U.S. patent application number 17/832837 was filed with the patent office on 2022-09-22 for plant activator and a method of manufacturing the same.
The applicant listed for this patent is IBIDEN CO., LTD.. Invention is credited to Toru Nakai, Tomohiro Nohara, Katsuya Ohno, Kumiko Takada, Hiroko Takagi, Kenta Uemura, Teruaki Yokota.
Application Number | 20220295713 17/832837 |
Document ID | / |
Family ID | 1000006377851 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220295713 |
Kind Code |
A1 |
Ohno; Katsuya ; et
al. |
September 22, 2022 |
PLANT ACTIVATOR AND A METHOD OF MANUFACTURING THE SAME
Abstract
The objective of the invention is to provide a plant activator
with superior resistance-inducing activity and growth promoting
activity and low toxicity and soil contamination. A plant activator
comprising a fatty acid metabolite obtainable by a metabolism of a
fatty acid with 4 to 30 carbon atoms by a proteobacteria under a
dissolved oxygen concentration of 0.1-8 mg/L, and a method for
manufacturing a plant activator comprising a fatty acid metabolite,
comprising a step for fatty acid metabolism wherein a fatty acid
with 4 to 30 carbon atoms is subjected to a proteobacterial
metabolization under a dissolved oxygen concentration of 0.1-8
mg/L. A method for manufacturing a plant activator comprising a
fatty acid metabolite, comprising a step for fatty acid metabolism
wherein a fatty acid with 4 to 30 carbon atoms is subjected to a
proteobacterial metabolization under a dissolved oxygen
concentration of 0.1-8 mg/L.
Inventors: |
Ohno; Katsuya; (Gifu,
JP) ; Takada; Kumiko; (Gifu, JP) ; Nohara;
Tomohiro; (Gifu, JP) ; Yokota; Teruaki; (Gifu,
JP) ; Uemura; Kenta; (Gifu, JP) ; Takagi;
Hiroko; (Gifu, JP) ; Nakai; Toru; (Gifu,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IBIDEN CO., LTD. |
Gifu |
|
JP |
|
|
Family ID: |
1000006377851 |
Appl. No.: |
17/832837 |
Filed: |
June 6, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16331300 |
Mar 7, 2019 |
11382281 |
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PCT/JP2017/032354 |
Sep 7, 2017 |
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17832837 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/20 20200101;
A01G 7/06 20130101; A01N 63/50 20200101 |
International
Class: |
A01G 7/06 20060101
A01G007/06; A01N 63/50 20060101 A01N063/50; A01N 63/20 20060101
A01N063/20 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 8, 2016 |
JP |
2016-175861 |
Claims
1. A plant activator comprising a fatty acid metabolite obtainable
by a metabolism of a fatty acid with 4 to 30 carbon atoms by a
proteobacteria under a dissolved oxygen concentration of 0.1 to 8
mg/L.
2. The plant activator of claim 1, wherein the fatty acid is a
liquid fatty acid at a temperature of 20.degree. C.
3. The plant activator of claim 1, wherein the proteobacteria is a
proteobacteria pre-cultured to 1.times.10.sup.8 to
9.times.10.sup.10 cells/mL.
4. The plant activator of claim 1, wherein the metabolism is a
metabolism in the presence of at least one type of mineral selected
from Mg, P, Na and K.
5. The plant activator of claim 1, wherein the plant activator
comprises a biosurfactant.
6. The plant activator of claim 1, wherein the metabolism is a
metabolism under a condition of temperature from 10 to 40.degree.
C.
7. The plant activator of claim 1, wherein the plant activator
serves as a resistance-inducing agent.
8. The plant activator of claim 1, wherein the plant activator
serves as a prophylactic agent for a wilt disease in solanaceous
plants.
9. A method for manufacturing a plant activator comprising a fatty
acid metabolite, comprising a step for fatty acid metabolism
wherein a fatty acid with 4 to 30 carbon atoms is subjected to a
proteobacterial metabolization under a dissolved oxygen
concentration of 0.1 to 8 mg/L.
10. The method for manufacturing a plant activator of claim 9,
wherein the fatty acid is liquid at a temperature of 20.degree.
C.
11. The method for manufacturing a plant activator of claim 9,
wherein the proteobacteria is a proteobacteria pre-cultured to
1.times.10.sup.8 to 9.times.10.sup.10 cells/mL.
12. The method for manufacturing a plant activator of claim 9,
wherein the step for fatty acid metabolism is performed in the
presence of at least one type of mineral selected from Mg, P, Na
and K.
13. The method for manufacturing a plant activator of claim 9,
wherein the plant activator comprises a biosurfactant.
14. The method for manufacturing a plant activator of claim 9,
wherein the step for fatty acid metabolism is performed under a
condition of temperature from 10 to 40.degree. C.
15. The method for manufacturing a plant activator of claim 9,
wherein the plant activator serves as a resistance-inducing
agent.
16. The method for manufacturing a plant activator of claim 9,
wherein the plant activator serves as a prophylactic agent for a
wilt disease in solanaceous plants.
Description
TECHNICAL FIELD
[0001] The present invention relates to a plant activator and a
method of manufacturing the same.
BACKGROUND ART
[0002] For the purpose of promoting the plant growth, some
measures, such as an optimization of temperature conditions or
daylight conditions, or a fertilization, have been implemented for
a long time. However, those measures have their limitations. For
example, increasing an amount of a fertilizer to be used for a
fertilization neither provides a further desirable growth-promoting
effect beyond a certain level, nor, applying too much fertilizer
would cause a plant growth disorder and may result in a
contamination of the soil.
[0003] Therefore, in addition to those measures, there has been
some reports including a growth promotion using a plant activator
having, for example, a plant growth control activity such as growth
promotion (which refers to the concept that includes an enlargement
of leaves and stems and a growth promotion of tubers and tuberous
roots), sleep suppression, imparting a stress resistance to plant,
and anti-aging. Reference 1 describes a plant activator comprising
a ketol fatty acid with 4 to 24 carbon atoms as an active
ingredient.
