JP2023001843A - Method for improving plant disease resistance - Google Patents

Abstract

Kind Code: A1 An object of the present invention is to provide a novel technique for improving the disease resistance of plants without genetic modification or the use of pesticides.
SOLUTION: A method for improving disease resistance of a target plant includes a freezing step of freezing plant tissue, wherein the lowest freezing temperature in the freezing step is −20° C. or lower, and the temperature drop in the freezing step is less than or equal to −20° C. The rate is 0.8 ° C./day or less, the period of the freezing step is 100 days or more, and the disease resistance is resistance to downy mildew, resistance to diseases caused by flagellated bacteria, Pseudomonas A method for improving disease resistance of a plant, characterized in that it is resistant to one or more kinds of diseases selected from syringae infectious disease resistance and rust resistance, and is carried out according to the following (A) or (B): .
(A) The disease resistance of the target plant is improved by subjecting the plant tissue of the target plant whose disease resistance is to be improved to the freezing step.
(B) subjecting a plant tissue different from the target plant to the freezing step, and bringing the target plant whose disease resistance is to be improved into contact with the extract obtained from the different plant tissue, thereby improve the disease resistance;
[Selection figure] None

Description

The present invention relates to a method for improving plant disease resistance without genetic manipulation.

Since ancient times, mankind has produced plants with advantageous properties by selective breeding techniques. Conventional breeding methods take a long time to fix certain characteristics, but with the advent of generation promotion technology, it is now possible to shorten the time required for fixing. However, there is a problem that it takes several years for fixation even with the generation promoting technology. Therefore, biotechnology such as anther culture, which does not require fixation work, has been developed.

Gene recombination technology is also known as a method for producing plants with advantageous properties. Herbicide-tolerant crops, pest-resistant crops, disease-resistant crops, and crops with increased storability have been produced by genetic recombination technology.

On the other hand, methods have been proposed to induce mutations and enhance the characteristics of plants by subjecting them to specific treatments. For example, Patent Literature 1 discloses a breed improvement method for imparting cold resistance, including the steps of gamma ray irradiation and chromosome doubling treatment.

Methods have also been devised to control plant characteristics without altering gene sequences. For example, in Patent Document 2, in the vegetative growth period of plants, salt stress, low light stress, high light stress, drought stress, excessive humidity stress, high temperature stress, low temperature stress, nutritional stress, heavy metal stress, A method is disclosed for controlling flowering time in the next generation of plants by applying stress treatments such as disease stress, anoxic stress, ozone stress, _CO2 stress, and strong wind stress.

By the way, most of Japan belongs to the temperate zone, and Hokkaido and the Tohoku region belong to the subarctic zone (cold zone). Therefore, we are dependent on imports for crops that are not suitable for cultivation in Japan's climate, such as those grown in subtropical and tropical regions.
As an epoch-making technology to solve this problem, the technology called "freeze-thaw awakening method" (Patent Document 3) and its improved technology called "enzyme solution method" (Patent Documents 4 and 5) are the present inventors. It has been developed by and has achieved a number of excellent results so far. For example, domestic pesticide-free bananas are produced by applying the freeze-thaw awakening method. The product is sold under the name of "Miracle Banana" (registered trademark). In addition, grains such as wheat and rice are produced by applying the enzyme solution method.

The freeze-thaw awakening method is a method for enhancing the characteristics of plants by cultivating frozen and thawed plant tissues. The enzymatic solution method is a method for enhancing the characteristics of plants by bringing other plant tissues into contact with extracts obtained from frozen plant tissues. That is, the enzymatic liquid method involves contacting (immersing or spraying) the plant tissue with an extract of the plant or its tissue that has undergone the freeze-thaw awakening method, thereby enhancing the plant characteristics enhanced by the freeze-thaw awakening method. It can be said that it is a method of propagating to Actually, plant characteristics that can be enhanced by the freeze-thaw awakening method and the enzyme solution method are common, specifically, environmental adaptation characteristics such as growth speed, cold resistance, heat resistance, high altitude adaptation characteristics, and low altitude adaptation characteristics. , the amount and size of fruits and seeds, sweetness, disease and pest resistance, drought resistance, etc. can be enhanced. Moreover, the freeze-thaw awakening method and the enzyme solution method are not limited to plants of a certain family, genus, or species, and can be applied to all plants. Furthermore, as a result of gene expression analysis, it has been confirmed that the gene expression profiles that fluctuate by applying the freeze-thaw awakening method and the enzyme solution method are very similar regardless of the plant species.

JP-A-2006-25632 JP 2016-182094 A Japanese Patent Application Laid-Open No. 2018-183112 Japanese Patent No. 6830591 Japanese Patent No. 6864304

Infectious diseases caused by pathogens such as bacteria are a serious problem in the cultivation of crops.
For example, downy mildew (Dawny mildew) is known as a plant disease. Downy mildew is a disease caused by downy mildew, a contagious (air- and water-borne) disease caused by several genera of the Mycobacterium family. Each of these genera includes several species, but representative genera that cause damage to crops in Japan are Pseudoperonospora, Peronospora, Plasmopara, and Bremia. four genera.

The symptoms of downy mildew vary slightly depending on the vegetable, but in general, fading green spots with unclear outlines are formed on the leaves, which gradually enlarge and become polygonal gray-white lesions, new lesions and high humidity. Under environmental conditions, a grayish-white frosty mold develops on the reverse side of the lesions. Later this becomes a black to purplish-black irregular macular.

There are two types of conidial germination methods for downy mildew fungi, that is, conidial germination of Cucurbitaceous vegetables is zoospore germination, and conidial germination of other vegetables is mycelial germination of direct germination. Zoospores in the former are stomatal infections, and mycelial germination in the latter are stomatal and cuticular infections. The optimum temperature for germination of conidia is approximately 10°C, and the optimum temperature for disease development (symptom onset temperature) is 13-20°C, which is relatively low. Therefore, it rarely occurs during the high summer season. Pathogenic bacteria that invade the host create haustoria in the cell and absorb nutrients. Under adverse environmental conditions or at the end of disease symptoms, oospores, which are durable organs, are formed in tissues, which play a role in primary infection. In spinach downy mildew, oospores play a major role as seed transmission and are involved in transmission and development. The formation of conidia is affected by the pathogenic environment such as temperature, humidity and light, and the freshness of the host.

Pesticides against downy mildew include Signum WDG (pyraclostrobin/boscalid wettable powder), zimandaisen wettable powder (mancozeb wettable powder), strobe flowable (kresoxime methyl wettable powder), and Daconil 1000 (TPN wettable powder). , Horizon Dry Flowable (cymoxanil/famoxadone wettable powder), Limay flowable (amisulbrom wettable powder), and the like are known.

As a disease of agricultural crops, a rust disease caused by a rust fungus is known. Diseases caused by rust fungi are air- and water-borne diseases caused by molds. The verbal rust that occurs in vegetables includes white rust and rust. The white rust fungi that are their pathogens are taxonomically different from rust fungi, the former belonging to the genus Albugo and the latter belonging to the genus Puccinia. Rust fungi are obligate parasites, filamentous fungi that can live only in living vegetable cells, that is, cannot be artificially cultured.

The lesions are spindle-shaped or elliptical, and produce orange-yellow, slightly raised, small spots. In late autumn, dark brown spots (teliospore layers) are formed in contact with the orange-yellow lesions. When the disease is severe, the leaves turn dirty white and eventually die. Rust disease occurs from spring to autumn, but temporarily stops in summer. Usually, there are many outbreaks around April, May and September, October. Overyears on damaged plants in the form of teliospores and uredinial spores, and the next spring uredinous spores are scattered and transmitted. It occurs frequently in spring, and when the summer is cold and rainy, the occurrence in autumn is encouraged. Densely planted and poorly ventilated, and in a humid environment, disease outbreaks and contagion are actively carried out.

Agricultural chemicals against rust include Amistar 20 flowable (azoxystrobin wettable powder), Signum WDG (pyraclostrobin/boscalid wettable powder), Dimandaisen wettable powder (mancozeb wettable powder), Strobe flowable (kresoxime methyl water powder). hydrating powder), trihumin wettable powder (triflumizole wettable powder), Berkut wettable powder (iminoctadine albesilate wettable powder), and the like.

Pseudomonas syringae is known as a plant pathogen. Pseudomonas is a genus of Gram-negative, aerobic gammaproteobacteria belonging to the Pseudomonadaceae family. The genus contains 191 taxonomically recognized species, many of which are plant pathogens. Among the Pseudomonas spp., P. syringae is a fertile plant pathogen, existing as over 50 different pathogenic types (pv.), many of which are highly host plant specificity. Many other Pseudomonas species can also act as plant pathogens, among other members the P. syringae subgroup. For example, commercially important diseases caused by P. syringae pathogens include stone fruit bacterial blast, tomato bacterial spot and pea blight. It is also widely known that infection with Pseudomonas syringae causes damage to various plant species, such as rust disease, due to decreased immunity.

Known drugs against Pseudomonas syringae include, for example, acibenzolar-S-methyl (Actigard/Bion, Syngenta) and antibiotics such as streptomycin sulfate and kasugamycin.

In addition, plant diseases caused by fungi or fungi include rice blast, powdery mildew, mildew, dry smut, aspergillus, sesame leaf blight, pod blight, spot rot, stem leaf blight, and aphids. Focal disease, catfish smut, sleeping disease, leopard disease, bacterial bacterial disease, chlorosis, wilt, epidemic, yellow wilt, yellow wilt, yellow wilt, yellow spot, gray mold, ash star disease , gray spot, horn spot, brown streak, brown snow rot, brown ring spot, brown spot, brown spot, brown spot, stock blight, stock rot, dry black rot, dry rot Disease, Eye spot, Sclerotinia, Stem plague, Stem blight, Red snow rot, Black soot, Black rot, Black wilt, Black root disease, Black root rot, Black stain, Black spot, Black spot Disease, black rot, black rot, black eye grain, black leaf blight, koshibori disease, root rot, root rot, root rot, purpura, purple feather disease, small sclerotia Disease, small sclerotium rot, streak, red head rot, red sclerotia, red rot, small snow rot, large sclerotium snow rot, anthracnose, anthracnose, brown rot , soft rot, white silk disease, white plague, white rot, white rot, white leaf blight, ergot disease, spot disease, seedling rot, seedling blight, rot, rot, sheath blight, Leaf blight, leaf scorch, leaf sheath browning, leaf rot, bare smut, defoliation, damping off, and ring blight are known. Various agricultural chemicals are generally used for the prevention of these diseases.

By the way, it is estimated that more than 14,000 types of plant pathogenic fungi exist in the natural world. Plants without voluntary locomotion have opportunities to come into contact with many plant pathogens in the natural world, but this contact rarely leads to infection. This is because plants recognize pathogens and induce their own immune response to prevent invasion of pathogens. When plant pathogens invade, plants recognize molecular groups widely present in plant pathogens called pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns (MAMPs), PTI (PAMP-triggered immunity) induce an immune response called

On the other hand, plant pathogens are known to secrete effector proteins into plant cells via the type III secretion apparatus, one of the secretion systems, thereby suppressing PTI. Interestingly, this effector protein may also function as a recognition substance that induces a stronger immune response called ETI (effector-triggered immunity) outside the host plant of the pathogen. That is, when this effector suppresses PTI, infection is established in many cases, but when the effector does not suppress PTI and newly induces ETI, infection is not established. Such duality of effectors and the two immune systems of PTI and ETI present in plants are considered to be the result of co-evolution of plants and pathogenic bacteria.

