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(11) Patent Number: KE 604

(45)Dateofgrant: 10/09/2013

(51) Int.Cl.8:A OlN 37/30, 43/38, A 01P 21/00

(2l)Application Number: KElP/ 2012/001587

(22) Filing Date: 09/12/2010

(30) Priority data: 2009-281348  11112/2009  JP

(86)  PCT data PCT/JP201 0/072599 09/12/2010 wo 2011/071187 16/06/2011

(54) Title:

(73) Owner:SUMITOMO CHEMICAL COMPANY LIMITEDof 27-1, Shinkawa 2-chome, Chuo-ku, Tokyo,1048260, Japan

(72) Inventors:NAGASAW A, Asako, 3-28-56, Kamokogahara,Higashinada-ku, Kobe-shi, Hyogo, 6580064, Japanand MUKUMOTO, Fujio, 2-11-15-209,Minamihibarigaoka, Takarazuka-shi, Hyogo,6650811, Japan

(74)    Agent/address for correspondence: J. K. Muchae & Company Advocates, P.O. Box 60664- 00200, Nairobi METHOD FOR REDUCING TEMPERATURE STRESS OF PLANTS

(57) Abstract:The present invention provides: a method for reducing temperature stress of plants which comprises applying an effective amount of one or more compounds selected from the group consisting of a compound represented by the formula (1) and an agriculturally acceptable salt thereof to a plant that has been exposed to or to be exposed to a temperature stress factor; and so on.

DESCRIPTION

METHOD  FOR  REDUCING  TEMPERATURE  STRESS  OF  PLANTS
echnical  Field

5 The present invention relates to•a method for reducing temperature stress of plants.

Background  Art

When  plants  encounter  an  environment  where  the

10    temperature exceeds the upper limit or the lower limit of an optimal temperature for growth or germination, what is called temperature stress factor, physiological functions of cells decline slowly or rapidly and thus various disorders may arise. While it has been known that phytohormones and some


15    chemical substances such as plant growth regulators have an effect of reducing temperature stress of plants, these substances cannot be said to be sufficient in terms of their effects. It has also been known that 4-oxo-4-[(2-phenylethyl)amino]-butyric acid and derivatives thereof have


20    activity to promote growth of roots (see, for example, Japanese Patent Publication No. 4,087,942, Japanese Unexamined Patent Publication No. 2001-139405, and Plant and Soil, 255: 67-75, (2003)). However, an effect of reducing temperature stress of plants has never been known.


25

Disclosure  of  Invention

An object of the present invention is to provide a method for reducing temperature stress of plants, and so on.

The present invention is based on the finding that plants that has been applied with a specified compound has a reduction in temperature stress even when the plants are exposed to a temperature stress factor.

5 That is, the present invention includes the following constitutions.


[1]    A  method  for  reducing  temperature  stress  of  plants

which comprises applying an effective amount of one or more compounds selected from the group consisting of a compound

10    represented by the formula (I) and an agriculturally acceptable salt thereof (hereinafter, sometimes referred to as the present compound) to a plant that has been exposed to or to be exposed to a temperature stress factor (hereinafter, sometimes referred to as the method of the present invention) :





(I)

15

wherein

R1 represents a phenyl group, a naphthyl group or an aromatic heterocyclic group, and these groups are optionally substituted with 1 to 5 members selected from among a halogen

20    atom, a hydroxyl group, a cyano group, a nitro group, a C1-C6 alkyl group optionally substituted with one or more halogen

atoms, a C1-C6 alkoxy group optionally substituted with one or more halogen atoms, a C1-C6 alkylthio group optionally substituted with one or more halogen atoms, a C2-C6 alkenyl

25    group qptionally substituted with one or more halogen atoms, a C2-C6 alkynyl group optionally substituted with one or more
 

3

halogen atoms, an amino group, a C1-C6 alkylamino group and a di(C1-C6 alkyl)amino group;
R2 represents a hydroxyl group, an amino group, or a C1-C6 alkoxy group;


5    X  represents  a  linear  or  branched  C1-C6  alkylene  group;

and

Y represents a linear or branched C1-C6 alkylene group, or a linear or branched C1-C6 alkenylene group.

[2]    The  method  according  to   [1],  wherein  in  the  formula

10    (I),

R1 is a phenyl group, a 1-naphthyl group or a 3-indolyl group, wherein one or more hydrogen atoms in these groups are optionally replaced by 1 to 5 members selected from among a halogen atom, a hydroxyl group, a nitro group, a Cl-C6 alkyl


15    group  and  a  Cl-C6  alkoxy  group;

R2   is  a  hydroxyl  group,   an  amino  group  or  a  Cl-C6  alkoxy

group;

X is a linear or branched Cl-C6 alkylene group; and Y is a linear or branched Cl-C6 alkylene group, or a

20    linear  or  branched  Cl-C6  alkenylene  group.

[3]    The  method  according  to   [1],  wherein  in  the  formula

(I) '

R1 is a phenyl group, a 4-iodophenyl group, a 1-naphthyl group or a 3-indolyl group;
25    R2   is  a  hydroxyl  group  or  a  methoxy  group;

X is an ethylene group or a tetramethy~ene group; and Y is an ethylene group or a trimethylene group.

[4]    The  method  according  to   [1],  wherein  the  compound  of
 

4

the formula (I) is a compound selected from among the following compounds:


(1) 4-oxo-4-(2-phenylethyl)aminobutyric acid (hereinafter, sometimes referred to as the compound A),

5 (2) methyl 4-oxo-4-(4-phenylbutyl)aminobutyrate (hereinafter, sometimes referred to as the compound B),

(3)    methyl 4-oxo-4-(2-phenylethyl)aminobutyrate (hereinafter, sometimes referred to as the compound C),

(4)    4-oxo-4-(4-phenylbutyl)aminobutyric  acid

10    (hereinafter,   sometimes  referred  to  as  the  compound  D),

(5)    5-oxo-5-[2-(3-indolyl)ethyl]aminovaleric acid (hereinafter, sometimes referred to as the compound E),

(6)    5-oxo-5-[(1-naphthyl)methyl]aminovaleric  acid

(hereinafter, sometimes referred to as the compound F), and 15 (7) methyl 4-oxo-4-[2-(4-iodophenyl)ethyl]aminobutyrate

(hereinafter, sometimes referred to as the compound G). [5] The method according to any one of [1] to [4],

wherein the application is a soil irrigation treatment, a spraying treatment, a hydroponic treatment or a seed

20    treatment.

[6]    The method according to any one of [1] to [5], wherein the application is a seed treatment.

[7]    The  method  according  to   [6],  wherein  an  application

amount  of  the  compound  of  the  formula   (I)   in  the  seed

25    treatment  is  from  30g  to  500  g  per  100  kg  of  seeds.

[8]    The method according to any one of [1] to [7], wherein the plant is rice, corn, soybean, wheat or tomato.

[9]    The  method  according  to  any  one  of  [1]   to   [8],
 

5

wherein  the  plant  is  a  transgenic  plant.

[10]    The  method  according  to  any  one  of   [1]   to   [9],

wherein  the  temperature  stress  is  high  temperature  stress.

[11]    The  method  according  to  any  one  of   [1]   to   [9],

5    wherein  the  temperature  stress  is  low  temperature  stress.

[12]    The  method  according  to  any  one  of   [1]   to   [11],

wherein the temperature stress is indicated by a change in one or more of the following plant phenotypes:•

(1)    germination  percentage,

10    (2)   seedling  est~blishment rate,

(3)    number  of  healthy  leaves,

(4)    plant  length,

(5)    plant  weight,

(6)    leaf  area,

15    (7)   leaf  color,

(8)    number  or  weight  of  seeds  or  fruits,

(9)    quality  of  harvests,

(10)    flower  setting  rate  or  fruit  setting  rate,   and

(11)    chlorophyll  fluorescence  yield;

20 [13] Use of one or more compounds selected from the group consisting of a compound represented by the formula (I) of [1] and an agriculturally acceptable salt thereof for reducing temperature stress of plants.

[14]    The  use  according  to   [13],  wherein  the  temperature

25    stress is indicated by a change in one or more of the following plant phenotypes:

(1)    germination  percentage,

(2)    seedling  establishment  rate,

(3)    number  of  healthy .leaves,

(4)    plant  length,

(5)    plant  weight,

(6)    leaf  area,

5    (7)   leaf  color,

(8)    number  or  weight  of  seeds  or  fruits,

(9)    quality  of  harvests,

(10)    flower  setting  rate  or  fruit  setting  rate,   and

(11)    chlorophyll  fluorescence  yield.

10

Use of the method of the present invention enables reduction of temperature stress of plants.


Mode  for  Carrying  Out  the  Invention

15 In the present invention, generically referred to as a "temperature stress" factor is an environmental factor which causes decline in a physiological function of plant cells as a result of exposure of a plant to a temperature environment that deviates from an optimal temperature where the

20    temperature exceeds the upper limit or the lower limit of an optimal temperature for growth or germination of the plant. The temperature stress fac~or is referred to as a high. temperature stress factor in the case where the temperature

exceeds  the  upper  limit  of  the  optimal  temperature,  while  the

25    temperature stress factor is referred to as a low temperature stress factor in the case where the temperature exceeds the lower limit of the optimal temperature. The optimal temperature for growth or optimal temperature for germination

of plants vary depending on plants and, generally, the optimal temperature for germination is often higher than the optimal temperature for growth.