[0004] Meanwhile, the plant disease and insect pest control depends
largely on synthesized agrochemicals, however, in view of the soil
contamination as well as human health damage, reducing the amount
of the agrochemical to be used has been required.
[0005] Some measures using a disease resistance inducing agent are
known as a method for controlling a plant disease or pests to
protect plants without using agrochemicals. Examples of resistance
inducing agents include, for example, probenazole, isotianil,
acibenzolar-S-methyl (ASM),
3'-chloro-4,4'-dimethyl-1,2,3-thiadiazole-5-carboxianilide
(tiadinil), and Validamycin.
PRIOR ART DOCUMENT
Patent Document
[0006] Patent Document 1: JP 2001-131006 A [0007] Patent Document
2: JP H6-305921 A
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0008] The effect of the plant activator disclosed in Patent
Document 1 is still not enough, and the plant activator having a
better activation effect is in need. Also, the conventional
resistance-inducing agents are chemically synthesized, so that
their toxicities are extremely high. Aforementioned agents
including probenazole, isotianil, acibenzolar-S-methyl (ASM),
3'-chloro-4,4'-dimethyl-1,2,3-thiadiazole-5-carboxianilide
(tiadinil) may induce the systemic acquired resistance that is
activated through the salicylic acid and induced by the exposure to
elicitors such as pathogenic bacteria and viruses (salicylic acid
mediated signaling pathway), but does not induce the resistance to
be activated thorough the jasmonic acid (and ethylene) that is
synthesized in response to insect herbivory or insect injury
(jasmonate mediated signaling pathway). Validamycin has been
reported to be effective against wilt disease in solanaceous
plants, however, the use of Validamycin in tomatoes has, as an
exception, the problem that it could cause phytotoxic damage, and
thus it should not be used for tomatoes. Patent Document 2
discloses the use of chemically synthesized linoleic acid peroxide
as a plant growth regulator, however, it did not exhibit enough
efficacy in preventing disease damage.
[0009] It has been known that the fatty acid oxide including a
peroxylipid exhibits antibacterial activity. It has also been known
that a jasmonic acid, for example, is biosynthezised in plants from
fatty acid such as linolenic acid. However, it is not known that
metabolites from fatty acid oxidation by microbial metabolization
process have resistance-inducing activity.
[0010] Further those resistance-inducing agents and peroxylipids
are only very slightly water soluble, and thus, one would normally
have to use strong emulsifiers or dispersants when using them for
treatment.
[0011] In view of such problems described above, it is the
intention of the present invention to provide a plant activator
having superior resistance-inducing activity and growth-promoting
activity and low toxicity and soil contamination and to provide a
manufacturing method thereof.
Means to Solve the Problem
[0012] The present invention relates to a plant activator
comprising a fatty acid metabolite obtainable by a metabolism of a
fatty acid with 4 to 30 carbon atoms by a proteobacteria under a
dissolved oxygen concentration of 0.1 to 8 mg/L.
[0013] It may be preferable for the plant activator that the fatty
acid is a liquid fatty acid at a temperature of 20.degree. C.
[0014] It may be preferable for the plant activator that the
proteobacteria is a proteobacteria that has been pre-cultured to
1.times.10.sup.8 to 9.times.10.sup.10 cells/mL.
[0015] It may be preferable for the plant activator that the
metabolism is a metabolism in the presence of at least one type of
mineral selected from Mg, P, Na and K.
[0016] It may be preferable that the plant activator comprises a
biosurfactant.
[0017] It may be preferable for the plant activator that the
metabolism is a metabolism under a condition of temperature from 10
to 40.degree. C.
[0018] It may be preferable that the plant activator serves as a
resistance-inducing agent.
[0019] It may be preferable that the plant activator serves as a
prophylactic agent for a wilt disease in solanaceous plants.
[0020] Further the present invention relates to a method for
manufacturing a plant activator comprising a fatty acid metabolite,
comprising a step for fatty acid metabolism wherein a fatty acid
with 4 to 30 carbon atoms is subjected to a proteobacterial
metabolization under a dissolved oxygen concentration of 0.1 to 8
mg/L.
[0021] It may be preferable for the method for manufacturing a
plant activator that the fatty acid is liquid at a temperature of
20.degree. C.
[0022] It may be preferable for the method for manufacturing a
plant activator that the proteobacteria has been pre-cultured to
1.times.10.sup.8 to 9.times.10.sup.10 cells/mL.
[0023] It may be preferable that the method for manufacturing a
plant activator comprises the step for fatty acid metabolism
performed in the presence of at least one type of mineral selected
from Mg, P, Na and K.
[0024] It may be preferable for the method for manufacturing a
plant activator that the plant activator comprises a
biosurfactant.
[0025] It may be preferable that the method for manufacturing a
plant activator comprises the step for fatty acid metabolism
performed under a condition of temperature from 10 to 40.degree.
C.
[0026] It may be preferable for the method for manufacturing a
plant activator that the plant activator serves as a
resistance-inducing agent.
[0027] It may be preferable for the method for manufacturing a
plant activator that the plant activator serves as a prophylactic
agent for a wilt disease in solanaceous plants.
Effects of the Invention
[0028] The plant activator of the present invention has superior
resistance-inducing activity and growth promoting activity and low
toxicity and soil contamination. Further, according to the method
for manufacturing a plant activator of the present invention, the
plant activator having superior resistance-inducing activity and
growth promoting activity and low toxicity and soil contamination
can be produced.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a table (formerly "Table 1" in the international
phase) that shows test results of the plant activator in an example
1 followed by five comparative examples 1 through 5.
[0030] FIG. 2 is a graph showing a gene expression level in
Arabidopsis thaliana.
[0031] FIG. 3 shows a real-time PCR amplification curve of PR1
gene.
