Identification of strains of Mesorhizobium huakuii, root nodule bacteria of Astragalus sinicus, by the polymerase chain reaction

Ishikawa Agricultural College

NII-Electronic Library Service

Ishikawa

4AgriculturalCollege

BuiLRIAR,IshikawaAgr.Coli.6:6T-66(]999)

Identification

of

strains

of

Mesothizobium

huakuii,

root

nodule

bacteria

of

Astragatus

sinicus,

by

the

_polymerase

chain

reaction

Masao

TAcHIMoTO

I.aboratory

of

Microbiological

Resources,

Research

Institute

of

Agrieultural

Resources

Ishikawa

Agricu]tural

College,

Nonoichi,

Ishikawa,

921-8836,

JAPAN

Strains

of

Mesof'hi.zohium

huakuii,

root nodule

bactcria

of

Astf'agaius

sinicus.. were

identified

from

DNA

_po]ymorphism

amplified

by

the

_polymerase

chain reaction

(PCR)

using somc

known

()rrandom

prim-ers.

Five

strains of

M.

huakuii

isolated

in

japan

and

China

were examined using

SPH

(a

random

_primer),

nit/HDK

(a

_part

of

the

sequence of n(f'

_gene)

and

ERIC

(both

ends of an repetitive

intergenic

consensus

DNA

sequence ofenteric

bacteria).

DNA

bands

amplified

with

_prirners

showed

distinctly

ditferent

band

_patterns

between

_groups

of strains

isolated

in

Japan

and

those

iso]uted

in

China.

Although

it

was sometimes

difficult

to

idcntify

strains

be[onging

to

the

same

_group

w'ith

on[y

one

_primer,

strains

of

a

_group

could

be

identit'ied

by

cornparison of

Lhc

resu]ts obtained with

difi'erent

PCR

_pi'imers.

Strains

of

M.

huakuii

iso]ated

randomly

from

a rice

field

soil were examincd

by

PCR,

and several

typcs

of strains

were

found

to

survive

at

the

same

site.

This

identifieation

method using

PCR

was

also

usefu1

to

investigate

the

infection

rate

of

inoculatcd

strains

in

a

pot

cultivation

experiment.

Key

words :

Mesorhizobittm

huakuii,

Astragaius

siniciis,

PCR,ERIC,

nifHDK

Introduction

The

taxonomy

of root nodule

bacteria

of

Chinese

milk vetch

(Aslragafus

sinicLts

L.)

i'emained unclear unti]

it

was classificd as

Rhizobittm

huakuii

by

Chen

et at.

in

1991'i'.

It

was reclassifiedi

as

Mesorhizobiuni

huaketii

according

to

its

_{physio]ogical}

and

_phylogenetic

_properties

to

distinguish

it

from

othcr rhi7obial

bacteria.

Chinese

rnilk

vetch

is

a

leguminous

_plant,

bcing

cu[tivated

in

the

temperate

area

of

Far

East

Asia

especially

in

China,

and

it

has

not

bcen

_paid

attention

by

agricu]tural researchers of

the

western world except

in

a

few

casesS'.

The

role of

Chincse

mi]k vetch as

_grecn

manure

in

rice

ficlds,

how-ever,

has

become

lower

in

China

with

the

increase

of oil erops sueh as oi]seed rape as cash

cropsi).

In

Japan

a]so,

Chinese

milk vetch was ",idely

_grown

as a winter

crop

on

rice

tlelds

as

_green

manure.

but

the

cultivation area

of

Ch{nese

milk

vetch

has

rapidly

decrcased

after

World

War

II

with

the

spread

of

chemical

fertilizers.

However,

thc

ro]e of

Chinese

milk vetch

is

now

being

reassessed since

the

soil

fertility

of arable

land

in

Japan

is

though{

to

be

continuously

lower

and

the

estab]ishment of a sustain-ab]e agriculture system

is

required,

The

root nodu]e

bacteria

ofA. sinictts

are

considered

to

survive

as

indigenous

heterotrophic

inhabitants

of

Japa-nese soils,

because

Chinese

milk vetch

has

been

grown on

e

rice

t'ic]ds

since

the

17

th

century.