As PAMPs that have been shown to induce plant PTI, bacterial flagellum protein flagellin, translation elongation factor EF-Tu, lipopolysaccharide (LPS), peptidoglycan, chitin, etc. have been reported so far. Transcriptome analysis using microarrays and the like has revealed that common genes are induced by the recognition of these different PAMPs. Although plants have different PAMP recognition systems, their signaling mechanisms and transcription factors It is believed that the gene expression control mechanism via

As mentioned above, flagellin, a recent flagellar protein, functions as a PAMP. A search for a recognition sequence in the N-terminal region, whose sequence is conserved among flagellins, revealed that a peptide (flg22) consisting of 22 amino acid residues in the N-terminal region induces an immune response. A recognition receptor for flg22 has been clarified by analysis of flg22-insensitive mutant fls2 (FLAGELLIN-SENSING 2) of Arabidopsis thaliana. FLS2, the identified flg22 receptor, has extracellular leucine-rich repeats (LRR: leucine-rich repeat) and intracellular serine / threonine kinase with a single transmembrane receptor kinase (RLK) and belongs to the LRR XII family. The extracellular domain is composed of 28 LRRs, and LRRs 9 to 15 have been shown to be involved in flagellin binding.

As described above, the use of agricultural chemicals as a countermeasure against diseases caused by bacteria and fungi is an example, but their use should be avoided because of their undeniable effects on environmental pollution and human health. However, there is no known method for imparting resistance to these pathogenic bacteria to plants without using pesticides or depending on methods such as genetic recombination.
In addition, there is no known method for enhancing the immune response inherent in plants without genetic recombination or the use of drugs.

In view of such problems, the problem to be solved by the present invention is to provide a novel technique for improving the disease resistance of plants without using genetic recombination or agricultural chemicals.

The present inventors performed transcriptome analysis on plants treated by the freeze-thaw awakening method and the enzymatic solution method. As a result, we found that significant changes in gene expression were observed that contributed to improved plant disease resistance. They also found that plants treated by these methods have high resistance to diseases. The present invention was completed based on these findings.
The present invention for solving the above problems is as follows.

[1] A method for improving disease resistance of a target plant, comprising a freezing step of freezing plant tissue,
The minimum freezing temperature in the freezing step is −20° C. or less, the rate of temperature drop in the freezing step is 0.8° C./day or less, and the period of the freezing step is 100 days or more;
The disease resistance is resistance to one or more selected from resistance to downy mildew, resistance to diseases caused by flagellated bacteria, resistance to Pseudomonas syringae infection, and resistance to rust. ,
A method for improving plant disease resistance, characterized by being carried out according to the following (A) or (B).

(A) The disease resistance of the target plant is improved by subjecting the plant tissue of the target plant whose disease resistance is to be improved to the freezing step.
(B) subjecting a plant tissue different from the target plant to the freezing step, and bringing the target plant whose disease resistance is to be improved into contact with the extract obtained from the different plant tissue, thereby improve the disease resistance;
[2] A method for inducing changes in gene expression levels that contribute to improved disease resistance in a target plant, comprising a freezing step of freezing plant tissue,
The minimum freezing temperature in the freezing step is −20° C. or less, the rate of temperature drop in the freezing step is 0.8° C./day or less, and the period of the freezing step is 100 days or more;
The gene expression level variation that contributes to the improvement of disease resistance in the target plant is an increase in expression of the RPPL1 (Putative disease resistance RPP13-like protein 1) gene or its ortholog, an increase in expression of the FLS2 (Flagellin sensing 2) gene or its ortholog, LRK10 (rust resistance kinase Lr10) gene or its orthologue increased expression and UGT74F2 (UDP-glucosyltransferase 74F2) gene or its orthologs selected from suppressed expression, gene expression level variation that contributes to disease resistance,
A method for inducing variation in gene expression that contributes to improved disease resistance of a plant, characterized by being carried out according to (A') or (B') below.

(A′) Gene expression level variation that contributes to the improvement of disease resistance of the target plant by subjecting the plant tissue of the target plant to the freezing step to induce the gene expression level variation that contributes to the improvement of disease resistance. to induce
(B') Subjecting a plant tissue different from the target plant to the freezing step, and inducing a gene expression level change that contributes to improvement of disease resistance in the extract obtained from the different plant tissue Said target plant is brought into contact with the target plant to induce variation in the gene expression level that contributes to the improvement of the disease resistance of the target plant.
[3] Resistance to downy mildew, resistance to diseases caused by flagellated bacteria, resistance to Pseudomonas syringae infection, and rust resistance, including the step of performing the method of [1] or [2] A method for producing a plant with improved disease resistance of one or more selected from disease resistance.
[4] containing as an active ingredient an extract extracted with an aqueous solvent from a plant tissue that has undergone a freezing process;
The minimum freezing temperature in the freezing step is −20° C. or less, the rate of temperature drop in the freezing step is 0.8° C./day or less, and the period of the freezing step is 100 days or more;
A composition for inducing variation in gene expression levels that contributes to improvement of disease resistance of plants and/or disease resistance of plants, characterized by being used for the following uses (i) and/or (ii).

(i) Use for improving resistance to disease selected from resistance to downy mildew, resistance to diseases caused by flagellated bacteria, resistance to Pseudomonas syringae infection, and resistance to rust.
(ii) increased expression of the RPPL1 (Putative disease resistance RPP13-like protein 1) gene or its ortholog, increased expression of the FLS2 (Flagellin sensing 2) gene or its ortholog, increased expression of the LRK10 (rust resistance kinase Lr10) gene or its ortholog and suppression of expression of UGT74F2 gene UGT74F2 (UDP-glucosyltransferase 74F2) or its ortholog, for inducing variation in gene expression that contributes to disease resistance.

According to the present invention, changes in gene expression that contribute to disease resistance, such as increased expression of the RPPL1 gene, FLS2 gene, and LRK10 gene, and suppressed expression of the UGT74F2 gene, are induced, resulting in downy mildew resistance, rust resistance, and flagella. It is possible to improve the disease resistance of plants by conferring resistance to bacteria having Pseudomonas syringae and the like.

Embodiments of the present invention will be described in detail below. Needless to say, the scope of the present invention is not limited to the embodiments described below.

There is no limitation on the plant species whose disease resistance can be improved by the present invention. For example, Papayaceae (Caricaceae), Pineapple (Bromeliaceae), Musaceae (Musaceae), Cucurbitaceae (Cucurbitaceae), Myrtaceae Myrtaceae, Oxalidaceae (Oxalidaceae), Moraceae (Moraceae), Malvaceae (Malvaceae), Rubiaceae ( Rubiaceae), Laureaceae, Passifloraceae, Sapindaceae, Clusiaceae, Ebenaceae, Rutaceae, Annonaceae, Palmaceae , Cactaceae, Rosaceae, Fabaceae, Poaceae, and Brassicaceae.

More specifically, the genus Papaya (Carica), the genus Ananas (Ananas), the genus Musa (Musa), the genus Siraitia, the genus Psidium, the genus Carambola (Averrhoa), the genus Figus (Ficus), the genus Cocoa (Theobroma) ), Coffea, Cinnamomum, Passiflora, Litchi, Garcinia, Diospyros, Casimiroa, Annona, belongs to the genus Phoenix, Hylocereus, Cerasus, Glycine, Hordeum, Triticum, Zea, Raphanus A plant etc. can be illustrated.

The present invention is a method for improving disease resistance. The term "disease resistance" as used herein means resistance to disease, and its content is not limited.
INDUSTRIAL APPLICABILITY The present invention can be applied to improve resistance to diseases caused by bacteria. It can improve resistance to diseases caused by either Gram-positive-Gram-negative, aerobe-anaerobe, or cocci-bacillus pathogens. In particular, the present invention is preferably used to improve resistance to diseases caused by flagellated bacteria.

The present invention can be applied to improve resistance to diseases caused by fungi. The present invention can improve resistance to diseases caused by both filamentous fungi and yeast. In particular, the present invention is preferably used for improving resistance to diseases caused by filamentous fungi among fungi.

Downy mildew, rust, rice blast, powdery mildew, wilt, smut, aspergillus, sesame leaf blight, pod wilt, spot rot, stem leaf blight, and pinhole disease , Catfish smut, sleeping disease, leopard disease, bacterial blight disease, wilt, wilt, late blight, yellow wilt, yellow wilt, yellow wilt, yellow spot, gray mold, ash, ash spot, horn spot, brown streak, brown snow rot, brown ring spot, brown spot, brown spot, brown spot, stock blight, stock rot, dry black rot, dry rot, Eye spot, sclerotia, stem plague, stem blight, red snow rot, black soot, black rot, black wilt, black root disease, black root rot, black stain, black spot, black spot, Black rot, black rot, black eye grain, black leaf blight, koshibori disease, root rot, root rot, root rot, purpura, purpura, small sclerotia, Small sclerotia, streak, red head rot, red sclerotia, red rot, small snow rot, large snow rot, anthracnose, anthracnose, brown rot, soft rot Diseases, shaft rot, heart rot, Antrodia cinnamomea, fruit heart black rot, root rot, Panama disease, sigatoka, white silk disease, white plague, white rot, white crest, white leaf blight, Ergot disease, spot disease, seedling rot, seedling damping, rot, rot, sheath blight, leaf blight, leaf scorch, leaf sheath browning, leaf rot, naked smut, defoliation, leaf rot It can be used to improve resistance to one or more diseases selected from blight and ring blight.

One embodiment of the present invention is a method for improving resistance to diseases other than root rot. This embodiment aims to improve resistance to diseases other than root rot, but as a result of its implementation, even if the resistance to root rot is concomitantly improved, the technical scope of this embodiment naturally included in

One embodiment of the present invention is a method for improving resistance to diseases other than Panama disease. Although this embodiment aims at improving resistance to diseases other than Panama disease, even if resistance to Panama disease is concomitantly improved as a result of its implementation, it is naturally within the technical scope of this embodiment. include.

In plants to which the present invention has been applied, a marked improvement in the expression of the RPPL1 (Putative disease resistance RPP13-like protein 1) gene is observed. Therefore, the present invention can be applied to enhance the expression of the RPPL1 gene or its ortholog.
RPP is an abbreviation of "Recognition of Peronospora Parasitica", and RPPL1 is a gene highly homologous to the RPP13 gene, which recognizes filamentous fungi that cause downy mildew, including Peronospora parasitica, and provides resistance to them. be. The RPPL1 gene is a gene with the same molecular function GO (ADP binding, ATP binding) and biological process GO (defense response) as the RPP13 gene, and is known as a defense-related gene (e.g., Journal of Agricultural and Food Chemistry, 2020, Vol.68, pp.11026-11037). The NB-ARC domains possessed by RPP13 and RPPL1 are functional ATPase domains. As a result of mutation analysis, it is known that the NB-ARC domain plays an important regulatory role in the process of regulating the activity of resistance proteins (Journal of Experimental Botany, Volume 59, Issue 6, April 2008, Pages 1383-1397.). .
The present invention exhibits an effect of improving resistance to downy mildew due to the increased expression of the RPPL1 gene having the NB-ARC domain that regulates the activity of the aforementioned resistance protein.

In plants to which the present invention has been applied, a marked improvement in the expression of the FLS2 (Flagellin sensing 2) gene is observed. Therefore, the present invention can be applied to enhance the expression of the FLS2 gene or its ortholog.
FLS2 recognizes flagellin, a bacterial flagellar protein PAMP, and induces an immune response (PTI). Improved expression of the FLS2 gene results in improved resistance to flagellated bacteria (for example, Chemistry and Biology Vol.50, No. 5, 2012, 363-369 and Plant Signal Behav. 2008 Jun;3(6):423 -426.).
Therefore, the present invention exhibits an effect of improving resistance to flagella-bearing bacteria resulting from the improvement in FLS2 gene expression.

