Plant Pathogenic Bacteria 36-72

8/14/2019 Plant Pathogenic Bacteria 36-72

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H. GRAM-NEGATIVE BACTERIA

B. Erwinia andPantoea

S. H. De Boer, D. L. Coplin, and A. L. Jones

I. INTRODUCTION

The taxonomic position, nomenclature, and interrelationships of the members of the genus

Erwinia hae been the sub!ect of dierse proposals. The broadest classification is that of D"e #$,

%, &, '(, )hich separatesErwinia into the pectol"tic soft rot "carotovora" group, the "ello)

pigmented "herbicold7group, the )hite nonpectol"tic )ilt*causing "amylovorrf' group, and the

+at"pical+Erwinia. These hae turned out to be alid clusterings based on DA*DA homolog"

studies and -%S seuence homolog" #-$,-&(, but do not completel" agree )ith purel" phenetic

groupings #/0(. Controers" remains on )hether or not the differences are great enough to

constitute ne) genera. 1n the basis of -%S r2A seuence homologies, Hauben et al. #-$(

recommended that these four groups be split into the generaPectobacterium emend,Pantoea gen.no.,Erwinia emend., andBrenneria gen. no., respectiel". 3n this manual, these groups are

recogni4ed as alid diisions of the species )ithin the traditionalErwinia genus but, except for

Pantoea, the recommended generic designations )ill not be adopted at this time. 5urther

taxonomic studies on the relationship of the plant associated enterobacteria*li6e bacteria to those

associated )ith humans and animals ma" reuire further changes in nomenclature.

The follo)ing species and subspecies are currentl" included in the pectol"tic soft rot or

"carotovora" group #p. $%(7Erwinia carotovora subsp. carotovora, E. carotovora subsp.

atroseptica, E. carotovora subsp. betavasculorum #//(,E. carotovora subsp. wasabiae #-8(,E.

carotovora subsp. odorifera #-0(,E. chrysanthemi, E. cypripedii, E. rhapontici #-9( andE.

cacticida (I. Subspecific diision ofE. chrysanthemi has been suggested. Although theheterogeneit" of the species is no) generall" recogni4ed, satisfactor" descriptions of subspecies or

pathoars hae not "et been designated #/, 0, 8, 9, -:, //(.

The +herbicola+ group of "ello) pigmented strains )hich consists of epiph"tic as )ell as

plant pathogenic bacteria, has no) been classified asPantoea #p. &0( together )ith some species of

the genusEnterobacter #-/, -%,-'(. 3nitiall", ;)ing and 5ife #--( recommended, on the basis of

DA*DA homologies, thatEnterobacter a!!lomerans, E. herbicola andE. milletiaebe

combined into the speciesEnterobacter a!!lomerans comb. no. Subseuentl"


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The species in the +am"loora+ #p. 8:( group )ill be considered together )ith the at"pical

er)inias and )ill includeE. amylovora, E. persicina, E. pyrifoliae, E. mallotivora, E. psidii, and

E. tracheiphila along )ithE. alni, E. ni!rifluens, E. paradisiaca, E. uercina, E. rubrifaciens,

andE. salicis #/8(. The latter group comprises the species included in the proposed genus

Brenneria, 3t should be noted that C ratios do not entirel" support the diision of theErwinia

species according to the presence or absence of pectol"tic abilities, since the" suggest a closersimilarit" bet)eenE. amylovora andE. chrysanthemi than bet)eenE. amylovora and the other

species of the "amylovora" group #/-(. eertheless, diision of theErwinia genus on the basis

of phenot"pic characteristics is useful for grouping together those species that reuire similar

protocols for their manipulation )ithin the laborator".

Due to the heterogeneit" of the genus, a general description ofErwinia is necessaril"

limited. As other members of the famil" ;nterobacteriaceae, the er)inia occur as straight rods

#:.$*-.: ? -*0 @@m( singl", in pairs, or sometimes in short chains, are


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'. D"e, D. E. -9%9. A taxonomic stud" of the genusErwinia. 3. The Gat"picalG er)inias. .

F. J. Sci. -/7'00*'09

9. D"e, D. E. -9'-. A numerical taxonomic stud" of the genus Erwinia.. F. J. Agric.

2es. /87//0*//9.

-:. D"e, D. E., J. 5. Bradbur", =.


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-9. Sell)ood, J., and 2. A. Lelliott. -9&'. 3nteraal bro)ning of h"acinth caused b"Erwiniarhapontici. lant athol. /&7-/:*-/8.

/:. S6erman, . B. D., . =c


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. GRAM-NEGATIVE BACTERIA

B-l. Erwinia amylovora Group

A. L. Jones and K. Geder

!. INTRODUCTION

The non*soft rot group in the genusErwinia corresponds to the +am"loora+ group as

proposed b" D"e #%( and to a number of plant pathogenic species described in the last -: to -$

"ears #-:, --, -$, /-, 0:(. The species of Erwinia included in this chapter includeE. alni #0:(,

E. amylovora, E. mallotivora, E. ni!rifluens, E. paradisicina #--(,E. persicinus #-:(,E. psidii

#/-(,E. pyrifoliae #-$(,E. uercina, E. rubrifacinens,E. salicis, andE. tracheiphila .