Therefore

the

nodules

ofA.

sinicus

are

usua]ly

formed

by

indi.g.enous

root nod-ulc

bacteria.

In

such

cases,

thc

et'fect of

the

inoeulation

of

M.

huakuii

must

be

evaluated

by

checking

if

the

root nod-u]es are

formed

by

the

inoculant

strains

or other native ones.

It

is

rather

difficult

to

identify

the

strains

of root nodule

bacteria

by

such conventional mcthods

as

intrinsic

antibiotic

resistance. serologica] reaction, enzymc

linked

immunosorbent

assays

_(ELISA}

or restriction

fragmcnt

lcngth

_{potymorphisms}

_<RFLP)

which require

complicated

procedures.

Recently,

the

_polymerase

chain reaction

(PCR)

using

random

_primers

has

become

one of

the

most

etTective methods

to

identify

bacterial

strains and

it

has

been

used

for

rhizobial

bacteria,

However,

PCR

has

not

been

applied

to

M.

htfakttii.

In

this

_paper,

I

report

the

utility and

detailed

conditions of

PCR

to

identify

strains

ofM.

huakt{ii.

This

method was also applied

to

cstimate

the

infection

rate

of

inocutated

strains on

the

root oi'

A.

sinicets

in

a

_pot

experimcnt.

Mcthods

and

Materials

Strains

_qf'M.

huakuii

Thc

strains used

in

thc

experiment wcre

the

five

strains

Iisted

in

Table

1.

Three

of

them

were

isolated

in

Japan

and the other

two

in

China.

The

strain

103T

is

the

Ishikawa Agricultural College

IshikawaAgriculturalCollege

62

Bullcttn

ot'

RIAR,Ishikawa

Agricuitural

College

Nc).

6

C

T999}

Table

1,Strains

used

iti

the

experinients

Stra]ns

Ongin

i03T(NAU)

1

so05

(ACCC)

B39C)1

([A(t･)

912

cEAC)

Type

stainof it4.

ht"tkttit

CNanjmg

A.gricultural

Universit.v.

China)

Stram

used in

China

tAgrieultural

Culture

Co]lectton.

Chtna)

IKoliLteb},

N{urooka,

Hirosh"na

Univ

.Jupan.

Isolatc

b},

Tokachi

Federation

ot'

Agr.

Coop

.

Japan

Isolute

in this.i'F.iF.a-rs/!.!s.bl

ls.eniL.As,r.

Coll,Japan

.

1'able

2,Primers

uscd

for

PCR

Ss,mbolSPH

1n]fHDKER]C

1RERIC

2

Base

seguenve

5'-GACGACGACCGACGAC-3.'

5'-GGTTATCGAAATCAGCAGCCACAGCGC-3'

5'

ATGTAAGCTCC.T(}GGGA']'1'CAC-3'

5'-AAGTAAG']'GACI'GGGGTGAGCG-3'

Rcmark

Random

_primer

Part

ei'mf

_gene

In{crgcmcconsensusrepelitn,c

scquence et'enteric

bacterui

Reference

6)

9)

4)

type

strain of iW.

ht.takttii.

Prci)at'ation

of'

l)iYTA

_,fbr

PCR

The

bacteria

to

be

tested

were cultured

in

TY

liquid

mcdium

(Bactotryptonc

<Difco)

5

_g,

_yeast

extract

CDit'co)

3

_g,

CaC12'6

H]O

1.3

_g.

disti11eci

water

1

L,

_pH

7.0)

at

26

℃

ft)r

aboul

t-･o

da>'s

until

the

logarithmic

_growth

stage,

The

cultured

bacteria]

body

was

colleeted

b>,

centrifuga-tion

(1O.OOOXg,

1O

mm),

and

washed with

_{ph>,siological}

saltne solution.

Tbe

eo]lectcd

bacterial

bodx,

was

sus-pended

with

an

adequate

amounr of s.terilized watcr and ciispensed

into

1.5

mL mlcrotubcs and centrifuged again.

To

the

_precipitat]t

was added

200

pl.

ot'steri]izcd water,

and

the

suspension was stocked ut

20'C.

