JP4940393B2 - Plant activator screening method - Google Patents

Description

  The present invention relates to a screening method for a plant activator. More specifically, the present invention relates to plant activator screening using plants that are sensitive to plant activators and utilizing the positive correlation between systemic acquired resistance and the occurrence of phytotoxicity by plant activators. Regarding the method.

  Plants are constantly exposed to phytopathogenic microorganisms that inhabit the soil, such as wilt fungus and bacterial wilt. Since diseases caused by these pathogens are transmitted through the soil, once they occur, it is difficult to control them with drugs. Moreover, since these diseases cause plant bodies to die, they will suffer destructive damage.

  Conventionally, fumigating fungicides (for example, chlorpicrin, methyl bromide) used to control these diseases are highly toxic, tearing, can only be treated prophylactically, and can easily be applied and disinfected. It was necessary to pay attention to the application, for example, the effects were often inadequate, plants would be damaged if they were not vented sufficiently, and damage to the surrounding fields would need to be considered. Moreover, because these drugs are non-selective, they kill even non-pathogenic soil microorganisms and other organisms, and as a result, the biota in the soil becomes simple, so the subsequent occurrence of disease is rather. In many cases, it was enormous. In addition, the use of these fumigants is refraining from the environmental impact.

  In addition, there is also a method for controlling soil diseases by killing pathogenic bacteria by mixing or irrigating a chemically synthesized pesticide having an effect of killing pathogenic bacteria (bactericidal). However, chemical pesticides tend to be adsorbed on the soil or decomposed by soil organisms and are generally less effective.

  On the other hand, chemical synthetic pesticides having generally bactericidal properties have been used for the above-ground diseases such as powdery mildew, plague and rice blast. Although these fungicides are indeed highly effective, they have not only suggested dangers such as persistence in the edible part and drift to surrounding fields, but continuous use can easily lead to the development of resistant bacteria. Due to the spread, the effect of drugs is often not recognized.

  For these reasons, there is a need for disease control agents having different mechanisms of action in place of conventional bactericidal chemically synthesized pesticides. There is a plant activator that imparts resistance to plants as an agrochemical that does not show the emergence of resistant bacteria and that efficiently controls soil diseases by a simple treatment method. Plant activators such as probenazole (PBZ), acibenzoral S-methyl (ASM or BTH), and thiazinyl have been put to practical use, but these drugs have a high control effect on rice blast, There is only a pesticide application registration for a small number of above-ground diseases in soil treatment as a disease, and an environment that exhibits high control effect on various diseases by inducing systemic acquired resistance to various plants with a simple treatment method Development of drugs with low load is desired.

  It is also known that the foliar spray of validamycin A (VMA) and validoxylamine A (VAA) has a high control effect against tomato wilt disease, a soil disease, and these effects are applied to plants in soil. Since it has been shown that resistance to diseases is induced, plant activators have been proposed as new control methods for soil diseases (Non-patent Document 1 and Non-patent Document 2).

  In addition, VMA and VAA also show disease control effects against other soil diseases such as bacterial wilt, powdery mildew, and epidemics, and this is due to induction of systemic acquired resistance. Is assumed (Non-Patent Document 1 and Non-Patent Document 2).

  On the other hand, in some cases, it has been reported that plant activators cause phytotoxicity such as plant growth suppression, and foliar application of validamycin A is sometimes reported to cause phytotoxicity on tomatoes (Non-patent Document 1). And Non-Patent Document 2)

  The plant activator is selected as a result of screening by a so-called “bukkake test” (ie, a biological test in which a plant is actually treated with a candidate compound and then inoculated with a pathogenic fungus to test and screen the disease-inhibiting effect). Although it has been obtained as a very small number of compounds included in candidate compounds having disease-inhibiting effects, this selection method generally requires a certain amount of time (usually several days to 10 days) until the control effect is manifested. The selection of the plant activator was inefficient because the beta was masked by many bactericidal substances selected at the same time, and it was difficult to find the beta. In recent years, molecular biological techniques have advanced, and it has been reported that plant activators activate signal transduction systems such as systemic aquired resistance (SAR) in plant tissues. Based on this, molecular markers ( Methods for screening plant activators using the expression of SAR-related genes and substances) as indicators and methods for synthesizing and screening candidate compounds with reference to the structure of known plant activators have been reported. However, these methods have drawbacks such as requiring expensive reagents and instruments, and inability to observe the reaction of the plant when the drug is actually sprayed.

R. Ishikawa, K. Shirouzu, H. Nakashita, H.-Y. Lee, T. Motoyama, I. Yamaguchi, T. Teraoka and T. Arie, Phytopathology 95 (10), 1209-1216 (2005) Ryo Ishikawa and Riki Arie, Plant Protection 60 (5), 219-223 (2006)

  In the above situation, if there is a method for screening plant activators at a high rate and in a short time, a plant activator that can induce systemic acquired resistance that can be expected to have a high control effect against diseases can be easily obtained. It will be possible to find out.

  Accordingly, an object of the present invention is to provide a method for screening plant activators at a high rate, easily and in a short time.

