H. B. I.

GRAM-NEGATIVE BACTERIA Erwinia and Pantoea S. H. De Boer, D. L. Coplin, and A. L. Jones INTRODUCTION

The taxonomic position, nomenclature, and interrelationships of the members of the genus Erwinia ha e been the sub!ect of di erse proposals. The broadest classification is that of D"e #$, %, &, '(, )hich separates Erwinia into the pectol"tic soft rot "carotovora" group, the "ello) pigmented "herbicold7 group, the )hite nonpectol"tic )ilt*causing "amylovorrf' group, and the +at"pical+ Erwinia. These ha e turned out to be alid clusterings based on D,A*D,A homolog" studies and -%S se.uence homolog" #-$,-&(, but do not completel" agree )ith purel" phenetic groupings #/0(. Contro ers" remains on )hether or not the differences are great enough to constitute ne) genera. 1n the basis of -%S r2,A se.uence homologies, Hauben et al. #-$( recommended that these four groups be split into the genera Pectobacterium emend, Pantoea gen. no ., Erwinia emend., and Brenneria gen. no ., respecti el". 3n this manual, these groups are recogni4ed as alid di isions of the species )ithin the traditional Erwinia 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" re.uire further changes in nomenclature. The follo)ing species and subspecies are currentl" included in the pectol"tic soft rot or "carotovora" group #p. $%(7 Erwinia 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( and E. cacticida (I . Subspecific di ision of E. chrysanthemi has been suggested. Although the heterogeneit" of the species is no) generall" recogni4ed, satisfactor" descriptions of subspecies or patho ars ha e 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 as Pantoea #p. &0( together )ith some species of the genus Enterobacter #-/, -%,-'(. 3nitiall", ;)ing and 5ife #--( recommended, on the basis of D,A*D,A homologies, that Enterobacter a!!lomerans, E. herbicola and E. milletiae be combined into the species Enterobacter a!!lomerans comb. no . Subse.uentl" <ra ini et al. #-/( proposed that E. a!!lomerans #including the t"pe species of E. herbicola and E. milletiae be transferred to a ne) genus Pantoea, )hich )ould consist of t)o species, P. a!!lomerans and P. dispersa. Later, =ergaert et al. #-'( placed E. ananas and E. stewartii into Pantoea and deemed E. uredovora to be s"non"mous )ith P. ananas. Subse.uentl", three ne) /, $ di6eto*D* gluconate producing bacteria isolated from fruit and soil )ere described as ne) Pantoea species7 P. citrea, P. punctata and P. terrea #-%(. The present genus Pantoea includes the ph"topathogens P. stewartii, P. ananas, P. citrea and P. a!!lomerans p s. milletiae, !ypsophilae and betae. 0%

The species in the +am"lo ora+ #p. 8:( group )ill be considered together )ith the at"pical er)inias and )ill include E. amylovora, E. persicina, E. pyrifoliae, E. mallotivora, E. psidii, and E. tracheiphila along )ith E. 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 di ision of the Erwinia species according to the presence or absence of pectol"tic abilities, since the" suggest a closer similarit" bet)een E. amylovora and E. chrysanthemi than bet)een E. amylovora and the other species of the "amylovora" group #/-(. ,e ertheless, di ision of the Erwinia genus on the basis of phenot"pic characteristics is useful for grouping together those species that re.uire similar protocols for their manipulation )ithin the laborator". Due to the heterogeneit" of the genus, a general description of Erwinia 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 <ram negati e and are motile b" peritrichous flagella. The" are facultati el" anaerobic, chemoorganotrophic, and gro) optimall" at /0*0:AC. All species are oxidase negati e but catalase positi eB nitrates are not reduced b" most species. All species cataboli4e glucose and arious other carboh"drates )ith the production of acid but usuall" )ithout gas formation. H. LITERATURE CITED

-. Alcorn, S. =., T. C. 1rum, A. <. Steiger)alt, J. L. =. 5oster, J. C. 5ogleman, and D. J. Brenner. -99-. Taxonom" and pathogenicit" of Erwinia cacticida subsp. no . 3nt. J. S"st. Bacterid. 8-7-9&*/-/. /. Dic6e", 2. S. -9&9. Erwinia chrysanthemi. A comparati e stud" of phenot"pic properties of strains from se eral hosts and other Erwinia species. Dh"topatholog" %970/8*0/9. 0. Dic6e", 2 S. -9'-. Erwinia chrysanthemi# reaction of eight plant species to strains from se eral hosts and to strains of other Erwinia species. Dh"topatholog" &-7/0*/9. 8. Dic6e", 2. S., and J. 3. Cictoria. -9':. Taxonom" and emended description of strains of Erwinia isolated from $usaparadisiaca Linnaeus. 3nt. J. S"st. Bacterid. 0:7-/9*-08. $. D"e, D. E. -9%'. A taxonomic stud" of the genus Erwinia. 3. The amylovora group. ,. F. J. Sci. --7$9:*%:&. %. D"e, D. E. -9%9. A taxonomic stud" of the genus Erwinia. 33. The caro to vora group. ,.F.J. Sci. -/7'-*9&. &. D"e, D. E. -9%9. A taxonomic stud" of the genus Erwinia. 333. The herbicola group. ,. F. J. Sci. -/7//0*/0%
37

'. D"e, D. E. -9%9. A taxonomic stud" of the genus Erwinia. 3C. 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", =. <oto, A. C. Ha")ard, 2. A. Lelliott, and =. ,. Schroth. -9':. 3nternational standards for naming patho ars of ph"topathogenic bacteria and a list of patho ar names and pathot"pe strains. 2e . Dlant Dathol. $97-$0*-%'. ;)ing, E. H., and =. A. 5ife. -9&/. Enterobacter a!!lomerans #Be"erinc6( comb. no . #the herbicola%lathyri bacteria(. 3nt. J. S"st. Bacteriol. //78*--. <a ini, 5., J. =ergaert, A. Be!i, C. =ielcare6, D. 34ard, H. Hersters, and J. De Le". -9'9. Transfer of Enterobacter a!!lomerans #Bei!erinc6 -'''( ;)ing and 5ife -9&/ to Pantoea gen. no . as Pantoea a!!lomerans comb. no . and description of Pantoea dispersa sp. no . 3nt. J. S"st. Bacteriol. 09700&*08$. <allois, A., 2. Samson, ;. Agaron, and D. A. D. <rimont. -99/. Erwinia carotovora subsp. odorifera subsp. no ., associated )ith odorous soft rot of chicor" (&hichorium intybus' . 3nt. J. S"st. Bacteriol. 8/7$'/*$''. <oto, =, andH. =atsumoto. -9'&. Erwinia carotovora subsp. wasabiae subsp. no . isolated from diseased rhi4omes and fibrous roots of Japanese horseradish (Eutrema wasabi =axim(. 3nt. J. S"st. Bacteriol. 0&7-0:*-0$. Hauben, L., ;. 2. B. =oore, L. Cauterin, =. Steenac6ers, J. =ergaert, L. Cerdonc6, and J. S)ings. -99'. Dh"logenetic position ofph"topathogens )ithin the ;nterobacteriaceae. S"stem. Appl. =icrobiol. /-70'8*09&. Hage"ama, B., =. ,a6ae, S. Iagi, and T. Sono"ama. -99/. Pantoea punctata sp. no ., Pantoea citrea sp. no ., and Pantoea terrea sp. no . isolated from fruit and soil samples. 3nt. J. S"st. Bacteriol. 8/7/:0*/-:. H)on, S. E., S. J. <o, H. E. Hang, J. C. 2"u, and J. H. Jo. -99&. Dh"logenetic anal"sis of Erwinia species based on -%S r2,A gene se.uences. 3nt. J. S"st. Bacteriol. 8&7-:%-* -:%&. =ergaert, J., L. Cerdonc6, and H. Hersters. -990. Transfer of Erwinia ananas #s"non"m, Erwinia uredovora and Erwinia stewartii to the genus Pantoea emend, as Pantoea ananas #Serrano -9/'( comb. no . and Pantoea stewartii #Smith -'9'( comb. no ., respecti el", and description of Pantoea stewartii subsp. indolo!enes. 3nt. J. S"st. Bacteriol. 807-%/*-&0.
38