Remarkable suppression of the expression of the UGT74F2 (UDP-glucosyltransferase 74F2) gene is observed in plants to which the present invention has been applied. Therefore, the present invention can be applied to suppress the expression of the UGT74F2 gene or its ortholog.

Salicylic acid (SA) is a signal molecule that plants use in response to various stresses. Salicylic acid combines with low-molecular-weight organic compounds such as glucose to form an inactive form of salicylic acid, which can be transported and stored in plant vacuoles. In plants, two homologous enzymes, UGT74F1 and UGT74F2, transfer glucose from UDP-glucose to SA to form the glucose conjugate of SA. UGT74F1 and UGT74F2 are 77% identical, and despite the conservation of active site residues, these enzymes catalyze the formation of different products. Genetic deletion of these two enzymes has been shown to result in different phenotypes against bacterial infection. Mutants of the UGT74F1 gene have lower SA levels and reduced resistance to bacterial infection from Pseudomonas syringae. On the other hand, mutants of the UGT74F2 gene have high levels of SA and high resistance to Pseudomonas syringae. Similarly, overexpression of the UGT74F2 gene reduces levels of SA and increases susceptibility to Pseudomonas syringae. Since UGT74F1 and UGT74F2 have the ability to glucate SA, they are involved in controlling the level of free SA and, in turn, controlling the response of plants to pathogenic fungi (for example, Scientific Reports volume 7, Article number: 46629 ( 2017), Physiology Plantum, Volume 132, Issue 4, April 2008, Pages 417-425, etc.).
Significant suppression of UGT74F2 expression is observed in plants to which the present invention has been applied. Therefore, the present invention exerts an effect of reducing susceptibility to Pseudomonas syringae resulting from significant suppression of UGT74F2 expression.

In addition, in plants to which the present invention has been applied, a significant increase in expression of the LRK10 (rust resistance kinase Lr10) gene is observed. Therefore, the present invention can be applied to enhance expression of the LRK10 gene or its ortholog.

LRK10 is a protein having an extracellular receptor domain and a kinase domain. LRK10 is a protein also called rust resistance kinase Lr10, which confers resistance to rust to plants (eg The Plant Journal (1997) 11(1), 45-52, etc.). Since the present invention exhibits a remarkable effect of improving the expression of the LRK10 gene, the present invention can be used for improving rust resistance.

The plants to which the present invention is applied have plant characteristics such as cold resistance, growth characteristics, high temperature adaptability, germination rate, growth uniformity, root tension, abundant production, pest resistance, and drought resistance. Remarkable improvement is seen (see Patent Document 3, Patent Document 4, Patent Document 5, etc.). Therefore, the present invention can be applied for these purposes in addition to the purpose of improving disease resistance.

In addition, as a useful effect of the freeze-thaw awakening method (Patent Document 3) and the enzyme solution method (Patent Documents 4 and 5), there is an effect of improving cold resistance. This action enables plants that are cultivated in subtropical to tropical regions to be cultivated in cool climates such as Japan.
On the other hand, the above-mentioned rust fungi, Pseudomonas syringae, and downy mildew pathogens have a relatively low symptom onset temperature. Therefore, when the freeze-thaw awakening method or the enzymatic method is applied to tropical plants and they are cultivated in cool climates, it is possible that these plants may be affected by diseases with relatively low symptom onset temperatures.
However, the application of the present invention, which shares common steps with the freeze-thaw awakening method and the enzyme solution method, imparts resistance to diseases that develop disease symptoms in a relatively low temperature range. Therefore, the above-mentioned concern due to the application of the freeze-thaw awakening method or the enzyme liquid method is eliminated.

Next-generation plants obtained by methods other than sexual reproduction from plants to which the method for improving disease resistance of the present invention has been applied inherit the enhanced characteristics. Therefore, if a plant with enhanced disease resistance can be obtained by the method for enhancing characteristics of the present invention, a plant tissue (eg, offspring) other than a seed that can generate a plant individual independent of the plant obtained from the plant can be used. Subsequent generations of offspring generated also have enhanced properties.

In addition, plants to which the method for improving disease resistance of the present invention has been applied exhibit enhanced disease resistance even when used as scions for grafting.

The steps of the present invention are described in detail below. The present invention includes a freezing step of freezing plant tissue. The plant tissue to be subjected to the freezing step is not limited, and plant seeds, roots, shoots, stems, leaves, petals and the like can be exemplified. When subjected to the freezing step, these tissues may be frozen as they are, or may be partially excised and frozen in the form of sections.

In the freezing step, it is preferable to freeze the plant tissue while immersed in a liquid. As the liquid in which the plant tissue is immersed, it is preferable to use a freeze protection agent comprising an aqueous solution of DMSO (dimethylsulfoxide), glycerin, ethylene glycol, saccharides, or the like. Among them, it is preferable to use an aqueous saccharide solution, particularly an aqueous trehalose solution.

The upper limit of the lowest freezing temperature in the freezing step is preferably −20° C. or less, more preferably −30° C. or less, still more preferably −40° C. or less, still more preferably −50° C. or less, still more preferably −55° C. or less. be.
The lower limit of the lowest freezing temperature is preferably −200° C. or higher, more preferably −150° C. or higher, still more preferably −100° C. or higher, still more preferably −80° C. or higher, further preferably −70° C. or higher. It is preferably -65°C or higher.

In the freezing step, it is preferable to lower the temperature slowly, rather than rapidly lowering the temperature to the minimum freezing temperature. From the viewpoint of the survival rate after thawing, the rate of temperature drop is preferably 0.8°C/day or less, more preferably 0.6°C/day or less, more preferably 0.5°C/day or less as an average value. It is more preferably 0.3°C/day or less, still more preferably 0.2°C/day, still more preferably 0.1°C/day.
When the temperature is lowered slowly like this, it is preferable to use a program freezer in the freezing step.

The lower limit of the period of the freezing step is preferably 100 days or longer, more preferably 120 days or longer, still more preferably 150 days or longer, still more preferably 160 days or longer, still more preferably 180 days or longer.
The “freezing step period” is the period from when the temperature of the plant tissue starts to drop until when the thawing step starts.

The method for improving disease resistance of the present invention is technically characterized by including a freezing step. Although having such technical features is common, the present invention can mainly take the following embodiments (A) or (B).

(A) An embodiment in which the disease resistance of the target plant is improved by subjecting the plant tissue of the target plant whose disease resistance is to be improved to the freezing step.
(B) subjecting a plant tissue different from the target plant to the freezing step, and bringing the target plant whose disease resistance is to be improved into contact with the extract obtained from the different plant tissue, thereby Embodiments that improve said disease resistance.

In addition, the methods of the present invention for inducing changes in gene expression levels that contribute to the improvement of disease resistance are commonly provided with the technical feature of including a freezing step, but mainly the following (A') or (B) ') can be taken.

(A′) Gene expression level variation that contributes to the improvement of disease resistance of the target plant by subjecting the plant tissue of the target plant to the freezing step to induce the gene expression level variation that contributes to the improvement of disease resistance. Embodiments that induce
(B') Subjecting a plant tissue different from the target plant to the freezing step, and inducing a gene expression level change that contributes to improvement of disease resistance in the extract obtained from the different plant tissue Said target plant is brought into contact with the target plant to induce a change in gene expression level that contributes to the improvement of the disease resistance of the target plant.

Each of the embodiment (A) and the embodiment (B) will be described in detail below. In addition, description regarding embodiment (A) can be applied mutatis mutandis as description of embodiment (A'). Also, the description of the embodiment (B) can be applied mutatis mutandis to the description of the embodiment (B').

Embodiment (A) is an embodiment in which the plant tissue of the target plant whose disease resistance is desired to be improved is frozen in the freezing step described above. As described above, there is no limitation on the plant species capable of improving disease resistance in the present embodiment.

In this embodiment, the plant tissue is thawed after the freezing step. The thawing method in the thawing step is not particularly limited. The frozen plant tissue may be left at normal temperature to thaw naturally, or the frozen plant tissue may be thawed while being rinsed with running water.

Plants that have undergone the freezing process acquire disease resistance. That is, plants developed from thawed plant tissues have higher disease resistance than plants that have not undergone the freezing process.

When the plant tissue subjected to the freezing step is a plant seed, it can be sown in a conventional manner to generate plant individuals.

When the plant tissue subjected to the freezing step is a plant part other than seeds, it may be transferred as it is to soil or a medium for germination, or it may be finely chopped and subjected to cell culture according to a conventional method to induce callus. , somatic embryo induction, and somatic bud induction, plant individuals can be generated.

Next, embodiment (B) will be described in detail. Embodiment (B) is an embodiment in which a target plant whose disease resistance is desired to be improved or its plant tissue is brought into contact with an extract obtained from the plant tissue that has undergone the freezing step. A major difference from embodiment (B) is that the target plant whose disease resistance is to be improved is not subjected to the freezing step. A major feature of embodiment (B) is that it comprises a contacting step of contacting a target plant whose disease resistance is desired to be improved or its plant tissue with a plant tissue extract that has undergone the freezing step.

Embodiment (B) comprises an extraction step of subjecting a plant tissue different from the target plant to the freezing step and obtaining an extract from the plant tissue that has undergone the freezing step.
In the present specification, "a plant tissue different from the target plant" in the embodiment (B) means "a plant tissue of the target plant whose disease resistance is to be improved, and gene expression level variation that contributes to improvement of disease resistance is induced. It means "a plant tissue different from the plant tissue of the target plant to be grown". In other words, the plant tissue subjected to the freezing step is different from the plant tissue subjected to the contacting step described below.
In the embodiment (B), the plant species of “a plant tissue different from the target plant” subjected to the freezing step, the “target plant whose disease resistance is to be improved”, and the “gene expression level variation that contributes to improvement of disease resistance” are induced. The "target plant to be cultivated" may be of the same species or of a different species.

The plant tissue that has undergone the freezing step may be subjected to the extraction step in a frozen state, but is preferably thawed before being subjected to the extraction step. The thawing method is not particularly limited. The frozen plant tissue may be left at normal temperature to thaw naturally, or the frozen plant tissue may be thawed while being rinsed with running water. Natural thawing at room temperature is preferred.

A selection step is preferably included between the freezing step and the later-described extraction step. The selection process is a process of selecting live plant tissue from frozen plant tissue.
Specific aspects of the selection process are not limited as long as living plant tissues can be selected.

A preferred form of the selection process includes a method comprising fermenting the plant tissue that has undergone the freezing process. This method utilizes the difference in resistance to fermentation by microorganisms between living and dead plant tissues.
A living plant tissue maintains a tangible state without undergoing decomposition by microorganisms. On the other hand, dead plant tissues are softened or liquefied due to decomposition by microorganisms. Therefore, after the fermentation process, the living plant tissue and the dead plant tissue can be easily sorted out.

The fermentation treatment method is not particularly limited. A preferred example is a method in which the plant tissue that has undergone the freezing step is left in the open air.
In this case, the lower limit of the ambient temperature is preferably 0° C. or higher, more preferably 10° C. or higher, and even more preferably 20° C. or higher. The upper limit is preferably 50°C or lower, more preferably 40°C or lower.

The period of leaving the mixture in the open air is not particularly limited as long as the fermentation can be performed, and is preferably several days to several weeks, specifically one day to four weeks.

After the fermentation treatment, it is preferable to separate the dead plant tissue from the living plant tissue. The specific embodiment of the separation treatment is not particularly limited as long as the living plant tissue can be separated from a mixture of dead plant tissue and living plant tissue.
As mentioned above, after undergoing a fermentation process, dead plant tissue is decomposed by microorganisms and softened or liquefied. Therefore, by washing plant tissue that has undergone fermentation treatment, dead plant tissue can be easily washed away and removed. A preferred example of washing is washing with water.