Although most species in this group are found on different hosts, the" occur primaril" on

trees causing arious blights, can6ers, leaf spots, )ilts, and rots. E. amylovora, the fire blight

pathogen, is not onl" the t"pe species of the genus but it also the most important economicall".

". I#OLATION TECHNI$UE# U#ING DI%%ERENTIAL AND #EMI#ELECTIVE

MEDIA

3solation of non*soft rotErwinia species is often not difficult )hen aboe ground portions

of plant material are isibl" infected. The exception isE. paradisiaca, a pathogen on the roots of

banana. Bacteria occur in high titer in ne)l" infected tissues titers decline and secondar"

microorganisms increase as lesions age. Small pieces of ne)l" inaded )ater*soa6ed tissues from

the leading edge of lesions on leaes, shoots, spurs, and fruits can be plated directl" on suitable

culture media. Alternatiel", infected tissue is comminuted in sterile )ater and a small amount of

suspension strea6ed or dilution plated on suitable culture media. After 0*8 da"s, bacteriaconsistentl" associated )ith infected tissues should be strea6ed on fresh media. Bacterial oo4e

can be strea6ed directl" on the isolation medium. SomeErwinia exist as an epiph"tic bacterium

on flo)ers. ;piph"tic bacteria are detected b" )ashing blossoms in )ater and plating serial

dilutions on culture media.

;ither a standard medium or a semiselectie medium can be used for the recoer" of

Erwinia%li)ebacteria. Standard media such as Luria*Bertani #LB( agar, ing et al.Gs medium B

#B(, or nutrient agar are normall" used )hen pathogen populations are expected to be high in

relation to populations of secondar" microorganisms. These media are normall" supplemented

)ith $: to -:: ug@ml c"cloheximide to inhibit fungal contaminants. Coalescing of colonies can be

a problem )hen plating on B or onto media )ith sucrose. Strea6ing is done in a pattern to

obtain isolated colonies. Dilution of samples in )ater should be sufficient to obtain approximatel"

-:: colonies on a standard agar plate. After incubation at /'C for /*0 da"s, indiidual colonies

can be transferred to fresh plates. Eith experience, an inestigator generall" can distinguish

colonies of a pathogen from miscellaneous saproph"tic bacteria proided the numbers of

contaminating bacteria are relatiel" fe).

40


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Semiselectie media are used for the recoer" ofErwinia from specimens that potentiall"

harbor a significant mixture of attending saproph"tic bacteria. Some media contain chemicals that

inhibit the gro)th of certain bacterial species #selectie(, and someErwinia species ma" form

colonies on the media that can be differentiated from colonies of other bacteria b" their

characteristic color and colon" formation.

a. Re&pes 'or d''eren(al )eda*

!+ Lura-Ber(an ,LB+ aar

perL

Tr"ptone -:.: g

aCl $.: g

Ieast extract $.: g

Agar -$.: g

"+ Nu(ren( aar #A(

perL

Beef extract 0.: g

eptone $.: g

Agar -$.: g

+ Kn e( al./s )edu) B ,KB+ ,see d0 p. 1+

1+ 2eas( e3(ra&( - de3(rose - CaC4aar )edu) ,2DC+ ,see 50 p. 1+

This medium can be used for general isolation from plant tissues and is

recommended for examination of pigmentation #Table -(.

A )ater soluble pin6 pigment is present around colonies ofE. rubrifaciens on

IDC medium #0/(.

5. Re&pes 'or se)sele&(6e )eda*

!+ CG )edu) ,7+

perL

Sucrose -%:.: g

utrient agar /.: g

Cr"stal iolet :.' ml

C"cloheximide /:.: ml

Distilled )ater 0':.: ml

8-


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:.-K in absolute alcohol

:.-K in &:K alcohol

Colonies ofE. amylovora are distinguished b" formation of small craters or

depressions on the surface of the shin", opaue colonies. Some strains ma" not

produce craters, or the craters onl" exist for a short period of time.

"+ CCT )edu) ,!1+

perL

Sucrose -::.: g

Sorbitol -:.: g

Cr"stal iolet /.: ml

utrient agar /0.: g

Tergitol anionic & 0:.: ml

Distilled )ater to -.: L

After autoclaing add7

Thallium nitrate #-K )@( /.: ml

C"cloheximide $:.: mg

** :.-K solution in absolute ethanol

-K aueous solution

After 0 da"s,E. canylovora colonies are translucent, smooth, large #8*& mm(, light

blue )ith entire margins #late /, 5ig. /(. Blue striatums radiating from the centerof colonies are seen )hen ie)ed from the underside. 5luorescent Pseudomonas

syrin!aepathoars are detected under light.