--. -/.

-0.

-8.

-$.

-%.

-&.

-'.

-9. /:. /-. //.

Sell)ood, J., and 2. A. Lelliott. -9&'. 3nteraal bro)ning of h"acinth caused b" Erwinia rhapontici. Dlant Dathol. /&7-/:*-/8. S6erman, C. B. D., C. =c<o)an, and D. H. A. Sneath #;ds.(. -9':. Appro ed list of bacterial names. 3nt. J. S"st. Bacteriol. 0:7//$*8/:. Starr, =.D. and =. =andel. -9%9. D,A base composition and taxonom" of ph"topathogenic and other enterobacteria. J. <en. =icrobiol. $%7--0*-/0. Thomson, S. C., D. C. Hildebrand, and =. ,. Schroth. -9'-. 3dentification and nutritional differentiation of the Erwinia sugar beet pathogen from members of Erwinia carotovora and Erwinia chrysanthemi. Dh"topatholog" &-7-:0&*-:8/. Cerdonc6, L., J. =ergaert, C. 2uc6aert, J. S)ings, H. Hersters, and J. De Le". -9'&. <enus Erwinia# ,umerical anal"sis of phenot"pic features. 3nt. J. S"st. Bacteriol. 0&A8* -'. Ioung, J. =., I. Ta6i6a)a, L. <ardan, and D. ;. Stead. -99/. Changing concepts in the taxonom" of plant pathogenic bacteria. Annu. 2e . Dh"topathol. 0:7%&*-:$.

/0.

/8.

09

. B-l. !.

GRAM-NEGATIVE BACTERIA Erwinia amylovora Group A. L. Jones and K. Ge der INTRODUCTION

The non*soft rot group in the genus Erwinia corresponds to the +am"lo ora+ group as proposed b" D"e #%( and to a number of plant pathogenic species described in the last -: to -$ "ears #-:, --, -$, /-, 0:(. The species of Erwinia included in this chapter include E. alni( #0:(, E. amylovora, E. mallotivora, E. ni!rifluens(, E. paradisicina #--(, E. persicinus #-:(, E. psidii #/-(, E. pyrifoliae #-$(, E. "uercina(, E. rubrifacinens(, E. salicis(, and E. 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 rot Erwinia species is often not difficult )hen abo e ground portions of plant material are isibl" infected. The exception is E. paradisiaca, a pathogen on the roots of banana. Bacteria occur in high titer in ne)l" infected tissuesB titers decline and secondar" microorganisms increase as lesions age. Small pieces of ne)l" in aded )ater*soa6ed tissues from the leading edge of lesions on lea es, shoots, spurs, and fruits can be plated directl" on suitable culture media. Alternati el", 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, bacteria consistentl" associated )ith infected tissues should be strea6ed on fresh media. Bacterial oo4e can be strea6ed directl" on the isolation medium. Some Erwinia 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 semiselecti e medium can be used for the reco er" of Erwinia%li)e bacteria. Standard media such as Luria*Bertani #LB( agar, Hing et al.Gs medium B #HB(, 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 HB 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 /'AC for /*0 da"s, indi idual colonies can be transferred to fresh plates. Eith experience, an in estigator generall" can distinguish colonies of a pathogen from miscellaneous saproph"tic bacteria pro ided the numbers of contaminating bacteria are relati el" fe).

40

Semiselecti e media are used for the reco er" of Erwinia 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 #selecti e(, and some Erwinia 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 )ed a* !+ Lur a-Ber(a n ,LB+ a-ar perL -:.: g $.: g $.: g -$.: g perL 0.: g $.: g -$.: g

Tr"ptone ,aCl Ieast extract Agar "+ Nu(r en( a-ar #,A(

Beef extract Deptone Agar .+ 1+ K n- e( al./s )ed u) B ,KB+ ,see d0 p. 1+

2eas( e3(ra&( - de3(rose - CaC4. a-ar )ed u) ,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 of E. rubrifaciens on IDC medium #0/(. 5. Re& pes 'or se) sele&( 6e )ed a* !+ CG )ed u) ,7+ Sucrose ,utrient agar Cr"stal iolet C"cloheximide Distilled )ater perL -%:.: g /.: g :.' ml J /:.: ml J J 0':.: ml

8-

J JJ

:.-K in absolute alcohol :.-K in &:K alcohol

Colonies of E. amylovora are distinguished b" formation of small craters or depressions on the surface of the shin", opa.ue colonies. Some strains ma" not produce craters, or the craters onl" exist for a short period of time. "+ CCT )ed u) ,!1+ Sucrose Sorbitol Cr"stal iolet ,utrient agar Tergitol anionic & Distilled )ater to After autocla ing add7 Thallium nitrate #-K )@ ( C"cloheximide
**

perL -::.: g -:.: g /.: ml J /0.: g 0:.: ml JJ -.: L

/.: ml $:.: mg

:.-K solution in absolute ethanol -K a.ueous solution

After 0 da"s, E. canylovora colonies are translucent, smooth, large #8*& mm(, light blue )ith entire margins #Dlate /, 5ig. /(. Blue striatums radiating from the center of colonies are seen )hen ie)ed from the underside. 5luorescent Pseudomonas syrin!ae patho ars are detected under LC light. .+ =S )ed u) ,"4+ Agar =annitol L*asparagine Sodium taurocholate H/HD:8 ,icotinic acid =gS:8 ,itrilotriacetic acid #,T A( Sodium heptadec"l sulfate Bromth"mol blue ,eutral red perL /:.: g -:.: g 0.: g /*$ g /.: g :.$ g :./ g :.$ gJ :.-mlJJ 8.: mg JJJ -:.: mg JJJJ