[0032] FIG. 4 is a picture showing a result of a growth-promoting
activity in eggplant root.
[0033] FIG. 5 is a picture showing a result of a growth-promoting
activity in eggplant root.
EMBODIMENT FOR CARRYING OUT THE INVENTION
Plant Activator
[0034] A plant activator of the present invention is characterized
in that it comprises a fatty acid metabolite obtainable by a
metabolism of a fatty acid with 4 to 30 carbon atoms by a
proteobacteria under a dissolved oxygen concentration of 0.1 to 8
mg/L.
[0035] A fatty acid metabolite of the present invention can render
plants resistance and promote plant growth by being sprayed or
applied to a plant root, stem or leaf. This may be because the
fatty acid metabolite includes a substance or a precursor of the
substance that activates the salicylic acid mediated signaling
pathway or jasmonate mediated signaling pathway associated with
resistance induction. Further, the fatty acid metabolite may also
include a substance that activates plant growth, given that plant
growth-promoting effect can be seen.
[0036] A metabolism in the context of the present invention
involves performing a decomposition or synthesis using a fatty acid
with 4 to 30 carbon atoms as a starting material by, for example,
enzymes secreted via an endocrine or exocrine pathway by
proteobacteria under a predetermined dissolved oxygen
concentration. Examples include a method for culturing the
proteobacteria in a culture media including the fatty acid in the
context of the present invention under a predetermined dissolved
oxygen concentration.
[0037] The number of carbon atoms in the fatty acid to be used in
the present invention is from 4 to 30, preferably 10 to 20. When
the number of carbon atoms is less than 4, the melting point and/or
boiling point of the fatty acid is low, so that it tends to become
highly volatile at a culture temperature and can hardly remain in
the culture media. When the number of carbon atoms is more than 30,
the melting point and/or boiling point of the fatty acid is high,
so that it tends to become a solid at a culture temperature and
cannot be mixed with a culture media, resulting in the separation
of the fatty acid from the culture media. However, it should be
noted that the melting point of the fatty acid does not always
depend on only the number of carbon atoms based on the number of
hydrogen bonds.
[0038] The fatty acid to be used in the present invention is
preferably a liquid fatty acid at a temperature from 20 to
30.degree. C. in terms of their metabolism efficiency as well as to
prevent solidification in the culture media.
[0039] The fatty acid in the context of the present invention may
be either a saturated fatty acid or a unsaturated fatty acid, or a
mixture including both fatty acids. Further a vegetable oil, a
triglyceride form, or a free fatty acid may be used. Preferably,
the fatty acid is a free fatty acid (monocarboxylic acid) in terms
of its superior decomposition rate.
[0040] Exemplary examples of free fatty acid with 4 to 30 carbon
atoms include for instance, butanoic acid (butyric acid), pentanoic
acid (valeric acid), caproic acid, enanthic acid (heptylic acid),
caprylic acid, pelargonic acid, capric acid, lauric acid, myristic
acid, pentadecylic acid, palmitic acid, palmitoleic acid, margaric
acid, stearic acid, oleic acid, vaccenic acid, linoleic acid,
.alpha.-linolenic acid, .gamma.-linolenic acid, eleostearic acid,
arachidic acid, mead acid, arachidonic acid, behenic acid,
lignoceric acid, nervonic acid, cerotic acid, montanic acid, and
melissic acid. Preferably, the fatty acid is a fatty acid which has
10 to 20 carbon atoms such as capric acid, lauric acid, myristic
acid, pentadecylic acid, palmitic acid, palmitoleic acid, margaric
acid, stearic acid, oleic acid, vaccenic acid, linoleic acid,
.alpha.-linolenic acid, .gamma.-linolenic acid, eleostearic acid,
arachidic acid, mead acid, or arachidonic acid. More preferably,
the fatty acid is a fatty acid which has 18 carbon atoms such as
oleic acid, linoleic acid, .alpha.-linolenic acid, or
.gamma.-linolenic acid.
[0041] Preferably, a fatty acid content in the culture media is 100
g/L or less, more preferably 60 g/L or less, still more preferably
12 g/L or less when the culture media containing the fatty acid is
used. When the fatty acid content is over 100 g/L, emulsification
of the fatty acid with water in the culture media may become
difficult, deteriorating metabolism efficiency or resulting in an
inhibition of proteobacteria growth. Further, although the lower
limit of the fatty acid content is not particularly limited, the
fatty acid content is preferably 1.0 g/L or more.
[0042] Preferably, the culture media containing a fatty acid in the
context of the present invention also contains a mineral component.
Exemplary examples of mineral components include for instance, but
not particularly limited to, a mineral component which is used for
culturing microorganism. Examples include for instance a component
containing magnesium (Mg), phosphorous (P), sodium (Na), or
potassium (K). These components can be used alone or in
combination. A mineral component content in the culture media is
not particularly limited, and can be adapted to the content used
with a conventional method for culturing an aerobic bacteria.
[0043] A proteobacteria which can be used in the context of the
present invention is not particularly limited unless it provides a
desirable effect of the present invention. Preferably, the
proteobacteria is a proteobacteria which has a suitable growth
temperature (an optimum temperature) of from 10 to 40.degree. C.,
more preferably 20 to 30.degree. C. in terms of its fatty acid
metabolism efficiency as well as growth efficiency.
[0044] Preferably, a proteobacteria in the context of the present
invention is a pre-cultured proteobacteria, preferably, to the cell
concentration at the end of pre-culture of 1.times.10.sup.8 to
9.times.10.sup.10 cells/mL, in such case the proteobacteria can
have superior fatty acid metabolism efficiency.