The

suspen-sion

was melted

just

befoi'e

use, and

tO

_,uL

of

Proteinase

K

solution

(1

mg mL i) and

50

yl.

of

BL

buffcr

solution

(Tris

40

mM,

Tween-20

1

9E･,

Nonidet

P-40

O.5%,

EDTA

･

2

Na

1

mM.

_pH

8.0)

were aclded

and

incubated

at

60

℃

for

20

mm

to

dissolvc

the

bacterial

body.

The

reaction was

-stopped

by

heating

at

95

℃

t'or

IO

min and

the

supernatant was

co]lected

by

centrifugation and used as crude

DNA

so]ution.

The

c)ptica]

densjty

at

260

nm of

the

crude

DNA

solutton w'as measured and

the

concentration

of

DNA

was adjusted

to

150

_yg

_uL

i

_tbrPCR,

1]CR

The

reaction

so]ution contained

lO

uL of

10

×

buffer

i

(Takara

Bio),

1.5rnM

rvls,C]!,

O.2mM

dNTP,

1pM

Primer<s),

2.S

unlts ot'

Taq

DNA

po]ymerase

CTakara

Bi())

and

5

pL

oi'

the

diluted

erude

DNA

solution

c750

_pg

as

template

DNA).

the

totah,olumc

ol'which was adjusted

to

100

_pL.

The

_primers

uscd m

PCR

were

as

listed

in

Table

2.

SPH

1

was

a

random singlc

_primer

and

the

oth-ers were

directed

_primers

that

are a

_part

of a

known

sequence

(nifHDK)

or of a conscnsus sequence

(ERIC).

The

reaction mixture was

taken

into

a

1.5

ml. microtube.

over[aid with

]OO

pL

ef mineral oil and

set

in

a

thermal

c.vcler

(Astec

PC-700).

The

te;nperature

condition

ef

PCR

with

SPH

1

and

nitl-IDK was as

follows

i

pre-run

at

94

℃

for

1

min,

30

cycles of

denaturjng

at

94

℃

for

1

min, annealing at

SO

℃

for

1

min,

_{polymeriLation}

at

72'(.;

for

1,)'

min, and

_posl-run

at

65

℃

for

2

min,

The

tempera-ture

condition of

PCR

with

ERIC

_priiners

was as

{'ollows

_;

pre-run

at

95

℃

foi'

5

min,

30

c>,cles

of

denature

at

94

℃

for

min, annealing

at

52

℃

for

]

min,

_{po]ymerization}

aL

65

℃

for

8

min,

and

_post-run

at

65"(:

for

16

min.

The

PCR

products

werc separated

from

minera] oi]

by

addition ot'

100

uL

of

chloroform

isoamyl

alcohol mixture

(24

:

1

vl

v)

and

electi'ophorcscd

with

2`7c,

agarose

(.ttmplisize

aga-rese,

Bio-Rad)

in

[XTBE

(pH

8.2)

under a

1(}O

V

for

about

45

tnin.

As

DNA

size markers, a

IOObase-pair

ladder

and

Kilo

base-pair

[adder

(Pharmacia)

wer'c

elec-trophoresed

at

the

sume

time.

The

agarose

s,e]

was

staincd with

ethidium

bremide

solution

(2

_,ug

L-i)

and

DNA

fragments

wcre

detected

under

UV

light

of

26e

nm.

inoc'ulation

e.xl?eritnei'tt }i,ith

the

strains of root Jtoduie

hacteJ'ia

Air-dried

soi]

_(gray

low[and

soil, sand.v

cluy

toam)

was mixed with river sancl at

the

i'atioof

1

:

1,

and

2.5

kg

of

the

mixture

_pcr

Wagner

_pot

of

]15,OOO

a was used

i'or

the

_pot

cultivation cxperimen{.

Nodu]e

bacteria

shown

in

Table

1

k-'ereculturcd

tn

TY

liquid

medium

at

26

℃

for

about

two

days

and

the

suspension of

bacteria

(3'--9

×

1OS

CFU

mL-i,

2

inL

_pe;'

_pot)

was

inoculated

to

the

seecllings ofA. sinic'us

(cv.