42

Add -: ml of /K a.ueous solution neutrali4ed )ith :.&0 g of potassium h"droxide per gram of ,T A. JJ ,ote7 The Tergitol & is optional and can be eliminated, if not a ailable. JJJ As9mlofa:.$K a.ueous solution. JJJJ As /.$ ml of a :.$K solution. ;nterobacteriaceae produce red*orange colonies #Dlate /, 5ig. 0(B pseudomonads and other bacteria are distinguished b" the bluish color of their colonies. Sorbitol is substituted for mannitol )hen isolating E. amylovora. Pantoea a!!lomerans #herbicola strains( cannot be distinguished fromM amylovora because of relati el" similar colon" morpholog" and color. Erwinia "uericina #-0( also gro)s on =S medium and produces colonies of distinct color and morpholog" #/'(. 1+ MM"Cu ,.+ L*asparagine H/HD:8 =gS:8 ,aCl ,icotinic acid Thiamine h"drochloride Sorbitol Cupric sulfate #/ ra=( Agar perL 8.: g /.: g :./ g 0.: g :./ g :./ g -:.: g :.$ g -$.: g

Erwinia amylovora forms "ello), highl" mucoid colonies after 8 da"s at /' AC #Dlate /, 5ig. 8(B 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 )ed u) ,"8+

<l"cerol ,H8S:8 H/HD:8 ;osin I =eth"lene blue Agar

perL -:.: ml $.: g /.: g :.8 g %$.: -$.: g 43

After autocla ing asepticall" add the follo)ing antibiotics7 C"clohexamideJ ,o obiocin ,eom"cin sulfate #%': ug@mg( +Lse a stoc6 solution #-:: ug@ml of &:K ethanol( Lseful for isolating Erwinia rubrifaciens from soil. The bacterium forms isible colonies after 8 da"s at /&AC. After % da"s colonies are -*/ mm in diameter, circular, )ith entire margins and a con ex surface. .. DI%%ERENTIATION O% COMMONL2 I#OLATED #9ECIE# /.$ ml 8:.: mg 8:.: mg

=ost species in the non*soft rot Erwinia%!rovp are found on different hosts. Therefore, the identification of Erwinia%*)e, bacteria from plant material is relati el" eas" pro ided un6no)n strains are tested in parallel )ith control strains of 6no)n identit" from the same or closel" related host plants #/9(. The genus Erwinia, in the ;nterobacteriaceae, )as proposed b" Einslo) et al. #00( for <ram*negati e plant*associated bacteria )ith the follo)ing characteristics7 facultati e anaerobes, peritrichous flagella, rod shaped, and acid produced from fructose, glucose, galactose, and sucrose. All species exhibit motilit". 3dentification to species is based on the tests listed in Table -. 1. DIAGNO#TIC MEDIA AND TE#T# a. H:persens ( 6e rea&( on ,see e, p. ;<+

Approximatel" -:9 colon" forming units #C5L(@ml )ater are in!ected into the intercellular space of a leaf of tobacco c . +Burle"+ )ith a /$ gauge needle and s"ringe #for high bacterial densities, false positi e reactions ma" occur(. Complete collapse of the tissue after /8 h is recorded as positi e. Since plant pathogenic bacteria elicit the h"persensiti e response but saproph"tic bacteria do not, a positi e reaction indicates that the suspect bacterium should be identified further using other tests listed in this section.

44

Ta5le !. =a!or diagnostic tests differentiating species in the non*soft rot group of Erwinia.++
E. "uercina E. alni E. mallotivora E. pyrifoliae E. salicis
-

E.psidii

Tobacco h"persensiti it" Dectate degradation: <ro)th factors re.uired+ Din6 pigment on IDC <ro)th at 0%AC <ro)th at 09AC H/S from c"steine Lrease 3ndole test ,itrate reduction <elatin li.uification Acid production from%7 Salicin H*meth"l glucoside =elibiose 3nositol L*arabinoise

=5

+
-

+
-

* *

ND ,D > ND ND > >

*

+
-

,D
*

* * *

> > >

*

> >
* * *

>
*

>
* * * *

+
-

+
-

ND ,D

,D ,D
* * * * * *

>
* *

>D
*

>
*

> >D
* * *

> >
* *

>
* *

+
-

>
* * *

>
* * *

>
* * *

> ND

>

> ,D
* *

* * * * C

> ND > > >

>
*

>
*

+ + > >

,D
*

> >

>
*

>
*

> ,D >

ND ,D ND ND ND ,D ND > ND ND >
*

,D ,D ,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 positi eB >D, ':K or more strains dela"ed positi eB *, ':K or more strains negati eB ,D, not determinedB , ariable. c

c Tests )ere made on DatonGs media #//(. Ditting after 0 da"s represents a positi e test. d All positi e species re.uired "east extract to gro) in basal media )ith glucose. E. amylovora re.uires nicotinic acid for good gro)th in some minimal media. e After & da"s gro)th at /&CC in unsha6en a.ueous solution of -K organic compound and -K peptone )ith bromcresol purple as indicator.

17

E. tracheiphila

E. ni!rifluens

E. rubrifaciens

E. amylovora

E.paradisiaca

E. persicinus

Test

>
* * *

>
* * *

* *

5.

N (ra(e redu&( on ,itrate broth H,:0 Deptone Ieast extract 1xoid ion agar ,o. / pH &.:*&./ perL -.: g $.: g 8.: g 0.: g

-( /( 0(

Seed 8 ml broth )ith inoculum and gro) /8*8' hr. ;xamine for foam, )hich indicates gas production. 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 acetic acid to /.$ parts )ater(, to each tube. A distinct pin6 or red color in the broth indicates the presence of nitrite and is considered positi e. 3f )ithin - hr a pin6 or 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 negati e test. Ho)e er, if the tube does not turn pin6 then complete denitrification has occurred and the test is considered positi e. Also test a sterile control tube 6ept under the same conditions to guard against errors due to absorption of nitrous acid from the air. CALT31,7 The naphth"lamine reagents are carcinogenic *handle )ith care. Gela( n l ?ue'a&( on ,<+

c.

Add -/: g of gelatin #bacteriological grade( to - L )ater and ad!ust the pH to &.:. Add -: ml to each tube, autocla e, and cool #do not slant( immediatel". 3noculate b" stabbing a loop )ith inoculum into the center of the medium, then incubate the tubes at /:*//AC or at room temperature. 1bser e )hether rapid or slo) li.uefaction occurs. Also obser e shape produced )hen the gelatin begins to li.uef". d. Mo( l (: (es(s

=otilit" agar is prepared in tubes )ithout slanting. Dissol e tr"ptone -: g, sodium chloride $ g, and agar $ g in - L )ater and ad!ust pH to %.' * &.:. 3noculate the sterile semi*solid medium b" stabbing the center of the medium as abo e for gelatin. 3ncubate and obser e after ', /8, and 8' hr. =otilit" is sho)n b" a diffused 4one of gro)th spreading from the line of inoculation. Diffuse gro)th ma" extend throughout the entire medium or onl" from one or t)o points. 8%

e.