[0045] With the context of the present invention a dissolved oxygen
concentration during metabolism is 0.1 mg/L or more. When the
dissolved oxygen concentration is less than 0.1 mg/L, the
proteobacteria activity tends to become low, resulting in a
significantly low fatty acid metabolism efficiency. Preferably, the
dissolved oxygen concentration is 8 mg/L or less, more preferably 5
mg/L or less, still more preferably 3 mg/L or less. When the
dissolved oxygen concentration is over 8 mg/L, the fatty acid tends
to be oxidized by oxygen in the culture media, resulting in the
reduced plant activation activity. In this context, dissolved
oxygen levels are values detected by using PO electrodes by a
diaphragm galvanic electrode method or diaphragm polarographic
method using a dissolved oxygen meter from HORIBA, Ltd.
[0046] The temperature during metabolism can be adapted suitably
according to the proteobacteria to be used, and is preferably from
10 to 40.degree. C., more preferably from 20 to 30.degree. C. in
terms of fatty acid metabolism efficiency.
[0047] Preferably, in terms of the fatty acid metabolism efficiency
and the handleability, the plant activator of the present invention
includes a biosurfactant in addition to the fatty acid metabolite,
so that the fatty acid as well as the fatty acid metabolite becomes
water soluble. In this context, the biosurfactant according to the
present invention is a surfactant produced by proteobacteria.
[0048] The plant activator of the present invention is
significantly effective as a resistance-inducing agent and growth
promoting agent with a low toxicity and soil contamination. The
plant activator of the present invention is particularly effective
as a resistance-inducing agent compared to the conventional
resistance-inducing agents in that the plant activator of the
present invention can activate both of respective resistance
inductions through a salicylic acid mediated signaling pathway and
through a jasmonate mediated signaling pathway. Further, the plant
activator of the present invention can activate a resistance
induction and plant growth in all parts of the plant by being
applied to a part of the plant root, stem or leaf.
[0049] While, as described above, Validamycin, which is known as a
resistance-inducing agent against wilt disease in solanaceous
plants, should not be used in tomatoes, as an exception, because it
could cause phytotoxic damage, the plant activator of the present
invention can exhibit a resistance-inducing effect even on the wilt
disease in tomatoes without causing any phytotoxic damages.
[0050] The plant activator of the present invention can be widely
applied to the plants, regardless of their species. Examples of the
plants include dicotyledonous plants of Cucurbitaceae family or
Solanaceae family, and monocotyledonous plants of Gramineae family.
Further, Methods for application includes, for example, a method of
applying or spraying to, for example, a plant root, stem or leaf,
and a method of spraying on the soil or watering.
Manufacturing Method
[0051] A method for manufacturing a plant activator containing a
fatty acid metabolite of the present invention is characterized in
that it comprises a step for fatty acid metabolism wherein a fatty
acid with 4 to 30 carbon atoms is subjected to a proteobacterial
metabolization under a dissolved oxygen concentration of 0.1 to 8
mg/L.
[0052] The step for fatty acid metabolism in the context of the
present invention is a step wherein a decomposition or synthesis is
performed using a fatty acid with 4 to 30 carbon atoms as a
starting material by, for example, enzymes secreted via an
endocrine or exocrine pathway by proteobacteria under a
predetermined dissolved oxygen concentration. Examples include a
method for culturing the proteobacteria in a culture media
including the fatty acid in the context of the present invention
under a predetermined dissolved oxygen concentration.
[0053] The dissolved oxygen concentration during metabolism in the
step for fatty acid metabolism in the context of the present
invention is 0.1 mg/L or more. When the dissolved oxygen
concentration is less than 0.1 mg/L, the proteobacteria activity
tends to become low, resulting in a significantly low fatty acid
metabolism efficiency. Preferably, the dissolved oxygen
concentration is 8 mg/L or less, more preferably 5 mg/L or less,
still more preferably 3 mg/L or less. When the dissolved oxygen
concentration is over 8 mg/L, the fatty acid tends to be oxidized
by oxygen in the culture media, resulting in the reduced plant
activation activity. In this context, dissolved oxygen levels are
values detected by using PO electrodes by a diaphragm galvanic
electrode method or diaphragm polarographic method using a
dissolved oxygen meter from HORIBA, Ltd.
[0054] The dissolved oxygen concentration in the context of the
present invention may be adjusted according to, for example, a
culture vessel, shaking speed, and an aeration volume.
[0055] The culture condition in the step for fatty acid metabolism
in the context of the present invention may be a similar condition
as any of conventional conditions applied for culturing the
conventional aerobic bacteria, other than the dissolved oxygen
concentration, which is, in the present invention, within the
predetermined range. Exemplary conditions include for instance a
culturing method wherein an aerating cultivation is employed for 3
to 7 days by shaking a culture flask, or using a spinner flask or
jar fermentor.
[0056] Preferably, the culture period may be a period during which,
for example, an emulsification or a decomposition of the fatty acid
is sufficiently employed, however, the culture period can be
altered according to the shaking condition or amount of
microorganisms to be employed. In this context, preferably the end
point of the step for fatty acid metabolism is determined by
detecting the degree of decomposition of fatty acid from, for
example, the absorbance at wavelength 230 nm, thin layer
chromatography (TLC), high performance liquid chromatography
(HPLC), gas chromatography-mass spectrometry (GC-MS), or liquid
chromatography-mass spectrometry (LC-MS).
[0057] The temperature in the step for fatty acid metabolism in the
context of the present invention may be adapted according to the
proteobacteria to be used, and the step is preferably performed
under the condition of the temperature of 10 to 40.degree. C., more
preferably 20 to 30.degree. C. in terms of fatty acid metabolism
efficiency.
[0058] The fatty acid and proteobacteria described herein in the
context of plant activator of the present invention can be adapted
for the fatty acid and proteobacteria for the step for fatty acid
metabolism in the context of the present invention.
[0059] In this context, a step for pre-culturing proteobacteria is
not particularly limited, and may be any conventional methods for
culturing aerobic bacteria. Preferably, after the preculture, only
the proteobacterial cells may be collected by, for example,
centrifugation, and subjected to the step for fatty acid
metabolism.