Gifu-hinode-wasc).

which were

sown

at

Ishikawa Agricultural College

NII-Electronic Library Service

Ishikawa

'AgriculturalCollege

63

TACHTMOTO

:Identi

ficanon

ofstrains of

Meso

nhizobium

huakuii

by

PCR

the

density

of nine

_per

_pot,

Calcium

superphesphate and

potassium

chloride were supplied as

basal

fertilizers

at

the

rate of

O.5

_g

_per

_pot

as

P20s

and

KiO,

respectively.

A.

sinictts was

_grown

in

a

_greenhouse

at about

20

℃

for

2

months.

The

cultivation

experiment was carried out with

three

rep]icates.

The

_growth

and

nitrogen content ofA. sinicus were measured

after

harvesting,

and

the

root

nodules were counted and randomly sampled at

_the

rate

of

ten

nodules

_per

treatment.

The

isolated

bacteria

from

colonies

fbrmed

on

_yeast

extraet mannitol agar medium

(YM)

were

subjected

to

PCR

and examined

fbr

nodule

i'c)rmution

by

inocugated

strains

or other

indigenous

strains of

M.

huakuii,

Results

and

Discussion

DNA

fragments

were amptified with any

_primers

used

in

this

experiment

as shown

in

Fig.

1.

Electophore-sed

DNA

band

_pattems

were similar

in

each

_group

of

Chinese

strains and

Japanese

strains,

and

it

was easy

to

determine

to

which

_group

isolated

strains

be]onged,

Within

a

_group,

identifieation

with

on]y

one

_primer

such

NcSSSi

LSPst/Sfi,g･cs'/di3'

siss'.6S.IStb{}iesi/di3f.cS5'

';tt.t'

t.t

ERiC

nifHDK

Fig.

1,

_DNA

_bancl

_patterns

of testetistrains of

Mesorhi.zohit{m

huakuii

by

PCR

.

Le

ft,

ERIC

primers

;

Right,

nitHDK

_primer,

M

1OO,

1OO

base-pair

[adder

DNA

sizc marker ;

Mk,

kilo

base-pair

iadder

DNA

size marker

;

NC,

negative control,

s8"gr

g

i

2

3

4

s

6

7

s

g

w

$8

Fig

2.DNA

band

_patterns

oi' strains

iso[ated

t'rom

a

ficld

soil

by

PCR

with nifHDK

_primer.

M

100.

1OO

basc-pair

ladder

DNA

size rnarker

_;

Mk.

k][o

base-pair

ladder

DNA

stzemarker ;

NC,

negative eonLrol,

as nifHDK was sometimes

difficutt,

but

the

strains

cou]d

be

identified

by

comparison of

the

resu]ts obtained with

different

_primers.

This

PCR

method

is

considered

to

be

useful

to

investigate

the

int'ection

rate of

inoculated

nod-ule

bacteria

in

A.

sinicus.

The

nodule

bacteria

isolated

from

nodules

which were randomly sampled

frotn

A.

sinicus

_grown

in

one

rice

field

showed

various

DNA

_{po]ymorphisms}

by

PCR,

This

suggestes

that

various

types

of

indigenous

strains of

M,

huakuii

inhabit

even

in

the

soi]

of

the

same site.

The

inoculation

experiment

was

carried

out with

Wagner

_pots

of

lf5,OO()

a

to

confirm

the

effect of strains of

M.

h"akttii

as

inoculants

in

a condition where

indige-nous nodule

bacteria

survived.

As

shown

in

Table

3,

the

growth

and nitrogen content

were

significantly

stimulated

by

the

inocu]ation

of

13005,

B3

and

901

except

I03T,

The

investigation

of

isolates

from

nodules of

A.

sinicus

by

PCR

(Figs,

3

and

4)

suggested

that

a

high

_percentage

of

the

nodules

were

infected

with

the

inoculants

excepL

103T,

As

summarized

in

Tablc

4,

the

infection

rate with

103T,

13005,

B3

and

901

was

O%,

100%,

639e

and

88%,

respectively.