Urease produ&( on ,)os( use'ul 'or E. nigrifluens) =odified "east salts #IS( broth per ':: ml :.$ g :.$ g :./ g $.: g !.4 g -%.: mg ,H8H/D:8 H/HD:8 =gS:8N&H/: ,aCl Ieast extract Cresol red After autocla ing add7 Lrea #stoc6 solution( J Add /:.: g to -': ml )ater and filter sterili4e. /:: ml J

Disperse $.: ml into sterile /$ cm test tubes. These are inoculated and placed on an incubator sha6er at approximatel" /' AC. Control tubes containing the basal medium )ithout urea should also be included. An increase in al6alinit" indicated b" a magenta red color #pH approx. 9.:( is e idence of urease acti it". '. A& d produ&( on 'ro) &ar5o@:dra(es

This should be obser ed from agar slants of )ed u) C of D"e #%( O,H8H/D:8, :.$ g, H/HD:8, :.$ gB =gSC"&H/:, :./ gB ,aCl, $ gB "east extract #Difco(, - gB agar, -/ gB add )ater, - LB bromcresol purple, :.& ml of -.$K alcohol solutionB pH %.'B and heat sterili4ed. Add carbon source, :.$K # @ ( asepticall" from filter*sterili4ed, stoc6 solutions. Because of the difficult" of dissol ing and filter sterili4ing some carbon sources, substances such as salicin should be prepared b" dissol ing in a large olume of )ater added to medium C, and sterili4ing in steam on three successi e da"s. Cultures should be examined for acid production after /,8, %,/-, and /' da"s. A "ello) color indicates production of acid. g. GroA(@ a( .<BC 1bser e gro)th in tubes of li.uid $/0 medium #see -(, p. /&( or IS broth. @. H"# 'ro) &:s(e ne

Cultures are sha6er incubated in IS broth > c"steine h"drochloride, :.- g@L, or IS broth > peptone, :.$ g@L for 0, %, and -8 da"s #%(. Strips of filter paper moistened )ith a -:K solution of neutral lead acetate are held in place o er the medium to maintain the lo)er end of the paper about $ mm abo e the surface of the li.uid medium. The

47

presence of H/S is indicated b" a blac6ening of the paper. .9e&(a(e de-rada( on !+ H lde5rand/s )ed u) ,!"+. A basal pol"pectate medium is ad!usted to three pH le els. 3ngredients for the basal medium are added in the follo)ing order7 -,::: ml distilled )ater #heated to near boiling(B - ml bromth"mol blue #-.$K alcoholic solution(B % ml -:K CaCl/NH/: #freshl" prepared( and // g of sodium pol"pectate. This mixture is stirred )ith a mechanical stirrer )hile being 6ept )arm b" a hot plate or other means. After the sodium pol"pectate is dissol ed, add -:: ml of sterile 8K agar solution to each medium #A, B, C( and ad!ust the pH as follo)s7 =edium A. Ad!ust to pH 8.$*8.& )ith - , HC- and autocla e. =edium B. Ad!ust to pH %.9*&.- )ith - , HC- and autocla e. =edium C. Autocla e and then ad!ust to pH '.0*'.$ )ith sterile - , ,a1H. Detri dishes must be poured before the temperature falls belo) &: AC. The plates are stored at room temperature for se eral 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 /' AC before the occurrence of pitting is ascertained. "+. 9a(on/s )ed u) ,""+.

A /K pectate gel is poured onto a calcium agar base #-0(. The agar base is composed of - L tap )ater plus - g H/HD:8 or - g #,H8(/S:8 or ,H8C- added after $ g CaCl/PH/:. The calcium agar base is poured into a Detri dish, allo)ed to solidif" and an e.ual amount of the pectate solution poured on top. C+ D+ 7. 9 -)en( on 2DC ,see 50 p. 1+. Indole produ&( on ,see 0 p. <!+.

9ATHOGENICIT2 T;STS

All of the non*soft*rot Erwinia pathogens 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 * -:J C5L@ml( should be tested. <ro) bacteria as described #see $, p. //%(. Care should be ta6en in e aluating results obtained )ith high inoculum concentrations #-:Q=A C5L@ml(B high concentrations can result in false positi es. Sterile )ater and 6no)n 8'

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(. Dathogenicit" tests ma" also be done b" inoculating slices of "oung, green pear or apple fruit #0-( or b" inoculating germinating apple or pear seedlings #/$(. The inoculated tissues are incubated in a moist chamber at /' AC. 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 positi e test on fruit is e ident b" a bro)ning around the )ound site and oo4ing of bacteria in about $ da"s. Seedlings inoculated )ith E. 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 )ith E. amylovora. Erwinia pyrifoliae barel" 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 effecti e, test is made b" inoculating tender, "oung shoots of ,uercus a!rifolia ,ee #California field oa6, California li e oa6, coast li e oa6( or ,. wisli-enii A. DC #interior li e 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 techni.ues. Eith E. 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 inoculum containing -:% C5L@ml #/&(. <. MOLECULAR0 #EROLOGICAL0 AND COMMERCIAL AUTOMATED TECHNI$UE# a. Mole&ular (e&@n ?ues

Although some highl" specific molecular tests are a ailable for the identification of se eral )ell*characteri4ed species of Erwinia, classical biochemical tests as described in Ber!ey's $anual are still the standard in microbiolog" for identif"ing Erwinia spp. Carious D,A h"bridi4ation and pol"merase chain reaction #DC2( techni.ues are a ailable for the identification of E. amylovora but not the other non*soft rot Erwinia species #Table 8(. H"bridi4ation techni.ues based on D,A from p;A/9 #&, -9( or the chromosome #9( can be used to identif" E. amylovora colonies. The most )idel" used, fast, specific, and sensiti e DC2 method to identif" colonies of E. amylovora and to detect E. amylovora in extracts from s"mptomatic and as"mptomatic plants is based on the 89

amplification of a PstI fragment from uni ersal plasmid p;A/9 #8,-9(. Lse of nested DC2 #-9( can increase sensiti it" of detection. Se eral DC2 primers based on chromosomal D,A can be used to supplement those based on plasmid p;A/9 D,A. These include DC2 assa"s based on primers deri ed from a -'& bp .ina.l fragment from E. amylovora genomic D,A #'(, from the chromosomal ams region #/(, from the /0S r2,A gene #-'(, and from the -%S r2,A gene follo)ed b" restriction anal"sis #/(. DC2 methods in ol ing chromosomal D,A ma" be slightl" less sensiti e than those in ol ing p;A/9. Dulsed*field gel electrophoresis of genomic D,A digested )ith arious restriction en4"mes ma" be useful, also #08(.