+ =S )edu) ,"4+

perL

A ar /:.:=annitol -:.: g

L*asparagine 0.: g

Sodium taurocholate /*$ g/H:8 /.: g

icotinic acid :.$ g=gS:8 :./ g

itrilotriacetic acid #T A( :.$ g

Sodium heptadec"l sulfate :.-ml

Bromth"mol blue 8.: mg

eutral red -:.: mg

42


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Add -: ml of /K aueous solution neutrali4ed )ith :.&0 g of potassium

h"droxide per gram of T A.

ote7 The Tergitol & is optional and can be eliminated, if not aailable.

As9mlofa:.$K aueous solution.

As /.$ ml of a :.$K solution.

;nterobacteriaceae produce red*orange colonies #late /, 5ig. 0( pseudomonads

and other bacteria are distinguished b" the bluish color of their colonies. Sorbitol

is substituted for mannitol )hen isolatingE. amylovora. Pantoea a!!lomerans

#herbicola strains( cannot be distinguished fromM amylovorabecause of relatiel"

similar colon" morpholog" and color.

Erwinia uericina #-0( also gro)s on =S medium and produces colonies of

distinct color and morpholog" #/'(.

1+ MM"Cu ,+

perL

L*asparagine 8.: g

/H:8 /.: g

=gS:8 :./ g

aCl 0.: g

icotinic acid :./ g

Thiamine h"drochloride :./ g

Sorbitol -:.: g

Cupric sulfate #/ ra=( :.$ g

Agar -$.: g

Erwinia amylovora forms "ello), highl" mucoid colonies after 8 da"s at /' C

#late /, 5ig. 8( some strains are less mucoid. Erwinia pyrifoliae is also mucoid,

but slightl" "ello) on the agar. =an" other species )ill not gro) on this medium.

==/Cu medium should not be used for primar" isolation and about /$ colonies

are transferred to a ==/Cu agar plate for screening.

$( Mod'ed EMB )edu) ,"8+


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Ta5le !. =a!or diagnostic tests differentiating species in the non*soft rot group ofErwinia.++

Test

E.alni

E.amylovora

E.m

allotivora

E.ni!rifluens

E.paradisiaca

E.

persicinus

E.psidii

E.

pyrifoliae

E."uercina

E.ru

brifaciens

E.salicis

E.tracheiphila

Tobacco h"persensitiit" =5 + + - ND D - + - - - D

ectate degradation: * - - - > > - - > > > *

* * >

in6 pigment on IDC * - - * ND > - D * > * *

D - - > ND > - * * * * *

* - * * * * *

H/S from c"steine > - - > > > + * > > > >

rease >D - - > * * - * * * * *

3ndole test * - - * > * - * * * * *

itrate reduction * - - * ND > - * * * * *

* -

> > > + D >*

>*

*meth"l glucoside D * - ND * * + * > > * *

=elibiose * * - > > ND D ND ND ND D D

3nositol * * - > D ND > ND ND * > D

L*arabinoise > - > > > > > * > * D

a Data ta6en from Hao et al. #-:(, Hauben et al. #--(, eto et al. #/-(, Schroth and Hildebrand #/'(, Surico et al. #0:(.

b>, ':K or more strains positie >D, ':K or more strains dela"ed positie *, ':K or more strains negatie D,

not determined , ariable. cc Tests )ere made on atonGs media #//(. itting after 0 da"s represents a positie test.dAll positie species reuired "east extract to gro) in basal media )ith glucose.E. amylovora reuires nicotinic acid for good

gro)th in some minimal media.eAfter & da"s gro)th at /&CC in unsha6en aueous solution of -K organic compound and -K peptone )ith bromcresol

purple as indicator.

17


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5. N(ra(e redu&(on

itrate brothperL

:0 -.: g

eptone $.: gIeast extract 8.: g1xoid ion agar o. / 0.: g

pH &.:*&./

-( Seed 8 ml broth )ith inoculum and gro) /8*8' hr.

/( ;xamine for foam, )hich indicates gas production.

0( Test cultures on arious da"s. Add - ml of a :.%K #@( solution of ,*

dimeth"l*- naphth"lamine #or :.$K solution alpha naphth"lamine( and - ml of a:.'K #@( solution of sulfanilic acid, both in $ acetic acid #- part glacial aceticacid to /.$ parts )ater(, to each tube. A distinct pin6 or red color in the brothindicates the presence of nitrite and is considered positie. 3f )ithin - hr a pin6or red color does not appear, add 4inc dust to the tube. The 4inc dust )ill react)ith the nitrate, producing a pin6 color if present. This )ill confirm a negatietest. Ho)eer, if the tube does not turn pin6 then complete denitrification hasoccurred and the test is considered positie. Also test a sterile control tube 6eptunder the same conditions to guard against errors due to absorption of nitrousacid from the air. CAT317 The naphth"lamine reagents are carcinogenic*handle )ith care.

c. Gela(n l?ue'a&(on ,


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e. Urease produ&(on ,)os( use'ul 'orE. nigrifluens)

=odified "east salts #IS( broth

H8H/:8/H:8=gS:8N&H/:

aClIeast extract

Cresol red

After autoclaing add7

rea #stoc6 solution( /:: ml

Add /:.: g to -': ml )ater and filter sterili4e.

