JP7621784B2 - Plant activators - Google Patents

Description

The present invention relates to a plant activator.

Technologies for regulating plant growth have been developed with the aim of improving the supply efficiency of grain plants and horticultural plants. In addition to measures such as optimizing temperature and sunlight conditions and fertilization, methods have been reported for activating plants using plant activators that have plant growth regulating effects such as promoting growth, suppressing dormancy, and suppressing stress.

Patent Document 1 reports a plant activator containing fatty acid metabolites obtained by metabolizing fatty acids having 4 to 30 carbon atoms with Proteobacteria in an environment with a dissolved oxygen concentration of 0.1 to 8 mg/L. The plant activator described in Patent Document 1 has excellent resistance induction and growth promotion effects, but has the problem of being very unstable and quickly decomposing.

The decomposition is believed to be caused by oxidation, but there is no effective way to inhibit this decomposition, and there is a strong need for technology to prevent oxidation.

International Publication No. WO 2018/47918

The plant activator produced by the method described in Patent Document 1 uses saturated fatty acids and unsaturated fatty acids as raw materials, and has the problem of being easily oxidized in the atmosphere. In order to stably exert its effect as a plant activator over the long term, there is a demand for a plant activator that is not easily oxidized and can maintain its effect as a plant activator.

The present invention aims to provide a plant activator that contains an antioxidant to prevent oxidation and has excellent disease resistance and growth promotion effects.

The present invention relates to a plant activator that contains a fatty acid metabolite obtained by metabolizing fatty acids having 4 to 30 carbon atoms with Proteobacteria in an environment with a dissolved oxygen concentration of 0.1 to 8 mg/L, and a phenolic antioxidant.

Preferably, the plant activator is characterized in that the phenolic antioxidant is butyl hydroxyanisole.

The phenol-based antioxidant is preferably butylhydroxyanisole, and is added in a weight ratio of 1/2 to 5 times the amount of the active ingredient.

It is preferable that the plant activator is a plant activator used as a spray or immersion agent that is brought into contact with the stems, leaves, or roots of a plant, or as a soil drenching agent.

The plant activator is preferably a plant activator used for plants of the Brassicaceae family.

The plant activator of the present invention has excellent stability of the active ingredient and has excellent disease resistance and growth promotion effects.

FIG. 1 shows the stability of active ingredients in a plant activator containing an antioxidant. 1 is a graph showing the growth-promoting effect on plant weight of a Brassicaceae plant. 1 is a graph showing the growth-promoting effect on leaf length of a Brassicaceae plant.

Plant Activator The plant activator of the present invention is characterized by comprising a fatty acid metabolite obtained by metabolizing a fatty acid having 4 to 30 carbon atoms with Proteobacteria in an environment with a dissolved oxygen concentration of 0.1 to 8 mg/L, and a phenolic antioxidant.

In the present invention, "plant activation" means adjusting the plant's growth activity in some way to activate or maintain it, and includes plant growth regulation effects such as growth promotion (a concept that includes the expansion of stems and leaves, and the promotion of tuber and root growth), dormancy suppression, induction and conferring of resistance to plant stress (e.g., disease), and anti-aging.

The plant activator of the present invention contains a fatty acid metabolite as an active ingredient for activating a plant. This fatty acid metabolite is a metabolite obtained by metabolizing a mixture containing either saturated or unsaturated fatty acids, or both, having 4 to 30 carbon atoms, with Proteobacteria in an environment with a dissolved oxygen concentration of 0.1 to 8 mg/L. In this specification, the term "active ingredient" refers to such a fatty acid metabolite. This fatty acid metabolite contains a substance that activates plant growth, and therefore, by contacting the plant activator of the present invention with a part of the stem, leaf, or root of a plant, a growth-promoting effect can be imparted to the plant. In addition, this fatty acid metabolite also contains a substance or a precursor of this substance that activates the salicylic acid pathway or the jasmonic acid pathway, which are related to resistance induction, and therefore, by applying it to a plant, resistance can be induced in the plant.