AIthough

103T

is

the

type

strain

of

M.

huakuii,

it

seemed

to

]ack

a

_plasmid

containing nod

_gene

which

is

related

to

the

nodulation

of

leguminous

_plantsi'.

The

ability of

103T

to

form

nodules

in

A.

sinicus was not

confirmed

also

in

this

experiment.

Grewth

and

tota]

nitrogen

content

of

the

_plants

{noculated

with

the

strains

except

103T

were

significantly

higher

than

the

eontrol without

inocu]ation,

but

there

was no significant

differ-ence

between

the

three

_inoculants

of

l3005,

B3

and

901.

There

was no significant

diderence

in

the

number of

big

nodules

(>3

mm

in

diameter)

between

the

treatments,

but

the

total

number of nodules was significantly

in-creased

by

the

inoculation

treatments

except

103T.

It

is

estimated

that

inoculation

treatments

caused

_the

increase

of small nodu]es

in

A,

sinicus.

The

soil

used

in

this

experiment was estimated

_to

have

ahistory

of

_growingA.

sinicuh'.

However,

as

it

was used

in

an air-dried condjtion.

the

density

of

indigenous

M,

huakuii

was rather

low.

Iess

than

10]

CFU

_g-i

and

this

might

have

caused

the

high

inoculation

effect

in

this

experiment,

It

may

be

necessary

to

confirm

the

effecL

of

inocu]ation

in

fresh

soil.

The

inoculation

of some

strains

of

M.

huakuii

was

_quite

effective

for

Chinese

milk vetch

in

soils which

had

no

history

of

_growing

Chinese

milk

vetch

in

such countries as

U.

S.

A.

and

Nepal]'").

How-ever,

in

China

where

Chinese

milk vetch

had

been

_grown

for

a

long

_period.

the

effect

of

inoculation

was sometimes not so clear2) and

it

has

not

been

cont'irmed whether nod-ules

of

Chinese

milk vetch were

really

formed

by

Ishikawa Agricultural College

エshlkawa 　Agrloultural 　College

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　arld 　dno 、、 丶md ［cule 　Lhご】neculatcd 、［lam、_〔

130

〔〕

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、

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t

’

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尋

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3

τ

ぱ

13

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go

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pattern

丶ol 匸s〔、

lutc

〜in　the　

p

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b

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p

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Ml

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100

　

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DNA

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Mk

、

　

klk

〕

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丶

e

−

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I

．

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DN

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ke

［

、

NC

、

　ncg

、

虹nvc し〔｝nU 〔｝

l

　

The

Ishikawa Agricultural College

NII-Electronic Library Service

IshikawaAgriculturalCollege

65

TAcH[MoTo

/

Identification

ofstrainsof

n4esorhi.7obi"m

huakuii

by

PCR

Table

3.

Inoculation

test

by

pot

cultivation

-. ･-

=.･

StrainsShoot

growth

N

content mg

_per

_potRoot

nodules

per

plantInt'ecnonrate

D.W.

(g)

perpot

big<>S

mm) totalinfccted1tolal(`",)

Control103T1300S901B3

3.3ttO,18a'

3.82

±

O.13a

5,29

±

O,7eb

6.59

±

O.30b

620

±

･

O.77

b

107

±

1a

153

±

8

ab

244

±

30be

300

±

.

17

c

276

±

27c

2.96.01.23.54.5

8a

18a1OO

b146c103

bc

O/8

₍

_O)

10tlO(100>

5f8

(

63)

7f8

₍

88)

*

_Same

_]etters

_indieate

_{no significant}

_difference

_by

_Duncan's

_{rnultip]e range}

_test

_at

p=O.OS

(n=3).

Tabie

4.

Infection

rates of

inoculated

strains

in

the

_pot

expcriment

Pnmer

Inoculated

strain nitHDK

ERIC

+

±

+

±

Infection

rate

infected1totat{%.)