Disperse $.: ml into sterile /$ cm test tubes. These are inoculated and placed on

an incubator sha6er at approximatel" /' C. Control tubes containing the basal medium

)ithout urea should also be included. An increase in al6alinit" indicated b" a magenta redcolor #pH approx. 9.:( is eidence of urease actiit".

'. A&d produ&(on 'ro) &ar5o@:dra(es

This should be obsered from agar slants of )edu) C of D"e #%( OH8H/:8, :.$

g, /H:8, :.$ g =gSC"&H/:, :./ g aCl, $ g "east extract #Difco(, - g agar, -/ g add

)ater, - L bromcresol purple, :.& ml of -.$K alcohol solution pH %.' and heat

sterili4ed. Add carbon source, :.$K #@( asepticall" from filter*sterili4ed, stoc6 solutions.

Because of the difficult" of dissoling and filter sterili4ing some carbon sources,

substances such as salicin should be prepared b" dissoling in a large olume of )ateradded to medium C, and sterili4ing in steam on three successie da"s. Cultures should be

examined for acid production after /,8, %,/-, and /' da"s. A "ello) color indicates

production of acid.

g. Gro(@ a(


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presence of H/S is indicated b" a blac6ening of the paper.

.9e&(a(e derada(on

!+ Hlde5rand/s )edu) ,!"+.

A basal pol"pectate medium is ad!usted to three pH leels. 3ngredients for

the basal medium are added in the follo)ing order7 -,::: ml distilled )ater #heated

to near boiling( - ml bromth"mol blue #-.$K alcoholic solution( % ml -:K

CaCl/NH/: #freshl" prepared( and // g of sodium pol"pectate. This mixture is

stirred )ith a mechanical stirrer )hile being 6ept )arm b" a hot plate or other

means. After the sodium pol"pectate is dissoled, add -:: ml of sterile 8K agar

solution to each medium #A, B, C( and ad!ust the pH as follo)s7

=edium A. Ad!ust to pH 8.$*8.& )ith - HC- and autoclae.

=edium B. Ad!ust to pH %.9*&.- )ith - HC- and autoclae.

=edium C. Autoclae and then ad!ust to pH '.0*'.$ )ith sterile - a1H.

etri dishes must be poured before the temperature falls belo) &: C. The

plates are stored at room temperature for seeral da"s until the surface of the gel is

dr". =asses of bacteria #/*8 mm in diameter( are spotted using a sterile toothpic6

or transfer loop #four samples@plate( on the gel surface and the plates incubated for

-*% da"s at /' C before the occurrence of pitting is ascertained.

"+. 9a(on/s )edu) ,""+.

A /K pectate gel is poured onto a calcium agar base #-0(. The agar base is

composed of - L tap )ater plus - g /H:8or - g #H8(/S:8or H8C- added after

$ g CaCl/PH/:. The calcium agar base is poured into a etri dish, allo)ed to solidif"

and an eual amount of the pectate solution poured on top.

+ 9)en( on 2DC ,see 50 p. 1+.

+ Indole produ&(on ,see 0 p.

7. 9ATHOGENICIT2 T;STS

All of the non*soft*rotErwiniapathogens ma" be inoculated b" in!uring tissue, then

appl"ing inoculum b" atomi4ing or in!ecting inoculum )ith a needle and s"ringe #/'(. A range of

inoculum concentrations #-:8* -: C5@ml( should be tested.


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pathogenic strains of the suspect pathogen or are included as controls. 2e*isolations should be

done to confirm the identit" of the bacterium.

Erwinia amylovora ma" be inoculated )ith a needle b" )ounding succulent gro)ing

tissue laden )ith the test strain or b" atomi4ing pomaceous flo)ers )ith a suspension of bacteria

#/0(. athogenicit" tests ma" also be done b" inoculating slices of "oung, green pear or applefruit #0-( or b" inoculating germinating apple or pear seedlings #/$(. The inoculated tissues are

incubated in a moist chamber at /' C. 5lo)ers appear )ater*soa6ed often )ith oo4e droplets on

the surface, then )hole flo)ers turn bro)n to blac6, collapsing in 8 to % da"s. Tender shoots )ill

produce t"pical fire blight s"mptoms, oo4e, )ilting, necrosis, and hoo6ing at the tip, in $ to &

da"s. A positie test on fruit is eident b" a bro)ning around the )ound site and oo4ing of

bacteria in about $ da"s. Seedlings inoculated )ithE. amylovora exhibit )ilting, discolored and

necrotic tissue, and copious amounts of oo4e after 0 da"s. 1ther pathogens such as

Pseudomonas syrin!ae )ill produce necrotic s"mptoms on seedlings but not the discoloration and

oo4e production associated )ithE. amylovora. Erwinia pyrifoliaebarel" forms s"mptoms on

apple plantlets and should be assa"ed on ashi pear seedlings #/8(.

Erwinia uercina ma" be inoculated )ith a needle carr"ing the pathogen b" )ounding

"oung acorns attached to the tree. An alternate, perhaps more effectie, test is made b"

inoculating tender, "oung shoots of uercus a!rifoliaee #California field oa6, California lie oa6,

coast lie oa6( or . wisli-enii A. DC #interior lie oa6( #-0( )ith a needle carr"ing the

bacterium. Eithin 8 to $ da"s, shoots )ilt and a froth" oo4e occurs at the point of inoculation.

