AU2020395007B2

AU2020395007B2 - Chickpea nodule formation promoter

Info

Publication number: AU2020395007B2
Application number: AU2020395007A
Authority: AU
Inventors: Keiji Endo; Teruhisa FUJIMATSU; Haruka MAEDA; Atsuki OHNISHI; Yuhei TSUNO
Original assignee: Kao Corp
Current assignee: Kao Corp
Priority date: 2019-12-02
Filing date: 2020-12-02
Publication date: 2023-12-07
Anticipated expiration: 2040-12-02
Also published as: WO2021112139A1; AU2020395007A1; JP2021091676A; JP7355723B2

Abstract

Provided is a technique for promoting nodule formation by nodule bacteria that coexist with chickpeas and increasing the yield of chickpeas.　This chickpea nodule formation promoter contains, as an active ingredient, methylated isoflavones represented by formula (1) (in the formula, R

Description

KS1685

Description

CHICKPEA NODULE FORMATION PROMOTER

Field of the Invention

[0001] The present invention relates to a technique for

promoting chickpea nodule formation and enhancing the

yield of chickpea.

Background of the Invention

[0002] Chickpea (Cicer arietinum L.) is an annual herb in

the legume family, and grains (beans), young stems and

leaves, and pods are edible. The dried seeds of chickpea

contain 25% protein, 5% fat, 59% carbohydrate, and 1.2%

dietary fiber, as well as rich mineral components, such

as phosphorus, calcium, and iron. To ingest them has the

effect of supplementing essential amino acids, which tend

to be deficient when refraining from animal protein in

diets, or deficient in cereals (Non-Patent Literature 1).

[0003] Chickpea ranks second in terms of the harvested

area and third in terms of the production volume among

pulses in the whole world, and is the most productive and

in-demand pulse in India, where the population is growing

rapidly. However, the unit yield of chickpea in India is

said to diverge between the pilot field level and the

actual producer level (Non-Patent Literature 2).

[0004] The yield of chickpea is known to increase due to

nodule formation by rhizobia (Mesorhizobium ciceri, etc.)

KS1685 -2

(Non-Patent Literature 3), and it is considered important

to ensure productivity by strengthening the nodules.

Conventionally, there have been reports of technologies

such as the addition of specific compounds to grow and

activate rhizobia. For chickpea, however, flavonoids

such as daidzein and naringenin, which are empirically

used to activate rhizobia symbiotic with soybean, kidney

bean, and the like, are used, and their effects are not

sufficient.

[0005] On the other hand, methylated isoflavones such as

biochanin A and formononetin, which are abundant in red

clover, have long been known to have female hormone-like

effects. Recently, it has been reported that they have a

positive effect on alleviating dementia (Patent

Literature 1) and preventing or treating sarcopenia

(Patent Literature 2).

However, the detailed effects of methylated

isoflavones on nodules have not been known at all.

[0006] [Patent Literature 1] JP-A-2019-526612

[Patent Literature 2] JP-A-2013-507361

[0007] [Non-Patent Literature 1] Coastal Bioenvironment

Center Saga University Volume 8 (2006) 35-40

[Non-Patent Literature 2] Outlook on Agriculture,

Volume 46, Issue 3 (2017) 1-6

[Non-Patent Literature 3] European Journal of

Agronomy, Volume 19, Issue 1 (2003) 15-21

Summary of the Invention

[0008] The present invention relates to the following 1)

to 8).

1) A methylated isoflavone of the following formula

(1') when used in promoting chickpea nodule formation:

HO 0

R1 O0 OCH3 R2 (1'), wherein R1 and R 2 both

represent a hydroxy group.

2) A methylated isoflavone of the following formula

(1') when used in enhancing the yield of chickpea:

HO 0

R1 O OCH3 R2 (1'), wherein R1 and R 2 both

represent a hydroxy group.

3) A composition for promoting chickpea nodule

formation or enhancing the yield of chickpea, comprising

a methylated isoflavone of the following formula (1) and

a rhizobium using chickpea as a host.

HO O

(1) R1 0/ OCH3 R2

wherein R1 and R 2 both represent a hydrogen atom or a

hydroxy group.

4) A method for promoting chickpea nodule formation,

comprising the step of applying a methylated isoflavone

of the following formula (1') to soil or a plant:

HO O

R1 0/ OCH3 R2 (1'), wherein R1 and R 2 both

represent a hydroxy group.

5) A method for enhancing the yield of chickpea,

comprising the step of applying a methylated isoflavone

of the following formula (1') to soil or a plant:

HO O

R1 0/ OCH3 R2 (1'), wherein R1 and R 2 both

represent a hydroxy group.

- 4a

6) A chickpea cultivation method, comprising the

step of applying a methylated isoflavone of the following

formula (1') to soil or a plant:

HO 0

R1 O0 OCH3 R2 (1'), wherein R1 and R 2 both

represent a hydroxy group.

6a) A method for promoting chickpea nodule

formation, a method for enhancing the yield of chickpea,

or a method for cultivating chickpea, comprising the step

of applying a methylated isoflavone of the following

formula (1):

HO O

(1) R1 O OCH3 R2

wherein R1 and R 2 both represent a hydrogen atom or a

hydroxy group, in combination with a rhizobium using

chickpea as a host to soil or a plant.

- 4b

[00091 Where any or all of the terms "comprise",

"comprises", "comprised" or "comprising" are used in this

specification (including the claims) they are to be

interpreted as specifying the presence of the stated

features, integers, steps or components, but not

precluding the presence of one or more other features,

integers, steps or components.

[0010] A reference herein to a patent document or any

other matter identified as prior art, is not to be taken

as an admission that the document or other matter was

known or that the information it contains was part of the

common general knowledge as at the priority date of any

of the claims.

Brief Description of the Drawings

[0011] [Figure 1] Nodule formation-promoting effect of

methylated isoflavones (nodule weight).

[Figure 2] Nodule formation-promoting effect of

methylated isoflavones (number of nodules).

[Figure 3] Initial growth-promoting effect of

methylated isoflavones (above-ground part weight).

[Figure 4] Initial growth-promoting effect of

methylated isoflavones (below-ground part weight).

[Figure 5] Yield-enhancing effect of methylated

isoflavones (grain weight).