The present inventors have found for the first time that the degree of phytotoxicity of each variety that appears when a plant activator is applied to a plant correlates with the degree of systemic acquired resistance to phytopathogenic microorganisms inoculated after the treatment. Based on this, the present inventors have found a method of treating a sensitive plant with a test compound and screening a compound that induces strong phytotoxicity as a substance having a high ability to induce disease resistance.
In the present specification, the “test compound” refers to a candidate compound for a plant activator for screening according to the present invention.

Therefore, in summary, the present invention has the following features.
The present invention applies a different test compound to a plant sensitive to a plant activator, and no phytotoxicity due to the test compound is detected against an insensitive plant control of the same species. On the other hand, when a phytotoxicity is detected, a screening method for a plant activator comprising determining that the test compound has an ability to induce systemic acquired resistance to a disease is provided.

  The term “plant activator” as used herein has the activity of inducing systemic resistance to pathogens by pre-treating the plant and often avoiding the occurrence of multiple types of diseases. Means a drug that does not have bactericidal properties against pathogens.

  As used herein, the term “sensitivity” means that a phytotoxicity is likely to occur. The term “insensitive” means that no or little phytotoxicity occurs.

  As used herein, the term “whole body acquired resistance” refers to endogenous resistance to disease, ie, immune-like resistance, by activation of a plant cell's associated signaling system by a plant activator. Means the state assigned to.

  According to the present invention, in one embodiment, the screening is performed based on a positive correlation between the degree of systemic acquired resistance by a plant activator and the degree of phytotoxicity.

  Here, the “positive correlation” means that a higher degree of phytotoxicity caused by a plant activator has a higher degree of systemic acquired resistance (effect of suppressing disease).

  According to the present invention, in another embodiment, the method of the present invention comprises the steps of (1) providing a plant activator sensitive plant and an insensitive allogeneic plant, and (2) a test compound for each plant. (3) optionally inoculating each plant with a phytopathogenic microorganism, (4) measuring the phytotoxicity of the test compound, and (5) sensitive plants compared to insensitive plant controls The method includes the step of selecting, as a plant activator, a test compound from which a phytotoxicity is detected.

  In another embodiment thereof, the method of the present invention further comprises using different concentrations of plant activator to determine a plant activator concentration that can suppress the phytotoxicity while retaining the systemic acquired resistance. It is characterized by including.

  According to the present invention, in yet another embodiment, the method of the present invention comprises treating the target plant or plant cultivar with the test compound to confirm the effectiveness of the test compound (i.e., disease-inhibiting ability). The method further includes:

  According to the invention, the plants that can be used in the method are selected from dicotyledonous plants or monocotyledonous plants. Examples of such plants include, but are not limited to: Solanum (tomato, tobacco, etc.), Cucurbitaceae (cucumber, melon, etc.), Asteraceae (Chisha, lettuce, etc.), Brassicaceae (Arabidopsis, etc.), A plant made of rice, wheat, barley, corn, and the like.

  The method of the present invention does not require a special device as long as a plant sensitive to a plant activator is prepared, and the test can be easily and quickly determined simply by treating the plant with the test compound and then observing it. In addition, it is advantageous in that a compound having a simple herbicidal activity (in this case, phytotoxicity occurs in both) can be eliminated by using a plant that is difficult to cause phytotoxicity.

In the screening method of the present invention, it is necessary to prepare a plant or plant variety that is sensitive to a plant activator (that is, causing phytotoxicity), preferably highly sensitive, and conversely, an insensitive same plant or plant variety. There is. Such sensitive plants can be determined by the following procedures (1) to (6).
(1) Prepare an appropriate test compound.
(2) A plurality of varieties of a specific plant are prepared and cultivated. Here, the plant body may be obtained through germination of seeds, or may be planted in a container filled with soil by obtaining seedlings. The plant that can be used in the test may be in any stage from seeds, bulbs, ears and seedlings to adults, but plants with seedlings and true leaves in the 2-15 leaf stage are preferred.
(3) Each plant variety is treated with the test compound. Here, the treatment method includes, for example, a method of spraying or applying a drug to the foliage of a plant, a method of adding a drug to soil or a nutrient medium, a method of immersing or dressing seeds, bulbs, ears, etc. .
(4) Usually, 3 to 10 days after treatment with the test compound, each plant variety is inoculated with a phytopathogenic microorganism desired to be controlled. Inoculation methods include, for example, irrigating microorganisms in soil where plants are cultivated, inoculating plants, mixing contaminated microorganisms with soil to create contaminated soil, and planting plants, plant stems Alternatively, a method in which a part of a leaf is damaged and a microorganism is brought into contact with the site to be infected, a method in which a plant is spray-inoculated with a microorganism suspension is included.
(5) Measure the degree of disease caused by microorganisms and the degree of chemical damage. At this time, when the degree of disease is low, it indicates that the induction of systemic acquired resistance by the test compound is high, and conversely, when the degree of disease is high, this indicates that the induction of resistance is low. Yes. The degree of onset and the degree of phytotoxicity can be quantified based on the degree of symptoms in comparison with healthy plants, such as the degree of browning, the degree of necrosis, and the death. Specifically, see Example 1 described later.