-'&

The si4e of the amplified fragment ma" differ slightl" from strain to strain due to the number of '*bp repeat se.uences )ithin the fragment. b Can be combined in the same reaction )ith primers AJ&$@AJ&% to "ield the t)o expected amplification products. These primers cross react )ith E. pyrifoliae D,A se.uence anal"sis of the -%S rD,A and the ad!acent intergenic transcribed spacer #3TS( region can be used to confirm the identit" of a plant pathogenic Erwinia $:

species #--,-$,-%(. See Appendix A and C for details. 5. #erolo- &al (e&@n ?ues

Serological tests are sometimes used for the identification of E. amylovora in areas )here the bacterium does not occur and reference strains cannot be maintained. Antisera prepared 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 of E. amylovora #-&(. See Appendix B for details. &. !+ Co))er& al au(o)a(ed (e&@n ?ues Car5on sour&e u( l Ea( ons

An identification s"stem consisting of a 9%*)ell microtiter plate #Biolog <, =icroplate, Biolog, 3nc., Ha")ard, CA( tests for the abilit" of a bacterium to utili4e 9$ different carbon sources in the presence of an indicator d"e. A disad antage of this identification techni.ue is that metabolic profiles are not a ailable for all Erwinia species. See Appendix C for description and details. &. CULTURE 9RE#ERVATION

Cultures of most Erwinia species )ill remain iable for about / "ears on nutrient agar or LB agar slants co ered )ith sterile mineral oil and stored at $ *-:AC. The" can be stored at room temperature b" stabbing heart infusion agar and co ering )ith mineral oil #personal communication, ,. E. Schaad(. 5or long*term storage at *':AC, mix o ernight cultures in LB )ith an e.ual amount of sterile LB containing /:K gl"cerol #final gl"cerol concentration -:K(. Dreser ation can also be done b" l"ophili4ation. F. LITERATURE CITED

-. Burton, B., L. =elcher, T. Fitter, S. Dair, J. 5letcher, and 5. =itchell. -999. Dol"merase chain reaction for detection of Erwinia tracheiphila in cucurbits. Dh"topatholog" '97S-:. #Abstr.(. /. Beres)ill, S., D. Bugert, 3. Bruchmuller, and H. <eider. -99$. 3dentification of the fire blight pathogen, Erwinia amylovora, b" DC2 assa"s )ith chromosomal D,A. App. ;n iron. =icrobiol. $'70$//*0$/%. 0. Beres)ill, S., S. Joc6, D. Bellemann, and H. <eider. -99'. 3dentification of Erwinia amylovora b" gro)th morpholog" on agar containing copper sulfate and b" capsule staining )ith lectin. Dlant Dis. '/7-$'*-%8. $-

8.

Beres)ill, S., A. Dahl, D. Bellemann, E. Feller, and H. <eider. -99/. Sensiti e and species*specific detection of Erwinia amylovora b" pol"merase chain reaction anal"sis. App. ;n iron. =icrobiol. $'70$//*0$/%. Crosse, J. ;. and 2. ,. <oodman. -9&0. A selecti e medium for and a definiti e colon" characteristic of Erwinia amylovora. Dh"topatholog" %07-8/$*-8/%. D"e, D. E. -9%'. A taxonomic stud" of the genus Erwinia. 3. the "amylovora" group. ,e) Fealand J. Sci. --7$9:*%:&. 5al6enstein, H, D. Bellemann, S. Ealter, E. Feller, and H. <eider. -9''. 3dentification of Erwinia amylovora, the fire blight pathogen, b" colon" h"bridi4ation )ith D,A from plasmid p;A/9. App. ;n iron. =icrobiol. $87/&9'*/':/. <uilford, D. 3, 2. H. Ta"lor, 2. <. Clar6, C. ,. Hale, and 2. L. S. 5orster. -99%. DC2* based techni.ues for the detection of Erwinia amylovora. Acta Horticulturae 8--7$0*$%. Hale, C. ,., 2 <. Clar6, and D. Harte. -990. A D,A approach to Erwinia amylovora detection in large scale apple testing and in epidemiological studies. Acta Horticulturae 00'7$9*%%. Hao, =. C., D. J. Brenner, A. <. Steiger)alt, I. Hosa6o, and H. Homagata. -99:. Erwiniapersicinus, a ne) species isolated from plants. 3nt. J. S"st. Bacteriol. 8:70&9* 0'0. Hauben, L., ;. 2. B. =oore, L. Cauterin, =. Steenac6ers, J. =ergaert, L. Cerdonc6, and J. S)ings. -99'. Dh"logenetic position of ph"topathogens )ithin the Enterobacteriaceae. S"st. Appl. =icrobiol. /-70'8*09&. Hildebrand, DC. -9&-. Dectate and pectin gels for differentiation ofPseudomonas sp. and other bacterial plant pathogens. Dh"topatholog" %-7-80:*-80%. Hildebrand, D. C. and =. ,. Schroth. -9%&. A ne) species of Erwinia causing the dripp" nut disease of li e oa6s. Dh"topatholog" $&7/$:*/$0. 3shimaru, C. and ;. J. Hlos. -9'8. ,e) medium for detection of Erwinia amylovora and its use in epidemiological studies. Dh"topatholog" &87-08/*-08$. Him, E.*S., L. <ardan, S.*L. 2him, and H. <eider. -999. Erwinia pyrifoliae sp. no ., a no el pathogen that affects Asian pear trees (Pyruspyrifolia ,a6ai(. 3nt. J. S"st. Bacteriol 897'99*9:%. H)on, S.*E., S.*J. <o, H.*E. Hang, J.*C. 2"u, and J.*H. Jo. -99&. Dh"logenetic $/

$. %. &.

'. 9.

-:.

--.

-/. -0. -8. -$.

-%.

anal"sis of Erwinia species based on -%S r2,A gene se.uences. 3nt. J. S"st. Bacteriol. 8&7-:%-*-:%&. -&. -'. Lin, C. D., T. A. Chen, J. =. Eells, and T. an der F)et. -9'&. 3dentification and detection oiErwinia amylovora )ith monoclonal antibodies. Dh"topatholog" &&70&%*0':. =aes, =, D. <arbe a, and C. Crepel. -99&. 3dentification and sensiti e endoph"tic detection of the fire blight pathogen Erwinia amylovora )ith /0 S ribosomal D,A se.uences and the pol"merase chain reaction. Dlant Datholog" $87--09*--89. =c=anus, D. S. and A. L. Jones. -99$. Detection oiErwinia amylovora b" nested DC2 and DC2*dot*blot and re erse*blot h"bridi4ations. Dh"topatholog" '$7%-'*%/0. =iller, T. D. and =. D. Schroth. -9&/. =onitoring the epiph"tic population oiErwinia amylovora on pear )ith a selecti e medium. Dh"topatholog". %/7--&$*--'/. ,eto, J. 2, C. 5. 2obbs, and T. Iamashiro. -9'&. A bacterial disease of <ua a (Psidium !ua/ava caused b" Erwiniapsidii sp. no . 5itopatol. Bras. -/708$*0$:. Daton, A. =. -9$'. Dectin*decomposing strains of Psendomonas. ,ature -'-7%-*%/. Duse", D. L. -99&. Crab apple blossoms as a model for research on biological control of fire blight. Dh"topatholog" '&7-:9%*--:/. 2him, S.*L., B. Col6sch L. <ardan, J.*D. Daulin, C. Langlot4, E.*S. Him, and H. <eider. -999. Erwinia pyrifoliae, an Erwinia species, different from Erwinia amylovora, causes a necrotic disease of Asian pear trees. Dlant Dathol. 8'7$-8*$/:. 2itchie, D. 5. and ;. J. Hlos. -9&8. A laborator" method of testing pathogenicit" of suspected Erwinia amylovora isolates. Dlant Dis. 2ep $'7-'-*-'0. 2oberts, D. -9':. Droblems encountered during immunofluorescence diagnosis of fire blight. Dlant Dath. /9790*9&. Schaad, ,. E. and ;. ;. Eilson. -9&:. Sur i al oiErwinia rubrifaciens in soil. Dh"topath. %:7$$&*$$'. Schroth, =. ,. and D. C. 5lildebrand. -998. Erwinia amylovora or non*soft rot group. Dages 0&* 80 in7 ,. E. Schaad, ed., Laborator" <uide for 3dentification of Dlant Dathogenic Bacteria, /nd ;dition. American Dh"topathological Societ", St. Daul, =,. Slade, =. B. and A. 3. Tiffin. -9'8. Biochemical and serological characteri4ation of $0

-9. /:. /-. //. /0. /8.