I03T13005B3901

o*103s 7o53

2ooo

o1075 oel2

8ooo

_0f8<

_O)

1OAO

_(1OO)

518

(

63)

718

(

88)

+,

same asthe

inoculated

strain

±, almost

thc

same

but

part]y

different

from

the

ino

¢uluted strain

--,

different

from

theinoculated strain

' _{number of}

_iso]ates

_C=

_nodules)

The

PCR

method

with adequate

_primers

is

thought

to

be

usefu]

_to

identify

the

strains

of

M.

huakuii,

but

there

were some cases

in

which

accurate

identification

was

dif-ficult

because

of

low

reproducibi1ity

of

DNA

band

_pattern.

It

was necessary

to

_perform

PCR

amplification

with

stan-dard

strains under

the

same conditions.

To

increase

the

reliability

of

the

PCR

method, we must consider

the

cul-ture

conditions,

refining

_procedure

of

DNA

templates,

temperature

set

of

PCR

and

so

on.

As

it

is

known

that

M.

huakuii

easily

lose

_plasmids

during

culturc]']3),

the

change

of

_plasmid

composition

dur-ing

the

culture or

in

the

soil

may

be

one

of

the

factors

causing

the

variation of

DNA

band

_putterns.

Acknowledgements

The

author

is

_gratefu1

to

Prof.

Murooka,

Hiroshima

University

_(currently

Osaka

University)

for

_providjng

the

strains

of

103T

and

B3

of

M,

huakuii,

and

the

_Renge

Society

of

Japan

fbr

supplying

the

strain of

13005.

Literature

Cited

1)

Chan,

C.

L.,

T,

A.

Lumpkin

and

C.

Characterization

of

Brad.vrhizobiumS.sP'Root

1988.

(Astragatus

sinicus

L,)

using

serological agglutination,

intrinsic

antibiotic resistance,

_plasmid

visualization, and

fie]d

peifbrrnance.

Plant

and

Soil

109

:85-91.

2

₎

Chan,

H.

K.,

F.

D.

Li

and

Y.

Z.

Cao

1992.

istics,

distribution,

ecology and utilization

of

galus

sinicus-rhizobia symbiosis.

in

"The nitrogen

fixation

and

its

research

in

China,"

Springer-Verlag,

Shanghai

Scientific

and

Technical

Publishers,

hai,

439-455.

3

₎

Chen,

W.

X.,

G.

S.

Li,

Y.

L.

_Qi,

E.

T.

Wang,

H.

L,

Yuan

and

J.

L.

Li

1991.

Rhizobium

httakuii

sp. nos,.

iselated

from

the

root

nodules

ol'

Astiugalus

sinicus.

Int.

J.

Syst

Bacteriol,

41(2)

:

275-280.

4)

de

Bruijin,

F.

J.

1992.

Use

of repetitive

(repetitive

extragenic

palindromic

and enterobacterial repetitive

intergeneric

consensus)

sequences and

the

erase chain

reaction

to

fingerprint

the

_genomes

of

Rhi.7.ohium

meliloti

isolates

and

other soil

bacLeria.

App.

Environ.

Microbiol.

58(7)

/

2180-2]87,

5

)

Dooley,

J,

J.,

Hanison

S.

P.,

L.

R,

Mytton,

M.

Dye,

A,

Gresswell,

L.

Skot

and

J,

B.

Beeching

1993.

Phylo.oenetic

grouping

and

identification

of

hittm

isolates

on

the

basis

of

random

amp]ified

morphic

DNA

_profiles,

Can.

J.

Microbiol.

39

:

Ishikawa Agricultural College

エshikawa 　Agrioultural 　College

66

Bullctin

　Df 　

RIAR

，

且shlkaw こ虹

A

_呂11icultural 　

College

　

No

．

6

_〔

1999

_）

6

）

Harrison

．

　

S．

　

P

．

，

　

L

．

　

R

．

　

My

宅

toll

，

　

L，

　

Skot，

　

M

，

　

Dye

，

　and

　 　

A

，

　

Gressw・

eH 　

l992

．

　

Characterizalion

　oll　

R

’z’ぞo わ此〃ア1

　　is

〔｝

lates

　

by 　

amphfica τ

ioll

　of　

DNA 　polymorphtsms

　　

using 　1

』

alldom 　

primcrs

、

　