[Figure 6] Yield-enhancing effect of methylated

isoflavones (number of grains).

- 4c

[Figure 71 Synergistic effect of combined use of a

rhizobium and pratensein (number of nodules).

[Figure 81 Synergistic effect of combined use of a

rhizobium and pratensein (nodule weight).

KS1685 -5

Detailed Description of the Invention

[0012] The present invention relates to the provision of

a technique for promoting nodule formation by rhizobia

symbiotic with chickpea and enhancing the yield of

chickpea.

[0013] The present inventors examined natural materials

harmless to the soil and environment, and consequently

found that specific methylated isoflavones have the

effect of promoting nodule formation by rhizobia

symbiotic with chickpea, and are useful for enhancing the

yield of chickpea.

[0014] The present invention makes it possible to promote

nodule formation by rhizobia symbiotic with chickpea, and

to enhance the yield of chickpea.

[0015] In the present invention, "chickpea" refers to

Cicer arietinum L. belonging to the genus Cicer of the

family Fabaceae. Known species are kabuli species and

desi species, both of which can be used.

In the present invention, "nodule" means a mass

formed on the root of a plant by coexistence with

bacteria (rhizobia). Rhizobia perform so-called

symbiotic nitrogen fixation, in which nitrogen in the air

is reduced to convert it into ammonium nitrogen in

nodules, and supplied to the host.

Rhizobia are said to have specificity to symbiotic

plants, and Mesorhizobium ciceri, Mesorhizobium

mediterraneum, and the like are known as rhizobia using

KS1685

-6

chickpea as a host. In the present invention, "rhizobia"

are not limited as long as they can live symbiotically

with chickpea; however, Mesorhizobium ciceri is

preferred.

[0016] In the present invention, methylated isoflavones

are isoflavones in which the hydroxy group at position 4'

is methylated, as shown in the following formula (1).

[0017]

HK) 7 O

5 4| R1 O 3, OCH3 R2

[0018] wherein R1 and R 2 may be the same or different and

each represents a hydrogen atom or a hydroxy group.

[0019] A compound wherein R1 and R 2 are both hydroxy

groups (5,7,3'-trihydroxy-4'-methoxyisoflavone) is known

as "pratensein," a compound wherein R1 is a hydroxy group

and R 2 is a hydrogen atom (5,7-dihydroxy-4'

methoxyisoflavone) is known as "biochanin A," and a

compound wherein R1 and R 2 are both hydrogen atoms (7

hydroxy-4'-methoxyisoflavone) is known as "formononetin."

Among these, pratensein is preferred in terms of the

effect of promoting nodule formation.

[0020] All of such methylated isoflavones of the present

invention are reportedly contained in plants, such as red

clover (Trifolium pratense), white clover (Trifolium

KS1685 -7

repens), beggarweed (Podocarpium podocarpum), and

Capillary Thread-moss (.Bryum capillare). In general,

they can be extracted, isolated, and purified from these

plants by known methods; however, commercial products

(pratensein: FUJIFILM Wako Pure Chemical Corporation and

Chromadex; biochanin A: Chromadex; and formononetin:

Sigma-Aldrich) can also be used. Alternatively,

chemically synthesized products produced by the reported

method (e.g., Wong, E. 1963. "Pratensein. 5,7,3'

Trihydroxy-4'-Methoxyisoflavone." Journal of Organic

Chemistry 28(9): 2336-39) or a method similar thereto may

also be used.

[0021] As shown in the Examples described later, in the

case of cultivating chickpea after sowing chickpea seeds

and inoculating rhizobia, when the methylated isoflavones

of the present invention are added, the nodule weight and

the number of nodules increase, and the grain weight, the

number of grains, and the protein yield per individual

plant also increase. The effect thereof is superior to

naringenin (4',5,7-trihydroxyflavanone).

Therefore, the methylated isoflavones of the present

invention can serve as chickpea nodule formation

promoters or chickpea yield enhancers, and can be used

for promoting chickpea nodule formation or enhancing the

yield of chickpea, and also for producing chickpea nodule

formation promoters or chickpea yield enhancers.

KS1685 -8

[0022] In the present invention, the phrase "promoting

chickpea nodule formation" means that the ability of

rhizobia to form nodules by coexistence with chickpea

increases. Specifically, it means that the nodule weight

or the number of nodules, or both of them, increase more

than the methylated isoflavone untreated group.

The phrase "enhancing the yield of chickpea" means

that the number of grains (beans) obtained from chickpea

increases. For example, it means that the grain dry

weight or the number of grains per individual plant, or

both of them, increase more than the methylated

isoflavone untreated group.

The phrase "enhancing the yield of chickpea" also

includes an increase in the yield of protein obtained

from chickpea. The phrase "increase in the yield of

protein" means that the mass of protein contained in

grains (beans) obtained from chickpea increases. For

example, it means that the mass of protein contained in

grains (beans) per individual plant increases more than

the methylated isoflavone untreated group.

[0023] The chickpea nodule formation promoter or chickpea

yield enhancer described above can serve as a composition

for promoting chickpea nodule formation or yield

enhancement (composition for promoting chickpea nodule

formation or for enhancing the yield of chickpea) (e.g.,

various agricultural or horticultural materials), or a

material (simple substance) or formulation for addition

KS1685 -9

or blending directly or after dilution with water into

cultivation substrates for cultivating chickpea, such as

soil, media, and solutions for hydroponic cultivation.

The above composition may be in the form of a liquid

or gel composition, or a solid composition (block,

powder, granules, etc.). In the case of a liquid

composition, it can be used as it is or as a

concentration type used after dilution. In the case of a

solid composition, it can be used after being dissolved

in water.