Next, a graph is created by plotting the degree of disease onset (the larger the value, the more severe the disease is) on the vertical axis and the degree of drug damage (the higher the number is, the more harmful the drug is) on the horizontal axis. (In other words, there is a positive correlation between systemic acquired resistance and occurrence of phytotoxicity) (FIG. 1).
(6) From the graph showing the correlation of (5) above, a plant variety having a relatively high degree of phytotoxicity is selected as a sensitive plant.

  Sensitive plants and non-sensitive plants that can be used in the present invention as determined by the above-described method can be obtained from plants such as wild type plants, mutant plants (including plant varieties), and transformed plants.

  Mutant plants are plants obtained by mutation by natural mating, treatment with radiation such as UV, X-rays, gamma rays and neutron rays. Many plant varieties are created by such mutations. For these existing or newly created plant varieties, a graph showing the correlation between the degree of illness caused by a specific known plant activator and the level of phytotoxicity is created. A variety having a relatively high degree can be selected as a sensitive variety.

  Transformed plants include, for example, plants genetically engineered to suppress systemic acquired resistance (SAR) induction, such as an enzyme that degrades salicylic acid that expresses SAR induction into catechol, ie, salicylate hydroxylase (NahG), And a plant into which a nahG gene derived from a bacterium encoding for example Pseudomonas fluorescence has been introduced. Although this plant can recognize plant activator, SAR is not induced because salicylic acid does not accumulate, and catechol accumulation is likely to cause phytotoxicity, and plant activator that acts upstream of salicylic acid in SAR High sensitivity, ie strong phytotoxicity. In this case, the transformed plant is a sensitive plant and the wild type plant is a non-sensitive plant. Examples of such sensitive and non-sensitive plants are tomato varieties Z617 (Plant J. 23: 305-318, 2000) and Moneymaker, respectively. Sensitive plants into which the nahG gene has been introduced and non-sensitive plants can also be used in the same manner for eggplant tobacco, cruciferous Arabidopsis thaliana, cucurbitaceae melon and gramineous rice.

  A transformed plant is obtained by transforming cells, tissues (eg, growth points, leaves, etc.), protoplasts, shoot primordia, polyblasts, and hairy roots of the target plant with a vector containing the nahG gene, and then tissue culture conditions. Vectors that can be created by forming callus, multi-buds, etc., and regenerating them into plants include vectors commonly used in Agrobacterium methods such as intermediate vectors and binary vectors. . Vectors can include multiple cloning sites for incorporating foreign DNA, promoters / enhancers, selectable markers, terminators, polyadenylation sites, ribosome binding sites, and the like. The vector used as the base of the intermediate vector or binary vector may be a vector for bacteria such as E. coli (for example, pBR series, pUC series, pBluescript, etc.). Examples of promoters include cauliflower mosaic virus 35S promoter, maize ubiquitin promoter, nopaline synthase (NOS) gene promoter, octopine synthase (OCT) gene promoter, rice actin (Act1) gene promoter, and the like. Examples of selectable markers include kanamycin resistance gene, neomycin resistance gene, hygromycin resistance gene, β-glucuronidase gene (GUS), luciferase gene, green fluorescent protein gene, chloramphenicol acetyltransferase gene, and the like. Examples of terminators include nos terminators.

  The intermediate vector is a vector containing T-DNA and is a vector that can be homologously recombined with the acceptor vector (Ti plasmid) via T-DNA. 95/016031 and the like.

  A binary vector is a vector containing a right border (RB) and a left border (LB) of a T-DNA region, and is a vector in which foreign DNA is incorporated between RB and LB, examples of which are pBI101, pBI121. , PBI221, pZH2B, pABH-Hm1 and the like.

  Methods for transformation include the Agrobacterium method, particle gun method, electroporation method, gene transfer method to protoplasts (eg, polyethylene glycol method), and the like. When a transformed monocotyledonous plant is produced by the Agrobacterium method, acetosyringone can be added to the transformation medium to increase transformation efficiency.

  Examples of Agrobacterium include Agrobacterium tumefaciens (eg, LBA 4404 strain, EHA101 strain, C58C1RifR strain).

  Plants that can be used in the present invention are not particularly limited, and include dicotyledonous plants, monocotyledonous plants, and the like. Plants are theoretically applicable to any species as long as the SAR moves. Since the specificity and degree of substance recognition are expected to vary depending on the plant species, screening with different species using the screening method of the present invention selects a plant activator suitable for the species. Preferred plants are dicotyledonous plants such as solanaceae (eg, tomatoes, tobacco), Brassicaceae (eg, Arabidopsis), asteraceae (eg, chisha, lettuce, etc.), cucurbitaceae (eg, cucumber, melon, etc.), rice, wheat Monocotyledonous plants such as barley and corn. In addition, examples of the species from which NahG introductions are easily available include tobacco, Arabidopsis (Brassicaceae), melon, and rice.