The  temperature  stress  of  plants  can  be  monitored  by  a

5    comparison in a change in the following plant phenotypes between plants which are not exposed to a temperature stress factor and plants exposed to the temperature stress factor. That is, the plant phenotypeB serve as indicators of the temperature stress of plants.


10    <plant  phenotypes>

(1)    germination  percentage

(2)    seedling  establishment  rate

(3)    number  of  healthy  leaves

(4)    plant  length

15    (5)   plant  weight

(6)    leaf  area

(7)    leaf  color

(8)    number  or  weight  of  seeds  or  fruits

(9)    quality  of  harvests

20    (10) flower setting rateor fruiting rate, and {11) chlorophyll fluorescence yield


In the present specification, the temperature stress may be quantified by determining the "intensity of stress"

25    represented  by  the  following  equation.



Equation: "Intensity of stress" = 100 x "any one of plant phenotypes in plants which are not exposed to a
 

8

temperature stress factor"/"the plant phenotype in plants exposed to a temperature stress factor"


The  method  of  the  present  invention  is  applied  to  plants

5    that have been exposed to or to. be exposed to a temperature stress factor whose "intensity of stress" represented by the above equation is from 105 to 200, preferably from 110 to 180, and more preferably from 120 to 160.

10 When plants are exposed to the temperature stress factor, one or more of the above phenotypes varies. That is, due to the temperature stress:•

(1)    decrease  in  germination  percentage,

(2)    decrease  in  seedling  establishment  rate,

15    ( 3)    decrease  in  number  of  healthy  leaves,
    ( 4)   decrease  in  plant  length,
    (5)   decrease    in  plant  weight,
    ( 6)    decrease    in  leaf  area  increasing  rate,
    ( 7)    leaf  color  fading,

20    (8)   decrease  in  number  or  weight  of  seeds  or  fruits,

(9)    decline  in  quality  of  harvests,

(10)    decrease  in  flower  setting  rate  or  fruit  setting  rate,

and

(11)    decrease  in  chlorophyll  fluorescence  yield;

25    are observed and the magnitude of.the temperature stress of plants can be measured using these as indicators. The present

invention  is  directed  to  a  method  for  reducing  an  influence  of

a    temperature  stress  factor  on  plants  that  have  been  exposed
 

9

to or to be exposed to the temperature stress factor by applying the compound represented by the formula (I) to the plants. The effect of reducing the temperature stress can be evaluated by a comparison of the indicator after the plants


5    are exposed to the temperature stress factor between plants treated with the compound represented by the formula (I) and plants which are not treated.

Stages  in  which  target  plants  in  the  present  invention

can  be  exposed  to  the  temperature  stress  factor  include  all

10    growth stages of plants, including a germination period, a vegetative growing period, a reproductive growing period and a harvesting period. The application period of the present compound used in the present invention may be any growth stage

of  plants,   and  examples  thereof  include  the  germination  period

15    such as before seeding, at the time of seeding, and after seeding and before or after emergence; the vegetative growing period such as at the time of seedling raising, at the time of

seedling  transplantation,   at  the  time  of  cuttage  or  sticking,

or  at  the  time  of  growing  after  settled  planting;  the

20    reproductive growing period such as before blooming, during blooming, after blooming, immediately before earing or during the earing period; and the harvesting period such as before harvesting plan, before ripening plan, or a coloration initiation period of fruits. Plants to which the present


25    compound is to be applied may be plants that have been exposed to or to be exposed to the temperature stress factor. That

is, the present compound can also be preventively applied to plants before being exposed to the temperature stress factor
 

10

in addition to plants exposed to the temperature stress factor.

The  present  compound  used  in  the  method  of  the  present

5    invention is one or more compounds selected from the group consisting of a compound represented by the following formula
wherein

10 R1 represents a phenyl group, a naphthyl group or an aromatic heterocyclic group, and these groups are optionally substituted with 1 to 5 members selected from among a halogen atom, a hydroxyl group, a cyano group, a nitro group, a Cl-C6 alkyl group optionally substituted with one or more halogen


15    atoms, a Cl-C6 alkoxy group optionally substituted with one or more halogen atoms, a Cl-C6 alkylthio group optionally substituted with one or more halogen atoms, a C2-C6 alkenyl group optionally substituted with one or more halogen atoms, a C2-C6 alkynyl group optionally substituted with one or more


20    halogen atoms, an amino group, a Cl-C6 alkylamine group and a di(Cl-C6 alkyl)amino group;

R2 represents a hydroxyl group, an amino group, or a Cl-C6 alkoxy group;


X  represents  a  linear  or  branched  Cl-C6  alkylene  group;

25    and

Y  represents  a  linear  or  branched  Cl-C6  alkylene  group,
 

11


or a linear or branched Cl-C6 alkenylene group; and an agriculturally acceptabl~ salt thereof.

The compound represented by the formula (I) is a compound described in Japanese Patent Publication No. 4087942

5    or Japanese Unexamined Patent Publication No. 2001-139405 and can be synthesized, for example, by the method described in the publications.



The  present  compound  is  preferably  one  or  more  compounds

10    selected from the group consisting of the compound of the formula (I), wherein in the formula (I),

R1 is a phenyl group, a 1-naphthyl group or a 3-indolyl group, wherein one or more hydrogen atoms in these groups are optionally replaced by 1 to 5 members selected from among a•

15    halogen atom, a hydroxyl group, a nitro group, a Cl-C6 alkyl group and a Cl-C6 alkoxy group;
R2    is  a  hydroxyl  group,   an  amino  group  or  a  Cl-C6  alkoxy

group;

X  is  a  linear  or  branched  Cl-C6  alkylene  group;   and

20 Y is a linear or branched Cl-C6 alkylene group, or a linear or branched Cl-C6 alkenylene group;


and  an  agriculturally  acceptable  salt  thereof.

The present compound is more preferably one or more compounds selected from the group consisting of the compound

25    of  the  formula   (I),   wherein  in  the  formula   (I),

R1 is a phenyl group, a 4-iodophenyl group, a 1-naphthyl group or a 3-indolyl group;
R2   is  a  hydroxyl  group  or  a  methoxy  group;
 

12

X is an ethylene group or a tetramethylene group; and y is an ethylene group or a trimethylene group;

and  an  agriculturally  acceptable  salt  thereof.

Specific  examples  of  the  present  compound  include:

5    (1)   4-oxo-4-(2-phenylethyl)aminobutyric  acid,

(2)    methyl  4-oxo-4-(4-phenylbutyl)aminobutyrate,

(3)    methyl  4-oxo-4-(2-phenylethyl)aminobutyrate,

(4)    4-oxo-4-(4-phenylbutyl)aminobutyric  acid,

(5)    5-oxo-5-[2-(3-indolyl)ethyl]aminovaleric  acid,

10    (6)   5-oxo-5-[(1-naphthyl)methyl]aminovaleric  acid,   and

(7)    methyl  4-oxo-4-[2-(4-iodophenyl)ethyl]aminobutyrate,

and the compound is preferable from the viewpoint that . it is capable of effectively reducing temperature stress of plants.


15    The  compound  represented    by  the  formula   (I)  may  be  a
    salt  with  a  base.    Examples  of  a  basic  salt  of  the  compound
    represented    by  the  formula    (I)    include  the  followings:
    metal    salts  such  as  alkali  metal  salts  and  alkaline
    earth  metal  salts,   including  salts  of  sodium,  potassium  or
20    magnesium;           
    salts    with  ammonia;    and   
    salts    with  organic  amines  such  as  morpholine,

piperidine, pyrrolidine, mono-lower alkylamine, di-lower alkylamine, tri-lower alkylamine, monohydroxy lower

25    alkylamine, dihydroxy lower alkylamine and trihydroxy lower alkylamine.
 





The  present  compound  used  in  the  method  of  the  present
 

13

invention can be used alone, or used after being formulated using various inert ingredients as described hereinafter.

Examples of the solid carrier used in formulation include fine powders or granules such as minerals such as

5    kaolin clay, attapulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and calcite; natural organic materials such as corn rachis powder and walnut husk powder; synthetic organic materials such as urea; salts such as calcium carbonate and ammonium sulfate;


10    synthetic inorganic materials such as synthetic hydrated silicon oxide; and as a liquid carrier, aromatic hydrocarbons such as xylene, alkylbenzene and methylnaphthalene; alcohols such as 2-propanol, ethyleneglycol, propylene glycol, and ethylene glycol monoethyl ether; ketones such as acetone,


15    cyclohexanone and isophorone; vegetable oil such as soybean oil and cotton seed oil; petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonitrile and water.

Examples  of  the  surfactant  include  anionic  surfactants

such  as  alkyl  sulfate  ester  salts,   alkylaryl  sulfonate  salts,

20    dialkyl sulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfonate formaldehyde polycondensates; and nonionic surfactants such as polyoxyethylene alkyl aryl ethers,

polyoxyethylene  alkylpolyoxypropylene  block  copolymers  and  •

25    sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.