[0024] The above composition may contain any components,

in addition to the methylated isoflavones of the present

invention. Examples of such components include solvents

(e.g., water, buffers, media, and solutions for

hydroponic cultivation), carriers (diatomaceous earth,

vermiculite, perlite, peat moss, activated carbon, humus,

talc, zeolite, clay, carbon black, pulp, straw, soybean

cake, bentonite, kaolin, montmorillonite, alumina, etc.),

pH adjusters for promoting the dissolution of the above

compounds, spreading agents for increasing the ability to

spread to plant bodies or soil, fertilizer components for

increasing fertilizer effects, agrichemical components,

binders, extenders, plant growth-promoting microorganisms

such as rhizobia (e.g., rhizobia belonging to the genus

Mesorhizobium using chickpea as a host, such as

Mesorhizobium ciceri and Mesorhizobium mediterraneum) and

mycorrhizal fungi, essential nutrients for plants,

KS1685 - 10

flavonoids (daidzein, genistein, etc.), organic acids,

amino acids, peptides, nucleosides, nucleotides,

nucleobases, sugars, monohydric alcohols, non-ionic

surfactants, food additives, microorganism extracts,

plant hormones, Nod factors, i.e., lipo

chitooligosaccharides, synthetic lipo

chitooligosaccharides, and chitooligosaccharides,

chitinous compounds, linoleic acid or derivatives

thereof, linolenic acid or derivatives thereof,

karrikins, acyl-homoserine lactone derivatives, betaine

compounds, phenolic compounds (ferulic acid, chlorogenic

acid, etc.), antioxidants, and the like.

[0025] Examples of the above composition include, but are

not limited to, cultivation substrates containing the

methylated isoflavones of the present invention as active

ingredients (e.g., agricultural or horticultural soil,

culture soil, media, solutions for hydroponic

cultivation, and water), fertilizers, water for watering,

microbial materials such as rhizobium materials, soil

conditioners, agricultural chemicals, sowing materials,

plant supplements (e.g., activating agents and

nutritional supplements), and the like.

[0026] The fertilizers, microbial materials, soil

conditioners, sowing materials, and plant supplements are

preferred because they contribute to the improvement of

soil for cultivating plants such as leguminous plants.

The fertilizers, microbial materials, soil conditioners,

KS1685

- 11

sowing materials, and plant supplements may be solid or

liquid. In the case of solids, they may be blocks,

powders, granules, or the like, but are preferably

powders or granules. The fertilizers, microbial

materials, soil conditioners, sowing materials, and plant

supplements may contain components of fertilizers,

microbial materials, soil conditioners, sowing materials,

and plant supplements generally used for cultivation of

chickpea, in addition to the methylated isoflavones of

the present invention, which are contained as active

ingredients.

[0027] The cultivation substrates, fertilizers, microbial

materials such as rhizobium materials, soil conditioners,

agricultural chemicals, sowing materials, and plant

supplements may be prepared by adding the methylated

isoflavones of the present invention to general

cultivation substrates (e.g., agricultural or

horticultural soil, culture soil, media, solutions for

hydroponic cultivation, and water), fertilizers,

microbial materials such as rhizobium materials, soil

conditioners, agricultural chemicals, sowing materials,

plant supplements (e.g., activating agents and

nutritional supplements), and the like.

[0028] The content of the methylated isoflavones of the

present invention in the above composition for promoting

chickpea nodule formation or enhancing the yield of

chickpea can be appropriately set so as to be suitable

KS1685 - 12

for the amount of application. For example, in the total

mass of the composition, the content of the methylated

isoflavones is preferably 0.000001 mass% (0.01 pg/g) or

more, more preferably 0.00001 mass% (0.1 pg/g) or more,

and further more preferably 0.0001 mass% (1 pg/g) or

more, as well as preferably 1 mass% (10,000 pg/g) or

less, more preferably 0.1 mass% (1,000 pg/g) or less,

further more preferably 0.01 mass% (100 pg/g) or less,

and even more preferably 0.001 mass% (10 pg/g) or less.

Further, the content of the methylated isoflavones is

preferably from 0.000001 to 1 mass%, more preferably from

0.00001 to 0.1 mass%, further more preferably from 0.0001

to 0.01 mass%, and even more preferably from 0.0001 to

0.001 mass%.

[0029] The above composition for promoting chickpea

nodule formation or enhancing the yield of chickpea is

preferably a composition as a microbial material

containing the methylated isoflavone of the present

invention and a rhizobium using chickpea as a host (e.g.,

rhizobia belonging to the genus Mesorhizobium, such as

Mesorhizobium ciceri and Mesorhizobium mediterraneum),

and particularly preferably a material containing

Mesorhizobium ciceri, in terms of promoting chickpea

nodule formation.

[0030] The rhizobial count can be appropriately set so as

to be suitable for the amount of application. For

example, the rhizobial count per gram of the composition

KS1685 - 13

is preferably 104 cfu or more, more preferably 105 cfu or

more, and further more preferably 106 cfu or more, as

well as preferably 1011 cfu or less, more preferably 1010

cfu or less, further more preferably 109 cfu or less, and

even more preferably 5 x 108 cfu or less. Further, the

rhizobial count per gram of the composition is preferably

from 104 to 1011 cfu, more preferably from 105 to 1010 cfu,

further more preferably from 106 to 109 cfu, and even

more preferably from 2 x 106 to 5 x 108 cfu.

[0031] The contents of the rhizobia and the methylated

isoflavones per gram of the composition are, for example,

such that the rhizobial count is preferably from 104 to

1011 cfu and the methylated isoflavone content is from

0.01 to 10,000 pg, more preferably the rhizobial count is

from 105 to 1010 cfu and the methylated isoflavone content

is from 0.1 to 1,000 pg, further more preferably the

rhizobial count is from 106 to 109 cfu and the methylated

isoflavone content is from 1 to 100 pg, and even more

preferably the rhizobial count is from 2 x 106 to 5 x 108

cfu and the methylated isoflavone content is from 1 to 10

pg. The content ratio of the rhizobia to the methylated

isoflavones (the rhizobial count cfu per pg of the

methylated isoflavones) is preferably from 105 to 5 x

109, more preferably from 106 to 5 x 109, further more

preferably from 2 x 106 to 5 x 109, and even more

preferably from 2 x 106 to 5 x 108.

KS1685 - 14

[0032] The method for supplying the chickpea nodule

formation promoter or chickpea yield enhancer of the

present invention is not particularly limited, as long as

they are applied to be able to exhibit the effect of

promoting nodule formation in chickpea and enhancing the

yield of chickpea.