Embodiment (B) includes an extraction step of obtaining an extract from the plant tissue that has undergone the freezing step. The extraction method is not particularly limited. The extractant used for extraction is preferably an aqueous solvent, more preferably water or an aqueous solution.

The extraction agent preferably contains sugars or sugar alcohols. More specifically, monosaccharides (glucose, lactose, threose, arabinose, xylose, galactose, ribose, glucose, sorbose, fructose, mannose), disaccharides (sucrose (sucrose), lactose (milk sugar), maltose (maltose) , trehalose, cellobiose, isomaltose, isotrehalose, neotrehalose, neolactose, turanose, palatinose), other polysaccharides (trisaccharides: raffinose, melezitose, maltotriose, tetrasaccharides: acarbose, stachyose, glycogen, solubilized starch , amylose, dextrin, glucan, β1,3-glucan, fructan, N-acetylglucosamine, chitin, chitosan), sugar alcohols (xylitol, sorbitol, erythritol, mannitol, maltitol), oligosaccharides (raffinose, panose, maltotri Extraction agent containing one or two or more saccharides or sugar alcohols selected from oligosaccharides, melezitose, gentianose, stachyose, cyclodextrin, xylooligosaccharides, cellulose oligosaccharides, lactooligosaccharides, fructooligosaccharides, galactooligosaccharides, and mannan oligosaccharides) is preferably used.
More preferably, as the saccharides or sugar alcohols, an extract containing one or a combination of sucralose and trehalose is used.
Moreover, when using an extractant that does not contain the above sugars or sugar alcohols, the above sugars or sugar alcohols may be added to the extract obtained after the extraction step.

It is preferable to perform a crushing treatment for crushing the plant tissue that has undergone the freezing step, more specifically, the living plant tissue that has undergone the freezing step. As a method of crushing treatment, grinding treatment can be preferably exemplified.
For the grinding treatment, a crusher such as a mixer or a ball mill may be used, but crushing by using a mortar can be preferably exemplified.

The stress applied to the plant tissue in the crushing treatment is not particularly limited, but it is preferable to crush gently without applying too much stress. Particularly preferably, it is gently ground using a mortar and a pestle.

The time for the crushing treatment is not particularly limited, but preferably from several tens of seconds to several hours. Specifically, the lower limit is preferably 10 seconds or longer, more preferably 30 seconds or longer. The upper limit is preferably 10 hours or less, more preferably 5 hours or less, and even more preferably 3 hours or less.

The crushing treatment of the plant tissue may be performed while the plant tissue is immersed in the extractant, but is preferably performed before adding the extractant. That is, it is preferable to extract by bringing the crushed plant tissue into contact with an extractant.

The plant tissue that has undergone the freezing step is brought into contact with the extractant, more specifically, the plant tissue is immersed in the extractant to transfer components contained in the plant tissue to the extractant to obtain an extract.
After the extraction step, a step of filtering the extract to remove plant tissue residues may be provided.

The amount of the extractant used in the extraction step is not particularly limited, but is preferably 0.5 parts by mass or more, more preferably 1 part by mass or more, and still more preferably 3 parts by mass or more, relative to 1 part by mass of the plant tissue. More preferably, it is 5 parts by mass or more.
The upper limit is also not particularly limited, and the amount of the extractant is preferably 100 parts by mass or less, more preferably 50 parts by mass or less, and still more preferably 20 parts by mass or less per 1 part by mass of the plant tissue.

The immersion time in the extraction agent is not particularly limited. Although the lower limit of the immersion time is not limited, it is preferably 1 minute or longer, more preferably 10 minutes or longer, and still more preferably 30 minutes or longer. Although the upper limit of the immersion time is not limited, it is preferably 2 days or less, more preferably 1 day or less, still more preferably 12 hours or less, and still more preferably 6 hours or less.

The temperature of the extractant during immersion is also not particularly limited. The lower limit is preferably 0°C or higher, more preferably 10°C or higher. The upper limit is preferably 60° C. or lower, more preferably 50° C. or lower, still more preferably 45° C. or lower, still more preferably 40° C. or lower.

When a liquid or paste in which components contained in the plant tissue are dissolved or dispersed is obtained by the crushing treatment, the liquid or paste is also included in the "extract".
In this case, the crushing process itself constitutes the extraction process.

In the present specification, the term "extract" does not only refer to an extract obtained primarily through an extraction process. The "extract" includes a diluted liquid obtained by diluting a primarily obtained extract with an arbitrary liquid and a concentrated liquid obtained by concentrating a primarily obtained extract. The "extract" also includes a solution obtained by dissolving the dried extract in any solution, in other words, a solution obtained by exchanging the solvent of the extract obtained primarily.
Further, as described above, when a liquid or paste in which components contained in the plant tissue are dissolved or dispersed is obtained by the crushing treatment, the liquid or paste is also included in the "extract".

Embodiment (B) includes a contacting step of contacting the target plant whose disease resistance is desired to be improved or the plant tissue thereof with the above extract. As described above, the plant whose disease resistance is improved by subjecting it to the contacting step may be any plant species.

The plant from which the extract used in the contact step is derived and the plant to be subjected to the contact step may be of the same or different species. That is, an extract obtained by freezing and extracting a plant tissue of a specific plant species may be brought into contact with a plant species other than the specific plant species or its plant tissue. Even in such a heterogeneous application, the desired effect can be obtained according to the property enhancement method of the present invention.

Embodiment (B) can be further classified into the following embodiments (B-1) and (B-2) according to the specific aspects of the contacting step.

(B-1) An embodiment in which the plant tissue of the target plant whose disease resistance is to be improved is immersed in the extract obtained from the plant tissue that has undergone the freezing step (B-2) The extract obtained from the plant tissue that has undergone the freezing step is sprayed on a target plant whose disease resistance is to be improved.

Each embodiment will be described below.

First, the embodiment (B-1) including an immersion step will be described. The plant tissue to be subjected to the immersion step is not limited, and plant seeds, roots, buds, stems, leaves, petals and the like can be exemplified. When subjected to the immersion step, these tissues may be immersed as they are, or may be partially excised and immersed in the form of slices.

It is also preferred that the plant tissue whose disease resistance is to be improved is dried prior to the soaking step. Since it is sufficient to dry the surface of the plant tissue, for example, the plant tissue may be dried in the sun for a few days. In addition, drying using a dryer such as a dryer may be performed. As a result, it is possible to increase the efficiency of permeation of the liquid extract into the plant tissue in the immersion step.

When a seed having a seed coat is subjected to the soaking step, the thickness of the seed coat is preferably 3 cm or less, more preferably 1 cm or less, more preferably 5 mm or less, more preferably 3 mm or less. When the thickness of the seed coat is within the above range, the liquid extract permeates efficiently and the seed can be permeated with the liquid extract in a short period of time.
As such seeds, cereal seeds such as barley, wheat, soybeans, and rice are preferred examples of seeds to which the present invention is applied.

The immersion time is not particularly limited, but is preferably 30 minutes or longer, more preferably 1 hour or longer, more preferably 6 hours or longer, more preferably 12 hours or longer, more preferably 24 hours or longer, still more preferably 48 hours or longer, and more preferably 48 hours or longer. It is preferably 60 hours or longer. The upper limit of the immersion time is preferably 300 hours or less, more preferably 200 hours or less, and even more preferably 100 hours or less.

It is preferable that the temperature of the extract in the immersion step is such that germs do not grow during the step. Preferably, the temperature is 0°C or higher, more preferably 5°C or higher, and still more preferably 10°C or higher. The upper limit is preferably 50° C. or lower, more preferably 45° C. or lower, still more preferably 40° C. or lower, still more preferably 35° C. or lower, and still more preferably 30° C. or lower.

The extract used in the immersion step is preferably a diluted solution obtained by diluting the extract obtained primarily in the extraction step with an arbitrary liquid. By diluting the liquid extract temporarily obtained in the extraction process, many plant tissues can be subjected to the immersion process at one time, and the production efficiency can be improved.

A dilution ratio is not particularly limited. The volume of the diluted solution after dilution is preferably 100 times or more, more preferably 1000 times or more, still more preferably 5000 times or more, and still more preferably 8000 times or more the volume of the plant tissue used for extraction. Even when diluted at such a high dilution rate, the effect of the present invention can be sufficiently obtained.
Although the upper limit of the dilution ratio is not particularly limited, it is preferably 100,000 times or less, more preferably 50,000 times or less, even more preferably 20,000 times or less, and still more preferably 10,000 times or less.

In addition, when a diluted solution is obtained by diluting the liquid or paste obtained by crushing the plant tissue without adding an extractant, the dilution ratio is preferably 100 times or more, more preferably. It can be 1000 times or more, more preferably 5000 times or more, more preferably 8000 times or more. Even when diluted at such a high dilution rate, the effect of the present invention can be sufficiently obtained.
Although the upper limit of the dilution ratio in this case is not particularly limited, it is preferably 100,000 times or less, more preferably 50,000 times or less, even more preferably 20,000 times or less, and still more preferably 10,000 times or less.

The liquid used for dilution is preferably a liquid containing sugars or sugar alcohols, like the extractant. More specifically, monosaccharides (glucose, lactose, threose, arabinose, xylose, galactose, ribose, glucose, sorbose, fructose, mannose), disaccharides (sucrose (sucrose), lactose (milk sugar), maltose (maltose) , trehalose, cellobiose, isomaltose, isotrehalose, neotrehalose, neolactose, turanose, palatinose), other polysaccharides (trisaccharides: raffinose, melezitose, maltotriose, tetrasaccharides: acarbose, stachyose, glycogen, solubilized starch , amylose, dextrin, glucan, β1,3-glucan, fructan, N-acetylglucosamine, chitin, chitosan), sugar alcohols (xylitol, sorbitol, erythritol, mannitol, maltitol), oligosaccharides (raffinose, panose, maltotri A liquid containing one or more saccharides or sugar alcohols selected from oligosaccharides, melezitose, gentianose, stachyose, cyclodextrin, xylooligosaccharides, cellulose oligosaccharides, lactooligosaccharides, fructooligosaccharides, galactooligosaccharides, and mannan oligosaccharides) It is preferably used for dilution.
More preferably, as sugars or sugar alcohols, a liquid containing one or a combination of sucralose and trehalose is used for dilution.

In the immersion step, preferably 0.5 kg or more, more preferably 1 kg or more, and even more preferably 1.5 kg or more of plant tissue is immersed per liter of the extract.
The upper limit of the weight of the plant tissue to be immersed per liter of the extract is not particularly limited, but is preferably 3 kg or less, more preferably 2.5 kg or less.

In the immersion step, it is preferable that the entire plant tissue is immersed in the extract. If the entire plant tissue is not immersed in the extract at one time, the extract can be brought into contact with the entire plant tissue by rolling or stirring the plant tissue in the extract during the immersion process. I don't mind.

Plants with improved disease resistance can be obtained by cultivating the plant tissue that has undergone the immersion step. The plant tissue after the immersion step can be sown as it is without any treatment.
The cultivation method is not particularly limited. When the plant tissue subjected to the immersion step is a plant seed, it can be sown in a conventional manner to generate plant individuals and cultivated in a conventional manner.

When the plant tissue subjected to the immersion step is a plant part other than a seed, it may be transferred as it is to soil or a medium for germination, or it may be finely chopped and subjected to cell culture according to a conventional method to induce callus. , adventitious embryo induction, and adventitious bud induction, plant individuals can be generated and cultivated.