/$. /%. /&. /'.

/9.

Erwinia. =ethods in =icrobiolog" -$7//'*/90. 0:. Surico, <., L. =ugnai, 2. Dastorelli, L. <io annetti, and D. ;. Stead. -99%. Erwinia alni, a ne) species causing bar6 can6ers of alder (0lnus =iller( species. 3nt. J. S"st. Bacterid. 8%7&/:*&/%. Canneste, J. L, J. Iu, <. ;. Harper, and J. H. Derr". -99%. Dlugs of immature pear fruit to assess the irulence of Erwinia amylovora and to stud" in the laborator" the interaction bet)een biological control agents and the fire blight pathogen. Acta Horticulturae. 8--70:0*0:$. Eilson, ;. ;., 5. =. Feitoun, and D. L. 5redric6son. -9%&. Bacterial phloem can6er, a ne) disease of Dersian )alnut trees. Dh"topatholog" $&7%-'*%/-. Einslo), ;. ;. A., J. Broadhurst, 2. ;. Buchanan, C. Hrum)iede, Jr., L. A. 2ogers, and <. H. Smith. -9/:. The families and genera of the bacteria. 5inal report of the Committee of the Societ" of American Bacteriologists on characteri4ation and classification of bacterial t"pes. J. Bacterid. $7-9-*//9. Fhang, I. and H. <eider. -99&. Differentiation of Erwinia amylovora strains b" pulsed* field gel electrophoresis. Appl. ;n iron. =icrobiol. %0788/-*88/%. CHEMICAL LI#T #our&e

0-.

0/. 00.

08.

;.

C@e) &al

Lnless stated other)ise, all chemicals in this list can be obtained from Sigma Chemical Co., D.1. Box -8$:', St. Louis, =1 %0-&'.

Difco Difco Acetic acid Agar L*arabinose L*asparagine Beef extract Bromcresol purple Bromth"mol blue Cobalt chloride Cr"stal iolet Cresol red Cupric sulfate C"cloheximide L*c"steine h"drochloride ;osin I ;thanol $8

<elatin Difco <l"cerol 3nositol HCH/HD:8 H,:0 H1H =annitol =gS:8 =gSC"&H/: =elbiose =eth"lene blue N =eth"l glucoside =ineral oil ,H8C,H8H/D:8 #,H8(/S:8 ,aCl ,a1H ,eom"cin sulfate ,eutral red ,icotinic acid ,itrilotriacetic acid ,,,*dimeth"l*- *naphth"lamine ,o obiocin ,utrient agar ,utrient broth Difco Deptone Difco Salicin Sodium heptadec"l sulfate #Terigitol &( Source presentl" un6no)n Sodium pol"pectate Dr. =. Burger, ;ton 2idge 2oad, =adison, E3. LSA Sodium taurocholate Sorbitol Sucrose Tergitol anion & #sodium heptadec"l sulfate( Lnion Carbide Thallium nitrate H R H 2are Chemicals Thiamine h"drochloride Tr"ptone Difco Lrea Ieast extract Difco

55

H. B-" !.

GRAM-NEGATIVE BACTERIA Erwinia #o'( Ro( Group #. H. De Boer and A. Kel)an INTRODUCTION

The proposal presented $8 "ears ago b" Ealdee #$'( that the soft*rot Erwinia species be placed in a separate genus, Pectobacterium, has not been generall" accepted. 1ther similar recommendations to place the pectol"tic Erwinia into a separate genus ha e also lac6ed support #-0, 0/, 0$(. Ho)e er, 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 perspecti e. 3n this manual, the follo)ing designations are used for the "carotovord( group7 Erwinia carotovora su5sp. caratavora, E. carotovora su5sp. atroseptica, E. carotovora su5sp. betavasculorum, E. carotovora su5sp. wasabiae ,!;+0 E. carotovora su5sp. odorifera ,!8+0 E. chrysanthemi, E cypripedii, E. rhapontici #-0, -8, 8', $:, $$( and E. cacticida #-(. 1nl" the general characteristics for E. chrysanthemi are included in this manual, although patho ars ha e been designated #-0,-8( and described in se eral references #', 9, -:, $$(. ; en thought. cypripedii and E. 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. Dhenot"pic characteristics of species in the genus Erwinia are presented b" Starr #$0( and Starr and Chatter!ee #$8(, and more specificall" for E. chrysanthemi b" Dic6e" #', 9(. ;colog" of the soft rot Erwinia )as re ie)ed b" Derombelon and Helman #8-( and b" Stanghellini #$/(. The status of research on the potato blac6leg disease is summari4ed in the 2eport on the 3nternational Conference on potato Blac6leg Disease #/-(. Details of methods generall" applicable for identification of bacteria in the famil", Enterobacteriaceae are gi en in <erhardt #-'(. Drocedures for specific identification of soft*rot bacteria )ere outlined b" Cother and Si asithamparam #0( and a manual on methods for the detection and .uantification of E. carotovora subsp. atroseptica on potatoes )as published b" Derombelon and an der Eolf #8/(. ". I#OLATION TECHNI$UE# U#ING #EMI#ELECTTVE MEDIA

Standard procedures for isolating ph"topathogenic bacteria from plant and soil samples can be used for the pectol"tic Erwinia #0-(. Detailed procedures for isolation of soft rot bacteria are found in Helman and Dic6e" #0:(. 3solation from deca"ed plant tissue and soil is facilitated b" the use of selecti e and enrichment media #/, 8, /', 08, 0&, 09, 8:, 88, $%(. To isolate E. chrysanthemi and the subspecies of E. carotovora, homogeni4e a small piece of tissue from the peripher" of the deca" lesion in sterile )ater, and strea6 or spread $%

appropriate dilutions on a selecti e medium. The selecti e medium and temperature of incubation ma" affect the efficienc" of reco er" and gro)th of specific strains #09(. Derhaps one of the most effecti e and easil" prepared selecti e media is cr"stal iolet pectate medium #CCD, 8(. 3solation plates should be incubated at /0*/&AC. 1n CCD the soft*rot Erwinia species form iridescent, cross*hatched, translucent colonies in deep, cup*li6e depressions or pits, )hereas pectol"tic Pseudomonas spp. form shallo) pits #Dlate /, 5ig. $(. Colonies are most clearl" obser ed under a lo) po)ered dissecting microscope illuminated from belo) )ith obli.ue illumination. Dure cultures can often be obtained b" strea6ing directl" from the deep pits in CCD onto CD< medium #/9(. To isolate pectol"tic bacteria from en ironmental samples, prior enrichment of the sample in a li.uid enrichment medium greatl" enhances isolation of lo) populations of the bacteria. Samples are incubated in enrichment medium #08( for 0*$ da"s at /0*/&AC before the" are strea6ed onto CCD. Selecti e media for isolating the other members of this group, E. cacticida, E. cypripedii, and E. rhapontici ha e not been de eloped. Ho)e er, these bacteria can be isolated on non*selecti e media such as nutrient agar or CD< medium. a. CV9 )ed u) #8( 3,,a1H -:K CaCl/*/H/: ,a,10, Agar Sodium pol"pectate SDS :.:&$K cr"stal iolet per $:: ml )ater 8.$ ml 0.: ml -.: g -.$ g -:.: g :.$ ml -.: ml