Examples of the other formulation auxiliary agents include water-soluble polymers such as polyvinyl alcohol and
 

14

polyvinylpyrrolidone, polysaccharides such as Arabic gum, alginic acid and the salt thereof, CMC (carboxymethyl-cellulose), Xanthan gum, inorganic materials such as aluminum magnesium silicate and alumina sol, preservatives, coloring


5    agents and stabilization agents such as PAP (acid phosphate isopropyl) and BHT.





The  method  of  the  present  invention  is  usually  carried

out  by  applying  an  effective  amount  of  the  present  compound  to

10    plants or growing sites of plants. Examples of the plant to which the present compound is to be applied include foliages, buds, flowers, fruits, ears or spikes, seeds, bulbs, stern

tubers,    roots  and  seedlings.   As  used  herein,  bulbs  mean

discoid  stern,   corm,   rhizorna,   root  tuber  and  rhizophore.    In

15    the present specification, the seedlings include cutting and sugar cane stern cutting. Examples of growing sites of plants

include soil before or. after sowing plants. When the. present compound is applied to plants or growing sites of plants, the compound is applied to the target plants once or more than

20    once.



Specific examples of the application method in the method of the present invention include treatment of foliages, floral organs or ears or spikes of plants, such as foliage

25    spraying; treatment of cultivation lands of plants such as soil treatment; treatment of seeds such as seed sterilization, seed immersion or seed coating; treatment of seedlings; and treatment of bulbs such as seed tuber.
 

15




Specific examples of the treatment of foliages, floral organs or ears or spikes of plants in the method of the • present invention include the treatment method of applying the

5    compound to the surface of plants, such as foliage spraying or trunk spraying. Examples of the treatment also include a method of spraying the compound to the floral organ or entire

plants  in  the  blooming  season  including  before  blooming,

during  blooming  and  after  blooming.    Examples  of  the  treatment

10    in cereals and the like include a method of spraying the compound to the ear or. spikes or entire plants in the earing season.



Examples  of  the  soil  treatment  method  in  the  method  of

15    the present invention include spraying onto the soil, soil incorporation, and perfusion of a chemical liquid into the soil (irrigation of chemical liquid, soil injection, and

dripping  of  chemical  liquid) .   Examples  of  the  place  to  be

treated  include  planting  hole,   furrow,   around  a  planting  hole,

20    around a furrow, entire surface of cultivation lands, the parts between the soil and the plant, area between roots, area beneath the trunk, main furrow, growing soil, seedling raising box, seedling raising tray and seedbed. Examples of the

treating  period  include  before  seeding,   at  the  time  of

25    seeding, immediately after seeding, raising period, before settled planting, at the time of settled planting, and growing period after settled planting. In the above soil treatment, more than one kinds of the present compounds may be
 

16

simultaneously applied to the plant, or a solid fertilizer such as a paste fertilizer containing the present compound may be applied to the soil. Also, the present compound may be mixed in an irrigation liquid, and, examples thereof include


5    injecting to irrigation facilities such as irrigation tube, irrigation pipe and sprinkler, mixing into the flooding liquid between furrows and mixing into a hydroponic medium. Alternatively, an irrigation liquid may be mixed with the

present  compound  in  advance  and,   for  example,   used  for

10    treatment by an appropriate irrigating method including the irrigating method mentioned above and the other methods such as sprinkling and flooding. Alternatively, the present

compound  can  be  applied  by  winding  a  crop  with  a  sheet  or  a

string  of  a  resin  formulation,   putting  a  string  of  the  resin

15    formulation around a crop so that the crop is surrounded by the string, and/or laying a sheet of the resin formulation on the soil surface near the root of a crop.




Examples  of  the  method  of  treating  seeds  in  the  method

20    of the present invention include a method for treating seeds or bulbs of a plant to be protected from temperature stress with the present compound and specific examples thereof include a spraying treatment in which a suspension of the present compound is atomized and sprayed on the seed surface


25    or the bulb surface, a smearing treatment in which a wettable powder, an emulsion or a flowable agent of the present compound is applied to seeds or bulbs with a small amount of water added or applied as it is without dilution, an immersing
 

17

treatment in which seeds are immersed in a solution of the present compound for a certain period of time, film coating treatment, and pellet coating treatment.



5    Examples  of  the  treatment  of  seedlings  in  the  method  of
the  present  invention  include  spraying  treatment  of  spraying
to  the    entire    seedlings    a  dilution  having  a  proper
concentration    of  active    ingredients  prepared  by  diluting  the

present  compound  with  water,   immersing  treatment  of  immersing

10    seedlings in the dilution, and coating treatment of adhering the present compound formulated into a dust formulation to the entire seedlings. Examples of the method of treating the soil

before  or  after  sowing  seedlings  include  a  method  of  spraying

a  dilution  having  a  proper  concentration  of  active  ingredients

15    prepared by diluting the present compound with water to seedlings or the soil around seedlings after sowing seedlings,

and a method of spraying the present compound formulated into • a solid formulation such as a granule to soil around seedlings after sowing seedlings.

20 The present compound may be mixed with a hydroponic medium in hydroponics, and may also be used as one of culture medium components in tissue culture. When the present compound is used for hydroponics, it can be dissolved or suspended in a conventionally used culture medium for


25    hydroponics, such as ENSHI, at a concentration within a range from 0.001 to 10,000 ppm. When the present compound is used at the time of tissue culture or cell culture, it can be dissolved or suspended in a conventionally used culture medium
 

18

for plant tissue culture, such as an MS culture medium, at a concentration within a. range from 0.001 to 10,000 ppm. In this case, in accordance with a usual method, saccharides as a carbon source, various phytohormones and the like can be


5    appropriately  added.

When the present compound is used for treatment of plants or growing sites of plants, the treatment amount can vary depending on the kind of plants to be treated, formulation form, treating period and meteorological


10    conditions, but is usually within a rang from 0.1 to 1,000 g, and preferably from 1 to 500 g, in terms of an active ingredient amount, per 1,000 m2 • When the present compound is incorporated into the entire soil, the treatment amount is

usually  within  a  range  from  0.1  to  1,000  g,   and  preferably

15    from 1 to 500 g, in terms of an active ingredient amount, per 1,000 m2 •


An  emulsion,   a  wettable  powder,   a  flowable  agent  and  a

microcapsule  are  usually  used  for  the  treatment  by  spraying

after  dilution  with  water.    In  this  case,   the  concentration  of

20    the active ingredient is usually within a range from 0.01 to 10,000 ppm, and preferably from 1 to 5,000 ppm. A dust formulation and a granule are usually used for the treatment as they are without dilution.

In  the  treatment  of  seeds,   the  weight  of  the  present

25    compound per seed is usually within a range from 0.0001 to 5 mg, and preferably from 0.005 to 1 mg, and the weight of the present compound per 100 kg of seeds is usually within a range from 5 to 1,000 g, and preferably from 30 to 500 g, and more
 

24

A-451  878,  WO  03/052073,   etc.

Toxins contained in such genetically engineered plants are able to confer resistance particularly to insect pests belonging to Coleoptera, Hemiptera, Diptera, Lepidoptera and

5    Nematodes,   to  the  plants.

Genetically engineered plants, which comprise one or multiple insecticidal pest-resistant genes and which express one or multiple toxins, have already been known, and some of such genetically engineered plants have already been on the


10    market. Examples of such genetically engineered plants include YieldGard (registered trademark) (a corn variety for expressing CrylAb toxin), YieldGard Rootworm (registered trademark) (a corn variety for expressing Cry3Bbl toxin),

YieldGard  Plus   (registered  trademark)   (a  corn  variety  for

15    expressing CrylAb and Cry3Bbl toxins), Herculex I (registered trademark) (a corn variety for expressing phosphinotricine N-acetyl transferase (PAT) so as to confer resistance to Cry1Fa2 toxin and glufosinate), NuCOTN33B (registered trademark) (a cotton variety for expressing CrylAc toxin), Bollgard I


20 (registered trademark) (a cotton variety for expressing CrylAc toxin), Bollgard II (registered trademark) (a cotton variety for expressing CrylAc and Cry2Ab toxins), VIPCOT (registered trademark) (a cotton variety for expressing VIP toxin), NewLeaf (registered trademark) (a potato variety for


25    expressing Cry3A toxin), NatureGard (registered trademark) Agrisure (registered trademark) GT Advantage (GA21 glyphosate-resistant trait), Agrisure (registered trademark) CB Advantage (Btll corn borer (CB) trait), and Protecta (registered
 

25

trademark).

The aforementioned "plants" also include crops produced using a genetic engineering technique, which have ability to generate antipathogenic substances having selective action.

5 A PR protein and the like have• been known as such antipathogenic substances (PRPs, EP-A-0 392 225). Such antipathogenic substances and genetically engineered crops that generate them are described in EP-A-0 392 225, WO 95/33818, EP-A-0 353 191, etc.




10 Examples of such antipathogenic substances expressed in genetically engineered crops include: ion channel inhibitors such as a sodium channel inhibitor or a calcium channel inhibitor (KP1, KP4 and KP6 toxins, etc., which are produced by viruses, have been known); stilbene synthase; bibenzyl


15    synthase; chitinase; glucanase; a PR protein; and antipathogenic substances generated by microorganisms, such as a peptide antibioti~, an antibiotic having a hetero ring, a protein factor associated with resistance to plant diseases (which is called a plant disease-resistant gene and is


20    described in WO 03/000906) . These antipathogenic substances and genetically engineered plants producing such substances are described in EP-A-0392225, W095/33818, EP-A-0353191, etc.