Next, the embodiment (B-2) will be described in detail. Embodiment (B-2) is an embodiment including a spraying step of spraying the above-mentioned extract onto a plant whose disease resistance is to be improved.

The plant species of the plant tissue from which the extract used in the spraying step is derived and the plant species of the plant subjected to the spraying step may be the same or different. That is, an extract obtained by freezing and extracting plant tissue of a specific plant species may be sprayed on plant species other than the specific plant species. Even in such cross-species application, an effect of improving disease resistance can be obtained.

In the spraying step, the state of the plant to which the liquid extract is sprayed is not particularly limited. Plants to be sprayed may be plants cultivated in soil such as fields, plants cultivated in pots or planters, and plants cultivated on hydroponic culture media.

The method of spraying the liquid extract is not particularly limited, and can be carried out using a watering can or an existing sprayer. Also, the extract can be sprayed on any part of the planted plant, such as buds, flowers, leaves, stems, tree branches, and soil (roots).
When the extract is sprayed on the above-ground part of the plant, it may be sprayed on the soil at the same time. By spraying the extract not only on the ground but also on the soil, the plant can absorb the extract through its roots, and the effect of enhancing the properties of the extract can be exhibited.

The extract used in the spraying step is preferably a diluted solution obtained by diluting the extract obtained primarily in the extraction step with an arbitrary liquid. By diluting the liquid extract primarily obtained in the extraction process, the liquid extract can be sprayed on many plants.

The dilution ratio of the extract used in the spraying step is not particularly limited. The volume of the diluted solution after dilution is preferably 100 times or more, more preferably 250 times or more, still more preferably 2500 times or more, still more preferably 12500 times or more, and still more preferably 20000 times the volume of the plant tissue used for extraction. It can be as above. Even when diluted at such a high dilution rate, the effect of the present invention can be sufficiently obtained.
The upper limit of the dilution ratio is not particularly limited, but is preferably 1,000,000 times or less, more preferably 500,000 times or less, still more preferably 250,000 times or less, still more preferably 125,000 times or less, even more preferably 50,000 times or less, further preferably 25,000 times or less. can be used as a guideline.

In addition, when a diluted solution is obtained by diluting the liquid or paste obtained by crushing the plant tissue without adding an extractant, the dilution ratio is preferably 100 times or more, more preferably. It can be 250 times or more, more preferably 2500 times or more, more preferably 12500 times or more, further preferably 20000 times or more. Even when diluted at such a high dilution rate, the effect of the present invention can be sufficiently obtained.
The upper limit of the dilution ratio in this case is not particularly limited, but is preferably 1,000,000 times or less, more preferably 500,000 times or less, still more preferably 250,000 times or less, still more preferably 125,000 times or less, even more preferably 50,000 times or less, and still more preferably 50,000 times or less. 25000 times or less can be used as a guideline.

The spraying amount in the spraying step is not particularly limited. For example, as a guideline, preferably 0.01 liter or more, more preferably 0.1 liter or more, more preferably 0.5 liter or more, and even more preferably 1 liter or more of the extract per 1 m ² of planted area is sprayed. can be done.
In addition, preferably 1000 liters or less, more preferably 100 liters or less, and even more preferably 10 liters or less of the extract can be sprayed per ^square meter of planted area.

The spraying treatment may be performed only once during the cultivation period, or may be performed multiple times during the cultivation period. When spraying is carried out multiple times during the cultivation period, the spraying can be carried out, for example, every 1 day to 1 month, preferably every 2 days to 1 week.

In addition, the description of the embodiment (B-1) applies as it is to the plant species to which the embodiment (B-2) including the spraying step can be applied and the liquid used for diluting the extract.
Further, an embodiment (an embodiment including both the immersion step and the spraying step) in which the above-described embodiment (B-1) and embodiment (B-2) are combined may be employed.

As described above, according to the present invention having the embodiment (B), disease resistance of plants can be improved. In other words, the plant extract that has undergone the freezing process can be used for improving disease resistance. That is, the invention also resides in a composition for improving disease resistance of plants containing an extract extracted from frozen plant tissue.

The composition for improving disease resistance of the present invention contains an extract produced by the production method described above. The present invention also includes a dried extract obtained by drying the extract to remove the solvent. Although the method for obtaining the dried extract is not particularly limited, examples thereof include spray drying and freeze drying.

In a preferred embodiment of the present invention, the composition for improving disease resistance is used for improving resistance to diseases caused by bacteria or fungi. In addition, the composition for improving disease resistance of the present invention is selected from increased expression of the RPPL1 gene, increased expression of the FLS2 gene, increased expression of the LRK10 gene, and suppressed expression of the UGT74F2 gene. used for In addition, the composition for improving disease resistance of the present invention is used for improving disease resistance selected from downy mildew resistance, rust resistance, resistance to flagellated bacteria, and resistance to Pseudomonas syringae.

<Test Example A1> Application to papaya Papaya seeds were immersed in an aqueous solution of trehalose, left to stand in a program freezer, and frozen. Freezing was carried out slowly over 180 days at a rate of temperature drop of 0.5°C/day so that the lowest freezing temperature was -60°C. Frozen papaya seeds were thawed by rinsing with running water and then sown and cultivated. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.
パパイアにおいては、パパイア軸腐病（病原：Ｂｏｔｒｙｏｄｉｐｌｏｄｉａ ｔｈｅｏｂｒｏｍａｅ、Ｐｈｏｍｏｐｓｉｓ ｓｐ．、Ｃｏｌｌｅｔｏｔｒｉｃｈｕｍ ｇｌｏｅｏｓｐｏｒｉｏｉｄｅｓ、Ｆｕｓａｒｉｕｍ ｓｏｌａｎｉ、Ａｓｃｏｃｈｙｔａ ｃａｒｉｃａｅ）、パパイア黒腐病（病原：Ｅｒｗｉｎｉａ ｓｐ．）、軟腐病（病原：Ｐｈｙｔｏｐｈｔｈｏｒａ ｎｉｃｏｔｉａｎａｅ Ｂｒｅｄａ ｄｅ Ｈａａｎ ) and papaya brown spot (pathogen: Corynespora cassiicola).
In the papaya of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of the above-described diseases caused by bacteria and filamentous fungi was observed at a certain rate. On the other hand, in this test example to which the present invention was applied, none of these diseases, downy mildew, rust, Pseudomonas syringae infection, etc. occurred, although no antibacterial pesticide was used.

<Test Example A2> Application to Pineapple Pineapple seeds were freeze-thawed in the same manner as in Test Example A1, and then sown and cultivated. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

パイナップルにおいては心腐病（病原：Ｐｈｙｔｏｐｈｔｈｏｒａ ｃｉｎｎａｍｏｍｉ、Ｐｈｙｔｏｐｈｔｈｏｒａ ｐａｌｍｉｖｏｒａ、Ｐｈｙｔｏｐｈｔｈｏｒａ ｎｉｃｏｔｉａｎａｅ）、花樟病（病原：Ａｃｅｔｏｂａｃｔｅｒ ｐａｓｔｅｕｒｉｎａｎｕｓ、Ａｃｅｔｏｂａｃｔｅｒ ｐｅｒｏｘｙｄａｎｓ、Ｅｒｗｉｎｉａ ａｎａｎａｓ）、果心黒変病（病原：Ｆｕｓａｒｉｕｍ ｓｐｐ．、Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐｐ． ) and root rot (pathogen: Ceratosystis paradoxa).
In the pineapples of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of the above-described diseases caused by bacteria and filamentous fungi was observed at a certain rate. On the other hand, in this test example to which the present invention was applied, none of these diseases, downy mildew, rust, Pseudomonas syringae infection, etc. occurred, although no antibacterial pesticide was used.

<Test Example A3> Application to Banana Roots of banana seedlings were cut into round slices and freeze-thawed in the same manner as in Test Example A1. The roots of the offspring after freezing and thawing were chopped, and the fragmented growing cell clusters were cultured on the medium for germination. Seedlings grown to some extent were transferred to soil and cultivated. Cultivation was performed in Okayama Prefecture, Japan. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In bananas, it is known that diseases such as Panama disease (pathogen: Fusarium oxysporum f. sp. cubense), sigatoka (pathogen: Mycosphaerella fijiensis), anthracnose (pathogen: Colletotrichum musae) and the like are caused. there is
In the bananas of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of the above-described diseases caused by bacteria and filamentous fungi was observed at a certain rate. On the other hand, in this test example to which the present invention was applied, none of these diseases, downy mildew, rust, Pseudomonas syringae infection, etc. occurred, although no antibacterial pesticide was used.

<Test Example A4> Application to Banana Side shoots growing from the base of the banana plant were cut off, the leaves and roots were cut off, and processed into a bamboo shoot shape. This was freeze-thawed by the same method as in Test Example A1. The axillary buds after thawing were planted in pots. After that, the stem rotted and disappeared, but it was confirmed that new shoots sprouted. This sprout was cultivated.
In the bananas of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a certain rate. On the other hand, in this test example to which the present invention was applied, none of these diseases, downy mildew, rust, Pseudomonas syringae infection, etc. occurred, although no antibacterial pesticide was used.

<Test Example A5> Enhancement of coffee properties Coffee is said to be a very difficult plant to grow. Unless all of the four conditions of rainfall, sunshine, temperature, and soil conditions are appropriate, the tree will not grow, and it takes about three years for the tree to mature and bear fruit. Arabica is vulnerable to rust, and it is known that Arabica cultivated in Ceylon (Sri Lanka), Indonesia, etc. has been wiped out. Among them, Typica is the oldest of the Arabica varieties, and because of its low yield and low disease resistance, it is particularly difficult to grow among many coffee species. In addition, the distribution volume is extremely low at 0.01% of the total coffee.
In this test example, the seeds of Typica coffee were freeze-thawed by the same procedure as in test example A1, and the seeds were sown and cultivated in Okayama Prefecture. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

As mentioned above, rust caused by rust bacteria is the worst disease in coffee, and countermeasures against it are essential. However, in this test example, no pesticides including antibacterial pesticides were used in the cultivation of Typica coffee, which is known to have particularly low disease resistance. No occurrence was observed. Usually, it takes about 3 years for the tree to mature and bear fruit, but in this test example, flowering and fruiting were observed about 1 year after seeding.
In the Typica coffee of Comparative Example cultivated under the same conditions except that the present invention was not applied, the occurrence of the above-mentioned diseases caused by bacteria and filamentous fungi was observed at a considerable rate.

<Test Example A6> Application to Other Plant Species The seeds of the plants listed below were freeze-thawed in the same manner as in Test Example A1, and plant individuals were generated and cultivated from the treated plant tissues. No pesticides were used in the cultivation of any of the plant species, but no disease was observed.
Lokan fruit, guava, star fruit, fig, cacao, Ceylon cinnamon, passion fruit, lychee, mangosteen, black sapote, white sapote, spiny leaf sugar apple, date palm, red dragon fruit, almond, blue neck radish. In the plant species of the comparative example cultivated under the same conditions except for the above, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate.

<Test Example B1> Improvement of disease resistance of wheat using papaya extract Commercially available papaya seeds were immersed in an aqueous trehalose solution, left to stand in a program freezer, and frozen. Freezing was carried out slowly over 180 days at a rate of temperature drop of 0.5°C/day so that the lowest freezing temperature was -60°C.

Frozen papaya seeds were naturally thawed at room temperature (25°C). This was left in the open air (25° C.) for one week. Seeds that died during the freezing process fermented and softened or liquefied when left in the open air. By placing the seeds in a colander and washing with water, the fermented seeds (that is, dead seeds) were washed away and only live seeds were selected.