The Proteobacteria that produce the fatty acid metabolites of the present invention from fatty acids are not particularly limited. A single species of Proteobacteria may be used, or multiple species of Proteobacteria may be used, and the bacterial flora is also not limited. For example, the bacterial flora may be composed of α-proteobacteria, β-proteobacteria, or may contain both. For example, Proteobacteria derived from activated sludge may be suitably used. Activated sludge is widely used in water treatment (sewage and wastewater treatment). The bacterial flora has been analyzed by the FISH method, the clone library method, and the like, and it has been found that the main constituent bacteria (priority group) in the activated sludge are Proteobacteria (see, for example, Sadaie Yoshito et al., Analysis of Environmental Purification Microorganisms, Bulletin of Saitama University Regional Cooperative Research Center, 2007, Vol. 7, pp. 35-36; Akiyama Takashi et al., Analysis of Bacterial Community Structure in Sewage Treatment Plant Activated Sludge Using the FISH Method, Journal of Japan Society on Water Environment, 2000, Vol. 23, No. 5, pp. 271-278, and the like). For example, the activated sludge-derived Proteobacteria include Azoarcus buckelii, Propionivibrio pelophilus, Thauera selenatis, Pandoraea pulmonicola, Pusillimonas noertemannii, Rhodovulum kholense, Haematobacter massiliensis, Hyphomicrobium hollandicum, Chelatovorus multitrophus, Nitrosococcus halophilus, Thioalkalivibrio thiocyanodenitrificans, Marinobacter hydrocarbonoclasticus, Halomonas xinjiangensis, or Pseudomonas pertucinogena, or a complex flora containing two or more of these.

The plant activator of the present invention contains an antioxidant, which inhibits the decomposition of the active ingredients in the plant activator and stabilizes the plant activator. In other words, the inclusion of an antioxidant prevents the activation effect and storage stability of the plant activator from decreasing during processing, distribution, and storage when the plant activator is formulated, and ensures its effectiveness as a plant activator when applied. For example, a preferred example of an antioxidant that contributes to the stabilization of the plant activator is a phenol-based antioxidant. An example of a phenol-based antioxidant is butyl hydroxyanisole. However, other phenolic antioxidants that have antioxidant properties can also be used.

The addition of the antioxidant of the present invention does not reduce or eliminate the excellent disease resistance and growth promoting effects of the plant activator containing fatty acid metabolites. Furthermore, the addition of the antioxidant does not have any adverse effects on the plant body to which it is applied. Therefore, by adding the antioxidant of the present invention, the excellent activating effect of the plant activator containing fatty acid metabolites is maintained, and by suppressing the decomposition of the active ingredients in the plant activator, it is possible to achieve the maintenance of a stable activating effect of the plant activator over a long period of time.

In one embodiment of the present invention, butylhydroxyanisole is used as the phenol-based antioxidant. Butylhydroxyanisole can be added as an antioxidant in a ratio of about 5 times or less by weight relative to the amount of the active ingredient in the plant activator, i.e., the amount of fatty acid metabolites. The preferred concentration of butylhydroxyanisole that can be contained in the plant activator of the present invention depends on the plant species and its condition to which it is applied, but if the ratio exceeds 5 times, there is a risk of causing phytotoxicity to the plant to which it is applied. There is no particular limit to the lower limit of the concentration of butylhydroxyanisole, but it is preferably about 1/2 or more by weight relative to the amount of the active ingredient. In a preferred embodiment of the present invention, the ratio of butylhydroxyanisole is 1/2 to 5 times by weight relative to the amount of the active ingredient.

The amount of active ingredient in the present invention refers to the amount of substance contained in the plant activator of the present invention, which corresponds to the peak with the shortest retention time among the four peaks observed between 16 and 20 minutes under the following liquid chromatography separation conditions (mobile phase: solution A (100% acetonitrile solution), solution B (0.1% acetic acid solution), Accucore PR-MS column (Thermo Fisher Scientific, column size φ2.1×150 mm, particle size 5 μm), flow rate 0.25 mL/min, column temperature 40° C., detection wavelength 272 nm, gradient conditions: mobile phase B concentration 80% (0 min) → mobile phase B concentration 60% (10 min) → 60% (20 min)). The amount of active ingredient is measured by separating the component with the shortest retention time through the column and drying and removing the mobile phase.