[0060] Preferably, in terms of the fatty acid metabolism efficiency
and the handleability, the plant activator obtainable from a
manufacturing method of the present invention includes a
biosurfactant in addition to the fatty acid metabolite, so that the
fatty acid as well as the fatty acid metabolite become water
soluble. In this context, the biosurfactant according to the
present invention is a surfactant produced by proteobacteria.
[0061] The plant activator of the present invention may be obtained
as a culture solution which is a mixture of, for example, culture
media, substances secreted by proteobacteria containing a
biosurfactant, and bacterial cells. This culture solution may be
adapted for the plant activator of the present invention as it is
or after a removal of bacterial cells from this culture solution
by, for example, a centrifugation. Although the culture solution
can be used without dilution, preferably the culture solution is
used after dilution because it may cause a shrinking of a plant
part to which the culture solution is applied due to the osmotic
effect caused by an evaporation of solution and a concentration of
mineral components at a high temperature. A dilution ratio is not
particularly limited unless it provides a desirable effect of the
present invention, and preferably it would be 100 to 500 times
dilution. In this context, the bacterial cells, once removed from
the culture solution, may be cultured again in the culture media
containing fatty acid, and then the step for fatty acid metabolism
can be repeated.
EXAMPLES
[0062] The present invention will be illustrated in detail by way
of the Examples below, although the present invention shall be not
limited to those specific Examples.
Preparation of Plant Activator for Test
<Pre-Culturing Step>
[0063] To 1 L of water in the glass Erlenmeyer flask 20 g of
peptone (Difco, enzymatic digest of protein), 1.5 g of magnesium
sulfate heptahydrate, and 1.5 g of dipotassium hydrogenphosphate
were dissolved, autoclaved at 121.degree. C. for 20 min to render
it sterile, and after cooled to room temperature, the suspension
containing proteobacterial cells was inoculated. The mouth of the
Erlenmeyer flask was sealed with a silicone closure. After
inoculation, cells in the flask were cultured at 20.degree. C. for
24 hours with shaking (120 rpm) using Bioshaker (Taitec, BR-23UM).
The number of bacterial cells in the culture broth was
5.times.10.sup.8 cells/mL. After the culturing, the culture broth
was subjected to the centrifugation at 15,000.times.g, at
temperature of 20.degree. C., isolating the bacterial cells from
the culture broth, and then the bacterial cells were collected.
<Fatty Acid Metabolism Step>
[0064] To 1 L of sterilized water in the glass Erlenmeyer flask 12
g of linoleic acid (Wako, first grade), 1.5 g of magnesium sulfate
heptahydrate, 1.5 g of dipotassium hydrogenphosphate, and the whole
amount of the bacterial cells obtained from the pre-culturing step
were added. The bacterial cells in the flask were cultured at
20.degree. C. for 4 days with shaking (120 rpm) using Bioshaker
(Taitec, BR-23UM) under the condition of a dissolved oxygen
concentration of 4 mg/L. The decomposition of linoleic acid was
determined through the analysis of the culture broth by measuring
the absorbance at wavelength 230 nm using BioSpec-mini
spectrophotometer (Shimadzu Scientific Instruments) and by
estimating the amount of peroxylipid formation, which is one of the
intermediate products from the degradation of linoleic acid. After
the culturing, the culture broth containing the bacterial cells was
evaluated as a plant activator for test as described below.
Gray Mold Caused by Botrytis cinerea in Cucumber Leaves
Example 1
[0065] The plant activator for test, which was 500 times diluted
with water, was sprayed to the true leaves of cucumber plant grown
in the pot. 24 hours after spraying, pathogenic bacteria was
inoculated to the spray treated true leaves by making a contact
with a filter paper impregnated with Botrytis cinerea spore
suspension. Then the cucumber plant was grown at 23.+-.2.degree. C.
in a high humidity environment for 5 days, and the pathogenic
bacteria inoculated part was evaluated. The results are shown in
FIG. 1 (formerly Table 1). The prevention effect was determined
according to the following criteria.
.circleincircle.: no disease symptoms .smallcircle.: disease
symptoms such as browning caused by, for example, necrosis was
slightly recognized. .DELTA.: disease symptoms such as browning
symptoms caused by, for example, necrosis or yellowing of the whole
pathogenic bacteria inoculated part was clearly recognized. X:
browning caused by, for example, necrosis was observed on the whole
pathogenic bacteria inoculated part, or the disease symptoms which
made a hole at the pathogenic bacteria inoculated part was
recognized even though the browning area was not large.
Comparative Examples 1 to 5
[0066] The cucumber plants were treated similar to Example 1 except
that the water, 500 times diluted pyroligneous acid, 0.04% aqueous
sorbitan fatty acid solution, a mixed aqueous solution of 0.2%
potassium carbonate and 0.01% polyoxyethylene nonylphenylether, or
emulsified 0.08% tetrachloroisophthalonitrile aqueous solution was
used instead of the plant activator for test. The results are shown
in FIG. 1.
[0067] It should be appreciated from the results shown in FIG. 1
(formerly Table 1) that the plant activator of the present
invention induces resistance against the gray mold disease in
cucumber plants.
Arabidopsis thaliana Root
Examples 2 to 4
[0068] The roots of Arabidopsis thaliana grown in the soil were
treated by applying the 10 times, 100 times or 500 times diluted
solution of the plant activator for test using the watering. 24
hours after the treatment, bacterial soft rot causing bacteria
(Erwinia carotovora subsp. Carotovora) was inoculated on the leaves
and stems above the ground. The plants were then grown at
23.+-.2.degree. C. in a high humidity environment for 3 days, and
the disease symptoms at the pathogenic bacteria inoculated part had
been evaluated up to one week after. The results are shown in Table
2. The disease prevention effect was determined according to the
following criteria.