The live seeds left in the colander were gently ground and crushed using a mortar and pestle to obtain a paste. 1 cc of the paste was diluted to 10 L with an aqueous solution of sucralose and trehalose (approximately 8000-fold to 10000-fold dilution) to prepare a diluted extract.

20 kg of wheat "Fukuhonoka" seeds were immersed in 10 L of the diluted extract and allowed to stand for 72 hours. The soaked seeds were dried naturally in the sun for a few days before soaking. Seeds that had undergone the soaking process were sown and cultivated at a farm in Okayama Prefecture. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In wheat, wheat black blight (pathogen: Pseudomonas syringae pv. japonica), wheat head blight (pathogen: Gibberella zeae, Gibberella avenacea Cook, Fusarium culmorum, Fusarium nivale, Casati f. spol.gramic rust) Pathogen: Puccinia recondita Roberge ex Desmazieres), wheat ergot disease (pathogen: Claviceps purpurea), wheat eye spot (pathogen: Pseudocercospoorella herpotrichoides), wheat bare smut (pathogen: Ustilago nuda), wheat leaf blight (pathogen: Roberge ex Desmazieres), wheat spot disease (pathogen: Cochliobolus sativus), wheat blight disease (pathogen: Leptosphaeria nodorum Mueller), wheat blast disease (pathogen: Pyricularia oryzae Cavara), wheat stock rot (pathogen: Ceratobasidioma) Horn spot (pathogen: Selenophoma donacis), blight from wheat (pathogen: Mycosphaerella hordeicola Hara), smut from wheat (pathogen: Urocystis agropyri), wheat brown snow rot (pathogen: Pythium iwayamai Ito, Pythium paddicum Hidenum Hidenum) ｈｏｒｉｎｏｕｃｈｉｅｎｓｅ Ｈｉｒａｎｅ、Ｐｙｔｈｉｕｍ ｇｒａｍｉｎｉｃｏｌａ Ｓｕｂｒａｍａｎｉａｍ、Ｐｙｔｈｉｕｍ ｏｋａｎｏｇａｎｅｎｓｅ Ｌｉｐｐｓ、Ｐｙｔｈｉｕｍ ｖａｎｔｅｒｐｏｏｌｉｉ Ｖ． Ｋｏｕｙｅａｓ ｅｔ Ｈ． Ｋｏｕｙｅａｓ、Ｐｙｔｈｉｕｍ ｖｏｌｕｔｕｍ Ｖａｎｔｅｒｐｏｏｌ ｅｔ Ｔｒｕｓｃｏｔｔ）、コムギ黄枯病（病原：Ｐｙｔｈｉｕｍ ｓｐｐ．）、コムギ黄さび病（病原：Ｐｕｃｃｉｎｉａ ｓｔｒｉｉｆｏｒｍｉｓ Ｗｅｓｔｅｎｄｏｒｐ var. striiformis), wheat black spot (pathogen: Cladosporium herbarum), wheat black spot (pathogen: Epicoccum purpu rascens Ehrenberg ex Schlechtendahl), wheat red snow rot (pathogen: Micronectriella nivalis), wheat black eye grain (pathogen: Cochliobolus sativus, Alternaria sp. ), wheat black rust (pathogen: Puccinia graminis Persoon subsp. graminis), wheat forage smut (pathogen: Tilletia caries, Tilletia foetida), wheat yellow spot (pathogen: Pyrenophora tritici-repentis), wheat yellow dwarf disease (pathogen: : Sclerophthora macrospora), wheat leaf blight (pathogen: Ascochyta tritici Hori et Enjoji), wheat anthracnose (pathogen: Colletotrichum graminicola), wheat damping disease (pathogen: Gaeumannomyces graminis), wheat powdery mildew (pathogen: Erys graminis de Candolle f. sp. tritici Marchal), wheat snow rot (pathogen: Typula incarnata Lasch ex Fries, Typula ishikariensis Imai), wheat snow rot large grain rot (pathogen: Sclerotinia borealis et Bubelle) It is known that diseases such as streak (pathogen: Cephalosporium gramineum Nisikado et Ikata) occur.
In the wheat of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of the above-mentioned diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, none of these diseases, downy mildew, Pseudomonas syringae infection, etc. occurred, although no antibacterial pesticide was used.

<Test Example B2> Improvement of Disease Resistance of Wheat Using Papaya Extract Using the same procedure as in Test Example B1, wheat seeds soaked in the papaya seed extract were obtained. This was sown and cultivated in a farm in Miyazaki City, Miyazaki Prefecture. Also in this test example, no pesticides including antibacterial pesticides were used.
In the wheat of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B3> Improving Disease Resistance of Wheat Using Wheat Extract A test was performed in the same manner as in Test Example B1, except that wheat seeds were used instead of papaya seeds for the preparation of the extract solution. That is, an extract was prepared from wheat seeds that had undergone a freezing process, wheat seeds were immersed in the extract, and the seeds were sown in a field for cultivation.
In the wheat of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B4> Improvement of disease resistance of corn using papaya extract An extract is prepared from papaya seeds in the same manner as in Test Example B1, and corn seeds are added to the extract in the same manner as in Test Example B1. Soaked. The maize seed was cultivated in Hainan, China (Hainan base). No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In maize, smut (pathogen: Ustilago maydis), sesame leaf blight (pathogen: Cochliobolus heterostrophus), soot (Setosphaeria turcica), brown spot (pathogen: Kabatiella zeae), sheath blight (pathogen: Thanatephorus cucumeris ), bacterial streak (pathogen: Burkholderia andropogonis), brown streak (pathogen: Acidovorax avenae subsp. avenae), leopard disease (pathogen: Gloeocercospora sorghi), blast (pathogen: Pyricularia grisea, Pyricularia sp.)立枯病（病原：Ｆｕｓａｒｉｕｍ ａｖｅｎａｃｅｕｍ、Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ．）、ピシウム苗立枯病（病原：Ｐｙｔｈｉｕｍ ｓｙｌｖａｔｉｃｕｍ、Ｐｙｔｈｉｕｍ ｄｅｂａｒｙａｎｕｍ、Ｐｙｔｈｉｕｍ ｕｌｔｉｍｕｍ、Ｐｙｔｈｉｕｍ ｓｐｉｎｏｓｕｍ、Ｐｙｔｈｉｕｍ ｐａｒｏｅｃａｎｄｒｕｍ）、根腐病（病原：Ｐｙｔｈｉｕｍ ｇｒａｍｉｎｃｏｌａ）、根朽It is known that diseases such as disease (pathogen: Rhizoctonia solani) occur.
In the small corn of Comparative Example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, none of the diseases including the above-mentioned diseases occurred even though no antibacterial pesticide was used.

<Test Example B5> Improvement of disease resistance of soybeans using papaya extract An extract is prepared from papaya seeds in the same manner as in Test Example B1, and soybean seeds are added to the extract in the same manner as in Test Example B1. Soaked. This soybean seed was cultivated in China. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In soybeans, soybean follicle disease (pathogen: Phytoplasma), soybean spot bacterial disease (pathogen: Pseudomonas syringae pv. glycinea), soybean leaf scorch (pathogen: Xanthomonas campestris pv. glycines), soybean Fusarium head blight (pathogen: Fusarium ｒｏｓｅｕｍ Ｌｉｎｋ、Ｆｕｓａｒｉｕｍ ｏｘｙｓｐｏｒｕｍ Ｓｃｈｌｅｃｈｔｅｎｄａｈｌ）、ダイズべと病（病原：Ｐｅｒｏｎｏｓｐｏｒａ ｍａｎｓｈｕｒｉｃａ）、ダイズ葉腐病（病原：Ｔｈａｎａｔｅｐｈｏｒｕｓ ｃｕｃｕｍｅｒｉｓ）、ダイズ灰星病（病原：Ｐｈｙｌｌｏｓｔｉｃｔａ ｓｏｊａｅｃｏｌａ Ｍａｓｓａｌｏｎｇｏ）、ダイズ斑点病（病原：Ｃｅｒｃｏｓｐｏｒａ ｓｏｊｉｎａ Hara), soybean wilt (pathogen: Verticillium dahliae Klebahn), soybean stock blight (pathogen: Ophionectria sojae Hara), soybean gray spot (pathogen: Pleosphaerulina americana), soybean brown spot (pathogen: Mycosphaerella sojae Hori) Soybean brown spot (pathogen: Corynespora cassiicola), soybean brown spot (pathogen: Alternaria sp.), soybean brown spot (pathogen: Septoria glycines Hemmi), soybean sclerotia (pathogen: Sclerotinia sclerotiorum), soybean black spot (pathogen: Diaporthe phaselorum), soybean black rot (pathogen: Elsinoe glycines Jenkins), soybean stem blight (pathogen: Phytophthora sojae Kaufmann et Gerdemann), soybean stem blight (pathogen: Phoma sp.), soybean black wilt (pathogen: : Peckia sp.), soybean cochlear disease (pathogen: Thielaviopsis sp.), soybean black root rot (pathogen: Calonectria crotalariae), soybean purple leaf disease (pathogen: Helicobasidium mompa Tanaka), soybean night sickness (pathogen: Septogloeum sojae Yo et Nishizawa), soybean fomopsis rot (pathogen: Phomopsis longicolla Hobbs), soybean defoliation (pathogen: Phialophora gregata), soybean rhizoctonia root rot (pathogen: Rhizoctonia solani Kuhn), soybean ring spot (pathogen: Ascochyta phaseolorum Saccardo), soybean rust (pathogen: Phakopsora pachyrhizi Sydow), soybean pod wilt (pathogen: Phakopsora pachyrhizi Sydow) ), soybean purpura (pathogen: Cercospora kikuchii Matsumoto et Tomoyasu), soybean white silk disease (pathogen: Sclerotium rolfsii Curzi), soybean white leaf disease (pathogen: Rosellinia necatrix Prillieux), soybean anthracnose (pathogen: Macropholamina) , soybean anthracnose (cause: Colletotrichum truncatum, Colletotrichum trifolii Bain et Essary, Glomerella glycines (Hori) Lehman et Wolf, Gloeosporium sp. ), soybean wilt (pathogen: Fusarium oxysporum Schlechtendahl: Fries f. sp. tracheiphilum, Gibberella fujikuroi).
In the soybeans of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a certain rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B6> Improvement of disease resistance of wheat using papaya extract An extract is prepared from papaya seeds in the same manner as in Test Example B1, and wheat seeds are added to the extract in the same manner as in Test Example B1. Soaked. This wheat seed was cultivated in China.
In the wheat of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B7> Improvement of disease resistance of wheat using papaya extract An extract is prepared from papaya seeds in the same manner as in Test Example B1, and wheat seeds are added to the extract in the same manner as in Test Example B1. Soaked. The wheat seeds were sown and cultivated in Russian permafrost.
In the wheat of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B8> Improving Disease Resistance of Carrots Using Wheat Extract Wheat seeds were immersed in an aqueous trehalose solution, left to stand in a program freezer, and frozen. Freezing was carried out slowly over 180 days at a rate of temperature drop of 0.5°C/day so that the lowest freezing temperature was -60°C.

Frozen wheat seeds were naturally thawed at room temperature (25°C). This was left in the open air (25° C.) for one week. Seeds that died during the freezing process fermented and softened or liquefied when left in the open air. By placing the seeds in a colander and washing with water, the fermented seeds (that is, dead seeds) were washed away and only live seeds were selected.

The live seeds left in the colander were gently ground and crushed using a mortar and pestle to obtain a paste. 1 cc of the paste was diluted to 10 L with an aqueous solution of sucralose and trehalose (approximately 8000-fold to 10000-fold dilution) to prepare a diluted extract.