For example, the amount of active ingredient in the plant activator of the present invention is about 0.01 to 0.2 g/L. The plant activator of the present invention contains an antioxidant in an amount 1/2 to 5 times the amount of the active ingredient by weight. By adding an antioxidant in this amount, the decomposition of the active ingredient in the plant activator is significantly suppressed, and the activating effect can be maintained for a long period of time.

The plant activator of the present invention may contain, as necessary, a compatible surfactant and/or diluent or carrier suitable for use as a plant activator. These added components are not particularly limited as long as they are agriculturally acceptable agents. In addition, the plant activator may further contain components other than the surfactant, diluent, and carrier that are typically used in pesticide formulations.

The plant activator of the present invention can be applied to plants by any method. For example, it can be used as a spray or immersion agent that is brought into contact with the stems, leaves, or roots of a plant, or as a soil drenching agent. The plant activator of the present invention may also be contained in a porous structure or capsule, or impregnated in a sheet or the like, and used as a sustained-release agent. In the plant to which it is applied, the plant activator of the present invention imparts a plant growth-promoting effect and resistance to stress, such as disease.

The plants to which the plant activator of the present invention can be applied are not particularly limited, and can be used effectively on plants in general, but preferred plants include plants of the Brassicaceae family. However, the plant activator can also be applied to plants of the Poaceae family, Fabaceae family, Solanaceae family, Rosaceae family, Amaranthaceae family, or Malvaceae family. In addition, the plants to which the plant activator can be applied are not limited to wild-type plants, and may be, for example, mutants or transformants. In addition, the varieties of each plant are not particularly limited.

It has also been found that the plant activator of the present invention can be used as a plant activator that exhibits a strong resistance induction effect, and has been found to exhibit growth promotion effects, fruit yield increasing effects, and disease suppression effects in various plants. Specific examples of disease suppression effects include gray mold disease, vine splitting disease, vine wilt disease, and downy mildew in Cucurbitaceae plants such as cucumber, watermelon, melon, and pumpkin; bacterial wilt disease, wilt disease, half-leaf wilt disease, damping-off disease, and brown root rot disease in Solanaceae plants such as tomato, eggplant, and potato; powdery mildew, black spot disease, gray mold disease, and anthracnose in Rosaceae plants such as rose and strawberry; downy mildew in Amaranthaceae plants such as spinach; black rot disease, soft rot disease, bacterial spot disease, and Rhizoctonia disease in Brassica plants such as Chinese cabbage, cabbage, and Komatsuna; and southern blight in Apiaceae plants such as carrot.

The present invention will be described based on examples, but the present invention is not limited to only the examples.

Example 1
Preparation of plant activator <Pre-culture step>
20 g of peptone (protein hydrolysate manufactured by Difco), 1.5 g of magnesium sulfate heptahydrate, and 1.5 g of dipotassium hydrogen phosphate were dissolved in 1 L of water in a glass Erlenmeyer flask, sterilized by autoclave at 121°C for 20 minutes, cooled to room temperature, and then inoculated with a Proteobacteria solution derived from activated sludge. The mouth of the culture vessel was sealed with a silicone plug. After inoculation, the vessel was cultured for 24 hours under conditions of 20°C and 120 rpm using a Bioshaker (registered trademark) (BR-23UM manufactured by Taitec Co., Ltd.). The number of bacteria in the culture solution was 5 x 10 ⁸ cells/mL. After cultivation, the culture solution was centrifuged at 15,000 x G and 20°C to separate the bacteria from the culture solution, and the bacteria were collected.
<Fatty acid metabolism process>
12 g of linoleic acid (primary linoleic acid manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1.5 g of magnesium sulfate heptahydrate, 1.5 g of dipotassium hydrogen phosphate, and the total amount of the bacterial cells obtained in the pre-culture step were added to 1 L of sterilized water in a glass Erlenmeyer flask. This was cultured for 4 days under conditions of 20°C, 120 rpm, and a dissolved oxygen concentration of 4 mg/L using a Bioshaker (registered trademark) (BR-23UM manufactured by Taitec Co., Ltd.). The decomposition of linoleic acid was confirmed by the production of oxidized lipids, which are one of the intermediate products of linoleic acid, by analyzing the culture solution by measuring the absorbance at a wavelength of 230 nm using a spectrophotometer BioSpec-mini manufactured by Shimadzu Corporation. After the culture, the culture solution containing the bacterial cells was used as a test plant activator.

