively separated from corn and almonds,
Two strains having the ability to form ice nuclei belonging to Pseudomonas syringae, labeled with 31 rif 1 and 42B, were mutated according to a conventional method using ethyl methanesulfonate as a mutagen.

The mutagenized culture was inoculated on a rich medium such as King's medium B at a density of about 50 cells per plate. The colonies were allowed to grow for 24 hours, i.e. until the diameter of the colonies was about 1 mm, at which point the sterilized velvet pieces were used to replicate the colonies on the surface of aluminum foil coated with paraffin.

Next, the aluminum foil was placed at -5 ° C or -9 ° C.
It was placed on the frozen constant temperature bath surface. Colonies preserving the wild-type ice nucleation activity were immediately frozen at -5 ° C or -9 ° C.
Was identified from a mutant strain that lost the ability to form ice nuclei. Strains that lost ice nucleation ability remained liquid when the aluminum foil was sprayed with water droplets.

Thus, cells deficient in ice nucleation ability were isolated, re-cultured, purified, and it was confirmed that each of them completely lost ice nucleation ability at -5 ° C. Furthermore, an in vivo antagonistic test was also conducted by the above method.

The ice nucleation-deficient mutant strains of Pseudomonas syringae and Erwinia herbicola were detected and isolated at a frequency of about 4 × 10 ⁻³ from cells mutated with ethyl methanesulfonate by the replica freezing method. . Pseudomonas syringer mutant 53
The ice nucleation properties of the strain and 27 mutant strains of Erwinia herbicola were measured as the percentage of cells with ice nucleation activity as a function of temperature (ice nucleation frequency).

The mutant strain has a critical ice nucleation temperature (-) of the parent strain.
1.2 ° C.) lower limit ice nucleation temperature (−3 ° C. to −
8.4 ° C.) and a temperature below −5 ° C. or −
At 9 ° C, it showed a decrease in the frequency of ice nucleation at a rate of 10 ³ to 10 ⁸ as compared with the parent strain, that is, it showed any of these characteristics.

Plasmid R for Pseudomonas Syringa and Erwinia Herbicola
SF 1010 (10 ² to 10 ³ transformation / mg DN
A) and plasmid pRR322 were used for transformation to confer streptomycin resistance and oxytetracycline resistance, respectively.

The isolates were tested at local conditions to establish utilization of the isolates. In order to control frost damage during periods when test plants are most susceptible to frost damage, in most cases
It has been found that it is sufficient to apply one strain or a combination of two or more strains once at the earliest possible stage in the plant growth stage.

In most cases, it was found to be sufficient to apply the bacteria by foliar application to freshly germinating seedling leaves or deciduous flowers. Bacteria are about 10 ⁶ to 10 ⁸
Used as an aqueous suspension of individual vegetative cells / ml. In some cases where the ice nucleation deficient strain was antibiotic resistant, antibiotics such as streptomycin or oxytecycline were included in the aqueous suspension. The presence of these antibiotics helped the antagonist strains establish in the plant.

Instead of foliar application, the dry powder formulation was applied to seeds and bulbs and tubers. The formulation was prepared as follows. The vegetative cells of the antagonistic bacteria were made into a denser suspension than 10 ¹² cells / ml, this was mixed with 10 volumes of 0.1M magnesium sulfate solution, and this mixture was suspended in 20% aqueous solution of Xanthan gum. The liquid was added to 10 volumes. After thoroughly mixing the gum with the bacterial cell suspension, 4 parts by volume of talc was mixed with 1 part by volume of gum. This mixture was dried at 10 ° C. for 10 days and then pulverized to obtain a fine powder and uniform agent.

Potato tubers (slightly moist) were rolled in the dry powder formulation and planted. During germination from potato soil, bacteria were found to colonize the germinating stems and leaves, and frost damage mitigation was completed.

Furthermore, it was found that the bacteria also colonize the roots and promote the growth of shoots and the formation of daughter tubers in this plant. In addition, it has been shown to promote some growth. When the antagonistic bacteria are applied to fruits such as pears and almonds at the 10% flowering stage, Erwinia
It has been observed that disease symptoms such as rot caused by phytopathogenic fungi such as Erwinia amylovora are reduced.

The present invention provides novel methods, formulations and microorganisms for preventing frost damage to host plants.
This method is economical, effective and can be easily applied at various stages of plant growth such as seeds, bulbs or tubers, seedlings, shoots and flowers.

The presence of ice nucleation-deficient bacteria is equally beneficial not only for the survival of ice nucleating microorganisms but also for the inhibition of the survival of pathogenic microorganisms. Although the present invention has been described in detail by way of examples, this is for contributing to a clear understanding of the invention and does not limit the scope of the invention.

Claims (6)

[Claims] 1. A formulation containing an ice nucleation-deficient bacterium that antagonizes an ice nucleating bacterium by consuming at least one kind of restricted nutrient provided by a host plant, or a cell of a descendant of this bacterium. 2. The formulation according to claim 1, which is an aqueous formulation containing 10 ⁴ to 10 ¹¹ cells / ml of the antagonistic bacteria. 3. A formulation according to claim 2 which contains a sufficient number of cells to provide about 10 ⁴ or more cells per gram fresh weight of plant tissue. 4. A powder formulation containing a sufficient number of cells of the antagonistic bacteria to provide about 10 ⁴ or more cells per 1 g of fresh weight of plant tissue, and further containing an inert powder carrier. Item 1. The formulation according to Item 1. 5. The formulation according to any one of claims 1 to 4, wherein the antagonistic bacterium is resistant and contains an effective amount of an antibiotic that kills bacteria of the endophytic microbiota of the host plant. . 6. The composition according to claim 1, which contains an effective amount of at least one kind of chemical pesticide.