Can

．

　

J

．

　

Microbiol

、

38

：

iOO9

＿

　 　

1015

．

7

）

Jarvi

〜

，

　

B

．

　

D

．

　

W

，

，

　

P

．

　

Van

　

Bcrckum

，

　

W

．

　

X

．

　

Chen，

　

S，

　

M ．

　　

N

〔｝ur　and　

M

．

　

P

．

　

Fernadez

　

l

　

997，

　

Transfer

　

of

　

1

〜

hizo−

　　

∠フ

1

雄 η

loti

．

ノ〜

hio

，

，

θ

bit

・

tηi

．

　

ht

‘akt

・

tii

、

尺

Jzi

こ0わ

it

’

t〃1（

」

_iCE

．

厂

1

，

RhiN

　　

こob ’‘〃 ηiηedile ”rafteL〃 η

，

and　

Rhi

こθ

hii

〃11　

ti

αn

．

shanense

　　

：oMey θ 厂

hi

：oわ

ie

”n　

gen．

　

nov

．

　

ll

〕

t

．

　

J．

　

Sys

し

Bacterio1

．

47

　　 （

3

）

：

895−898．

8

）

Lumpkin

．

　

T

．

　

A

．

1993

．

　

In

しroduction 　of　

Chinese

　Inilk

　 　vetch 　

to

　coulltries 　 other 　

than



EasL

　

Asia

　and 　

its

　　

1

’

eSeal

−

Ch

．

〃t



’

‘

Renge



∠enShO



（

AII

　ab〔｝Ut　

Chinese

　　

mi 】

k



vetch ）∴



T

，

　

Yasue



（

Ed

．

）

，

　

N

〔，

sansongyoson

　　Kyokai

，

Tokyo

，

130

−

S43

（

in

　

japanese

）

．

9

）

Richardson

，

　

A

，

　

E

．

．

　

L

．

　

A ．

　

Vieca1

’

s

、

　

J．

　

M

．

　

Watso

冂 and

　 　

A

．

H

，

　

Gibson



1995

．

　

Differentiation

〔）

f

尺ん

izob

〜L，〃7

　　

strains　using 　tlle　

_polymcrase

　chain 　reaction 　with 　1

’

a11

−

　　

dom

　and　

directed

　

primers

．

　

Soil

　

Bio1

．

　

Blochcm

．

27

：

　 　

515−524．

10

，

Tan

，

　

Z

，

　

Y

．

，

　

X

．

1

．

）

．

　

Xu

，

　

E

．

　

T

．

　

Wang

，

　

J

．

　

L

　

Gao

，

　

E

　　

Martinc

∠

−

Romero

　and 　

W

．

　

X

．

　

CheD

　

l997

．

　

Phyloge

−

　　

netic



and



_genetjc



reiationship



of



IWesc冫厂

hi

．

zθ

hit

‘〃 ’

　　

ticlils11C

〃IEtils（t　

and

　

related

　

rhizobia

．

　

lnt

．

亅

．

Syst．

　

Bacte−

　　

riol

．

47

〔

3

）；

874−879．

lDYasue

_，

　

T

．

1991

．

Thc

　changc ⊂）

f

　cuhi ＞ation　and 　u1Hi

−

　　

zation 　of　

Chmese

　nlilk　vetch

_（

Astrag

‘ilus

．

s’nicl‘

．

s

’

L

，

）

，

　　

alld

　

the

　effect　of　

fertilizei

・

and　soil　

fcrtility

　

in

　

paddy

　　

ficld

　as　a　

grcen

　Inanure

．



Jap

．

　

J

、

　

Crop

　

Science

　

60

〔

4

_）

：

　　

583

−

592

（

1rl　

Japallese

_）

．

12

）

Yasu

じ

，

　

T

．

1993

．

The

　

history

　of　

Chinese

　milk 　vetch 　ln

　　

Japan

　

and



the



change



｛）

f

　

its

　

cultivatk ｝n

　

area

．

　

Il

・

t

　　

‘

‘

Rcngc

　

Zellsho

_〔

All

　

about

　

Chinese

　

milk

　vetc11 _）∴

T

．

　　