Addition of butylated hydroxyanisole and accelerated test 120 mg of butylated hydroxyanisole (Kishida Chemical Co., Ltd.), a phenolic antioxidant, was added to 1 L of the test plant activator obtained above (active ingredient amount 24 mg), to prepare the plant activator of Example 1. The obtained plant activator was subjected to an accelerated test at 54°C using a Bioshaker (registered trademark) (BR-23UM, Taitec Co., Ltd.), and left for 4 weeks (equivalent to about 2 years at 25°C. See "Stability of Pharmaceuticals" by Sumie Yoshioka). The active ingredient concentration was measured by UV before and after the accelerated test, and the ratio of the active ingredient concentration after the accelerated test to the active ingredient concentration before the accelerated test (active ingredient concentration change %) was calculated from the UV area ratio before and after the accelerated test.

Comparative Example 1
One liter of the test plant activator obtained above (active ingredient amount: 24 mg) was subjected to an accelerated test at 54°C using a Bioshaker (registered trademark) (BR-23UM manufactured by Taitec Co., Ltd.) and left for 4 weeks (equivalent to approximately 2 years at 25°C. See "Stability of Pharmaceuticals" by Sumie Yoshioka). The active ingredient concentration was measured by UV before and after the accelerated test, and the ratio of the active ingredient concentration after the accelerated test to the active ingredient concentration before the accelerated test (% change in active ingredient concentration) was calculated from the UV area ratio before and after the accelerated test.

Comparative Example 2
To 1 L of the test plant activator obtained above (active ingredient amount 24 mg), 170 mg of vitamin C (Kanto Chemical Co., Ltd.) and 120 mg of vitamin E (Nacalai Tesque, Inc.) were added. The obtained solution was subjected to an accelerated test at 54°C using a Bioshaker (BR-23UM, Taitec Co., Ltd.) and left for 4 weeks (equivalent to about 2 years at 25°C. See "Stability of Pharmaceuticals" by Sumie Yoshioka). The active ingredient concentration was measured by UV before and after the accelerated test, and the ratio of the active ingredient concentration after the accelerated test to the active ingredient concentration before the accelerated test (active ingredient concentration change %) was calculated from the UV area ratio before and after the accelerated test.

Comparative Example 3
To 1 L of the test plant activator obtained above (active ingredient amount 24 mg), 170 mg of vitamin C was added. The obtained solution was subjected to accelerated testing at 54°C using a Bioshaker (registered trademark) (BR-23UM manufactured by Taitec Co., Ltd.) and left for 4 weeks (equivalent to approximately 2 years at 25°C. See "Stability of Pharmaceuticals" by Sumie Yoshioka). The active ingredient concentration was measured by UV before and after the accelerated testing, and the ratio of the active ingredient concentration after the accelerated testing to the active ingredient concentration before the accelerated testing (% change in active ingredient concentration) was calculated from the UV area ratio before and after the accelerated testing.

Comparative Example 4
To 1 L of the test plant activator obtained above (active ingredient amount 24 mg), 120 mg of vitamin E was added. The obtained solution was subjected to an accelerated test at 54°C using a Bioshaker (registered trademark) (BR-23UM manufactured by Taitec Co., Ltd.) and left for 4 weeks (equivalent to about 2 years at 25°C. See "Stability of Pharmaceuticals" by Sumie Yoshioka). The active ingredient concentration was measured by UV before and after the accelerated test, and the ratio of the active ingredient concentration after the accelerated test to the active ingredient concentration before the accelerated test (% change in active ingredient concentration) was calculated from the UV area ratio before and after the accelerated test.

The results of the percentage change in active ingredient concentration obtained in Example 1 and Comparative Examples 1 to 4 are shown in Table 1 below.

As shown in Table 1, it can be seen that the plant activator containing the phenolic antioxidant of the present invention has a remarkable effect on stabilizing the active ingredient.