Carrot seeds were soaked in the diluted extract and left for 72 hours. The soaked seeds were sown in soil in Gifu Prefecture and cultivated. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In carrots, chlorosis (pathogen: Phytoplasma), root canker (pathogen: Agrobacterium tumefaciens), powdery mildew (pathogen: Erysiphe heraclei), black leaf blight (pathogen: Alternaria dauci), black spot (pathogen: Alternaria radicina), sclerotia (pathogen: Sclerotinia intermedia), dry rot (pathogen: Sclerotinia sclerotiorum), purple leaf disease (pathogen: Helicobasidium mompa), white silk disease (pathogen: Sclerotium rolfsii), spot rot It is known that diseases such as disease (pathogen: Pythium sulcatum) and root rot (pathogen: Rhizoctonia solani) occur.
In the comparative carrots cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B9> Improving disease resistance of Korean ginseng using wheat extract A wheat seed extract was obtained in the same manner as in Test Example B8. Ginseng roots were soaked in the diluted extract and left for 72 hours. The roots that had undergone this soaking process were planted and cultivated in Okayama Prefecture soil. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In Korean ginseng, spot disease (pathogen: Alternaria panax Whetzel), red rot (pathogen: Erwinia araliavora), white rot (pathogen: Erwinia sp.), koshibori disease (pathogen: Phytophthora cactorum), fusarium disease ( Pathogen: Fusarium solani), dry black rot (pathogen: Phoma panacicola), sclerotia (pathogen: Sclerotinia sp.), root rot (pathogen: Cylindrocarpon destructans), white leaf disease (pathogen: Rosellinia necatrix Prlieux), Diseases such as anthracnose (pathogen: Colletotrichum panacicola), seedling rot (pathogen: Pythium myriotylum), gray mold (pathogen: Botrytis cinerea) are known to occur.
In the comparative ginseng cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B10> Improving Disease Resistance of Other Plants The seeds of the plants listed below were treated with an extract extracted from papaya seeds in the same manner as in Test Example B1, and sown and cultivated. Cultivation was performed in Okayama Prefecture, Japan. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.
Coffee, chili hazelnut, golden egg fruit, banana, dwarf coconut, cacao, lychee, palm, Japanese pepper, durian, cashew, carob, pawpaw mango, acacia, cypress, pineapple, guava, acai, dates, bakpari, Danieli

As a result, none of the above-listed plant species suffered from diseases including downy mildew, rust, Pseudomonas syringae infection, etc., even though no antibacterial pesticide was used. In addition, in the plants of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate.

<Test Example B11> Enhancement of properties by spraying (green onion)
An extract was prepared from wheat seeds that had undergone the freezing step in the same manner as in Test Example B3. About 50 ml of this extract was sprayed on green onions 2 weeks after planting every 5 days for cultivation. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In green onions, chlorosis (pathogen: phytoplasma), downy mildew (pathogen: Peronospora destructor), wilt (pathogen: Fusarium oxysporum f. sp. cepae), plague (pathogen: Phytophthora nicotianae), white plague (pathogen: Phytophthora porri), yellow spot (pathogen: Heterosporium allii), black stain (pathogen: Mycosphaerella allicina), rust (pathogen: Puccinia allii), black spot (pathogen: Alternaria porri), leaf blight (pathogen: Pleospora herbarum , Pleospora allii, Stemphylium botryosum), black rot (pathogen: Sclerotium cepivorum), small sclerotia (pathogen: Ciborinia allii), small sclerotia (pathogen: Botrytis squamosa), white silk disease (pathogen: Sclerotium rolfsii), seedling damping (pathogen: Rhizoctonia solani), and soft rot (pathogen: Erwinia carotovora subsp. carotovora) are known to occur.
In the green onion of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B12> Improvement of disease resistance of rice by spraying An extract was prepared from wheat seeds that had undergone a freezing step in the same manner as in Test Example B3. This extract was sprayed on rice seedlings and cultivated. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In rice, bakanae disease (pathogen: Gibberella fujikuroi), sesame leaf blight (pathogen: Cochliobolus miyabeanus), seedling blight (pathogen: Rhizopus spp., Fusarium spp., Pythium spp., Trichoderma spp., Mucor spp. ), rice blast (cause: Pyricularia grisea), sheath blight (cause: Thanatephorus cucumeris), red sclerotia (cause: Waitea circinata), yellow dwarf disease (cause: Sclerophthora macrospora), rice blight (cause: Claviceps virens), brown streak (pathogen: Acidovorax avenae subsp. avenae), seedling bacterial disease (pathogen: Burkholderia plantarii), rice wilt disease (pathogen: Burkholderia glumae), bacterial leaf blight (pathogen: Xanthomonas oryvazae) oryzae), leaf sheath browning disease (pathogen: Pseudomonas fuscovaginae), lining brown disease (pathogen: Erwinia ananas), yellow wilt (pathogen: Phytoplasma), and other diseases are known to occur.
In the rice plants of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B13> Improvement of Disease Resistance of Broad Beans by Sprinkling An extract was prepared from wheat seeds that had undergone a freezing step in the same manner as in Test Example B3. This extract was sprayed and cultivated on broad beans cultivated in soil in Kure City, Hiroshima Prefecture. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In fava beans, diseases such as broad bean brown spot (pathogen: Ascochyta fabae), broad bean red spot (pathogen: Botrytis fabae), faba bean wilt (pathogen: Fusarium avenaceum, Fusarium oxysporum), broad bean rust (Uromyces viciae) known to occur.
In the broad beans of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B14> Improvement of Disease Resistance of Cabbage by Sprinkling An extract was prepared from wheat seeds that had undergone a freezing step in the same manner as in Test Example B3. This extract was sprayed on cabbage grown in a planter. No pesticides, including antibacterial pesticides against bacteria and fungi, were used during cultivation.

In cabbage, cabbage black rot (pathogen: Xanthomonas campestris pv.), cabbage downy mildew (pathogen: Peronospora brassicae Gaeumanncampestris), cabbage sclerotia (pathogen: Sclerotinia sclerotiorum), cabbage seedling blight (pathogen: Rhizoctonia solani Kuhn), cabbage blight (pathogen: Asteromella brassicicae), cabbage verticillium wilt (pathogen: Verticillium dahliae Klebahn), cabbage soft rot (pathogen: Erwinia carotovora subsp. carotovora), cabbage gray mold (pathogen: Botrytis cinerea), cabbage black spot (pathogen: Alternaria brassicae), cabbage clubroot (pathogen: Plasmodiophora brassicae Woronin), cabbage white rust (pathogen: Albugo macrospora), cabbage black spot (pathogen: Pseudomonas syringae pv. maculicola ), cabbage rot (pathogen: Pseudomonas marginalis pv. marginalis, Pseudomonas viridiflava), cabbage wilt (pathogen: Fusarium oxysporum Schlechtendahl), cabbage black soot (pathogen: Alternaria brassicicola), cabbage root rot (pathogen: Phingoma) , and cabbage powdery mildew (pathogen: Erysiphe polygoni de Candolle).
In the cabbage of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate. On the other hand, in this test example to which the present invention was applied, no disease occurred even though no antibacterial pesticide was used.

<Test Example B15> Characteristic enhancement by spraying (other plants)
It was extracted from wheat seeds by the same method as in Test Example B3, and tomatoes, green peppers, wheat, morning glory and watermelon were cultivated while spraying this extract. As a result, no disease was observed in any of the plants. In addition, in the plants of the comparative example cultivated under the same conditions except that the present invention was not applied, the occurrence of diseases caused by bacteria and filamentous fungi was observed at a constant rate.

<Test Example B16> Comparative test of downy mildew incidence in green onion cultivation An extract was prepared from wheat seeds that had undergone a freezing step in the same manner as in Test Example B3. In Kurahashi Town, Kure City, Hiroshima Prefecture, green onions were cultivated by spraying this extract. As a comparative example, green onions were cultivated under the same conditions except that the extract was not sprayed.

For green onions that reached harvest time in June and September when downy mildew tends to occur with a lot of rain, the incidence of downy mildew was aggregated, and the reduction rate of downy mildew occurrence in the example (reduction rate = (comparison The disease incidence in the example-the disease incidence in the example)×100/the disease incidence in the comparative example) was calculated. As a result, the welsh onions of Example had a 30% reduction in the incidence of downy mildew compared with the welsh onions of Comparative Example. In addition, almost all of the green onions of the comparative example rotted due to the propagation of bacteria and filamentous fungi under the influence of long rains, making it impossible to ship them. On the other hand, almost all of the green onions of Examples could be harvested in a state ready for shipment, despite the effects of long rains.

In addition, the reduction rate of downy mildew occurrence was also calculated for green onions that were harvested at a time other than June and September when there was a lot of rain. As a result, the welsh onions of the example showed a 70% reduction in the incidence of downy mildew compared to the welsh onions of the comparative example.

<Test Example B18> Comparative Test of Rust Attack Rate in Green Onion Cultivation An extract was prepared from wheat seeds that had undergone a freezing step in the same manner as in Test Example B3, and this extract was sprayed to cultivate green onions. The green onion of the comparative example was cultivated under the same conditions except that the extract was not sprayed. The rates of occurrence of red rust and white rust were counted for the green onions at harvest time, and the rate of reduction in the occurrence of red rust and white rust in the examples was calculated. As a result, the reduction rate of red rust and white rust in the green onion of Example was 100%.
During the cultivation process, some leaves and stems with symptoms of red rust and white rust were observed in the welsh onions of Example. However, by removing this, further spread of infection did not occur, and the above-mentioned 100% reduction rate of disease occurrence at harvest time was achieved. In addition, in the green onion of the comparative example, even if the leaves and stems in which the symptoms were observed were removed during the cultivation process, the spread of infection could not be stopped, and the green onion in a state ready for shipment at the harvest time could not be obtained.

<Test Example B19> Comparative Test of Scab and Echidna Disease Rates in Potato Cultivation An extract was prepared from wheat seeds that had undergone a freezing step in the same manner as in Test Example B3. Seed potatoes cut into pieces were immersed in this extract and planted in a field in Kurahashi-cho, Kure City, Hiroshima Prefecture in late February. Comparative potatoes were grown under the same conditions except that they were not immersed in the extract.

The rates of scab disease and equitic disease incidence were totaled for the potatoes at harvest time, and the reduction rate of scab and equitic disease incidence in the examples was calculated. As a result, the potatoes of the example showed a 100% reduction in the incidence of scab and epilepsy compared to the potatoes of the comparative example. That is, in the potatoes of the example, neither scab nor epilepsy occurred at all.

<Test Example B20> Comparative test of downy mildew incidence in garland chrysanthemum cultivation An extract was prepared from wheat seeds that had undergone the freezing process in the same manner as in Test Example B3, and the extract was sprayed to cultivate garland chrysanthemums. . The garland chrysanthemum of the comparative example was cultivated under the same conditions except that the extract was not sprayed. For the garland chrysanthemums at harvest time, the rate of occurrence of downy mildew was counted, and the reduction rate of the occurrence of downy mildew in the example was calculated. As a result, the reduction rate of downy mildew in the garland chrysanthemum of Example was 100%.

<Summary of cultivation experiments>
As described above, by applying the method for improving disease resistance of the present invention (embodiment (A) and embodiment (B)), the effect of improving disease resistance including resistance to diseases caused by bacteria and fungi as pathogens is obtained regardless of plant species. It was demonstrated that it can be obtained.
Gene expression analysis tests carried out for molecular biological analysis of the results of cultivation experiments will be described below.