The other species ma" be inoculated b" using similar techniues. EithE. ni!rifluens,best

results occur )hen the bacterium is in!ected into )alnut bar6 )ith a needle or s"ringe during

spring or summer.E. rubrifaciens is inoculated b" inserting a carpet*needle tangentiall" along the

cambium of a susceptible )alnut host such as c. Hartle" and in!ecting :.- to :./ ml of inoculumcontaining -:%C5@ml #/&(.


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amplification of aPstI fragment from uniersal plasmid p;A/9 #8,-9(. se of nested

C2 #-9( can increase sensitiit" of detection.

Seeral C2 primers based on chromosomal DA can be used to supplement those

based on plasmid p;A/9 DA. These include C2 assa"s based on primers deried from a -'&

bpinal fragment fromE. amylovora genomic DA #'(, from the chromosomal ams region#/(, from the /0S r2A gene #-'(, and from the -%S r2A gene follo)ed b" restriction

anal"sis #/(. C2 methods inoling chromosomal DA ma" be slightl" less sensitie than

those inoling p;A/9. ulsed*field gel electrophoresis of genomic DA digested )ith

arious restriction en4"mes ma" be useful, also #08(.

aThe si4e of the amplified fragment ma" differ slightl" from strain to strain due to the number of '*bp repeatseuences )ithin the fragment.

bCan be combined in the same reaction )ith primers AJ&$@AJ&% to "ield the t)o expected amplification products.These primers cross react )ithE. pyrifoliae

DA seuence anal"sis of the -%S rDA and the ad!acent intergenic transcribed

spacer #3TS( region can be used to confirm the identit" of a plant pathogenicErwinia

$:

-'&


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species #--,-$,-%(. See Appendix A and C for details.

5. #erolo&al (e&@n?ues

Serological tests are sometimes used for the identification ofE. amylovora in areas

)here the bacterium does not occur and reference strains cannot be maintained. Antiseraprepared in response to a alid reference strain ma" be used in agglutination tests, double*

diffusion tests, en4"me*lin6ed immunosorbent assa"s, and immunofluorescence tests #/9(.

Considerable care needs to be ta6en )hen utili4ing this approach because pol"clonal

antibodies often cross*react )ith other bacteria #/%( and monoclonal antibodies ma" be

too specific to identif" all strains ofE. amylovora #-&(. See Appendix B for details.

&. Co))er&al au(o)a(ed (e&@n?ues

!+ Car5on sour&e u(la(ons

An identification s"stem consisting of a 9%*)ell microtiter plate #Biolog


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anal"sis ofErwinia species based on -%S r2A gene seuences. 3nt. J. S"st. Bacteriol.

8&7-:%-*-:%&.

-&. Lin, C. ., T. A. Chen, J. =. Eells, and T. an der F)et. -9'&. 3dentification and

detection oiErwinia amylovora )ith monoclonal antibodies. h"topatholog" &&70&%*0':.

-'. =aes, =, .


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Erwinia. =ethods in =icrobiolog" -$7//'*/90.

0:. Surico,


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H. GRAM-NEGATIVE BACTERIA

B-" Erwinia #o'( Ro( Group

#. H. De Boer and A. Kel)an

!. INTRODUCTION

The proposal presented $8 "ears ago b" Ealdee #$'( that the soft*rotErwinia species be

placed in a separate genus,Pectobacterium, has not been generall" accepted. 1ther similar

recommendations to place the pectol"ticErwinia into a separate genus hae also lac6ed support

#-0, 0/, 0$(. Ho)eer, the soft rot or "carotovord' group comprises those species )hich incite

soft rot diseases of plants and hence form a logical group from a ph"topathological perspectie.

3n this manual, the follo)ing designations are used for the "carotovord group7Erwinia

carotovora su5sp. caratavora, E. carotovora su5sp. atroseptica, E. carotovora su5sp.

betavasculorum, E. carotovora su5sp. wasabiae ,!;+0E. carotovora su5sp. odorifera ,!8+0E.

chrysanthemi, E cypripedii, E. rhapontici #-0, -8, 8', $:, $$( andE. cacticida #-(. 1nl" the

general characteristics forE. chrysanthemi are included in this manual, although pathoars hae

been designated #-0,-8( and described in seeral references #', 9, -:, $$(. ;en thought.

cypripedii andE. rhapontici are non*pectol"tic, D"e #-/( considered them to be related to the

pectol"tic species and until further studies sho) other)ise, the" are treated as members of this

group.

henot"pic characteristics of species in the genusErwinia are presented b" Starr #$0(

and Starr and Chatter!ee #$8(, and more specificall" forE. chrysanthemib" Dic6e" #', 9(.