  The test compounds are not limited to the following, but compounds that act upstream of salicylic acid in SAR are selected by this screening method. For example, saccharides (polysaccharides present on the cell surface of fungi, bacteria, etc., or their Including oligosaccharides and disaccharides that are metabolites), compounds similar in steric structure to these saccharides, inhibitors of enzymes involved in saccharide metabolism, plant hormone related substances, plants, fungal extracts, etc. Specifically, for example, validamycin A (VMA), validoxylamine A (VAA), probenazole (PBZ), trehalose, sorbitol, saccharin, mannose, glucose, saccharin, aspartame, plant hormones (for example, auxins, gibberellins, ethylene , Jasmonic acids), plant hormone inhibitors (eg Uniconazole, AMO (AMO 01618 (Calbiochem-N ovabiochem, # 1712)) etc. The applied concentration may range from about 0.001 to 100000 μg / ml.

  The above correlation was found for the first time by the present inventors, but this correlation suggests that a compound with a high degree of phytotoxicity has a high ability to induce resistance to whole body acquisition and a high disease control effect. Is done. In fact, when screening test compounds using the selected sensitive plants, all of the selected test compounds had a high degree of phytotoxicity (Example 2 described later). At this time, if an insensitive plant is used as a plant control and the test compound causes no or little phytotoxicity to the control, the test compound can be regarded as a plant activator.

  Therefore, the present invention applies another test compound to a plant sensitive to a plant activator, and no phytotoxicity due to the test compound is detected against a non-sensitive plant control of the same species. A plant activator screening method comprising determining that the test compound has an ability to induce systemic acquired resistance to a disease when a phytotoxicity is detected for the plant of the present invention.

More specifically, the method of the present invention comprises:
(1) preparing a plant sensitive to a plant activator and a non-sensitive homologous plant;
(2) applying a test compound to each plant;
(3) optionally inoculating each plant with a phytopathogenic microorganism;
(4) measuring the phytotoxicity due to the test compound, and (5) selecting a test compound that can detect phytotoxicity in a sensitive plant as compared to an insensitive plant control as a plant activator,
including.

  In the above steps, the target plant, plant activator, microorganism inoculation method and the like are the same as described above.

  In the present invention, the test compound is not particularly limited, but is a substance expected to induce systemic acquired resistance-inducing ability for the target plant. For example, it can activate the signal transduction system downstream of salicylic acid. Substances that can activate signal transduction systems via substances, jasmonic acid and ethylene. Examples of test compounds and their application concentrations are as described above.

  The degree of phytotoxicity can be quantified based on symptoms such as, for example, the degree of browning, the degree of necrosis, and death in comparison with healthy plants. The phytotoxicity test can be performed, for example, by applying the test compound about 3 to 10 days later and visually observing the effect of the drug on the stem and leaves.

  Phytopathogenic microorganisms include bacteria, fungi, yeasts, chromistas (flagellates), protistas (deformers), nematodes, viruses, etc., and any microorganisms that are harmful to agriculture. Examples of microorganisms (or pathogens) include Fusarium oxysporum, Verticillium spp., Phytophthora spp., Downy mildew (such as Bremia spp.), And powdery mildew (such as Oidium). spp.), Blast fungus (Magnaporthe grisea), Gray mold fungus (Botrytis spp.), Alternaria spp., Soft rot fungus (Erwinia spp.), Black rot fungus (Xanthomonas spp.), Pseudomonas spp., Ralstonia solanacearum), but is not limited thereto.

The inoculation concentration of the microorganism is 10 ³ to 10 ¹⁰ cells (spore) / ml, but is not limited to this range.

  According to the present invention, using the test system of the present invention, no phytotoxicity is detected for non-sensitive plant controls, but the concentration of the test compound and the test compound at which phytotoxicity is detected for sensitive plants Thus, it is determined that the test compound functions as a plant activator, and candidate compounds can be selected.

  The plant activator thus screened can suppress the phytotoxicity while maintaining whole body acquired resistance by repeating the steps (1) to (5) using various concentrations. A practical level of optimal plant activator concentration can be determined.

  Furthermore, the plant activator that has been screened may be used to actually treat the plant of interest, its various varieties, or plants of the same genus or family to confirm the disease suppressive ability of the plant activator.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited by these examples.

<Example 1> Correlation between resistance induced by validamycin A (VMA) and occurrence of phytotoxicity 1) Experimental method Variety of tomato (Lycopersicon esculentum Mill.) Momotaro, World No. 1, Fukuju No. 2, Zuikou 102, Saturn, New World 1, Hikari, Mizuhide, House Momotaro, House Odoriko, Sun Road, Reishu, Mini Carol, First Power, Okitsu No.3, Powerful Ream VC, Homare 114, Zuikou 208, Vulcan, LS-89, Joint It was obtained from a seed company (for example, Sakata Seed, Takii Seed, Musashi Breeding Farm, Takayama Seed) and used in the following tests.