<Gene expression analysis>
The papaya of the example of Test Example A1, the coffee of the example of Test Example A5, the green neck radish of the example of Test Example A6, and the wheat of the example of Test Example B1, treated according to the present invention, and the treatment of the present invention. Leaves were collected from each plant (comparative example) cultivated under the same cultivation conditions as in the example without performing Total RNA was extracted from the collected leaves and subjected to RNA-seq analysis.

For the RNA-seq analysis, a De novo RNA sequencing method (hereinafter referred to as De novo RNA-seq) using no reference genome was performed. That is, by comparing the cDNA sequence of an unknown RNA obtained as a transcript with a known database cDNA sequence (hereinafter referred to as a reference cDNA sequence) using the Basic Local Alignment Search Tool (BLAST), it is possible to identify similarity to a known gene. We investigated the expression levels of gene transcripts with high potential. A highly accurate Arabidopsis thaliana cDNA sequence database was used as a model plant for the reference cDNA sequence, and the amounts of gene transcripts highly similar to these were calculated.

Specifically, total RNA was extracted from leaves of the plants of Examples and Comparative Examples using an RNA extraction kit (NucleoSpin ^R RNA Plant, Macherey Nagel). After quantifying total RNA using an Agilent 2200 TapeStation, a cDNA library was prepared using a library preparation kit (TruSeq Stranded mRNA Sample Prep Kit, Illumina). The prepared library was subjected to sequence analysis using a next-generation sequencer (HiSeq 2500, Illumina) to obtain sequence read data. After removing adapter sequences from the acquired read data using Cutadept software (v1.3), low-quality read sequences were removed using Sickle software (v1.200). The resulting data were used for de novo assembly into a reference Arabidopsis cDNA sequence by Trinity software (v2.0.6).
BLAST analysis was performed on the sequences of the transcripts obtained by assembling, and highly similar genes were extracted. Next, differentially expressed genes (DEGs) having a difference of 2-fold or more in the Fragments Per Kilobase of exon per Million mapped fragments Value (FPKM value) per gene between Examples and Comparative Examples were extracted. Finally, redundant genes were removed to confirm differential gene expression.

As a result, a 3.3-fold increase in the expression level of the RPPL1 gene involved in disease resistance and a 2.5-fold increase in the expression level of the FLS2 gene were observed in the papaya of Example compared to the papaya of Comparative Example.

A 3.6-fold increase in the expression level of the RPPL1 gene involved in disease resistance and a 6.9-fold increase in the expression level of the FLS2 gene were observed in the blue-necked radish of the example compared to the blue-necked radish of the comparative example. .

In the coffee of the example compared to the coffee of the comparative example, the expression level of the RPPL1 gene involved in disease resistance was increased by 584 times, the expression level of the FLS2 gene was increased by 3.9 times, and the expression of the UGT74F2 gene was 1/670. A decrease in volume was observed.

Compared to the wheat of the comparative example, in the wheat of the example, the expression level of the RPPL1 gene involved in disease resistance increased 36,107 times, the expression level of the FLS2 gene increased 66,913 times, and the expression level of the UGT74F2 gene decreased to 1/110. , a 6064-fold increase in the expression level of the LRK10 gene was observed.

<Summary of results>
By gene expression level analysis, gene expression level fluctuations contributing to improved disease resistance were observed in all plants to which the present invention was applied. Specifically, the RPPL1 gene, which recognizes filamentous fungi such as Peronospora parasitica and confers resistance to downy mildew, has a marked increase in expression, and FLS2, which recognizes the flagellar protein flagellin and induces an immune response. A significant increase in the expression level of genes, a significant decrease in the expression level of the UGT74F2 gene involved in susceptibility to Pseudomonas syringae, and a significant increase in the expression level of the LRK10 gene that provides rust resistance were observed.
The results of gene expression level analysis support the excellent disease resistance improvement effect observed in the cultivation experiments of Test Examples A1 to A6 and Test Examples B1 to B20 from a molecular biological point of view.

In Test Examples B1 to B20, extracts extracted from seeds that have undergone a freezing process are used. However, when the results of Test Examples A1 to A6 are also comprehensively considered, it can be seen that even extracts extracted from plant tissues other than seeds have the same effect of improving disease resistance as those shown in Test Examples B1 to B20. Naturally, it can be inferred.

At the time of the filing date of the present application, the freeze-thaw awakening method and the enzyme solution method were well known (Patent Documents 3 to 5). It has been found that the freeze-thaw awakening method and the enzyme solution method have similar effects, and similar changes in gene expression profiles are observed in plants to which both inventions have been applied. The gene expression analysis described above also reconfirms this.
Considering that the common point of the freeze-thaw awakening method and the enzyme solution method is the freezing process, it is reasonable that the freezing process triggers the occurrence of some specific factor that causes epigenetic changes and the resulting enhancement of properties. is led to In the freeze-thaw awakening method, it is considered that the specific factor generated through the freezing process acts on plant cells to induce epigenetic changes. On the other hand, in the enzymatic solution method, the specific factor generated by the freezing process is contained in the extract, and the specific factor acts on the cells constituting the plant tissue immersed in the extract. It can be understood that the freeze-thaw awakening method brings about the same epigenetic changes in plant cells.

Here, according to Test Examples A1 to A6, which were carried out in the same process as the freeze-thaw awakening method, the effect of improving disease resistance was the same in any case where various plant tissues such as plant seeds, roots, and axillary shoots were frozen. was gotten. In other words, the specific factor that causes the above-described enhancement of characteristics (improvement of disease resistance) does not depend on the type of plant tissue subjected to the freezing step, that is, it occurs in all types of plant tissue subjected to the freezing step. can understand.
In addition, if there is no "point of action" where the "plant property enhancing factor" directly or indirectly acts on the target plant tissue whose properties are to be enhanced, the property enhancement of the target plant tissue is induced. It is self-evident that it cannot be done. The "point of action" includes receptors present on the cell membrane, cytoplasm, or nucleus, proteins involved in signal transduction pathways controlling gene expression, transcription factors, and the like.
In this regard, Test Examples A1 to A6 demonstrate that similar property enhancement effects were obtained when any plant tissue was frozen. From this, it can be understood that all plant tissues have a "point of action" at which a "plant property-enhancing factor" acts.
Based on the results of Test Examples A1 to A6 described above and matters that can be understood from common technical knowledge, the mechanism by which the freeze-thaw awakening method and the enzyme solution method exhibit the effect of enhancing the characteristics of plant tissue will be described.

The freeze-thaw awakening method (embodiment (A)) is a method of imparting growth characteristics, cold resistance, and disease resistance to plant tissues that have undergone the freezing process, but as described above, the plant tissues subjected to the freezing process are not limited. . In other words, freezing plant tissue regardless of its type produces a factor that enhances plant characteristics, and the action of this factor causes an epigenetic change, resulting in a change in the genetic profile, which results in growth characteristics and cold resistance. an enhancement of

One enzyme liquid method (Embodiment (B)) also includes a freezing step like the freeze-thaw awakening method. In the enzymatic solution method as well, plant tissues that have undergone the freezing process produce "factors that enhance the characteristics of plants." As described above, "factors for enhancing plant characteristics" are generated in all plant tissues subjected to the freezing process, regardless of the type of plant tissue.
After that, the extraction process is performed to obtain an extract from the plant tissue that has undergone the freezing process. "factor of" is naturally included. When plant tissue is brought into contact with this extract, the "factors that enhance plant characteristics" act on the cells that make up the plant tissue, resulting in epigenetic changes similar to those of the freeze-thaw awakening method. can be understood as

In summary, when plants are treated by the enzyme solution method (Embodiment (B)), the extract extracted from the plant tissue that has undergone the freezing process can be used to enhance plant characteristics regardless of the type of plant tissue. Factors" are contained therein, and in the plant tissue brought into contact with one of the extracts, there exists a "point of action" where the "plant characteristics enhancing factor" acts regardless of the type.
Therefore, it can be naturally understood that in Embodiment (B), the type of plant tissue subjected to the freezing step is not limited, nor is the type of plant tissue subjected to the contacting step.

INDUSTRIAL APPLICABILITY The present invention can be applied to agricultural production technology.

Claims (4)

A method for improving disease resistance of a target plant, comprising a freezing step of freezing plant tissue,
The minimum freezing temperature in the freezing step is −20° C. or less, the rate of temperature drop in the freezing step is 0.8° C./day or less, and the period of the freezing step is 100 days or more;
The disease resistance is resistance to one or more selected from resistance to downy mildew, resistance to diseases caused by flagellated bacteria, resistance to Pseudomonas syringae infection, and resistance to rust. ,
A method for improving plant disease resistance, characterized by being carried out according to the following (A) or (B).

(A) The disease resistance of the target plant is improved by subjecting the plant tissue of the target plant whose disease resistance is to be improved to the freezing step.
(B) subjecting a plant tissue different from the target plant to the freezing step, and bringing the target plant whose disease resistance is to be improved into contact with the extract obtained from the different plant tissue, thereby improve the disease resistance; A method for inducing changes in gene expression levels that contribute to improved disease resistance in a target plant, comprising a freezing step of freezing plant tissue,
The minimum freezing temperature in the freezing step is −20° C. or less, the rate of temperature drop in the freezing step is 0.8° C./day or less, and the period of the freezing step is 100 days or more;
The gene expression level variation that contributes to the improvement of disease resistance in the target plant is an increase in expression of the RPPL1 (Putative disease resistance RPP13-like protein 1) gene or its ortholog, an increase in expression of the FLS2 (Flagellin sensing 2) gene or its ortholog, LRK10 (rust resistance kinase Lr10) gene or its orthologue increased expression and UGT74F2 (UDP-glucosyltransferase 74F2) gene or its orthologs selected from suppressed expression, gene expression level variation that contributes to disease resistance,
A method for inducing variation in gene expression that contributes to improved disease resistance of a plant, characterized by being carried out according to (A') or (B') below.

(A′) Gene expression level variation that contributes to the improvement of disease resistance of the target plant by subjecting the plant tissue of the target plant to the freezing step to induce the gene expression level variation that contributes to the improvement of disease resistance. to induce
(B') Subjecting a plant tissue different from the target plant to the freezing step, and inducing a gene expression level change that contributes to improvement of disease resistance in the extract obtained from the different plant tissue Said target plant is brought into contact with the target plant to induce changes in gene expression levels that contribute to the improvement of the disease resistance of the target plant. selected from resistance to downy mildew, resistance to diseases caused by flagellated bacteria, resistance to Pseudomonas syringae infection and rust resistance, comprising the step of performing the method of claim 1 or 2 A method for producing one or more plants with improved disease resistance. Contains as an active ingredient an extract extracted with an aqueous solvent from plant tissue that has undergone a freezing process,
The minimum freezing temperature in the freezing step is −20° C. or less, the rate of temperature drop in the freezing step is 0.8° C./day or less, and the period of the freezing step is 100 days or more;
A composition for inducing variation in gene expression levels that contributes to improvement of disease resistance of plants and/or disease resistance of plants, characterized by being used for the following uses (i) and/or (ii).

(i) Use for improving resistance to disease selected from resistance to downy mildew, resistance to diseases caused by flagellated bacteria, resistance to Pseudomonas syringae infection, and resistance to rust.
(ii) increased expression of the RPPL1 (Putative disease resistance RPP13-like protein 1) gene or its ortholog, increased expression of the FLS2 (Flagellin sensing 2) gene or its ortholog, increased expression of the LRK10 (rust resistance kinase Lr10) gene or its ortholog and suppression of expression of UGT74F2 gene UGT74F2 (UDP-glucosyltransferase 74F2) or its ortholog, for inducing variation in gene expression that contributes to disease resistance.