That is, it is not particularly limited as long as

the chickpea nodule formation promoter or chickpea yield

enhancer of the present invention is brought into contact

with or delivered to the plant body or soil in the plant

rhizosphere. Examples of the method include surface

spraying to the soil, irrigation, plowing-in, foliar

spraying to plants, application after mixing with

fertilizers, addition to hydroponic solutions, coating or

smearing of seeds before sowing (e.g. seed powder

coating), and the like.

As the soil, any soil which allows cultivation of

chickpea may be used, and soil supplemented with a soil

bacterial solution prepared from soil which allows

cultivation of chickpea can also be used. The soil

bacterial solution can be prepared as a filtrate by

filtering soil to which sterilized water with a mass of

0.1 to 10 times the soil mass is added.

[0033] In the cultivation of chickpea using the

methylated isoflavone of the present invention, the

methylated isoflavones can be combined with the above

mentioned rhizobia symbiotic with chickpea (using

KS1685 - 15

chickpea as a host) and appropriately inoculated in soil

or a plant. In this case, for example, rhizobia (e.g., a

rhizobium-containing solution) and the methylated

isoflavones are added to the soil in which plant seeds

are sown, or a composition (microbial material)

containing a rhizobium using chickpea as a host and the

methylated isoflavone of the present invention is

prepared in advance, and this material is added to

chickpea seeds or the soil for cultivating chickpea.

[0034] The amount of the chickpea nodule formation

promoter or chickpea yield enhancer of the present

invention applied can vary depending on the application

method, application time, type of plant, cultivation

density, growth stage, and the like; however, for

example, the amount of the methylated isoflavones of the

present invention used may be, as the concentration in

the above cultivation substrate for cultivating chickpea,

preferably from 0.000001 to 1 mass ppm, more preferably

from 0.00001 to 0.1 mass ppm, further more preferably

from 0.0001 to 0.01 mass ppm, and even more preferably

from 0.0001 to 0.001 mass ppm.

For example, the amount of the methylated

isoflavones of the present invention used per liter

volume of the cultivation substrate may be preferably

from 0.001 to 1,000 pg, more preferably from 0.01 to 100

pg, further more preferably from 0.1 to 10 pg, and even

more preferably from 0.1 to 1 pg. In the case of soil

KS1685 - 16

culture of chickpea, the methylated isoflavones of the

present invention may be added to the soil in an amount

of preferably from 0.0001 to 100 g, more preferably from

0.001 to 10 g, further more preferably from 0.01 to 1 g,

and even more preferably from 0.01 to 0.1 g, per 10 ares

of land.

That is, in the case of a composition containing the

methylated isoflavones of the present invention as active

ingredients, for example, a fertilizer, a microbial

material, a soil conditioner, a sowing material, or a

plant supplement, the amount of the composition used

depends on the concentration of the methylated

isoflavones contained in the composition. For example,

when the concentration of the methylated isoflavones

contained in the composition is 0.005 mass%, the amount

of the composition used per 10 ares of land is preferably

from 2 to 2,000,000 g, more preferably from 20 to 200,000

g, further more preferably from 200 to 20,000 g, and even

more preferably from 200 to 2,000 g.

The chickpea nodule formation promoter or chickpea

yield enhancer of the present invention may be applied in

an amount in the above range at one time or multiple

times.

[0035] When the methylated isoflavones are applied to

soil or a plant in combination with rhizobia symbiotic

with chickpea (using chickpea as a host), the rhizobial

count applied per chickpea seed is preferably 104 cfu or

KS1685 - 17

more, more preferably 105 cfu or more, further more

preferably 106 cfu or more, and even more preferably 107

cfu or more, as well as preferably 1011 cfu or less, more

preferably 1010 cfu or less, further more preferably 109

cfu or less, and even more preferably 5 x 108 cfu or

less. Further, the rhizobial count applied per chickpea

seed is preferably from 104 to 1011 cfu, more preferably

from 105 to 1010 cfu, further more preferably from 106 to

109 cfu, and even more preferably from 2 x 106 to 5 x 108

cfu.

In the combined application, the amount of the

methylated isoflavones applied per chickpea seed is

preferably 0.001 pg or more, more preferably 0.01 pg or

more, and further more preferably 0.1 pg or more, as well

as preferably 1,000 pg or less, more preferably 100 pg or

less, further more preferably 10 pg or less, and even

more preferably 5 pg or less. Further, the amount of the

methylated isoflavones applied per chickpea seed is

preferably from 0.001 to 1,000 pg, more preferably from

0.01 to 100 pg, further more preferably from 0.1 to 10

pg, and even more preferably from 0.1 to 5 pg. In the combined application, the amounts of the

rhizobia and methylated isoflavones applied per chickpea

seed are preferably such that the rhizobial count is from

104 to 1011 cfu and the methylated isoflavone content is

from 0.001 to 1,000 pg, more preferably the rhizobial

count is from 105 to 1010 cfu and the methylated

KS1685 - 18

isoflavone content is from 0.01 to 100 pg, further more

preferably the rhizobial count is from 106 to 109 cfu and

the methylated isoflavone content is from 0.1 to 10 pg,

and even more preferably the rhizobial count is from 2 x

106 to 5 x 108 cfu and the methylated isoflavone content

is from 0.1 to 5 pg.

In the combined application, the ratio of the

amounts of the rhizobia and methylated isoflavones

applied (the rhizobial count (cfu) per pg of the

methylated isoflavones) is preferably from 105 to 5 x

109, more preferably from 106 to 5 x 109, further more

preferably from 2 x 106 to 5 x 109, and even more

preferably from 2 x 106 to 5 x 108.

[0036] The timing and frequency of application of the

chickpea nodule formation promoter or chickpea yield

enhancer can vary depending on the type of plant etc.;

however, in general, in the case of application to a

cultivation substrate, such as soil, by surface spraying,

irrigation, plowing-in, or seed powder coating, it is

preferably applied once or 1 to 3 times before sowing or

at the same time as sowing. In the case of application

after sowing, the timing is preferably the early

nutritional growth phase before the reproductive growth

phase. Specifically, it is preferably applied once or 1

to 3 times between the day of sowing and 30 days, and

more preferably between the day of sowing and 14 days.