In the test, 6 plants were used per variety. The validamycin A used was a 99% pure drug substance (Sumika Takeda Agricultural Chemical Co., Ltd.). Tomato dwarf fungus (Fusarium oxysporum f. Sp. Lycopersici race 2 isolate 880621a-1) was used and cultured with shaking in a potato dextrose liquid medium at 28 ° C. for 4 days. The spore concentration in the culture solution of tomato wilt fungus was diluted to 1 to 2 × 10 ⁷ spores / ml with distilled water and used as an inoculation source.

  Tomato seeds were sown in a pot with a diameter of 9 cm filled with sterilized soil and cultivated in a growth chamber for 16 hours under light irradiation conditions at 28 ° C. and for 8 hours under dark conditions at 25 ° C. for 28 days. The validamycin A drug substance was dissolved in water and diluted with distilled water to 100 μg / ml. A sufficient amount of validamycin A aqueous solution was sprayed on tomato stems and leaves using a sprayer. Treated tomatoes were cultivated in a growth chamber for 7 days. Three 3 cm wide spatulas were inserted into the pot, the tomato roots were partially cut, and 5 ml of tomato wilt disease inoculation source was irrigated and inoculated. The inoculated tomato was cultivated for 28 days under the same conditions as before the inoculation.

  The tomato stalk was cut at the border, and the disease severity of 0-4 was investigated from the browning degree of the vascular bundle. Disease severity is 0; healthy, 1; browning is observed in 25% of stems, 2; browning is observed in 26 to 50% of stems, 3; browning is observed in 51 to 75% of stems, 4; stems Browning is observed in 76-100% of the sample. The average disease severity was determined using the following formula.

Average disease severity = (1 x A + 2 x B + 3 x C + 4 x D) / (4 x N1)
(In the formula, A, number of plants showing disease severity 1; B, number of plants showing disease severity 2; C, number of plants showing disease severity 3; D, number of plants showing disease severity 4; N1, Represents the number of plants used in the test.)

  The phytotoxicity was investigated on the basis of the following criteria from the degree of necrotic spots formed on the stem 3 to 10 days after spraying validamycin A. Degree of phytotoxicity 0; healthy, 0.5; slight browning of the stem, 1; browning of the stem, 2; necrotic spots formed on the stem, 3; The average phytotoxicity was determined using the following formula.

Average phytotoxicity = (0.5 x E + 1 x F + 2 x G + 3 x H) / (4 x N2)
(In the formula, E: number of plants showing a degree of phytotoxicity 0.5, F: number of plants showing a degree of phytotoxicity 1, G: number of plants showing a degree of phytotoxicity 2, H: number of plants showing a degree of phytotoxicity 3, N2; Represents the number of plants used in the test.)

2) Experimental results Table 1 shows the degree of onset of tomato wilt (DI-VMA) and the degree of phytotoxicity (Phytotoxicity) when validamycin A is sprayed on various tomato varieties. 1 is plotted on the horizontal axis and shown in FIG.

表1及び図１から、発病の程度（数値が大きいほど発病が激しい）は、薬害の程度（数値が大きいほど薬害が多い）と負の相関関係があることが認められた（相関係数；-0.57533、有意確率；0.0064）。

From Table 1 and FIG. 1, it was confirmed that the degree of disease onset (the larger the value, the more serious the disease) has a negative correlation with the degree of drug damage (the greater the value, the more drug damage) (correlation coefficient; -0.57533, significant probability; 0.0064).

  The above phytotoxicity is considered to be manifested by the accumulation of salicylic acid or its metabolites in plant tissues. In fact, when the salicylic acid concentration in the plant tissue used in the above experiment was measured, a high positive correlation was found between the phytotoxicity and the salicylic acid concentration. Salicylic acid has been reported to be a systemic acquired resistance (SAR) -induced signaling agent in plants (Ishikawa et al., Supra). In other words, when the salicylic acid concentration increases, the SAR pathway is expressed and the plant becomes resistant to disease. In summary, an increase in the salicylic acid concentration in the plant tissue leads to the expression of phytotoxic symptoms and the acquisition of resistance to diseases. Therefore, when a candidate compound is treated by using a plant that is susceptible to phytotoxicity (dot in the lower right in FIG. 1) as a tester and a compound with strong phytotoxicity is selected, the ability to induce resistance to disease (function as a plant activator) You can select the one with high.

<Example 2> Correlation between degree of occurrence of phytotoxicity and induction resistance of variety Z617 Validamycin-susceptible (susceptible to phytotoxicity when sprayed with validamycin A) tomato varieties, Zuikou 208 (Sakata Seed), Okitsu 3 ( Use LS-89 (Takii seedlings), Z617 (obtained from John Innes Laboratories, UK), etc. to spray candidate compounds and investigate the degree of phytotoxicity that appears. Especially sensitive variety Z617 (variety Moneymaker is transformed with nahG gene, so salicylic acid in tissue is quickly converted to catechol, so SAR induction is not required, but catechol accumulation is thought to cause phytotoxicity Are considered suitable as certified varieties. Compounds that cause strong (high) phytotoxicity to these tomato varieties as a result of foliar spraying are expected to have a high SAR-inducing ability and a high disease control effect by resistance induction. By using as a control a validamycin-insensitive variety (for example, Moneymaker for Z617) that is less susceptible to phytotoxicity than variidamycin-sensitive varieties, the phytotoxicity is controlled by mechanisms other than SAR induction (accumulation of salicylic acid, etc.), in other words, herbicidal activity. Can be eliminated (it can be judged that even non-sensitive varieties have herbicidal activity if they cause the same phytotoxicity).