The  "plant"  mentioned  above  includes  plants  on  which

advantageous  characters  such  as  characters  improved  in•  oil

25    stuff ingredients or characters having reinforced amino acid content have been conferred by genetically engineering technology. Examples thereof include VISTIVE (registered trademark) low linolenic soybean having reduced linolenic
 

26

content) or high-lysine (high-oil) corn (corn with increased lysine or oil content).


Stack varieties are also included in which a plurality of advantageous characters such as the classic herbicide

5    characters mentioned above or herbicide tolerance genes, harmful insect resistance genes, antipathogenic substance producing genes, characters improved in oil stuff ingredients or characters having reinforced amino acid content are


combined.

10

In the case where plants are exposed to a temperature higher than the optimal temperature for growth or the optimal temperature for germination, the physiological metabolism function in vivo declines and growth or germination is


15    inhibited to cause a decrease in vitality of the plants, resulting in a state of being exposed to a high temperature

stress factor. Specifically, in the case where plants are in a growing period, the conditions which lead to the high temperature stress may be conditions in which an average

20    cultivation temperature in an environment where plants are cultivated is 25°C or higher, more severely 30°C or higher, and still more severely 35°C or higher. The present invention is capable of providing a method for reducing temperature stress

of  plants  under  these  high  temperature  stress  conditions.

25    Reduction of the temperature stress of plants can be evaluated by measuring an improvement in the indicators which show the temperature stress.
 

27

In the case where plants are exposed to the temperature lower than the optimal temperature for growth or the optimal temperature for germination, physiological metabolism function in vivo declines and growth or germination is inhibited to


5    cause a decrease in vitality of the plants, resulting in a state of being exposed to a low temperature stress factor. Specifically, in the case where plants are in a growing period, the conditions which lead to the low temperature

stress  factor  may  be  conditions  in  which  an  average

10    cultivation temperature in an environment where plants are cultivated is 15°C or lower, more severely 10°C or lower, and still more severely 5°C or lower. The present invention is capable of providing a method for reducing temperature stress of plants under these low temperature stress conditions.


15    Reduction of the temperature stress of plants can be evaluated by measuring an improvement in the indicators which show the temperature stress.



In  the  present  invention,   it  is  possible  to  use,   as

20    indicators  of  the  temperature  stress,   plant  phenotypes  such  as

(1)    germination  percentage,   (2)   seedling  establishment  rate,

(3)    number  of  healthy  leaves,   (4)   plant  length,   (5)   plant

weight, (6) leaf area, (7) leaf color, (8) number or weight of seeds or fruits, (9) quality of harvests, (10) flower setting

25    rate or fruit setting rate and (11) chlorophyll fluorescence yield.
 





The  indicators  can  be  measured  in  the  following  manner.
 

28

{1)    Germination  percentage

Seeds of plants are sown, for example, in the soil, on a filter paper, on an agar culture medium or on sand, and allowed to undergo germination, and then the ratio of the

5    number  of  germinations  to  the  number  of  seeds  is  examined.

(2)    Seedling  establishment  rate

Seeds of plants are sown, for example, in the soil, on a filter paper, on an agar culture medium or on sand, and,then allowed to undergo cultivation for a given period of time.

10    During the entire or partial cultivation period, temperature stress is applied, and the percentage of surviving seedlings

is  examined.

(3)    Number  of  healthy  leaves

With  respect  to  plants,   the  number  of  healthy  leaves  is

15    counted and the total number of healthy leaves is examined. Alternatively, the ratio of the number of healthy leaves to the number of all leaves of plants is examined.

(4)    Plant  length

With  respect  to  plants,   the  length  from  the  base  of  the

20    stem of the above-ground part to the branches and leaves at the tip is measured.


(5)    Plant  weight

The above-ground part of each of plants is cut and the weight is measured to determine a fresh weight of plants.

25    Alternatively, the cut sample is dried and the weight is measured to determine a dry weight of plants.
 

(6)    Leaf  area

A  photograph  of  plants  is  taken  by  a  digital  camera  and
 

29

the area of a green portion in the photograph is determined by image analysis software, for example, Win ROOF (manufactured by MITANI CORPORATION) to obtain a leaf area of plants.

(7)    Leaf  color

5 After sampling leaves of plants, the chlorophyll content is measured using a chlorophyll gauge (for example, SPAD-502, manufactured by KONICA MINOLTA Holdings, Inc.) to determine the leaf color.

(8)    Number  or  weight  of  seeds  or  fruits

10 After cultivating plants until they bear fruits or fruits reach full maturity, the number of fruits per plant or the total fruit weight per plant is measured. After cultivating plants until seeds undergo ripening, elements constituting the yield such as the number of ears, ripening


15    rate  and  thousand  kernel  weight  are  examined.

(9)    Quality  of  harvests

After cultivating plants until fruits reach full maturity, the quality of harvests is evaluated, for example, by measuring the sugar content of fully matured fruits using a

20    saccharimeter.

(10)    Flower  setting  rate,   fruit  setting  rate

After cultivating plants until they bear fruits, the number of flower setting and the number of fruit setting are counted to determine the fruit setting rate % (number of fruit
25    setting/number  of  flower  setting  x  100).

(11)    Chlorophyll  fluorescence  yield

Using a pulse modulation chlorophyll fluorometer (for example, IMAGING-PAM manufactured by WALZ Company), the
 

30

chlorophyll fluorescence (Fv/Fm) of plants is determined to obtain the chlorophyll fluorescence yield.


Examples

5 While the present invention will be more specifically described by way of formulation examples, seed treatment examples, and test examples in the following, the present invention is not limited to the following examples. In the following examples, the part represents part by weight unless



10    otherwise  specified.



Formulation  example  1

Fully mixed are 3.75 parts of the present compound, 14 parts of polyoxyethylene styrylphenyl ether, 6 parts of

15    calcium dodecyl benzene sulfonate and 76.25 parts of xylene, so as to obtain an emulsion.





Formulation  example  2

Ten    (10)   parts  of  the  present  compound,   35  parts  of  a

20    mixture of white carbon and a polyoxyethylene alkyl ether sulfate ammonium salt (weight ratio 1:1) and 55 parts of-water are mixed, and the mixture is subjected to fine grinding according to a wet grinding method, ~o as to obtain a flowable

formulation.

25
 

Formulation  example  3

Fifteen (15) parts of the present compound, 1.5 parts of sorbitan trioleate and 28.5 parts of an aqueous solution
 

31

containing 2 parts of polyvinyl alcohol are mixed, and the mixture is subjected to fine grinding according to a wet grinding method. Thereafter, 45 parts of an aqueous solution containing 0.05 part of Xanthan gum and 0.1 part of aluminum


5    magnesium silicate is added to the resultant mixture, and 10 parts of propylene glycol is further added thereto. The obtained mixture is blended by stirring, so as to obtain a flowable formulation.




10    Formulation  example  4

Forty-five (45) parts of the present compound, 5 parts of propylene glycol (manufactured by Nacalai Tesque), 5 parts of SoprophorFLK (manufactured by Rhodia Nikka), 0.2 parts of an anti-form C emulsion (manufactured by Dow Corning), 0.3


15    parts of proxel GXL (manufactured by Arch Chemicals) and 49.5 parts of ion-exchange water are mixed so as to obtain a bulk slurry. One hundred and fifty (150) parts of glass beads

(diameter=  1  rnm)   are  put  into  100  parts  of  the  slurry,   and

the  slurry  is  ground  for  2  hours  while  being  cooled  with  a

20    cooling water. After ground, the resultant is filtered to remove the glass beads and flowable formulation is obtained.


Formulation  example  5

Mixed  to  obtain  an  AI  premix  are  50.5  parts  of  the

25    present compound, 38. 5 parts of NN kaolin clay (manufactured by Takehara Chemical Industrial), 10 parts of MorwetD425 and 1.5 parts of MorwerEFW (manufactured by Akzo Nobel Corp.).

This  premix  is  ground  with  a  jet  mill  so  as  to  obtain  a  powder
 

19

preferably from 50 to 200 g. When seeds are subjected to an immersing tre~tment, the present compound can be used after being dissolved or suspended at a concentration of the active ingredient within a range from 0.01 to 10,000 ppm.


5 In the treatment of seedlings, the weight of the present compound per seedling is usually within a range from 0.01 to 20 mg, and preferably from 0.1 to 10 mg. In the treatment of the soil before or after sowing seedlings, .the weight of the present compound per 1,000 m2 is usually within a range from


10    0.1  to  100  g,  and  preferably  from  1  to  50  g.