.circleincircle.: the ratio of the area of leaves with no disease
symptoms out of total area is 1.60 times or greater than the ratio
observed in untreated leaves. .smallcircle.: the ratio of the area
of leaves with no disease symptoms out of total area is 1.40 to
1.59 times than the ratio observed in untreated leaves. .DELTA.:
the ratio of the area of leaves with no disease symptoms out of
total area is 1.20 to 1.39 times than the ratio observed in
untreated leaves. X: the ratio of the area of leaves with no
disease symptoms out of total area is 1.20 times less than the
ratio observed in untreated leaves.
[0069] Further, the cDNA was synthesized from total RNA isolated
from each Arabidopsis thaliana plant 24 hours after treatment, and
the expression level of individual genes specific to the salicylic
acid mediated signaling pathway and the jasmonate mediated
signaling pathway was analyzed by real-time PCR. The gene
expression levels were respectively normalized using the house
keeping gene as negative control. FIG. 2 shows the expression
levels of PR1, PR2 and PR5 genes specific to the salicylic acid
mediated signaling pathway and PR3, PR4, and PDF1 genes specific to
the jasmonate mediated signaling pathway. FIG. 3 show a real-time
PCR amplification curve for PR1.
Comparative Examples 6 to 13
[0070] The roots of Arabidopsis thaliana were treated similar to
Example 2 except that the water, peroxylipid, probenazole,
isotianil, acibenzolar-S-methyl (ASM),
3'-chloro-4,4'-dimethyl-1,2,3-thiadiazole-5-carboxianilide
(tiadinil), ketol fatty acid solution 1, or ketol fatty acid
solution 2 was used instead of the plant activator for test. The
results are shown in Table 2.
[0071] The peroxylipid used in Comparative example 7 was prepared
according to the preparation method described below.
[0072] To Erlenmeyer flask 100 mL of distilled water and 1 mL of
32% sodium hydroxide solution were added and stirred with magnetic
stirrer, and then heated to 50.degree. C. in water bath. Then 1.2 g
of linoleic acid (Wako, first grade), and 0.53 mL of hydrogen
peroxide (35%) were added and the reaction mixture was refluxed at
50.degree. C. in water bath at atmospheric pressure for 3 days. The
reaction was monitored using a silica gel coated plate (Merck,
60F254 on glass plate), the solvent mixture
(chloroform:methanol=10:1) as a developing solvent, and sulfuric
acid as coupler. The spots on the glass plate were visualized by a
dark spot formed by sulfuric acid. The spot which was visualized by
UV light and sulfuric acid just below the spot of the starting
material was a spot for peroxylipid, and the reaction was stopped
when the formation of peroxylipid was visually observed to reach
about 40 to 50% of the starting material. The resulting reaction
mixture was neutralized with 5% hydrochloric acid and chloroform
was added, and then the chloroform layer was recovered, washed with
water and saturated brine using a separatory funnel, and then the
resulting chloroform layer was dried over anhydrous sodium sulfate,
filtered, and collected, and concentrated and vacuum-dried to
obtain linoleic acid oxide in the form of a syrup. The solution to
which 0.6 g of resulting linoleic acid oxide, 75 mg of dipotassium
hydrogenphosphate, and 75 mg of magnesium sulfate heptahydrate were
suspended was used as the peroxylipid.
[0073] The ketol fatty acid solution 1 used in Comparative example
12 was prepared according to the preparation method described
below.
[0074] 10 mg of soybean lipoxidase (Sigma) was added to the
linoleic acid suspension containing 1 g of linoleic acid, 0.15 g of
dipotassium hydrogenphosphate, and 100 mL of distilled water, and
the reaction mixture was stirred for 24 hours to form peroxylipid
1. The formation of peroxylipid was identified by TLC comparison
with a standard compound (developing solvent of
chloroform:methanol=20:1, visualized by sulfuric acid) and by the
increase in absorbance at 234 nm. It was also identified by NMR
that the main composition in the peroxylipid 1 was 13HPODE ((9Z,
11E)-13-hydroperoxy-9, 11-octadecadienoic acid).
[0075] To the resulting peroxylipid 1, 0.1 mg of allene oxide
synthase (Sigma-Aldrich) was added and stirred for 24 hours to form
ketol fatty acid, and the enzyme reaction was then stopped by
adding diluted hydrochloric acid on ice to adjust the pH of
reaction mixture to pH 3.0. The solution after whose pH was
adjusted to 6.5 was used as ketol fatty acid solution 1.
[0076] The ketol fatty acid solution 2 used in Comparative example
13 was prepared according to the preparation method described
below.
[0077] 10 g of maize embryos was grounded in a mortar, and then 30
mL of distilled water was added and the mixture was further
grounded in a mortar to make a suspension. The resulting suspension
was centrifuged at 16000 rpm for 15 min to obtain a supernatant,
and the resulting supernatant was used as a substance containing
lipoxygenase from maize embryos. 10 mg of the substance containing
lipoxygenase from maize embryos was added to the linoleic acid
suspension containing 1 g of linoleic acid, 0.15 g of dipotassium
hydrogenphosphate, and 100 mL of distilled water, and the reaction
mixture was stirred for 24 hours to form peroxylipid 2. The
formation of peroxylipid was identified by TLC comparison with a
standard compound (developing solvent of chloroform:methanol=20:1,
visualized by sulfuric acid) and by the increase in absorbance at
234 nm. It was also identified by NMR that the main composition in
the peroxylipid 2 was 9HPODE ((9S, 10E, 12Z)-9-hydroperoxy-10,
12-octadecadienoic acid).
[0078] To the resulting peroxylipid 2, 0.1 mg of allene oxide
synthase (Sigma-Aldrich) was added and stirred for 24 hours to form
ketol fatty acid, and the enzyme reaction was then stopped by
adding diluted hydrochloric acid on ice to adjust the pH of
reaction mixture to pH 3.0. The solution after whose pH was
adjusted to 6.5 was used as ketol fatty acid solution 2.