Dour 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. Dour medium in a / litre flas6 and add the SDS and cr"stal iolet. S)irl contents, cap flas6 )ith aluminum foil and autocla e for /$ min at -/:AC. Dour plates )hile medium is hot and entilate plates )hile cooling to pre ent condensation. 5. C9G )ed u) #/9( Casamino acids Deptone <lucose Agar
57

perL -.: g -:.: g -:.: g -'.: g

&.

9ol:pe&(a(e enr &@)en( )ed u) #08( Sodium pol"pectate -:K #,H8(/S:8 -:KH/HD18 $K =gS:8*&H/: perL -.$ g -:.: ml -:.: ml $.: ml

0.

D355;2;,T3AT31, 15 C1==1,LI 3S1LAT;D SD;CT;S A,D SLBSD;C3;S

The soft rot Erwinia, li6e other members of the Enterobacteriaceae are facultati el" anaerobic, peritrichousl" flagellated, <ram*negati e rods. All strains of the fi e species considered in this chapter are catalase positi e, oxidase negati e, ferment glucose, reduce nitrate, produce p*galactosidase and H/S, and utili4e L*arabinose, D*galactose, D*glucose, gl"cerol, D* mannose, D*ribose, and sucrose, but do not produce urease, or acid from adonitol. =ost strains utili4e L*rhamnose and D*mannitol but not dextrin. Holt et al. #/0( ha e noted in Berge"Gs =anual that a meaningful comparison of Erwinia )ith other genera of Enterobacteriaceae is still needed because er)iniae ha e not been full" tested )ith the complete range of standard tests used for other enterobacteria. 5or the pectol"tic Erwinia presumpti e identification can be made simpl" on the basis of pectol"tic acti it" and colon" characteristics on CCD medium obser ed )ith obli.ue illumination. 5or CD< medium, addition of :.::$K /,0,$ triphen"l tetra4olium chloride to the medium enhances the differences in colon" t"pes and ma6es presumpti e identification easier #0:(. 5urther identification to genus can be made on the basis of <ram negati e stain reaction, rod shape, facultati e anaerobic metabolism, and peritrichous flagellation. 5or the non*pectol"tic species, E. cypripedii and E. rhapontici, further tests to establish species identit" is re.uired to ensure correct genus designation. H"man et al. #/$( suggest that pectol"tic bacteria can be confirmed as Erwinia on the basis of being facultati el" anaerobic, catalase positi e and oxidase negati e. All the pectol"tic Erwinia species and subspecies can be differentiated from one another on the basis of a limited number of tests #Table -(. Although there are se eral different )a"s in )hich man" of these tests can be completed, onl" one procedure is described for each test. The methods ha e been selected as the most con enient and simplest for testing a number of isolates at one time. 3t is essential that positi e and negati e control strains be included in each test.

$'

Table -. Biochemical and ph"siological tests that differentiate pectol"tic species and subspecies of the Erwinia soft rot group0

>, ':K or more strains positi eB C, bet)een /- *&9K of strains positi eB *, ':K or more strains negati eB ,D, not determined.

;cc S E. carotovora subsp. carotovora ;ca S E. carotovora subsp. atroseptica ;cb S E. carotovora subsp. betavasculorum ;co S E. carotovora subsp. odorifera ;c) S E. carotovora subsp. wasabiae ;ch S E. chrysanthemi ;ct S E. cacticida b Some strains of E. carotovora subsp. carotovora ma" be )ea6l" positi e 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 fermentati e from oxidati e metabolism of carboh"drates #see 0, p. 9(

$9

5.

%la-ellar s(a n

The procedure of 2"u #8$( or the sil er stain #see -: a, p. -:( is er" effecti e in staining flagella ofErwinia carotovora. &. Ca(alase (es(

=ix a loopful of a 8'*h*old culture gro)n on nutrient agar )ith a drop of freshl" prepared 0K H/:/ on a glass slide. The formation of gas bubbles indicates a positi e reaction. d. O3 dase (es( ,see &, p. !4+

Lse a platinum loop to transfer bacteria from a 8'*h*old culture gro)n on nutrient agar onto filter paper impregnated )ith a drop of -K a.ueous tetrameth"l*p* phen"lenediamine dih"drochloride solution. De elopment of a purple color )ithin -: sec indicates a positi e reaction. e. GroA(@ a( .8BC

Lse CD< or nutrient agar medium at -$ ml per plastic Detri dish. Stab inoculate isolates from a dense a.ueous suspension in a grid pattern. Dlace plates in a plastic bag to pre ent dr"ing out of the medium, and incubate at 0&AC for three da"s. 1bser e inoculation point for bacterial gro)th. The presence of a bacterial colon" at the point of inoculation is considered a positi e test. '. Redu& n- su5s(an&es 'ro) su&rose

Dissol e -: g Bacto*peptone and -& g agar in ':: ml distilled )ater, autocla e, and add /:: ml of a filter sterili4ed sucrose solution #8: g sucrose in /:: ml distilled )ater( and pour into Detri dishes. Stab incubate isolates from a dense a.ueous suspension in a grid pattern and incubate for t)o da"s at /0 * /&AC. Then flood each plate )ith -: ml BenedictGs solution and incubate 0: * 8$ min at %:AC. A positi e test is an 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 dissol e. Separatel" dissol e 0.$ g of CuS:8*$H/: in 8: ml distilled )ater. Combine and mix the t)o solutions. The mixture is stable for se eral months at room temperature. g. 9@osp@a(ase a&( 6 (: ,".+ Drepare nutrient agar and )hen partiall" cooled after autocla ing add a filter* %:

sterili4ed solution of phenolphthalein diphosphate sodium salt to gi e a final concentration of :.:$K #)@ (, mix, and pour plates. Stab inoculate isolates from a dense a.ueous suspension in a grid pattern and incubate for 8' h at /0*/&AC. Then place a drop #ca. :.- ml( of concentrated ammonia in the lid of the Detri dish and in ert the medium o er it. Colonies that ha e phosphatase acti it" turn a bright pin6 color almost immediatel". Lse glass Detri dishes for this assa" because ammonia softens plastic dishes. @. #ens ( 6 (: (o er:(@ro):& n