;colog" of the soft rotErwinia )as reie)ed b" erombelon and elman #8-( and b"

Stanghellini #$/(. The status of research on the potato blac6leg disease is summari4ed in the2eport on the 3nternational Conference on potato Blac6leg Disease #/-(. Details of methods

generall" applicable for identification of bacteria in the famil",Enterobacteriaceae are gien in


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appropriate dilutions on a selectie medium. The selectie medium and temperature of

incubation ma" affect the efficienc" of recoer" and gro)th of specific strains #09(. erhaps

one of the most effectie and easil" prepared selectie media is cr"stal iolet pectate medium

#C, 8(. 3solation plates should be incubated at /0*/&C. 1n C the soft*rotErwinia

species form iridescent, cross*hatched, translucent colonies in deep, cup*li6e depressions or

pits, )hereas pectol"ticPseudomonas spp. form shallo) pits #late /, 5ig. $(. Colonies aremost clearl" obsered under a lo) po)ered dissecting microscope illuminated from belo)

)ith obliue illumination. ure cultures can often be obtained b" strea6ing directl" from the

deep pits in C onto C< medium #/9(.

To isolate pectol"tic bacteria from enironmental samples, prior enrichment of the

sample in a liuid enrichment medium greatl" enhances isolation of lo) populations of the

bacteria. Samples are incubated in enrichment medium #08( for 0*$ da"s at /0*/&C before

the" are strea6ed onto C.

Selectie media for isolating the other members of this group,E. cacticida, E.cypripedii, andE. rhapontici hae not been deeloped. Ho)eer, these bacteria can be

isolated on non*selectie media such as nutrient agar or C< medium.

a. CV9 )edu) #8(

per $:: ml )ater

3a1H 8.$ ml

-:K CaCl/*/H/: 0.: ml

a10, -.: g

Agar -.$ g

Sodium pol"pectate -:.: g

SDS :.$ ml:.:&$K cr"stal iolet -.: ml

our 0:: ml of boiling distilled )ater into a preheated Earing Blender !ar and then add

the a1H, CaCl/*/H/:, a:0, and agar. Blend at high speed for -$ sec. )hile

slo)l" adding the sodium pol"pectate #some sources of sodium pectate do not )or6

for this medium(. Blend for another -$ sec )hile adding another /:: ml of boiling

distilled )ater. our medium in a / litre flas6 and add the SDS and cr"stal iolet. S)irl

contents, cap flas6 )ith aluminum foil and autoclae for /$ min at -/:C. our plates

)hile medium is hot and entilate plates )hile cooling to preent condensation.

5. C9G )edu) #/9(

perL

Casamino acids -.: g

eptone -:.: g


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&. 9ol:pe&(a(e enr&@)en( )edu) #08(

perL

Sodium pol"pectate -.$ g

-:K #H8(/S:8 -:.: ml

-:K/H18 -:.: ml

$K =gS:8*&H/: $.: ml

0. D355;2;T3AT31 15 C1==1LI 3S1LAT;D S;CT;S AD SBS;C3;S

The soft rotErwinia, li6e other members of theEnterobacteriaceae are facultatiel"

anaerobic, peritrichousl" flagellated,


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Table -. Biochemical and ph"siological tests that differentiate pectol"tic species and

subspecies of theErwinia soft rot group0

>, ':K or more strains positie , bet)een /- *&9K of strains positie *, ':K or more strains negatie D, notdetermined.

a;cc E. carotovora subsp. carotovora

;ca E. carotovora subsp. atroseptica

;cb E. carotovora subsp. betavasculorum

;co E. carotovora subsp. odorifera

;c) E. carotovora subsp. wasabiae

;ch E. chrysanthemi

;ct E. cacticida bSome strains ofE. carotovora subsp. carotovora ma"

be )ea6l" positie

1 DIAGNO#TIC MEDIA AND TE#T#

a. %er)en(a(on o' lu&ose

The Hugh and Leifson #/8( 1*5 medium is used to differentiate fermentatie

from oxidatie metabolism of carboh"drates #see 0, p. 9(

$9


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5. %laellar s(an

The procedure of 2"u #8$( or the siler stain #see -: a, p. -:( is er" effectie instaining flagella ofErwinia carotovora.

&. Ca(alase (es(

=ix a loopful of a 8'*h*old culture gro)n on nutrient agar )ith a drop offreshl" prepared 0K H/:/on a glass slide. The formation of gas bubbles indicates a

positie reaction.

d. O3dase (es( ,see &, p. !4+

se a platinum loop to transfer bacteria from a 8'*h*old culture gro)n onnutrient agar onto filter paper impregnated )ith a drop of -K aueous tetrameth"l*p*

phen"lenediamine dih"drochloride solution. Deelopment of a purple color )ithin -:sec indicates a positie reaction.

e. Gro(@ a( 8C

se C< or nutrient agar medium at -$ ml per plastic etri dish. Stab inoculateisolates from a dense aueous suspension in a grid pattern. lace plates in a plastic bagto preent dr"ing out of the medium, and incubate at 0&C for three da"s. 1bsereinoculation point for bacterial gro)th. The presence of a bacterial colon" at the pointof inoculation is considered a positie test.