The active ingredients in the plant activator before the accelerated test, and the active ingredients in Example 1 and Comparative Example 1 without antioxidants after the accelerated test were analyzed by HPLC. The analysis was performed under the following conditions. Mobile phase: Solution A (100% acetonitrile solution), Solution B (0.1% acetic acid solution), Accucore PR-MS column (Thermo Fisher Scientific, column size φ2.1×150 mm, particle size 5 μm), flow rate 0.25 mL/min, column temperature 40°C, detection wavelength 272 nm, gradient conditions: mobile phase B concentration 80% (0 min) → mobile phase B concentration 60% (10 min) → 60% (20 min). Figure 1 shows the observed changes in concentration of active ingredients depending on whether or not an antioxidant was added.

As shown in Figure 1, in Comparative Example 1, where no antioxidant was added, most of the active ingredient observed before the accelerated test was almost decomposed after four weeks of the accelerated test. On the other hand, in Example 1, where a phenolic antioxidant was added, the active ingredient was well maintained even after four weeks of the accelerated test. This shows that in Example 1, the addition of the antioxidant significantly suppressed the decomposition of the active ingredient. From these results, it can be seen that the plant activator containing the phenolic antioxidant of the present invention has an excellent effect on stabilizing the active ingredient.

Application Examples to Plants Using the plant activators of Example 1 and Comparative Example 1, the effect of adding an antioxidant to the plant activator on a Brassicaceae plant was observed. That is, the plant activators of Example 1 and Comparative Example 1 were sprayed onto the leaves of the above-ground parts of the plants of the Brassicaceae plant after germination and before harvesting. After harvesting each plant, the wet weight of the above-ground parts per plant and the length of the leaves were measured. The results are shown in Figures 2 and 3, respectively.

As shown in Figure 2, the above-ground wet weight was 1.07 g/plant in Example 1 compared to 0.85 g/plant in Comparative Example 1. Also, as shown in Figure 3, the leaf length was 9.45 cm in Example 1 compared to 8.10 cm in Comparative Example 1. Both the average above-ground wet weight and leaf length of the plants treated with the plant activator of Example 1, which contains antioxidants, were increased compared to the plants treated with the plant activator of Comparative Example 1, which does not contain antioxidants.

As shown in Figures 2 and 3, in the plant activator to which an antioxidant has been added, the antioxidant does not adversely affect the plant body, and the antioxidant acts well to stabilize the plant activator, suppressing the decomposition of the active ingredient, thereby maintaining the activating effect of the plant activator, and it can be seen that the effectiveness of the plant activator as a plant activator is ensured for a long period of time.

The above results show that the plant activator of the present invention is a plant activator that has excellent activation effect and storage stability, and is highly effective as a plant activator when applied.

Claims (5)

A plant activator comprising a linoleic acid metabolite obtained by metabolizing linoleic acid with proteobacteria in an environment having a dissolved oxygen concentration of 0.1 to 8 mg/L, and a phenolic antioxidant , wherein the proteobacteria are derived from activated sludge and are selected from the group consisting of Azoarcus buckelii, Propionivibrio pelophilus, Thauera selenatis, Pandoraea pulmonicola, Pusillimonas noertemannii, Rhodovulum kholense, Haematobacter massiliensis, Hyphomicrobium hollandicum, Chelatovorus multitrophus, Nitrosococcus halophilus, Thioalkalivibrio thiocyanodenitrificans, Marinobacter hydrocarbonoclasticus, Halomonas xinjiangensis, and Pseudomonas pertucinogena, or a complex flora containing two or more of these . The plant activator according to claim 1, wherein the phenolic antioxidant is butylhydroxyanisole. The plant activator according to claim 1, wherein the phenolic antioxidant is butylhydroxyanisole, and is added in an amount 1/2 to 5 times the amount of the active ingredient by weight. The plant activator according to any one of claims 1 to 3, which is used as a spray or immersion agent to be brought into contact with the stems, leaves or roots of a plant, or as a soil drenching agent. The plant activator according to any one of claims 1 to 4 is used for plants of the Brassicaceae family.