Examples of plants whose temperature stress can be reduced by the present invention include the followings.

crops:    corn,  rice,  wheat,  barley,  rye,  oat,  sorghum,

15    cotton, soybean, peanut, buckwheat, beet, canola, rapeseed, sunflower, sugar cane, tobacco, and pea, etc.;

vegetables:  solanaceous  vegetables   (eggplant,  tomato,

pimento,    pepper,  potato,  etc.),  cucurbitaceous  vegetables

(cucumber,    pumpkin,   zucchini,  water  melon,  melon,  squash,

20    etc.), cruciferous vegetables (Japanese radish, white turnip, horseradish, kohlrabi, Chinese cabbage, cabbage, leaf mustard, broccoli, cauliflower, etc.), asteraceous vegetables (burdock, crown daisy, artichoke, lettuce, etc.), liliaceous vegetables


(green  onion,  onion,  garlic,  and  asparagus),  ammiaceous

25    vegetables (carrot, parsley, celery, parsnip, etc.), chenopodiaceous vegetables (spinach, Swiss chard, etc.), lamiaceous vegetables (Perilla frutescens, mint, basil, etc.), strawberry, sweet potato, Dioscorea japonica, colocasia, etc.;
 

20

flowers;

foliage  plants;

turf  grasses;

fruits:    pomaceous  fruits   (apple,  pear,   Japanese  pear,

5    Chinese quince, quince, etc.), stone fleshy fruits (peach, plum, nectarine, Prunus mume, cherry fruit, apricot, prune, etc.), citrus fruits (Citrus unshiu, orange, lemon, rime,

grapefruit,    etc.},   nuts   (chestnuts,  walnuts,  ha~elnuts,

almond, pistachio, cashew nuts, macadamia nuts, etc.), berries 10 (blueberry, cranberry, blackberry, raspberry, etc.), grape,

kaki fruit, olive, Japanese plum, banana, coffee, date palm, coconuts, etc.; and


trees other than fruit trees; tea, mulberry, flowering plant, roadside trees (ash, birch, dogwood, Eucalyptus, Ginkgo

15    biloba, lilac, maple, Quercus, poplar, Judas tree, Liquidambar formosana, plane tree, zelkova, Japanese arborvitae, fir wood, hemlock, juniper, Pinus, Picea, and Taxus cuspidate), etc.

Examples of plants whose temperature stress can be reduced by the present invention preferably include rice,

20    corn,   soybean,  wheat  and  tomato.



The aforementioned "plants" include plants, .to which resistance to HPPD inhibitors such as isoxaflutole, ALS inhibitors such as imazethapyr or thifensulfuron-methyl, EPSP

25    synthetase inhibitors such as glyphosate, glutamine synthetase inhibitors such as the glufosinate, acetyl-CoA carboxylase

inhibitors  such  as  sethoxydim,   and  herbicides  such  as

~
 

bromoxynil,    dicamba,   2,4-D,  etc.  has  been  conferred  by  a
 

21


classical breeding method or genetic engineering technique. Examples of a "plant" on which resistance has been

conferred by a classical breeding method include rape, wheat, sunflower and rice resistant to imidazolinone ALS inhibitory

5    herbicides such as imazethapyr, which are already co~ercially available under a product name of Clearfield (registered trademark). Similarly, there is soybean on which resistance

to sulfonylurea ALS inhibitory herbicides such as thifensulfuron-methyl has been conferred by a classical

10    breeding method, which is already commercially available under a product name of STS soybean. Similarly, examples on which resistance to acetyl-CoA carboxylase inhibitors such as trione oxime or aryloxy phenoxypropionic acid herbicides has been conferred by a classical breeding method include SR corn. The


15    plant on which resistance to acetyl-CoA carboxylase inhibitors has been conferred is described in Proceedings of the National Academy of Sciences of the United States of America (Proc.

Natl. Acad. Sci. USA), vol. 87, pp. 7175-7179 (1990). A variation of acetyl-CoA carboxylase resistant to an acetyl-CoA

20    carboxylase inhibitor is reported in Weed Science, vol. 53, pp. 728-746 (2005) and a plant resistant to acetyl-CoA carboxylase inhibitors can be generated by introducing a gene of such an acetyl-CoA carboxylase variation into a plant by

genetically  engineering  technology,   or  by  introducing  a

25    variation conferring resistance into a plant acetyl-CoA carboxylase. Furthermore, plants resistant to acetyl-CoA carboxylase inhibitors or ALS inhibitors or the like can be generated by introducing a site-directed amino acid
 

22

substitution variation into an acetyl-CoA carboxylase gene or the ALS gene of the plant by introduction a nucleic acid into which has been introduced a base substitution variation represented Chimeraplasty Technique (Gura T. 1999. Repairing


5    the Genome's Spelling Mistakes. Science 285: 316-318) into a plant cell.


Examples  of  a  plant  on  which  resistance  has  been

conferred  by  genetic  engineering  technology  include  corn,

soybean,    cotton,   rape,   sugar  beet  resistant  to  glyphosate,

10    which is already commercially available under a product name of RoundupReady (registered trademark), AgrisureGT, etc. Similarly, there are corn, soybean, cotton and rape which are made resistant to glufosinate by genetic engineering

technology,    a  kind,   which  is  already  commercially  available

15    under a product name of LibertyLink (registered trademark) . A cotton made resistant to bromoxynil by genetic engineering

t~chnology is  already  commercially  available  under  a  product

name  of  BXN  likewise.

The  aforementioned  "plants"  include  genetically

20    engineered crops produced using such genetic engineering techniques, which, for example, are able to synthesize selective toxins as known in genus Bacillus.

Examples of toxins expressed in such genetically engineered crops include: insecticidal proteins derived from

25    Bacillus cereus or Bacillus popilliae; o-endotoxins such as Cry1Ab, Cry1Ac, Cry1F, Cry1Fa2, Cry2Ab, Cry3A, Cry3Bb1 or Cry9C, derived from Bacillus thuringiensis; insecticidal proteins such as VIP1, VIP2, VIP3, or VIP3A; insecticidal
 

23

proteins derived from nematodes; toxins generated by animals, such as scorpion toxin, spider toxin, bee toxin, or insect-specific neurotoxins; mold fungi toxins; plant lectin; agglutinin; protease inhibitors such as a trypsin inhibitor, a


5    serine protease inhibitor, patatin, cystatin, or a papain inhibitor; ribosome-inactivating proteins (RIP) such as lycine, corn-RIP, abrin, luffin, saporin, or briodin; steroid-metabolizing enzymes such as 3-hydroxysteroid oxidase,

ecdysteroid-UDP-glucosyl  transferase,   or  cholesterol  oxidase;

10    an ecdysone inhibitor; HMG-COA reductase; ion channel inhibitors such as a sodium channel inhibitor or calcium channel inhibitor; juvenile hormone esterase; a diuretic hormone receptor; stilbene synthase; bibenzyl synthase;

chitinase;    and  glucanase.

15 Toxins expressed in such genetically engineered crops also include: hybrid toxins of 8-endotoxin proteins such as CrylAb, CrylAc, CrylF, Cry1Fa2, Cry2Ab, Cry3A, Cry3Bbl, Cry9C, Cry34Ab or Cry35Ab and insecticidal prot~ins such as VIPl, VIP2, VIP3 or VIP3A; partially deleted toxins; and modified


20    toxins. Such hybrid toxins are produced from a new combination of the different domains of such proteins, using a genetic engineering technique. As a partially deleted toxin, CrylAb comprising a deletion of•a portion of an amino acid


sequence  has  been  known.   A  modified  toxin  is  produced  by

25 substitution of one or multiple amino acids of natural toxins. Examples of such toxins and genetically engineered

plants capable of synthesizing such toxins are described in EP-A-0 374 753, WO 93/07278, WO 95/34656, EP-A-0 427 529, EP-
 

32

formulation.



Formulation  example  6

Five    (5)   parts  of  the  present  compound,   1  part  of

5    synthetic hydrated silicon oxide, 2 parts of calcium lignin sulfonate, 30 parts of bentonite and 62 parts of kaolin clay are fully ground and.mixed, and the resultant mixture is added with water and fully kneaded, and then subjected to granulation and drying so as to obtain a granule formulation.


10

Formulation  example  7

Three (3) parts of the present compound, 87 parts of kaolin clay and 10 parts of talc are fully ground and mixed so as to obtain a powder formulation.

15

Formulation  example  8

Twenty-two (22) parts of the present compound, 3 parts of calcium lignin sulfonate, 2 parts of sodium lauryl sulfate and 73 parts of synthetic hydrated silicon oxide are fully

20    ground  and  mixed  so  as  to  obtain  wettable  powders.



Seed  treatment  example  1

An emulsion prepared as in Formulation example 1 is used for smear treatment in an amount of 500 ml per 100 kg of dried

25    sorghum seeds using a rotary seed treatment machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.
 

33

Seed  treatment  example  2

A flowable formulation prepared as in Formulation example 2 is used for smear treatment in an amount of 50 ml per 10 kg of dried rape seeds using a rotary seed treatment

5    • machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.


Seed  treatment  example  3

A  flowable  formulation  prepared  as  in  Formulation

10    example 3 is used for smear treatment in an amount of 40 ml per 10 kg of dried corn seeds using a rotary seed treatment machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.




15    Seed  treatment  example  4

Five (5) parts of a flowable formulation prepared as in Formulation example 4, 5 parts of pigment BPD6135 (manufactured by Sun Chemical) and 35 parts of water are mixed to prepare a mixture. The mixture is used for smear treatment


20    in an amount of 60 ml per 10 kg of dried cotton seeds using a rotary seed treatment machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.




Seed  treatment  example  5

25 A powder agent prepared as in Formulation example 5 is used for powder coating treatment in an amount of 50 g per 10 kg of dried corn seeds so as to obtain treated seeds.
 