'. Redu&n su5s(an&es 'ro) su&rose

Dissole -: g Bacto*peptone and -& g agar in ':: ml distilled )ater, autoclae,and add /:: ml of a filter sterili4ed sucrose solution #8: g sucrose in /:: ml distilled)ater( and pour into etri dishes. Stab incubate isolates from a dense aueoussuspension in a grid pattern and incubate for t)o da"s at /0 * /&C. Then flood each

plate )ith -: ml BenedictGs solution and incubate 0: * 8$ min at %:C. A positie test isan orange 4one around the colon" against a blue bac6ground.

To prepare BenedictGs solution add 0$ g sodium citrate and /: g a/C:0H/: to-%: ml distilled )ater and heat to dissole. Separatel" dissole 0.$ g of CuS: 8*$H/: in8: ml distilled )ater. Combine and mix the t)o solutions. The mixture is stable forseeral months at room temperature.

g. 9@osp@a(ase a&(6(: ,"+

repare nutrient agar and )hen partiall" cooled after autoclaing add a filter*

%:


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After autoclaing cool to $: C and immediatel" add $: ml of filter sterili4ed/:K 6eto*meth"l glucoside, and / ml of filter sterili4ed -K tetra4olium chloride, and

pour into etri dishes. Stab inoculate isolates from a dense aueous suspension in agrid pattern and incubate for 8' h at /0*/&C.E. carotovora subsp. atroseptica gro)s)ell and produces colonies )ith red centers #late /, 5ig. &(.E. carotovora subsp.

carotovora gro)s poorl" and colonies are )hite although a little red color ma" deelopat the point )here the inoculum )as stabbed into the medium.

7. 9ATHOGENICIT2 TE#T#

a. Tssue )a&era(on

The abilit" of a bacterium to macerate plant tissue confirms its pectol"tic natureand proides an indication of pathogenicit". =aceration abilit", ho)eer, does not

proe pathogenicit" of the bacterium in a natural enironment. 5alse positie results

ma" be due to naturall" occurring endoph"tic or epiph"tic microorganisms associated)ith inoculated tissue.

arious plant tissues such as potato tubers #late 0, 5ig.l(, peppers or celer"stal6s can be used to test maceration abilit". Disinfect the surface of the tissue b"immersing in a -:K household bleach #$./$K sodium h"pochlorite(, solution for -:min and air*dr". 2epeat bleach treatment or alcohol flame. Cut tissue into conenientsi4e pieces, place in a etri dish on moist sterile filter paper, and inoculate )ith :.-*-ml of the bacterial suspension #ca. -:%C5@ml( from a /8*h*old*culture. 3ncubate at/:*/& C for 8' h and probe the tissue surrounding the inoculation site )ith a spatula orneedle to determine )hether deca" and tissue maceration has occurred.

5. 9a(@oen&(: (es(s

The most precise method for determination of pathogenicit" inoles the use ofinfectiit" titrations #-$(. repare bacteria as described #see $, p. //%(. 3noculate host

plants using a range of serial -:*fold dilutions from -:/*-: C5@ml of the testbacterium using a micropipette tip or s"ringe #00(. At least ' to -: plants perinoculum leel should be used for each of four to fie dilutions. Seal the in!ection pointafter inoculation b" application of a small amount of petroleum !ell" oer the puncture.3nfection of inoculated plants )ill be enhanced if the" are maintained at a high relatiehumidit" or een placed under an intermittent misting s"stem.

5or additional details on other inoculation procedures, reference should bemade to studies on host range ofE. chrysanthemi #9( and tests )ithE. rhapontici #8'(.

%/


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%. =1L;CLA2, S;21L1


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5. #erolo&al (e&@n?ues

Serological procedures are useful for preliminar" identification of purifiedcultures or bacteria in plant and soil samples if specific antibodies to commonl"occurring serogroups are aailable. Standard indirect immunofluorescence and triple

antibod" sand)ich ;L3SA protocols can be used for strain identification if it is 6no)nthat the antibod" reacts according to serogroup specificit" in the particular procedure

being used #//(.

Lipopol"saccharides are important antigenic determinants of most


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Serological techniues using +1+ antigens #--,8%,8&(, purified membraneprotein complex #$9(, or fimbrial*specific monoclonal antibodies #89( are er" usefulfor rapid presumptie identification of suspected cultures ofE. chrysanthemi. Seeralserological t"ping schemes of three or four seroars or serogroups hae been suggested

but these hae not been reconciled )ith one another or adopted for further studies #--,

/&(. ol"clonal and monoclonal antibodies produced to the lipopol"saccharide ofE.chrysanthemi do react )ith man" strains of the species but their use for identificationand detection is greatl" hampered b" their cross*reactiit" )ith a lipopol"saccharideepitope of some common soil pseudomonads #$&(. A monoclonal antibod" to afimbrial epitope ofE. chrysanthemi has potential for identification and perhapsdetection of strains isolated from potato and some other hosts in ;urope #89(.

c. Co))er&al au(o)a(ed (e&@n?ues

!. Car5on sour&e u(la(ons

The commerciall" aailable Biolog s"stem #Biolog, 3nc., Ha")ard, CA( forsubstrate utili4ation is helpful for rapid identification of those species and subspeciesincluded in the database. These currentl" include7E. carotovora subsp. atroseptica, E.carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. chrysanthemi,andE. cypripedii. See Appendix C for description and details.