KS1685 - 19

In another embodiment, the addition of the chickpea

nodule formation promoter or chickpea yield enhancer of

the present invention to a cultivation substrate (e.g.,

soil, culture soil, a medium, a solution for hydroponic

cultivation, or water) before sowing, at the same time as

sowing, and/or after sowing may be combined with the

coating or smearing (e.g. seed powder coating) of seeds

before sowing with the chickpea nodule formation promoter

or chickpea yield enhancer.

In the coating or smearing (e.g. seed powder

coating) of seeds before sowing with the chickpea nodule

formation promoter or chickpea yield enhancer, it is

preferable to use a carrier such as peat moss, as well as

a spreading agent, a surfactant, and the like.

[0037] Regarding the embodiment described above, the

present invention further discloses the following

aspects.

<1> A chickpea nodule formation promoter comprising,

as an active ingredient, a methylated isoflavone of the

following formula (1).

<2> A chickpea yield enhancer comprising, as an

active ingredient, a methylated isoflavone of the

following formula (1).

<3> A composition for promoting chickpea nodule

formation or enhancing the yield of chickpea, comprising

a methylated isoflavone of the following formula (1) and

a rhizobium using chickpea as a host.

KS1685 - 20

<4> A method for promoting chickpea nodule

formation, comprising the step of applying a methylated

isoflavone of the following formula (1) to soil or a

plant.

<5> A method for enhancing the yield of chickpea,

comprising the step of applying a methylated isoflavone

of the following formula (1) to soil or a plant.

<6> A chickpea cultivation method, comprising the

step of applying a methylated isoflavone of the following

formula (1) to soil or a plant.

<7> The method according to any one of <4> to <6>,

wherein a rhizobium using chickpea as a host is further

combined and applied to soil or a plant.

<8> The method according to <7>, wherein a microbial

material comprising the rhizobium and the methylated

isoflavone is previously prepared, and the material is

applied to chickpea seeds or soil for cultivating

chickpea.

<9> The method according to <7> or <8>, wherein the

rhizobium is a rhizobium belonging to the genus

Mesorhizobium, and preferably Mesorhizobium ciceri or

Mesorhizobium mediterraneum.

<10> The method according to any one of <4> to <9>,

wherein the soil is soil supplemented with a soil

bacterial solution prepared from soil which allows

cultivation of chickpea.

KS1685 - 21

[0038] <11> Use of a methylated isoflavone of the

following formula (1) for producing a chickpea nodule

formation promoter.

<12> Use of a methylated isoflavone of the following

formula (1) for producing a chickpea yield enhancer.

<13> Use of a methylated isoflavone of the following

formula (1) and a rhizobium using chickpea as a host for

producing a composition for promoting chickpea nodule

formation or enhancing the yield of chickpea.

[0039] <14> In <1> to <13>, the methylated isoflavone is

pratensein, biochanin A, or formononetin, and preferably

pratensein.

<15> In <1> to <14>, the chickpea is preferably

kabuli species.

<16> When the agent of <1>, <2>, <11>, or <12> is a

composition, the content of the methylated isoflavone in

the composition is preferably 0.000001 mass% or more,

more preferably 0.00001 mass% or more, and even more

preferably 0.0001 mass% or more, as well as preferably 1

mass% or less, preferably 0.1 mass% or less, more

preferably 0.01% or less, and further more preferably

0.001 mass% or less, in the total mass of the

composition. Further, the content of the methylated

isoflavone in the composition is preferably from 0.000001

to 1 mass%, more preferably from 0.00001 to 0.1 mass%,

further more preferably from 0.0001 to 0.01 mass%, and

preferably from 0.0001 to 0.001 mass%.

KS1685 - 22

<17> In the composition of <3>, the rhizobial count

per gram of the composition is preferably 104 cfu or

more, more preferably 105 cfu or more, and further more

preferably 106 cfu or more, as well as preferably 1011 cfu

or less, more preferably 1010 cfu or less, further more

preferably 109 cfu or less, and preferably 5 x 108 cfu or

less, or preferably from 104 to 1011 cfu, more preferably

from 105 to 1010 cfu, further more preferably from 106 to

109 cfu, and preferably from 2 x 106 to 5 x 108 cfu.

<18> In the composition of <3>, the contents of the

rhizobium and the methylated isoflavone per gram of the

composition are preferably such that the rhizobial count

is from 104 to 1011 cfu and the methylated isoflavone

content is from 0.01 to 10,000 pg, more preferably the

rhizobial count is from 105 to 1010 cfu and the methylated

isoflavone content is preferably from 0.1 to 1,000 pg,

more preferably the rhizobial count is from 106 to 109

cfu and the methylated isoflavone content is from 1 to

100 pg, and more preferably the rhizobial count is from 2

x 106 to 5 x 108 cfu and the methylated isoflavone

content is preferably from 1 to 10 pg.

<19> In the composition of <3>, the content ratio of

the rhizobium to the methylated isoflavone (the rhizobial

count (cfu) per pg of the methylated isoflavone) is

preferably from 105 to 5 x 109, more preferably from 106

to 5 x 109, further more preferably from 2 x 106 to 5 x

109, and even more preferably from 2 x 106 to 5 x 108.

KS1685 - 23

<20> In the method of any of <4> to <10>, the amount

of the methylated isoflavone used is, as the

concentration in a cultivation substrate for cultivating

chickpea, preferably from 0.000001 to 1 mass ppm, more

preferably from 0.00001 to 0.1 mass ppm, from 0.0001 to

0.01 mass ppm, or from 0.0001 to 0.001 mass ppm.

<21> In the method of <7> or <8>, the rhizobial

count applied per chickpea seed is preferably 104 cfu or

more, more preferably 105 cfu or more, further more

preferably 106 cfu or more, and preferably 107 cfu or

more, as well as preferably 1011 cfu or less, more

preferably 1010 cfu or less, further more preferably 109

cfu or less, and preferably 5 x 108 cfu or less.

Further, the rhizobial count applied per chickpea seed is

preferably from 104 to 1011 cfu, more preferably from 105

to 1010 cfu, further more preferably from 106 to 109 cfu,

and preferably from 2 x 106 to 5 x 108 cfu.