1) Experimental method Tomato varieties Z617 and Moneymaker 4 weeks after sowing were sprayed with the compounds in the table, and the degree of phytotoxicity that appeared 7 days later was tested and compared. In addition, the cultivar Moneymaker 4 weeks after sowing was sprayed with the stem pillar compound, and 7 days later, the wilt of the wilt disease was inoculated, and the wilt disease onset suppression effect 40 days later was tested. As a control for the latter experiment, a water spray treated Moneymaker subjected to the same inoculation test was used. Degree of phytotoxicity: 0; healthy (no phytotoxicity), 1; slight browning of stem and petiole, 2; browning of stem and petiole, 3; necrotic spots formed on stem and petiole, 4; Or it evaluated by remarkable inhibition of growth. 1 to 6 pots with 2 plants were used in each ward, and the average value of the degree of phytotoxicity of each strain was displayed. The symptom was evaluated in the same manner as in Example 1. The t-test showed a significant decrease in the average disease severity as compared to the subject group, and the negative sign that the significant symptom was not significantly decreased. The fact that strong phytotoxicity was caused by spraying foliage on the cultivar Moneymaker was noted because the disease severity test could not be performed correctly.

100 μg/ml バリダマイシンA(VMA)、100 μg/ml バリドキシルアミンA(VAA)、10000 μg/ml プロベナゾール（PBZ）、10000 μg/ml アシベンゾラルＳ−メチル（BTH）、10000 μg/ml トレハロースのZ617に対する茎葉散布で強い（４）薬害が見られ、抵抗性誘導活性を持つ可能性が示唆された。このうち、10000 μg/ml BTHはMoneymakerにも強い薬害を生じ、除草活性を示した。薬害のために発病検定に至らなかった10000 μg/ml BTHを除き、供試化合物のトマト萎凋病抑制効果を評価したところ、100 μg/ml VMA、100 μg/ml VAA、10000 μg/ml PBZ、10000 μg/ml トレハロースで発病抑制効果が見られた。これ以外の化合物は、Z617に対する薬害も、Moneymakerにおける萎凋病発病抑制効果も示さなかった。以上から、100 μg/ml VMA、100 μg/ml VAA、10000 μg/ml PBZ、10000 μg/ml トレハロースは、抵抗性誘導による発病抑制効果を示すと考えられ、プラントアクチベーターとして選抜された。
なお、PBZは既知のプラントアクチベーターであるが、SARにおいてサリチル酸より下流に作用することが判明しており、サリチル酸→カテコールの蓄積によらず薬害を生じたと理解され、これはZ617およびMoneymakerの両者で強い薬害を生じたこととも矛盾しない。ここで、ビビフルフロアブルは、1.0% プロヘキサジオンカルシウム塩（クミアイ化学工業）を、アスパルテームは、0.8% アスパルテーム・L-フェニルアラニン（味の素）を表す。 2) Experimental result The measured result was shown to Tables 2-4, and the evaluation was described under each table | surface.

100 μg / ml Validamycin A (VMA), 100 μg / ml Validoxylamine A (VAA), 10000 μg / ml Probenazole (PBZ), 10000 μg / ml Acibenzoral S-methyl (BTH), 10000 μg / ml Z617 of trehalose Strong (4) phytotoxicity was seen in the foliage spraying against, suggesting the possibility of having resistance-inducing activity. Of these, 10000 μg / ml BTH caused strong phytotoxicity on Moneymaker and showed herbicidal activity. With the exception of 10000 μg / ml BTH, which did not result in a pathogenesis test due to phytotoxicity, the tomato wilt suppression effect of the test compound was evaluated.100 μg / ml VMA, 100 μg / ml VAA, 10000 μg / ml PBZ, 10000 μg / ml trehalose showed a disease-suppressing effect. Other compounds did not show phytotoxicity against Z617 nor an inhibitory effect on the onset of wilt disease in Moneymaker. Based on the above, 100 μg / ml VMA, 100 μg / ml VAA, 10000 μg / ml PBZ, and 10000 μg / ml trehalose were considered to exhibit disease-inhibiting effects due to resistance induction, and were selected as plant activators.
Although PBZ is a known plant activator, it has been found that it acts downstream of salicylic acid in SAR, and it is understood that it caused phytotoxicity regardless of the accumulation of salicylic acid → catechol. This is consistent with the strong phytotoxicity. Here, Bibiful Flowable represents 1.0% prohexadione calcium salt (Kumiai Chemical Industry), and Aspartame represents 0.8% Aspartame L-phenylalanine (Ajinomoto).