"+ !


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Cell suspensions of some soft rotErwinia also presere )ell in )ater at room

temperature. Ho)eer, some species, notabl"E. chrysanthemi, lose iabilit" almost

immediatel" )hen stored in )ater.


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rot bacteria from soil and plant tissue. h"topatholog" %878%'*8&$.

$. De Boer, S. H, 2 J. Copeman, and H. ruggin6. -9&9. Serogroups ofErwinia

carotovorapotato strains determined )ith diffusible somatic antigens. h"topatholog"

%970-%*0-9.

%. De Boer, S. H. and =. ;. =caughton. -9'&. =onoclonal antibodies to the

lipopol"saccharide of Erwinia carotovora subsp. atroseptica serogroup 3.

h"topatholog" &&7'/'*'0/.

&. De Boer, S. H. and L. J. Eard. -99$. C2 detection of Erwinia carotovora subsp.

atroseptica associated )ith potato tissue. h"topatholog" '$7'$8*'$'.

'. Dic6e", 2. S. -9&9.Erwinia chrysanthemi# A comparatie stud" of phenot"pic

properties of strains from seeral hosts and otherErwinia species. h"topatholog" %97

0/8*0/9.

9. Dic6e", 2S. -9'-.Erwinia chrysanthemi. 2eaction of eight plant species to strains

from seeral hosts and to strains of otherErwinia species. h"topatholog" &-7/0*/9.

-:. Dic6e", 2. S., and J. 3. ictoria. -9':. Taxonom" and emended description of strains

of Erwinia isolated from$usa paradisiaca Linnaeus. 3nt. J. S"st. Bacteriol. 0:7-/9*

-08.

--. Dic6e", 2. S., C. H. Fumpoff, and J. . "emoto. -9'8.Erwinia chrysanthemi#

serological relationships among strains from seeral hosts. h"topatholog" &87-0''*

-098.

-/. D"e, D. E. -9%9. A taxonomic stud" of the genusErwinia. 33. The +carotoora+ group. .

F. J. Sci. -/7'-*9&.

-0. D"e, D. E. -9'-. A numerical taxonomic stud" of the genusErwinia.. F. J. Agric. 2es.

/87//0*//9.

-8. D"e, D. E., J. 5. Bradbur", =.


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atroseptica. 3nstitut ational de la 2echerche Agronomiue, 3nstitut ational Agronomiue

aris*


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efficienc" in the diagnosis and enumeration of soft rot coliform bacteria. J. Appl. Bacteriol.

097-09*-8%.

8:. erombelon, =. C. =. -9&-. A semi*selectie medium for estimating population densities of

pectol"ticErwinia spp. in soil and in plant material. otato 2es. -87-$'*-%:

8-. erombelon, =. C. =, and A. elman. -9':. ;colog" of the soft rot er)inias. Annu. 2e.

h"topathol. -'70%-*0'&.

8/. erombelon, =. C. =. and J. =. an Der Eolf. -99'. =ethods for the detection and

uantification of Erwinia carotovoa subsp. atroseptica on potatoes. Scottish Crop 2esearch

3nst. 1ccasional ublication o. -:. 3ergo)rie, Dundee, DD/ $DA, Scotland, .

80. hillips, J. A, and A. elman. -9'/. Direct fluorescent antibod" stain procedure applied to

insect transmission of Erwinia carotovora. h"topatholog" &/7'9'*9:-.

88. ierce, L. and A. H. =ccain. -99/. Selectie medium for isolation of pectol"ticErwinia sp.

lant Dis. &%70'/*0'8

8$. 2"u, ;. -90&. A simple method of staining bacterial flagella. itasato Arch. ;xp. =ed.

-87/-'*/-9.

8%. Samson, 2. -9&0. LesErwiniapectinol"ties. 33. 2echerches sur les antigenes somatiues

tfErwinia carotovora ar. chrysanthemi #Bur6holder( D"e -9%9. Ann. h"topathol. $70&&*

0''.

8&. Samson, 2., 2. outier, =. Saill" and B. Jouan. -9'9. Caracterisation desErwiniachrysanthemi isolees de 2olanum tuberosum et dGautres plantes*hotes selon les bioars et

serogroupes. ;1 Bull. -&7--*-%.

8'. Sell)ood, J., and 2. A. Lelliott. -9&'. 3nternal bro)ning of h"acinth caused b"Erwinia

rhapontici. lant athol. /&7-/:*-/8.

89. Singh, ., C. =. Treors, S. H. DeBoer, and J. D. Janse. -999. A fimbrial*specific

monoclonal antibod"*based ;L3SA test for ;uropean potato strains of Erwinia chrysanthemi

and comparison to C2. lant Dis. '87880*88'.

$:. S6erman, . B. D., . =c


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$/. Stanghellini, =. ;. -9'/. Soft rotting bacteria in the rhi4osphere. ages /89*/%0 in7 =.S.

=ount and


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