<22> In the method of <7> or <8>, the amount of the

methylated isoflavone applied per chickpea seed is

preferably 0.001 pg or more, more preferably 0.01 pg or

more, and further more preferably 0.1 pg or more, as well

as preferably 1,000 pg or less, more preferably 100 pg or

less, further more preferably 10 pg or less, and

preferably 5 pg or less, or preferably from 0.001 to

1,000 pg, more preferably from 0.01 to 100 pg, further

more preferably from 0.1 to 10 pg, and preferably from

0.1 to 5 pg.

KS1685

- 24

<23> In the method of <7> or <8>, the amounts of the

rhizobium and the methylated isoflavone applied per

chickpea seed are preferably such that the rhizobial

count is from 104 to 1011 cfu and the methylated

isoflavone content is from 0.001 to 1,000 tg, more

preferably the rhizobial count is from 105 to 1010 cfu and

the methylated isoflavone content is from 0.01 to 100 ptg,

further more preferably the rhizobial count is from 106

to 109 cfu and the methylated isoflavone content is from

0.1 to 10 tg, and preferably the rhizobial count is from

2 x 106 to 5 x 108 cfu and the methylated isoflavone

content is from 0.1 to 5 pg.

<24> In the method of <7> or <8>, the ratio of the

rhizobium and the methylated isoflavone applied (the

rhizobial count cfu per pg of the methylated isoflavone)

is preferably from 105 to 5 x 109, more preferably from

106 to 5 x 109, further more preferably from 2 x 106 to 5

x 109, and preferably from 2 x 106 to 5 x 108.

[0040]

HO 0

(1) R1 O OCH3 R2

[0041] wherein R1 and R 2 may be the same or different and

each represents a hydrogen atom or a hydroxy group.

KS1685 - 25

Examples

[0042] Example 1: Effect of methylated isoflavones on

chickpea nodule formation and early growth

Initial fertilizer effect type culture soil (Takii

cell culture soil TM-1, Takii & Co., Ltd.) and

vermiculite grains (Akagi-Engei Co., Ltd.) were mixed at

a volume ratio of 1:1, and about 1.1 L of the soil was

placed in a pot (long plastic pot 120, Nippon Polypot

Hanbai Co., Ltd.). Then, two seeds of chickpea (kabuli

species) were sown in the pot at a depth from about 1 to

2 cm from the soil surface.

[0043] Separately, 1.5% agar (Wako Pure Chemical

Corporation) was added to Yeast-Mannitol (YM) medium (0.5

g of K2HPO 4 , 0.2 g of MgSO 4 -7H20, 0.1 g of NaCl, 0.4 g of

yeast extract, 10 g of mannitol, and 1 L of distilled

water (pH: 6.8)) to prepare a solid medium, and a

chickpea rhizobium (Mesorhizobium ciceri) NBRC100389T

strain was stationary-cultured on the solid medium at

°C for about 72 hours. A platinum loop of the grown

rhizobium was taken and inoculated in 50 mL of YM liquid

medium in a Sakaguchi flask with a volume of 500 mL, and

shake-cultured at 30°C for about 72 hours. 1 mL of a

rhizobium culture solution whose value of turbidity

(OD6oo) was adjusted to 0.02 (7.0 x 106 CFU/ml) using

sterilized water was inoculated dropwise using a

micropipette on the seeds at the time of sowing, and then

covered with soil.

KS1685 - 26

[0044] Subsequently, 1 pg of methylated isoflavone and a

comparison compound were applied in the following manner.

Specifically, 1 mL of a solution of naringenin (Sigma

Aldrich, Cat. No. N5893), formononetin (Sigma-Aldrich,

Cat. No. 47752-5MG-F), biochanin A (Chromadex, Cat. No.

ASB-00002276-005), or pratensein (Chromadex, ASB

00016080-001) dissolved in a 0.1 (v/v)% dimethylsulfoxide

(DMSO) aqueous solution to 1 ppm (mg/L) was added

dropwise on the seeds in each test plot using a

micropipette. In addition, a test plot to which 1 mL of

a 0.1% DMSO aqueous solution alone was applied was used

as a control plot. The number of repeats in each test

plot was 5 (n = 5).

The cultivation was carried out indoor, and the

cultivation conditions were a photoperiod of 16 hours,

°C, an LED light source (Ohgets; model number: VGL

1200W), and a light intensity from 400 to 440 pmol/m 2 /s.

On day 7 after inoculation, the plants were thinned so

that there was one plant per pot. Watering was performed

by adding water to a vat placed under the pot so that

about 5 cm of the lower part of the pot was submerged

after the vat had run out of water. After cultivation

for 25 days, the plant body was taken out from the pot,

the root was washed with water, and then the nodules were

harvested. For the harvested nodules, the nodule fresh

weight and the number of nodules per individual plant

were measured. After the plant body was dried at 100°C

KS1685 - 27

for 3 days, the above-ground part dry weight and below

ground part dry weight of the plant body were measured.

[0045] The measurement results are shown in Figures 1 to

4. The graph of each figure shows mean ± standard

deviation.

Cont., Nar 1, For 1, Bio 1, and Pra 1 in each figure

represent the control plot and the application plots of 1

ppm solutions of naringenin, formononetin, biochanin A,

and pratensein, respectively.

[0046] The nodule weight showed an increasing tendency of

% in the formononetin plot, 26% in the biochanin A

plot, and 71% in the pratensein plot, with respect to the

control plot (Figure 1). The number of nodules showed an

increasing tendency of 15% in the formononetin plot, 25%

in the biochanin A plot, and 38% in the pratensein plot,

with respect to the control plot; however, no increasing

tendency was observed in the naringenin plot (Figure 2).

The above-ground part weight showed an increasing

tendency of 19% in the formononetin plot, 5% in the

biochanin A plot, and 23% in the pratensein plot, with

respect to the control plot (Figure 3). The below-ground

part weight showed an increasing tendency of 34% in the

formononetin plot, 5% in the biochanin A plot, and 11% in

the pratensein plot, with respect to the control plot;

however, no increasing tendency was observed in the

naringenin plot (Figure 4).