100 μg/ml バリダマイシンA(VMA)、1000 μg/mlサッカリン、10000 μg/mlカテコール、10000 μg/mlサリチル酸のZ617に対する茎葉散布で薬害が見られ、抵抗性誘導活性を持つ可能性が示唆された。このうち、10000 μg/mlカテコール、10000 μg/mlサリチル酸はMoneymakerにも強い薬害を生じ、除草活性を示した。薬害のために発病検定に至らなかった10000 μg/mlカテコール、10000 μg/mlサリチル酸を除き、供試化合物のトマト萎凋病抑制効果を評価したところ、100 μg/ml VMA、1000 μg/mlサッカリンで発病抑制効果が見られた。1000 μg/ml Sweet'n lowは、Z617に対する薬害も、Moneymakerにおける萎凋病発病抑制効果も示さなかった。以上から、100 μg/ml VMA、1000 μg/mlサッカリンは、抵抗性誘導による発病抑制効果を示すと考えられ、プラントアクチベーターとして選抜された。ここで、Sweet'n lowは3.6% サッカリンを表す。
なお、サリチル酸はSARにおけるシグナル伝達物質であるが、今回の供試は10000 μg/mlという高濃度であったため、Moneymakerにも薬害を生じた。

100 μg / ml validamycin A (VMA), 1000 μg / ml saccharin, 10000 μg / ml catechol, 10000 μg / ml salicylic acid sprayed against Z617 showed phytotoxicity, suggesting the possibility of having resistance-inducing activity . Of these, 10000 μg / ml catechol and 10000 μg / ml salicylic acid caused strong phytotoxicity to Moneymaker and showed herbicidal activity. Excluding 10000 μg / ml catechol and 10000 μg / ml salicylic acid, which did not result in pathogenesis due to phytotoxicity, the tomato wilt disease inhibitory effect of the test compound was evaluated, and 100 μg / ml VMA and 1000 μg / ml saccharin were used. The disease suppression effect was seen. 1000 μg / ml Sweet'n low did not show any phytotoxicity against Z617, nor did it cause the wilt pathogenesis of Moneymaker. Based on the above, 100 μg / ml VMA and 1000 μg / ml saccharin were considered to exhibit disease-suppressing effects by resistance induction and were selected as plant activators. Here, Sweet'n low represents 3.6% saccharin.
Salicylic acid is a SAR signal transducing substance, but since the concentration of this test was as high as 10000 μg / ml, it caused phytotoxicity to Moneymaker.

100 μg/ml バリダマイシンA(VMA)、10 μg/ml バリダマイシンA(VMA)、10000 μg/mlトレハロース、1000 μg/mlトレハロース、10000 μg/mlカテコール、1000 μg/mlカテコール、10000 μg/mlソルビトール、1000 μg/mlスクロースのZ617に対する茎葉散布で薬害が見られ、抵抗性誘導活性を持つ可能性が示唆された。このうち、10000 μg/mlカテコールはMoneymakerにも強い薬害を生じ、除草活性を示した。カテコールの濃度を1000 μg/mlに落とすと、薬害程度は低減したが、Z617およびMoneymakerでみられた薬害が同等であったため、カテコールは除草活性をもつものの、抵抗性誘導活性は持たないと判断できた。薬害のために発病検定に至らなかった10000 μg/mlカテコールを除き、供試化合物のトマト萎凋病抑制効果を評価したところ、1000 μg/ml カテコール以外で発病抑制効果が見られた。1000 μg/ml カテコールで発病抑制効果が見られなかったことは、上述と合致しており、本スクリーニング系がうまく機能したことを示す。以上から、100 μg/ml VMA、10 μg/ml VMA、10000 μg/mlトレハロース、1000 μg/mlトレハロース、10000 μg/mlソルビトール、1000 μg/mlスクロースは、抵抗性誘導による発病抑制効果を示すと考えられ、プラントアクチベーターとして選抜された。

100 μg / ml validamycin A (VMA), 10 μg / ml validamycin A (VMA), 10000 μg / ml trehalose, 1000 μg / ml trehalose, 10000 μg / ml catechol, 1000 μg / ml catechol, 10000 μg / ml sorbitol, The phytotoxicity was observed in the foliar application of 1000 μg / ml sucrose to Z617, suggesting the possibility of having resistance-inducing activity. Of these, 10000 μg / ml catechol caused strong phytotoxicity to Moneymaker and showed herbicidal activity. Decreasing the catechol concentration to 1000 μg / ml reduced the degree of phytotoxicity, but the phytotoxicity seen with Z617 and Moneymaker was comparable, so catechol was determined to have no herbicidal activity but no resistance-inducing activity. did it. With the exception of 10000 μg / ml catechol, which did not result in a pathogenesis test due to phytotoxicity, the tomato wilt disease inhibitory effect of the test compound was evaluated. The fact that the disease suppression effect was not observed with 1000 μg / ml catechol was consistent with the above, indicating that this screening system worked well. Based on the above, 100 μg / ml VMA, 10 μg / ml VMA, 10000 μg / ml trehalose, 1000 μg / ml trehalose, 10000 μg / ml sorbitol, 1000 μg / ml sucrose Conceived and selected as a plant activator.