34

Seed  treatment  example  6

A powder agent prepared as in Formulation example 7 is used for powder coating treatment in an amount of 40 g per 100 kg of dried rice seeds so as to obtain treated seeds.

5

Seed  treatment  example  7

A flowable formulation prepared as in Formulation example 2 is used for• smear treatment in an amount of 50 ml per 10 kg of dried soybean seeds using a rotary seed treatment

10    machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.


Seed  treatment  example  8

A  flowable  formulation  prepared  as  in  Formulation

15    example 3 is used for smear treatment in an amount of 50 ml per 10 kg of dried wheat seeds using a rotary seed treatment

machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.


20    Seed  treatment  example  9

Five (5) parts of a flowable formulation prepared as in Formulation example 4, 5 parts of pigment BPD6135 (manufactured by Sun Chemical) and 35 parts of water are mixed and the resultant mixture is used for smear treatment in an


25    amount of 70 ml per 10 kg of potato tuber pieces using a rotary seed treatment machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.
 

35

Seed  treatment  example  10

Five (5) parts of a flowable formulation prepared as in Formulation example 4, 5 parts of pigment BPD6135 (manufactured by Sun Chemical) and 35 parts of water are mixed

5    and the resultant mixture is used for smear treatment in an amount of 70 ml per 10 kg of sunflower seeds using a rotary

seed treatment machine (seed dresser, produced by Hans-Ulrich Hege GmbH) so as to obtain treated seeds.


10    Seed  treatment  example  11

A powder agent prepared as in Formulation example 5 is used for powder coating treatment in an amount of 40 g per 10 kg of dried sugar beet seeds so as to obtain treated seeds.


15    Test Example 1: Evaluation Test for Reduction of High Temperature Stress in Hydroponics of Tomato (Number of Healthy

Leaves)

<Test  Plants>

Tomato  seeds   (cultivar:   PATIO)   were  sown  on  a  hydroponic

20    sponge and then cultivated for 3 to 4 weeks under the conditions of a temperature of 22 to 25°C, a humidity of 55 to 75%, an illuminance of 5,000 lx and a day length of 16 hours, using 1,000-times diluted HYPONeX (HYPONeX JAPAN CORP., LTD.) as a hydroponic culture medium. A seedling of tomato at the


25    3rd  leaf  stage  was  tested.



<Application  of  the  Present  Compound>

With  respect  to  the  compound  A,   the  compound  B,   the
 

36

compound c, the compound D, the compound E, the compound F and the compound G, a DMSO solution having a concentration 10,000 times of each test concentration was prepared, and 10 pL of the obtained DMSO solution was added to 100 ml of distilled


5    water to obtain a test liquid. An aqueous solution having a concentration of 250,000 ppm of a sodium salt of the compound A was prepared and the obtained aqueous solution was added to 100 ml of distilled water so as to give each test concentration, and then, 10 pL of DMSO was added thereto to


10    obtain a test liquid. As a control, an aqueous solution as a test liquid was prepared by adding 10 pL of DMSO to 100 ml of

distilled  water.

Next, 100 ml of each test liquid and three of the above test plants were put in a square-shaped cup (C-AP square cup

15    88-200Mi, manufactured by Chuo Kagaku Co., Ltd.) and then• cultivated for 2 days under the conditions of a temperature of 22 to 25°C, a humidity of 55 to 75%, an illuminance of 5,000 lx, and a light period of 16 hours/a dark period of 8 hours.




20    <High  Temperature  Stress  Test>

The above test plants to which each test liquid was applied were put in an artificial climate chamber set to the following conditions, and a stress test was carried out. Temperature: 50°C, 5 .hours, Illuminance: 6,500 to 7,900 lx,

25    and  Humidity:   50%.



<Evaluation>

After  the  above  high  temperature  stress  test,   the  number
 

37

of healthy leaves (2 cotyledons and 3 true leaves) of each test plant was counted and each test plants was assigned a score from 0 to 5 with a score of 0 representing complete death and with a score of 5 representing having healthy leave


5    equally to the case before the stress treatment. After combining scores of three test plants, the test results of th group to which aqueou~ solutions of the respective compounds have been applied were compared with the test results of the group to which an aqueous solution containing only DMSO added


10    therein (control) has been applied. As a result, the number of healthy leaves of plants in the cups to which aqueous solutions of the respective compounds were applied (test grouJ of the present invention) was apparently larger than that in the case of control and temperature stress was reduced.


15

Test Example 2: Evaluation Test• for Reduction of High Temperature Stress by Wheat Seed Treatment (Plant Weight) <Seed Treatment>

A  Blank  slurry  solution  containing  5%   (V/V)   color  coat

20    red (Becker Underwood, Inc.), 5% (V/V) CF-Clear (Becker Underwood, Inc.) and 0.4% Maxim XL (Syngenta) was prepared. F

sodium salt of the compound A was dissolved in the Blank slurry to obtain a slurry solution so as to control the amoun1 of the sodium salt within a range from 0.5 mg to 2 mg per 1 g

25    of seeds. Using a seed treating machine (HEGEll, manufacturec by Hans-Ulrich Hege), seed coating was carried out by mixing

1    .3 ml of the slurry solution with 50 g of wheat seeds (cultivar: Apogee) and the seeds were dried. As a control,
 

38

seed coating was carried out using the Blank slurry solution in place of the slurry solution to obtain seeds for non-treated group. The coated seeds (5 seeds each) were sown in the growing soil (AISAI) in a plastic pot and then cultivated


5    for 18 days under the conditions of a temperature of 20 to 25°C, a humidity of 50 to 75%, an illuminance of 5,300 lx, and a day length of 16 hours. Before the stress test, thinning was carried out to control the number of seedlings to 3 per


pot.

10

<High  Temperature  Stress  Test>

The above test plants on the 18th day after seeding were cultivated for 14 days under the conditions of a temperature of 40°C (day)/30°C (night), a humidity of 63% (day)/70%

15 (night), an illuminance of 7,100 lx, and a day length of 16 hours.





<Evaluation  Method>

After  the  stress  test,   on  the  32nd  day  after  seeding,

20    fresh weight of the above-ground part of test plants was examined for 4 to 7 pots. The results are shown in Table 1. Table 1

                                Amount    of    Fresh  weight    of    Percentage   
                                        the    above-ground       
        Test        compound    chemical    (mg/g            relative  to  non-   
                                part  {mg/3           
                                of  seeds)                treated  group  (%)   
                                        seedlings)           
                                                           
        None    (=    non    -    0            0.57        100       
    treated    group)                               
                                       
                                                           
    Compound  A,    Na    0.5            0.87        150       
                                       
                                       
            1.0            0.77        140       
            salt                                   
                                               
                                               
                                2.0            0.81        140       
                                                           
 

39




Test Example 3: Evaluation Test for Reduction of High Temperature Stress by Wheat Foliage Spraying Treatment (Plant Weight)

5    <Test  Plants>

Wheat (cultivar: Apogee) seeds were sown in the growing soil (AISAI) in a plastic pot (~55 rnm x 58 mm in height) and then cultivated for one week under the conditions of a temperature of 20 to 25°C, a humidity of 50 to 75%, an

10    illuminance  of  5,300  lx,   and  a  day  length  of  16  hours.



<Application  of  the  Present  Compound>

A  flowable  formulation  of  the  compound  A  was  obtained  by

adding  about  120  mg  of  a  mixture  of  white  carbon  and  a

15    polyoxyethylene alkyl ether sulfate ammonium salt (weight ratio of 1 : 1) and 300 pl of water to 0.5 mg of the compound A, followed by fine grinding using a wet grinding method. The obtained flowable formulation was diluted with 50 ml of water to obtain a spray liquid. After adding 0.2% RINO as a sticker


20    to the spray liquid, a sufficient amount (45 ml per 6 pots) was sprayed using an automatic spraying machine. As a control, a flowable formulation not containing the compound A was prepared and then sprayed to the non-treated group. Under the conditions of a temperature of 20 to 25°C, a humidity of


25    50 to 75%, an illuminance of 5,300 lx, and a day length of 16 hours, cultivation was carried out for 2 days.


<High  Temperature  Stress  Test>
 

40

The above test plants having been subjected to the spraying treatment were put in an artificial climate chamber set to the following conditions, and a stress test was carried out.



5    Temperature: 45°C, 17 hours, Illuminance: 6,500 to 7,900 lx, and Humidity: 50%.





<Evaluation  Method>

After  the  stress  test,   test  plants  were  cultivated  for  4

10    days under the conditions of a temperature of 20 to 25°C, a humidity of 50 to 75%, an illuminance of 5,300 lx, and a day length of 16 hours, visual evaluation was carried out and a fresh weight of the above-ground part was examined. As a


result,    in  the  group  treated  with  the  compound  A,   as  compared

15    with the non-treated group, alleviation of high temperature stress-induced withering and dying was observed in visual evaluation, and also the fresh weight of the above-ground part increased.




20    Test Example 4: Evaluation Test for Reduction of High Temperature Stress by Tomato Soil Irrigation Treatment {Fruit

Setting  Rate)

<Test  Plants>

Tomato  seeds   {cultivar:  Micro-Tom)   were  sown  in  the

25    growing soil (AISAI) in a plastic pot and then cultivated in a greenhouse (set at a temperature of 25°C) for about 4 weeks.