KS1685 - 28

[0047] Example 2: Effect of methylated isoflavone on

yield of chickpea

Medium-term fertilizer effect type culture soil

(Takii water-containing cell culture soil, Takii & Co.,

Ltd.) and vermiculite (Akagi-Engei Co., Ltd.) were mixed

at a volume ratio of 1:1, and the soil was placed in a

pot (diameter: 30 cm, height: 30 cm). A total of 4 seeds

of chickpea (kabuli species) were each sown in 4 sowing

holes provided at a depth from about 1 to 2 cm near the

center of the pot. In the same manner as in Example 1, a

bacterial solution of a chickpea rhizobium (Mesorhizobium

ciceri) NBRC100389T strain whose value of turbidity

(OD6oo) was adjusted to 0.02 (7.0 x 106 CFU/ml) using

sterilized water was inoculated dropwise on the seeds.

[0048] Subsequently, 0.1 pg or 1 pg of methylated

isoflavone and a comparison compound were applied in the

following manner. Specifically, 1 mL of a 0.1 ppm or 1

ppm solution of naringenin or pratensein dissolved in a

0.1% DMSO aqueous solution was added dropwise on the

seeds in each test plot using a micropipette. In

addition, a test plot to which 1 mL of a 0.1% DMSO

aqueous solution alone was applied was used as a control

plot. The number of repeats in each test plot was 11 (n

= 11).

[0049] The cultivation was carried out in a glass

greenhouse with sunlight from November 2018 to May 2019.

The air conditioning in the greenhouse was set to 20°C.

KS1685 - 29

On day 11 after sowing, the plants were thinned so that

there was one plant per pot and so that individuals with

no apparent growth problems were left. As a rule,

watering was performed by irrigation with an appropriate

amount of water with a shower once a week, and additional

irrigation was performed as appropriate when the soil

surface became dry. After the pods dried naturally, they

were harvested, and the grains (beans) were taken out

from the pods. For the obtained grains, the grain dry

weight and the number of grains per individual plant were

measured.

[0050] The measurement results are shown in Figures 5 and

6.

The graph of each figure shows mean ± standard

deviation. The abbreviations in each figure are the same

as those in Example 1. The grain weight showed an

increasing tendency of 5% in the 0.1 ppm pratensein plot,

and 25% in the 1 ppm pratensein plot, with respect to the

control plot (Figure 5). The number of grains showed a

significant increase of 92% in the 0.1 ppm pratensein

plot, and 55% in the 1 ppm pratensein plot, with respect

to the control plot (Figure 6). A significant difference

test was performed using the Williams method, and an

asterisk (*) was added when a significant difference

(P<0.025) was observed.

[0051] Example 3

KS1685 - 30

Table 1 shows the results of measuring the protein

content of the chickpea grains harvested in Example 2,

and converting it into protein yield. The protein yield

showed an increasing tendency in the 1 ppm pratensein

plot. The protein yield (g/strain) was determined by

grain weight (g/strain) x {grain protein content (g/100

g)/100}. The protein content of the chickpea grains was

measured in Japan Food Research Laboratories. The

combustion method was used for the measurement, and the

protein content was calculated by multiplying the

quantified total nitrogen by 6.25 as a nitrogen-protein

conversion factor.

[0052] [Table 1]

Cont. Nar Nar Pra Pra (DMSO) 0.1 1 0.1 1

Grain protein content 22.4 22.6 22.8 21.0 21.3 (g/100g)

Protein yield 0.56 0.58 0.58 0.55 0.66 (g/strain)

[0053] Example 4: Synergistic effect of combined use of

rhizobium and pratensein

Various microorganisms are present in the actual

chickpea cultivation soil, and it is desired that the

effects of rhizobium inoculation and methylated

isoflavones are exhibited even under such conditions. In

this example, the influence of the combined use of

KS1685 - 31

rhizobium inoculation and a methylated isoflavone on

nodule formation was evaluated under conditions in which

a soil bacterial solution prepared by extracting

microorganisms from field soil, described below, was

mixed with the culture soil, namely conditions similar to

actual cultivation soil. The evaluated test plots 1 to 6

were as shown below.

Test plot 1. without soil bacterial solution

Test plot 2. with soil bacterial solution

Test plot 3. with soil bacterial solution + rhizobium

inoculation

Test plot 4. with soil bacterial solution + rhizobium

inoculation + pratensein application

Test plot 5. with soil bacterial solution + rhizobium

inoculation (pratensein was added to the medium on the

previous day)

Test plot 6. with soil bacterial solution + pratensein

application

[0054] (1) Preparation and sowing of soil bacterial

solution

Medium-term fertilizer effect type culture soil

(Takii water-containing cell culture soil, medium-term

fertilizer effect type, Takii & Co., Ltd.) and

vermiculite grains (Akagi-Engei Co., Ltd.) were mixed at

a volume ratio of 1:1, and the soil was placed in a

polypot (diameter: 10.5 cm, height: 9 cm). 500 mL of

sterile milliQ water was added to 500 g of field soil in

KS1685 - 32

Tochigi Prefecture, and the mixture was stirred, followed

by filtration through circular quantitative filter paper

No. 5A (ADVANTEC) to obtain a filtrate, which was used as

a soil bacterial solution, and the bacterial solution was

supplied in an amount of 45 mL per pot (test plots 2 to

6). In the test plot 1 without a soil bacterial

solution, only sterilized water was supplied in the same

amount. In order to adjust the water content of the

soil, tap water was separately supplied in an amount of

200 mL per pot. The number of repeats in each test plot

was 4 to 6 (n = 4 to 6). Two seeds of chickpea (kabuli

species) were sown in each pot at a depth from about 1 to

2 cm from the soil surface.

[0055] (2) Inoculation of rhizobium

1.5% agar (Wako Pure Chemical Industries, Ltd.) was

added to Yeast-Mannitol (YM) medium (0.5 g of K2HPO 4 , 0.2

g of MgSO 4 -7H20, 0.1 g of NaCl, 0.4 g of yeast extract, 10

g of mannitol, and 1 L of distilled water (pH: 6.8)) to

prepare a solid medium, and a chickpea rhizobium

(Mesorhizobium ciceri) NBRC100389T strain was stationary

cultured on the solid medium at 30°C for about 72 hours.