Yasue

（

Fd

，

）

，

　

N

〔〕sans 〔〕ngyos 〔｝［l　

Ky

〔，

kai

，

　

Tokyo

、

35

−

　　44

（

mJapan じse

_）

、

i3

）

Xian

，

　

X

，

　

X

．

，

　

J

．

　

C

．

　

Zhou

，

　

Z

．

　

M

，

　

Zhang

，

　

H

．

　

K

．

　

Zhan

　　

and 　

F

．

　

D

，

　

Li

　

l995

．．

Bchavior

〔）

f

　

plasmld

　

pJB5

　

Jhn

　　

1

ぞん

1

ぞでアわ

iL

〃η



httakttii

　under 　

free

−

living



and 　symbiotic

　　

conditiol1 〜

，

　

Current

　

Microbiol

〔｝

gy

　

31

：

97

−

1

〔｝

1

、

PCR

法

に

よ

る

レ ン

ゲ 根 粒 菌

の

菌 株 識 別

［

．

日知

本

［ト

1

夫 （石

1

「「

県

農

業 煙 期 大学 農 業 資

源

研 究 所 微 生 物 資 源 研 究 室

）

　

幾

つかの既 矢

lr

あ

るい は ラン ダ ム の

ブ

ラ

イ

マ

ー

を

_用

い て

DNA

の

．

一

・

_部

を

PCR

増 幅

し

、

その バ ン ドバ タ

・

一

ンを もとに レンゲ

根 粒 菌

の

菌 株 を識 別 す

る こ と を

試

みた。

PCR

反

応には

国 内 外

の レンゲ

根 粒

菌

5

菌 株

を 供 試 し

、

　

SPH

　

1

（ラン ダム

プ

ラ

イ

マ

ー

：

：1

、

_mfHDK

_〔

_{窒 素 固 定 遺 伝 子}

〃

1

_プ

』

の

郡 ：

1

_、

_{お よ び}

_ERIC



_I



_R

_と

_ERIC



2

_〔腸

_{内 細 菌}

の

遺 伝

子 闘

1

叉復 配

列

の

両端

II

の プラ プラ イマ

ー

セッ ト を

用

いた。 いず れの プ ラ イマ

ー

_で

_も

_PCR

_{によっ て}

_DNA

_の

_断

₁

［ 「

が 増 幅 され

、

中

国

産

と 日

本 産

び）レンゲ

根 粒 菌

は そ れ ぞ れ 類 似の バ ン ドパ _タ

・

．

・

ン

を示

し

、

両

グル

ー

プの

識 別

は

容 易

で

あ

っ た。

各

グル

ー

プ

内

の

菌株 闘

の識

別

は ひ とつ の プラ イマ

ー

あ

るいは プラ

イ

マ

ー

セ ッ

ト

だ

け

で は

困 難

な

場 含

も

あ

っ た が

、

各

フ ライマ

ー

に よ る

結 果

を 組 み 合 わ せ ると識 別 が口」

．

能であ り

、

接 種 根 粒 菌

の

感 染率

の

調 査 等

に

利 用

が 可

能

で ある と

考

．

え ら れ る

。

水

田 転 換 畑の 同

．

一

・

圃 場 に 栽 培 さ れた レンゲ の

根 粒

か ら ラ ンダム に

分 離

し たレン ゲ

根

粒

菌

は

．

多様

なバ ン ドパ タ

ー

ン

を

示 し

、

同

．

．

・

［

頭

陽

で

も

数

種

類の レ ン ゲ

根 粒 菌

が

存

：

_在

_{する こ と が}

_示

lift

_{さ れ た}

。

_{ポッ ト}

_{栽 培}

_{に よ る 根 粒 菌 接 種 栽 培 試 験 に お}い て

11

彡

成

さ れ た

根 粒

から

分 離

さ れ た

根

粒

菌

につ いて

PCR

法に よ

・

っ て菌 株 を 調べ た と こ ろ

、

　

・

部の

接 種 菌 株

を 除

き

、

接 種 菌

株

に よる

根 粒 形 成

が

確 認

さ れ た

／

t