<Application  of  the  Present  Compound>
 

41

A soil irrigation treatment was carried out twice, using an aqueous solution of a sodium salt of the compound A in an amount of 50 ml per seedling, on the 15th day ~nd the 22nd day after seeding. As a control, a soil irrigation treatment was


5    carried out using distilled water in an amount of 50 ml per seedling to form a non-treated group.


<High  Temperature  Stress  Test>

After  the  irrigation  treatment,  plants  cultivated  up  to

10    the period when a 1st flower cluster blooms (1 to 2 flowers bloom(s), on the 32nd day after seeding) were cultivated for 7

days under the conditions of a temperature of 40°C (day)/30°C (night), a humidity of 63% (day)/70% (night), an illuminance of 7,100 lx, and a day length of 16 hours in the stress

15    exposed group, while plants were cultivated for 7 days in' a greenhouse (set at a temperature of 25°C) in the stress non-exposed group.




<Evaluation>

20 After the stress test, the test plants were cultivated for 13 days in a greenhouse (set at a temperature of 25°C), the number of flower setting and the number of fruit setting were counted to determine a fruit setting rate (%) (number of fruit setting/number of flower setting x 100). While the


25    fruit setting rate decreased due to high temperature stress exposure in the non-treated group, a decrease in fruit setting rate was remarkably alleviated in the group treated with the compound A as compared with the non-treated group.
 

42




Test Example 5: Evaluation Test for Reduction of High Temperature Stress by Tomato Spraying Treatment (Number or Weight of Seeds or Fruits, Fruit Setting Rate)

5    <Test  Plants>

Tomato seeds (cultivar: Micro-Tom) are sown in the growing soil (AISAI) in a plastic pot and then cultivated for about 4 weeks in a greenhouse (set at a temperature of 25°C) up to the period when a 1st flower cluster blooms (one to two


10    flowers  bloom(s)).



<Application  of  the  Present  Compound>

After adding 0.2% RINO as a sticker to an aqueous solution of any one of the compounds A to G, the solution is

15    sprayed to the entire test plants in an amount of 10 ml per seedling.


The  above  compound  solution  is  sprayed  to  foliages  of

the test plants in an amount of 10 ml per seedling. After the spraying treatment, the test plants are air-dried for about 2

20    hours.

As a control, a spraying treatment is carried out in the same manner, using distilled water containing only 0.2% RINO as a sticker added therein to form a non-treated group.




25    <High  Temperature  Stress  Test>

The test plants having been subjected to the spraying treatment are cultivated for 7 days under the conditions of a temperature of 40°C (day)/30°C (night), a humidity of 63%
 

43

{day)/70% (night), an illuminance of 7,100 lx, and a day length of 16 hours.





<Evaluation>

5 After the stress test, the test plants are cultivated for about 2 weeks in a greenhouse (set at a temperature of 25°C). The number of flower setting and the number of fruit setting are counted to determine a fruit setting rate (%) (number of fruit setting/number of flower setting X 100). The




10    diameters of fruits are measured and the average 1s determined. After the stress test, the plants are cultivated until fruits are fully matured and the total weight of the

fruits    is  examined.

In  the  group  treated  with  the  present  compound,   as

15    compared with the non-treated group, an increase in the fruiting rate, an increase in the number of fr~its, an increase in the diameter of fruits, and an increase in the• total weight of fruits are observed.




20    Test Example 6: Evaluation Test for Reduction of High Temperature Stress by Wheat Spike Spraying Treatment (Number or Weight of Seeds or Fruits)

<Test  Plants>

Wheat  seeds   (cultivar:  Perigee  or  Apogee)   are  sown  in

25    the growing soil (AISAI) in a plastic pot, and cultivated for about 4 weeks in a greenhouse (set at a temperature of 25°C) up to an earing period.
 

44

<Application  of  the  Present  Compound>

After adding 0.2% RINO as a sticker to an aqueous solution of any one of the compounds A to G, the solution is sprayed to the entire plants several times every other week

5    starting from 7 days before blooming in an amount of 10 ml per seedling. As a control, a spraying treatment is carried out in the same manner, using distilled water containing only 0.2% RINO as a sticker added therein to form a non-treated group.




10    <Hi.gh  Temperature  Stress  Test>

The test plants having subjected to the spraying treatment are cultivated for 14 days under the conditions of a temperature of 32°C (day)/22°C (night), a humidity of 63% (day)/70% (night), an illuminance of 7,100 lx, and a day

15    length  of  16  hours.



<Evaluation  Method>

After the stress test, the test plants are cultivated in a greenhouse (set at a temperature of 25°C) until grain

20    filling and the grain weight is examined. In the group treated with the present compound, as compared with the non-treated group, an increase in the grain weight is observed.




Test  Example  7:  Evaluation  Test  for  Reduction  of  High

25    Temperature or Low Temperature Stress in Hydroponics of Arabidopsis (Seedling Establishment Rate, Leaf Area, Chlorophyll Fluorescence Yield)

<Test  Plants>
 

45

A hydroponic sponge piece (1 em x 1 em x 0.2 em) is immersed with an MS culture medium (containing 2.5 mM MES, 2% sucrose, and a 1,000 times diluted Gamborg vitamin solution Gl019 (manufactured by Sigma-Aldrich Corporation)) and 5 to 8


5    Arabidopsis (ecotype Columbia) seeds having been subjected to surface sterilization are sown on the sponge. After the low

temperature treatment (at 4°C for 2 to 4 days), cultivation is carried out for 6 days under the conditions of a temperature of 23°C, a humidity of 45%, an illuminance of 3,500 lx, and a

10    light  period  of  16  hours/a  dark  period  of  8  hours.



<Transplantation  and  Application  of  the  Present  Compound>

In a 24-well plate (SUMILON MS-80240), 0.5 ml each of an MS culture medium, which is a culture medium containing 2.5 mM

15    MES, 2% sucrose and a 1,000 times diluted Gamborg vitamin solution Gl019 (manufactured by Sigma-Aldrich Corporation), containing any one of the compounds A to G having a concentration of 0.01 to 100 ppm is dispensed, and then a

sterilized  cotton  wool  is  spread  on  each  well.   After  thinning

20    the above. Arabidopsis seedlings to 1 to 2 per sponge, the seedlings are transplanted to each well together with the sponge and then grown overnight.

The  ?bove  "MS  culture  medium  containing  the  test

compound"  is  prepared  in  the  following  manner.    That  is,   with

25    respect to the compound A, the compound B, the compound c, the compound.D, the compound E, the compound For the compound G, a DMSO solution having a concentration 1,000 times of each test concentration is prepared, and 0.5 pL of the obtained
 

46


DMSO solution is added to 0.5 ml of the MS culture medium. An aqueous solution (250,000 ppm) of a sodium salt of the compound A is prepared and the obtained aqueous solution is added to 0.5 ml of the MS culture medium so as to give each

5    test concentration, and then 0.5 pL of DMSO is added to 0.5 ml of the culture medium. As a control, an MS culture medium containing 0.1% DMSO added therein is prepared to form a non-treated group.




10    <High  Temperature  Stress  Test>

A lidded 24-well plate is sealed with a film, followed by immersion in a water incubator and further incubation at 45°C for 60 minutes.



15    <Low  Temperature  Stress  Test>

A lidded 24-well plate is sealed with surgical tape, followed by cultivation for 6 days under the conditions of a temperature of 0 to 1°C, a humidity of 40 to 70%, an illuminance of 3,000 lx, and a light period of 16 hours/a dark

20    period  of  8  hours.



<Evaluation>

After the stress test, the test plants are cultivated for 3 to 5 days under the conditions of a temperature of 23°C,

25    a humidity of 45%, an illuminance of 3,500 lx, and a light period of 16 hours/a dark period of 8 hours. A photograph of each well is taken by a digital camera and the area of the green portion of the photograph is determined by imageing
 

47

analysis software Win ROOF (manufactured by MITANI CORPORATION) to determine the size of the plant body. One day after the high temperature stress test, the chlorophyll fluorescence (Fv/Frn) of each well is measured using a pulse


5    modulation chlorophyll fluorometer (IMAGING-PAM, manufactured by WALZ Company) .


In each group treated with the present compound, as compared with the non-treated group, plants become large and

growth  acceleration  of  the  above-ground  part  is  observed.    In

10    each group treated with the present compound, as compared with the non-treated group, an increase in the chlorophyll fluorescence is observed.




Test  Example  8:  Evaluation  Test  for  Reduction  of  Low

15    Temperature Stress by Corn Perfusion Treatment (Plant Weight, Chlorophyll Fluorescence Yield, Leaf Color)

<Test  Plants>

Corn  seeds   (cultivar:   PIONEER  120  31P41)   are  sown  in  the

growing  soil   (AISAI)   in  a  plastic  pot  and  then  cultivated  for

20    7 days under the conditions of a temperature of 20 to 25°C, a humidity of 50 to 75%, an illuminance of 4,500 lx, and a day length of 16 hours.