A platinum loop of the grown rhizobium was taken and

inoculated in 50 mL of YM liquid medium in a Sakaguchi

flask with a volume of 500 mL, and shake-cultured at 30°C

for about 72 hours. On the day before sowing, 1 mL of a

rhizobium culture solution whose value of turbidity

(OD6oo) was adjusted to 0.1 (3.5 x 107 CFU/ml) using

KS1685 - 33

sterilized water was inoculated dropwise on the seeds

using a micropipette at the time of sowing (test plots 3

and 4). Further, on the day before sowing, the turbidity

of a solution prepared by adding 1.5 pg of pratensein per

mL of the culture solution to 1.5 ppm of pratensein

(containing a final concentration of 0.1 (v/v)%

dimethylsulfoxide), followed by culturing for 24 hours

was also adjusted in the same manner as describe above,

and 1 mL thereof was inoculated at the time of sowing

(test plot 5).

[0056] (3) Application of pratensein

1.5 pg of pratensein was applied in the following

manner. Specifically, 1 mL of a solution of pratensein

(Chromadex) prepared by dissolving pratensein to 1.5 ppm

(containing a final concentration of 0.1 (v/v)%

dimethylsulfoxide) was added dropwise on the seeds using

a micropipette (test plots 4 and 6).

[0057] (4) Cultivation conditions

The cultivation was carried out indoor, and the

cultivation conditions were a photoperiod of 16 hours,

°C, an LED light source, and a light intensity from 400

to 440 pmol/m 2 /s. On day 7 after sowing, the plants were

thinned so that there was one plant per pot. Watering

was performed by adding water to a vat placed under the

pot so that about 5 cm of the lower part of the pot was

submerged after the vat had run out of water. After

cultivation for 21 days, the plant body was taken out

KS1685 - 34

from the pot, the root was washed with water, and then

the nodules were harvested. For the harvested nodules,

the nodule fresh weight and the number of nodules per

individual plant were measured. After the plant body was

dried at 100°C for 3 days, the above-ground part dry

weight and below-ground part dry weight of the plant body

were measured.

[0058] (5) Results

The measurement results are shown in Figures 7 and

8. The graph of each figure shows mean ± standard

deviation. A significant difference test was performed

using the Tukey-Kramer method, and different characters

(a, ab, and b) between the groups indicate that a

significant difference (P<0.05) was observed.

In the test plots 1 and 2, and the test plot 6,

which was a single application plot of pratensein, almost

no nodules grew. In the test plot 3, the number of grown

nodules increased by rhizobium inoculation; however, the

effect was limited. In the test plot 4 in which

pratensein was applied at the same time as the rhizobium

at the time of sowing, and the test plot 5 in which a

product prepared by previously adding pratensein to a

rhizobium culture solution, followed by culturing for 24

hours was inoculated, the number of nodules was 2.3 times

and 2.5 times, respectively, with respect to the test

plot 3 (Figure 7). Similarly, the nodule weight was 2.3

KS1685 - 35

times and 2.2 times in the test plots 4 and 5,

respectively, with respect to the test plot 3 (Figure 8).

The above results revealed that in both the case

where pratensein was applied at the same time as the

rhizobium, and the case where pratensein was previously

added to a rhizobium culture solution, the nodule-growing

effect was synergistically enhanced in comparison to the

single inoculation of the rhizobium or the single

application of pratensein.

Claims

The claims defining the invention are as follows:

[Claim 1] A methylated isoflavone of the following

formula (1') when used in promoting chickpea nodule

formation:

HO O

(1') R1O/ OCH3 R2

wherein R1 and R 2 both represent a hydroxy group.
[Claim 2] A methylated isoflavone of the following

formula (1') when used in enhancing the yield of

chickpea:

HO O

(1') R1O/ OCH3 R2

wherein R1 and R 2 both represent a hydroxy group.
[Claim 3] A composition for promoting chickpea nodule

formation or enhancing the yield of chickpea, comprising

a methylated isoflavone of the following formula (1):

HO O

(1) Ri O OCH3 R2

wherein R1 and R 2 both represent a hydrogen atom or a

hydroxy group,

and a rhizobium using chickpea as a host.
[Claim 4] The composition according to claim 3,

wherein the rhizobial count cfu per tg of the methylated

isoflavone, which is the content ratio of the rhizobium

to the methylated isoflavone, is from 105 to 5 x 109.
[Claim 5] The composition according to claim 3 or

claim 4, wherein the contents of the rhizobium and the

methylated isoflavone per gram of the composition are

such that the rhizobial count is from 104 to 1011 cfu and

the methylated isoflavone content is from 0.01 to 10,000

pLg.
[Claim 6] A method for promoting chickpea nodule

formation, comprising the step of applying a methylated

isoflavone of the following formula (1'):

HO O

(1') R1 O OCH3 R2

wherein R1 and R 2 both represent a hydroxy group,

to soil or a plant.
[Claim 7] A method for enhancing the yield of

chickpea, comprising the step of applying a methylated

isoflavone of the following formula (1'):

HO O

(1') R1 O OCH3 R2

wherein R1 and R 2 both represent a hydroxy group,

to soil or a plant.
[Claim 8] A chickpea cultivation method, comprising

the step of applying a methylated isoflavone of the

following formula (1'):

HO O

(1') R' O OCH3 R2

wherein R1 and R2 both represent a hydroxy group, to soil or a plant.
[Claim 9] A method for promoting chickpea nodule

formation, a method for enhancing the yield of chickpea,

or a method for cultivating chickpea, comprising the step

of applying a methylated isoflavone of the following

formula (1):

HO O

(1) R1 O OCH3 R2

wherein R1 and R 2 both represent a hydrogen atom or a

hydroxy group, in combination with a rhizobium using

chickpea as a host to soil or a plant.
[Claim 10] The method according to claim 9, wherein a

composition comprising the methylated isoflavone and a

rhizobium using chickpea as a host is applied to soil or

a plant.
[Claim 11] The method according to claim 9 or claim

, wherein the rhizobial count cfu per tg of the

methylated isoflavone, which is the ratio of the amounts

of the rhizobium and the methylated isoflavone applied,

is from 105 to 5 x 109.
[Claim 12] The method according to any one of claims 6

to 11, wherein the soil is soil supplemented with a soil

bacterial solution prepared from soil which allows

cultivation of chickpea.