TABLE-US-00001 TABLE 2 disease agent for treatment prevention
Example 2 10 times diluted .circleincircle. solution of plant
activator for test 3 100 times diluted .circleincircle. solution of
plant activator for test 4 500 times diluted .largecircle. solution
of plant activator for test Comparative 6 water X example 7
peroxylipid .DELTA. 8 probenazole .DELTA. 9 isotianil .DELTA. 10
ASM .circleincircle. 11 tiadinil X 12 ketol fatty acid X solution 1
13 ketol fatty acid X solution 2
[0079] From the results shown in Table 2 and FIG. 2, it should be
appreciated that the plant activator of the present invention
induces resistance against the bacterial soft rot disease in
Arabidopsis thaliana. Further, it is demonstrated that the amount
of gene expression, related to the salicylic acid mediated
signaling pathway as well as the jasmonate mediated signaling
pathway, in the presence of the plant activator of the present
invention is greater than those in the presence of ASM or
probenazole.
Leaves and Stems of Tomatoes (Grown in Pot)
Example 5
[0080] The leaves and stems of tomatoes (cultivar "Momotaro") grown
in the pot for 3 weeks were treated by applying 10 mL of undiluted
solution of the plant activator for test using the watering. The
soil (about 100 g) in the pot was moistened thoroughly before the
watering. The bacterial wilt disease bacteria were inoculated on
the leaves and roots of tomatoes 1 hour and 24 hours after the
watering. The plants were then grown at 30.degree. C. in a high
humidity environment for 7 days, and the incidences of the
bacterial wilt disease and phytotoxic damage were evaluated. The
results are shown in Table 3.
[0081] Further, the cDNA was synthesized from total RNA isolated
from each tomato 24 hours and 72 hours after the inoculation, and
the expression level of individual genes specific to the salicylic
acid mediated signaling pathway and the jasmonate mediated
signaling pathway was analyzed by real-time PCR. In this example,
the amount of the gene expression is represented by an index, which
is a calculated ratio given that the amount of gene expression in
untreated tomatoes is 1. The results are shown in Table 3.
Comparative Examples 14 and 15
[0082] The leaves and stems of tomatoes were treated similar to
Example 5 except that the water and the fatty acid (linoleic acid)
were used instead of the plant activator for test. The results are
shown in Table 3.
TABLE-US-00002 TABLE 3 amount of gene expression salicylic acid
jasmonate incidences mediated mediated agent for incidences of
phytotoxic signaling pathway signaling pathway treatment of disease
damage 24 hours 72 hours 24 hours 72 hours Example 5 plant
activator 50% 0% 11.0 7 3 3.5 for test Comparative 14 untreated
100% 0% 1 1 1 1 example 15 fatty acid 100% 0% 0 0 0.5 1
[0083] It should be appreciated from the results shown in Table 3
that the plant activator of the present invention induces
resistance against the bacterial wilt disease in tomatoes without
causing any phytotoxic damages. Further, it is demonstrated that
the amount of gene expression related to the salicylic acid
mediated signaling pathway as well as the jasmonate mediated
signaling pathway is high.
Leaves and Stems of Tomatoes (Growing in Farm Field)
[0084] Tomato (cultivar "Reika") seedlings were planted with space
of 60 cm between plants in the farm field where an outburst of
bacterial wilt disease was observed, and the solution of the plant
activator for test, which was 50 times diluted with water, was
applied either by spraying (20 mL/plant) or watering it to soil
(200 mL plant) once every week. The incidences of the bacterial
wilt disease and phytotoxic damage were evaluated once every week
for 4 weeks. The results are shown in Table 4.
Comparative Examples 16 and 17
[0085] The leaves and stems of tomatoes were treated similar to
Example 6 except that the Validamycin or no agent was used instead
of the plant activator for test. The results are shown in Table
4.
TABLE-US-00003 TABLE 4 incidences of disease incidences of
phytotoxic damage agent for Week Week Week Week Week Week Week Week
treatment 1 2 3 4 1 2 3 4 Example 6 plant activator 0% 0% 0% 33% 0%
0% 0% 0% for test Comparative 16 untreated 0% 0% 42% 75% 0% 0% 0%
0% example 17 Validamycin 0% 0% 0% 67% 100% 100% 100% 100%
[0086] It should be appreciated from the results shown in Table 4
that the plant activator of the present invention induces
resistance against the bacterial wilt disease in tomatoes without
causing any phytotoxic damages.
Leaves and Stems of Miniature Roses (in a Planter)
Example 7
[0087] The solution of the plant activator for test, which was 100
times diluted with water, was applied to the leaves and stems of
miniature roses (cultivar Rouge) planted in an outdoor planter
right after the planting and once every week by spraying (about 50
mL/plant), and the naturally occurring damages due to disease
(powdery mildew and black point disease) were evaluated on first
week and fourth week. The results are shown in Table 5.
Comparative Examples 18 and 19
[0088] The leaves and stems of miniature roses were treated similar
to Example 7 except that 0.02% mepanipyrim or no agent was used
instead of the plant activator for test. The results are shown in
Table 5.
TABLE-US-00004 TABLE 5 incidences of disease black point agent for
powdery mildew disease treatment Week 1 Week 4 Week 1 Week 4
Example 7 plant activator 12.5% 20.8% 0% 0% for test Comparative 18
untreated 29.1% 25.0% 0% 0% example 19 mepanipyrim 20.0% 30.0% 0%
15%
[0089] It should be appreciated from the results shown in Table 5
that the plant activator of the present invention induces
resistance against the powdery mildew and black point disease in
miniature roses.
Leaves and Stems of Miniature Roses (in a Pot)
Examples 8 to 10
[0090] The solution of the plant activator for test, which was 10
times, 100 times or 500 times diluted with water, was applied to
the leaves and stems of miniature roses (cultivar Rouge) grown in a
pot right after the planting and one week after, two weeks after,
and three weeks after by spraying (about 50 mL/plant), and then the
plants were transferred to a sealed box inside which a wet paper
towel was put and grown under a high humidity condition. One week
after the naturally occurring damages due to disease (gray mold
disease) were evaluated based on the disease prevention rate in
comparison with that in the untreated, comparative section. The
results are shown in Table 6.