<Application  of  the  Present  Compound>

25 A DMSO solution having a concentration 1,000 times of each test concentration of any one of the compounds A to G is prepared and then diluted with distilled water. An aqueous solution (250,000 ppm) of a sodium salt of the compound A is
 

49

MINOLTA Holdings, Inc.), the chlorophyll content is measured. In each group treated with the present compound, as

compared with the non-treated group, the length of true leaves and the plant weight increase and growth acceleration of the

5    above-ground part is observed. In each group treated with the present compound, as compared with the non-treated group, an increase in the chlorophyll fluorescence and an increase in the chlorophyll content are observed as compared with the non-treated group.




10

Test Example 9: Evaluation Test for Reduction of Low Temperature Stress in Hydroponics of Rice (Plant Length, Plant Weight, Chlorophyll Fluorescence Yield)

<Test  Plants>

15 Rice seeds (cultivar: NIHOMBARE) are immersed in distilled water containing 1,000 ppm of Benlate and then incubated at 30°C for one day. The seeds are washed with distilled water, incubated in distilled water for one day and then subjected to a forced germination treatment. In the


20    bottom of a 288-well plug tray, cotton wool is spread and then the seeds subjected to the forced germination treatment are sown. A hydroponic culture medium (8-fold diluted Kimura B hydroponic culture solution) is added thereto, followed by

cultivation  for  3  to  10  days  under  the  conditions  of  a

25    temperature of 28°C (day)/23°C (night), a humidity of 60%, an illuminance of 7,100 lx, and a day length of 12 hours.


<Application  of  the  Present  Compound>
 

50

A DMSO solution having a concentration 1,000 times of each test concentration of any one of the compounds A to G is prepared and then diluted with a hydroponic culture medium. An aqueous solution {250,000 ppm) of a sodium salt of the


5    compound A is diluted with the hydroponic culture medium to prepare a liquid having a test concentration. The above rice seedling is transferred to these hydroponic culture media containing the compound, and then cultivated for 7 to 10 days


under  the  conditions  of  a  temperature  of  28°C   (day)/23°C

10 (night), a humidity of 60%, an illuminance of 7,100 lx, and a day length of 12 hours.


Using a hydroponic culture medium containing 0.1% DMSO added therein as a control of any one of the •compounds A to G and using a hydroponic culture medium as a control of the

15    sodium salt of the compound A, the above rice seedlings are cultivated to form a non-treated group.


<Low  Temperature  Stress  Test>

The  seedlings  during  the  treatment  with  the  present

20    compound are cultivated for 3 to 7 days under the conditions of a temperature of 2 to 4°C, a humidity of 40 to 70%, an illuminance of 3,500 lx, and a day length of 12 hours in the stress exposed group, and under the conditions of a temperature of 25 to 30°C, a humidity of 50 to 75%, an


25    illuminance of 4,500 lx, and a day length of 12 hours in the stress non-exposed group.





<Evaluation>
 

52

Blank slurry solution in place of the slurry solution to obtain seeds for non-treated group. The coated seeds (5 seeds each) were sown in the growing soil (AISAI) in a plastic pot and then cultivated in a greenhouse (set at a temperature of


5    18°C (day)/15°C (night)) for 17 days. Before the stress test, thinning was carried out to control the number of seedlings to 3 per pot.



<High  Temper~ture Stress  Test>

10 The above test plants on the 17th day after seeding were cultivated for 19 days in an artificial climate chamber under the conditions of a temperature of 36°C (day)/32°C (night), a humidity of 50% (day)/60% (night), an illuminance of 7,000 lx, and a day length of 12 hours.




15

<Evaluation  Method>

After the stress test, on the 36nd day after seeding, fresh weight of the above-ground part of test plants was examined for 7 to 9 pots. In the group treated with the

20    compound C or the compound E, as compared with the non-treated group, the fresh weight of the above-ground part increased.


Industrial  Applicability

Use  of  the  method  of  the  present  invention  enables

25    relief  of  temperature  stress  of  plants.
 

53

CLAIMS



1 A method for reducing temperature stress of plants which comprises applying an effective amount of one or more

5    compounds selected from the group consisting of a compound represented by the formula (I) and an agriculturally acceptable salt thereof to. a plant that has been exposed to or to be exposed to a temperature stress factor:





(I)


10    wherein

R1 represents a phenyl group, a naphthyl group or an aromatic heterocyclic group, and these groups are optionally substituted with 1 to 5 members selected from among a halogen atom, a hydroxyl group, a cyano group, a nitro group, a Cl-C6


15    alkyl group optionally substituted with one or more halogen atoms, a Cl-C6 alkoxy group optionally substituted with one or more halogen atoms, a Cl-C6 alkylthio group optionally substituted with one or more halogen atoms, a C2-C6 alkenyl

group  optionally  substituted  with  one  or  more  halogen.atoms,   a

20    C2-C6 alkynyl group optionally substituted with one or more halogen atoms, an amino group, a Cl-C6 alkylamine group and a

di(Cl-C6  alkyl)amino  group;

R2   represents  a  hydroxyl  group,  an  amino  group,   or  a  Cl-

C6    alkoxy  group;

25    X  represents  a  linear  or  branched  Cl-C6  alkylene  group;

and
 

54

Y represents a linear or branched C1-C6 alkylene group, or a linear or branched C1-C6 alkenylene group.


2. The method according to claim 1, whe~ein in the 5 formula (I),


R1 is a phenyl group, a 1-naphthyl group or a 3-indolyl group, wherein one or more hydrogen atoms in these groups are optionally replaced by 1 to 5 members selected from among a halogen atom, a hydroxyl group, a nitro group, a C1-C6 alkyl


LO    group  and  a  C1-C6  alkoxy  group;

R2   is  a  hydroxyl  group,   an  amino  group  or  a  C1-C6  alkoxy

group;

X is a linear or branched C1-C6 alkylene group; and y is a linear or branched C1-C6 alkylene group, or a

15    linear  or  branched  C1-C6  alkenylene  group.



~.    The  method  according  to  claim  1,  wherein  in  the

formula    (I)  r

Rl    is  a  phenyl  group,   a  4-iodophenyl  group,   a  1-naphthyl

20    group  or  a  3-indolyl  group;

Rz    is  a  hydroxyl  group  or  a  methoxy  group;

X  is  an  ethylene  group  or  a  tetramethylene  group;   and
y    is  an  ethylene  group  or  a  trimethylene  group.



25 ~- The method according to claim 1, wherein the compound of the formula (I) is a compound selected from•among the following compounds:

(1)    4-oxo-4-(2-phenylethyl)aminobutyric  acid,
 

55

(2)    methyl  4-oxo-4-(4-phenylbutyl)aminobutyrate,

(3)    methyl  4-oxo-4-(2-phenylethyl)aminobutyrate,

(4)    4-oxo-4-(4-phenylbutyl)aminobutyric  acid,

(5)    5-oxo-5-[2-(3-indolyl)ethyl]aminovaleric  acid,

5    (6)   5-oxo-5-[(1-naphthyl)methyl]aminovaleric  acid,   and

(7)    methyl  4-oxo-4-[2-(4-iodophenyl)ethyl]aminobutyrate.



5.    The  method  according  to  any  one  of  claims  1  to  4,

wherein  the  application  is  a  soil  irrigation  treatment,   a

10    spraying treatment, a hydroponic treatment or a seed treatment.





6 rhe method according to any one of claims 1 to 5, wherein ~ne application is a seed treatment.

15

~~ The method according to claim 6, wherein an application amount of the compound of the formula (I) in the seed treatment is from 30g to 500 g per 100 kg of seeds.



20 ~. The method according to any one of claims 1 to 7, wherein the plant is rice, corn, soybean, wheat or tomato.


~ The method according to any one of claims 1 to 8, wherein the plant is a transgenic plant.

25
 

~10 The method according to any one of claims 1 to 9, wherein the temperature stress is high temperature stress.
 
56

11. The method according to any one of claims 1 to 9, wherein the temperature stress is low temperature stress.


-~2  The  method  according  to  any  one  of  claims  1  to  11,

5    wherein the temperature stress is indicated by a change in one or more of the following plant phenotypes:

(1)    germination  percentage,

(2)    seedling  establishment  rate,

( 3)  number  of  healthy  leaves,

10    ( 4)  plant  length,

(5)    plant  weight,

(6)    leaf  area,

( 7)   leaf  color,

( 8)  number  o'r  weight  of  seeds  or  fruits,

15    (9)   quality  of  harvests,

(10)    flower  setting  rate  or  fruit  setting  rate,   and

(11)    chlorophyll  fluorescence  yield;



13    Use  of  one  or  more  compounds  selected  from  the  group


20    consisting of a compound represented by the formula (I) of claim 1 and an agriculturally acceptable salt thereof for reducing temperature stress of plants.



u4 ..  The  use  according  to  claims  13,  wherein  the

25    temperature stress is indicated by a change in one or more of the following plant phenotypes:

(1)    germination  percentage,

(2)    seedling  establishment  rate,
 

57

{3)    number  of  healthy  leaves,

(4)    plant  length,

(5)    plant  weight,

(6)    leaf  area,

5    (7)   leaf  color,

(8)    number  or  weight  of  seeds  or  fruits,

(9)    quality  of  harvests,

(10)    flower  setting  rate  or  fruit  setting  rate,  and

(11)    chlorophyll  fluorescence  yield.

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