  As is clear from the table, all of the compounds that caused phytotoxicity in tomato variety Z617 and did not cause phytotoxicity in Moneymaker showed an inhibitory effect against the onset of wilt disease. From this, it was found that plant activators can be screened using phytotoxicity to Z617 as an index.

  The tomato variety Z617 produces the enzyme salicylate hydroxylase in the tissue because the salicylic acid hydroxylase gene (nahG) derived from Pseudomonas fluorescence is introduced into the variety Moneymaker. Since this enzyme converts salicylic acid to catechol, even if salicylic acid is produced in the cultivar Z617, it is immediately converted to catechol. For this reason, salicylic acid does not accumulate in the variety Z617 tissue, and SAR via salicylic acid is not induced. On the other hand, since catechol is harmful to plants, phytotoxicity may occur when a large amount of catechol is accumulated in tissues.

<Example 3> Concentration dependence of induction resistance and degree of phytotoxicity Next, concentration dependence of induction resistance and degree of phytotoxicity was examined.
In the same procedure as in Example 2, the degree of induction resistance and the occurrence of phytotoxicity were measured using the concentrations of validamycin A, trehalose, catechol, sorbitol and sucrose within the range of 10 to 10,000 μg / ml, and the induction resistance was maintained. However, a practical plant activator concentration with low phytotoxicity was investigated.

  From Table 4 above, it was shown that the phytotoxicity seen in Z617 at 100 μg / ml VMA and 10 μg / ml VMA is concentration dependent. At both concentrations, a disease-suppressing effect was observed. From the above, when using VMA as a plant activator for disease control, 10 μg / ml is sufficient (under the same conditions as in this test environment), and phytotoxicity to all tomato varieties (phytotoxicity is important for this screening) Although it is effective, it can be judged that application at 10 μg / ml is preferable to 100 μg / ml from the viewpoint of avoiding phytotoxicity that damages production in normal cultivation. In this screening method, it is possible to suggest a preferable concentration that can be used as a plant activator by testing a compound having a different concentration. In addition, in a compound having both plant activator activity and herbicidal activity, the plant activator can be used at a lower concentration. If it is possible to demonstrate the concentration, it becomes possible to know the concentration.

  In the actual use of a plant activator, the above procedure makes it possible to set a concentration that does not cause phytotoxicity and allows for a safety factor showing a disease control effect.

  Furthermore, in the same procedure as in Example 2, using VMA, saccharin, salicylic acid and galactose as test compounds, phytotoxicity against tomato plants Moneymaker (insensitive) and Z617 (sensitive), and onset of tomato wilt and powdery mildew The inhibitory effect of was investigated. The results are shown in Table 5.

表から、バリダマイシンA(VMA)及びサッカリンは、Z617による薬害を受けるがMoneymakerによる薬害を受けない、一方、トマト萎凋病及びうどんこ病のいずれに対しても発病抑制効果を示したことがわかる。したがって、このことは、本発明の方法が、病害の種類によらず、プラントアクチベーターのスクリーニングのために使用できることを示している。

From the table, it can be seen that validamycin A (VMA) and saccharin are phytotoxic by Z617 but not toxic by Moneymaker, while exhibiting a disease-inhibiting effect on both tomato wilt and powdery mildew. This therefore indicates that the method of the invention can be used for screening of plant activators, regardless of the type of disease.

  According to the present invention, it becomes possible to screen a plant activator that induces systemic acquired resistance against plant pathogenic microorganisms in a simple, reliable and short day, and thus has a high control effect on various agricultural diseases. It is possible to easily and quickly find a drug that exerts.

This figure shows a negative correlation between the degree of onset of tomato wilt (DI-VMA) and the degree of phytotoxicity (Phytotoxicity) when validamycin A is sprayed on tomato varieties.

Claims (6)

  A different test compound is applied to a plant sensitive to a plant activator, and no phytotoxicity due to the test compound is detected against an insensitive plant control of the same species. A method for screening a plant activator, comprising determining that the test compound has an ability to induce systemic acquired resistance to a disease.   The method of claim 1, wherein the screening is performed based on a positive correlation between systemic acquired resistance by a plant activator and occurrence of phytotoxicity.   (1) a step of preparing a plant activator-sensitive plant and a non-sensitive homologous plant, (2) a step of applying a test compound to each plant, and (3) a phytopathogenic microorganism in each plant depending on the case. (4) measuring the phytotoxicity due to the test compound, and (5) selecting a test compound such that the phytotoxicity is detected in a susceptible plant compared to an insensitive plant control as a plant activator. The method of claim 1, comprising steps.   The method of claim 1, further comprising determining plant activator concentrations that can suppress the phytotoxicity while maintaining the systemic acquired resistance using different concentrations of plant activators.   The method according to claim 1, further comprising treating a target plant or plant cultivar with the screened plant activator to confirm the disease suppressive ability of the plant activator.   The method of claim 1, wherein the plant is selected from dicotyledonous or monocotyledonous plants.

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