TABLE-US-00005 TABLE 6 disease prevention agent for treatment rate
Example 8 10 times diluted 100% solution of plant activator for
test 9 100 times diluted 89% solution of plant activator for test
10 500 times diluted 100% solution of plant activator for test
[0091] It should be appreciated from the results shown in Table 6
that the plant activator of the present invention induces
resistance against the gray mold disease in miniature roses.
Promoting Turf Growth (Germination Rate)
Example 11
[0092] The wet filter paper was put on the petri dish and 30 of
Western turf seeds were arranged at regular intervals on the wet
filter paper. The solution of the plant activator for test, which
was 500 times diluted with water, was injected into the filter
paper, and plants were incubated with a cover closed at
20.+-.2.degree. C. using 16 hours under plant growing light
irradiation and 8 hours of darkness cycle repeatedly, and then, at
day 3 and day 5 the germination rates of lateral root ware
evaluated. The results are shown in Table 7.
Comparative Example 20
[0093] The Western turf seeds were treated similar to Example 11
except that the water was used instead of the plant activator for
test. The results are shown in Table 7.
TABLE-US-00006 TABLE 7 germination rate agent for treatment Day 3
Day 5 Example 11 plant activator for 53% 80% test Comparative 20
water 27% 47% example
[0094] It should be appreciated from the results shown in Table 7
that the plant activator of the present invention promotes the
germination of Western turf.
Promoting Turf Growth (Growth of Leaves and Stems)
Examples 12 to 14
[0095] To the 72 seed cell tray the culture soil for planting was
put, and the turf seeds were placed and lightly covered with the
red clay soil which had been grounded in mortar and sieved. The
cell tray was put in the stainless container and water was poured
into the container up to about 1 cm depth to provide a
bottom-watering tray. Plants were incubated at 20.+-.2.degree. C.
using 16 hours under plant growing light irradiation and 8 hours of
darkness cycle repeatedly. Water was supplied when the water at the
bottom of the container was used up completely. After the
germination was observed in each seed cell, plants were
spray-treated by spraying the solution of the plant activator for
test, which was 10 times, 100 times or 500 times diluted with
water, to each cell (5 mL per cell), and the length of the leaves
and stems growth in 14 days were measured and evaluated. The
results are shown in Table 8.
Comparative Example 21
[0096] The turf seeds were treated similar to Example 12 except
that the water was used instead of the plant activator for test.
The results are shown in Table 8.
TABLE-US-00007 TABLE 8 agent for length of leaves (cm) treatment
Day 7 Day 10 Day 12 Day 14 Example 12 10 times diluted 3.0 5.1 5.7
6.2 solution of plant activator for test 13 100 times diluted 3.8
4.8 5.2 5.7 solution of plant activator for test 14 500 times
diluted 2.8 4.1 4.6 5.5 solution of plant activator for test
Comparative 21 water 2.5 3.5 3.9 5.1 example
[0097] It should be appreciated from the results shown in Table 8
that the plant activator of the present invention promotes the
leaves and stems growth of Western turf.
Promoting Eggplant Growth
Example 15
[0098] 17 Eggplant seedlings (root stock: Solanum integrifolium;
scion: Solanum melongena L. cultivar "Senryo"), which had been
grown up to or over 15 cm in pot, were planted with space of 60 cm
between plants, and a chemical fertilizer, a mixed fertilizer, and
an organic fertilizer were applied accordingly as a basal
fertilizer, in addition to an additional fertilizer. The solution
of the plant activator for test, which was 50 to 100 times diluted
with water, was applied to the plants right after the planting and
once every week by spraying (about 20 to 50 mL/plant) and by
watering (about 100 to 300 mL/plant). Then, once every week the
fruits which were grown to 15 cm or more were harvested and the
number of the harvested fruits was evaluated. All eggplant plants
were pruned to three main branches, and the main stem length had
been measured and evaluated from one week after the planting. The
results are shown in Table 9.
[0099] Further, after 2 to 3 months after the planting, the ridge
was watered adequately to soften the soil, and then the plants were
pulled out from the soil while loosening the soil covering the
roots. After pulling the plant, the plant was cut at 1 cm above
ground level, and the root side was washed with water, dried for
one day at room temperature, and then dried in thermostatic drier
(SANYO Electric Co., Ltd., MOV-112U) at 60.degree. C. for 24 hours,
and the dried weight was weighed using an electronic balance
(Shimadzu Scientific Instruments, UX220H). The measurement results
obtained for optionally selected three plants are shown in FIGS. 3
and 4.
Comparative Example 22
[0100] The eggplant seedlings were treated similar to Example 15
except that the no treatment was applied. The results are shown in
Tables 9 and 10, and FIGS. 3 and 4.
TABLE-US-00008 TABLE 9 average length of number of the harvested
main stem (cm) fruits per plant agent for Week Week Week Week Week
Week Week Week Week Week treatment 1 2 3 4 5 1 2 3 4 5 Example 15
plant activator 35.8 47.2 54.3 65.2 76.2 0.9 0.2 0.6 0.8 2.0 for
test Comparative 22 untreated 35.6 45.5 51.7 58.4 70.4 0.9 0.1 0.5
0.5 0.9 example
TABLE-US-00009 TABLE 10 agent for standard treatment dried weight
(g) average deviation Example 15 plant activator 11.606 10.476
8.163 10.08 1.755 for test Comparative 22 untreated 6.313 5.721
6.561 6.20 0.4316 example
[0101] It should be appreciated from the results shown in Tables 9
and 10, and FIGS. 3 and 4 that the plant activator of the present
invention promotes the eggplant growth.
[0102] The above described results show that the plant activator of
the present invention and the plant activator obtainable by the
manufacturing method of the present invention have superior
resistance-inducing activity and growth promoting activity and low
toxicity and soil contamination.
* * * * *