Seed a +la)n+ of bacteria on nutrient agar plates b" pouring % ml of molten nutrient agar cooled to $: AC and inoculated )ith -:: ul of a dense a.ueous suspension of the test bacterium o er the base la"er of medium in the plate. After the medium has solidified place a commerciall" prepared dis6 impregnated )ith -$ ug of er"throm"cin per dis6 on the surface of the medium. 3ncubate at /0*/& AC for 8' h. A 4one of inhibition around the dis6 is recorded as a positi e test for sensiti it". . Indole produ&( on ,.<+

Drepare medium containing -: g tr"ptone, - g L*tr"ptophan, and -$ g agar per liter. Stab inoculate isolates from a dense a.ueous suspension in a grid pattern and incubate for 8' h at /0*/&AC. Ehen read" for testing, pipet t)o or three drops of p* dimeth"lamino*cinnamaldeh"de #- g in -:: ml of -:K h"drochloric acid( on filter paper in a Detri dish. Dlace a loopful of bacteria from a colon" on the treated filter paper. A blue*green coloration of the treated area )ithin -: sec is a positi e test. A negati e reaction is denoted b" a colorless or light pin6 coloration of the treated area. C. A& d 'ro) &ar5o@:dra(es ,"4+

Drepare -K peptone broth and use bromcresol purple #-:*0: mg@L( as an indicator. Add the carboh"drate to be tested to gi e a final concentration of -K, ad!ust pH to &.:, filter sterili4e, and dispense into sterile test tubes. 3noculate tubes )ith a loopful of cells from a 8'*h*old culture and incubate at /0*/&AC for 8' h. A color change to "ello) indicates acid production #Dlate /, 5ig. %(. D. U( l Ea( on o' De(o-)e(@:l -lu&os de ,)e(@:l-G-d--lu&os de+ ,1"+ perL HH/D:8 /.: g H/HD:8 &.: g ,H8C-.: g -:K =gS:8*&H/: /.: ml Casaminoacid -.: g Agar -$.: g %-

After autocla ing cool to $: AC and immediatel" add $: ml of filter sterili4ed /:K 6eto*meth"l glucoside, and / ml of filter sterili4ed -K tetra4olium chloride, and pour into Detri dishes. Stab inoculate isolates from a dense a.ueous suspension in a grid pattern and incubate for 8' h at /0*/&AC. E. carotovora subsp. atroseptica gro)s )ell and produces colonies )ith red centers #Dlate /, 5ig. &(. E. carotovora subsp. carotovora gro)s poorl" and colonies are )hite although a little red color ma" de elop at the point )here the inoculum )as stabbed into the medium. 7. 9ATHOGENICIT2 TE#T# a. T ssue )a&era( on

The abilit" of a bacterium to macerate plant tissue confirms its pectol"tic nature and pro ides an indication of pathogenicit". =aceration abilit", ho)e er, does not pro e pathogenicit" of the bacterium in a natural en ironment. 5alse positi e results ma" be due to naturall" occurring endoph"tic or epiph"tic microorganisms associated )ith inoculated tissue. Carious plant tissues such as potato tubers #Dlate 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 con enient si4e pieces, place in a Detri dish on moist sterile filter paper, and inoculate )ith :.-*ml of the bacterial suspension #ca. -:% C5L@ml( from a /8*h*old*culture. 3ncubate at /:*/& AC for 8' h and probe the tissue surrounding the inoculation site )ith a spatula or needle to determine )hether deca" and tissue maceration has occurred. 5. 9a(@o-en & (: (es(s

The most precise method for determination of pathogenicit" in ol es the use of infecti it" titrations #-$(. Drepare bacteria as described #see $, p. //%(. 3noculate host plants using a range of serial -:*fold dilutions from -:/ *-:J C5L@ml of the test bacterium using a micropipette tip or s"ringe #00(. At least ' to -: plants per inoculum le el should be used for each of four to fi e dilutions. Seal the in!ection point after inoculation b" application of a small amount of petroleum !ell" o er the puncture. 3nfection of inoculated plants )ill be enhanced if the" are maintained at a high relati e humidit" or e en placed under an intermittent misting s"stem. 5or additional details on other inoculation procedures, reference should be made to studies on host range of E. chrysanthemi #9( and tests )ith E. rhapontici #8'(.

%/

%.

=1L;CLLA2, S;21L1<3CAL, A,D C1==;2C3AL ALT1=AT;D T;CH,3TL;S a. Mole&ular (e&@n ?ues !+ 9ol:)erase C@a n Rea&( on

Three sets of specific primers exist for the identification of E. carotovora subsp. atroseptica and t)o sets for E. chrysanthemi #Table /(. Drimers, ;CAlr and ;CAlf for E. carotovora subsp. atroseptica )ere constructed b" De Boer and Eard #&(, primers ;2E512 and AT212;C )ere designed b" Smid et al. #$-( and primers, I8$ and I8% b" 5rechon et al. #-%(. Smid et al. #$-( also designed a primer set for E. chrysanthemi, ;2E512 and CH22;C )hile another set of primers for E. chrysanthemi, AD;l and AD;/ )as designed b" ,assar et al. #0'(. See Appendix A for details. Ta5le ". Se.uence of oligonucleotide primer pairs currentl" in use in DC2 anal"sis of Erwinia species.

Target ;ca ;ca

,ame ;2E512 AT212; C I8$ I8% ;CAlf ;CA/r AD;l AD;/ ;2E51 2 CH22;C

Drimer Se.uence
S'-ACGCATGAAATCGGCCATGC-S" S'-ATCGATATTTGATTGTC-S' 5'-TCACCGGACGCCGAACTGTGGCGT-3' 5'-TCGCCAACGTTCAGCAGAACAAGT-3'

Droduct si4e 0'9

438

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5'-CGGCATCATAAAAACACG-3' 5'-GCACACTTCATCCAGCGA-3' 5-GATCAGAAAGCCCGCAGCCAGAT-3' S-CTGTGGCCGATCAGGATGGTTTTGTCGTGC-S' 5'-ACGCATGAAATCGGCCATGC-3' 5'-AGTGCTGCCGTACAGCACGT-3'

690 420 450 38

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5.

#erolo- &al (e&@n ?ues

Serological procedures are useful for preliminar" identification of purified cultures or bacteria in plant and soil samples if specific antibodies to commonl" occurring serogroups are a ailable. Standard indirect immunofluorescence and triple antibod" sand)ich ;L3SA protocols can be used for strain identification if it is 6no)n that the antibod" reacts according to serogroup specificit" in the particular procedure being used #//(. Lipopol"saccharides are important antigenic determinants of most <ram negati e bacteria including the pectol"tic er)iniae and antibodies to them can be used to differentiate among strains #$, 8&(. The usefulness of serolog" for identification of pectol"tic Erwinia to species and subspecies is limited, ho)e er. Although antibodies to lipopol"saccharides of E. carotovora subsp. carotovora and atroseptica are highl" specific #$( the existence of man" E. carotovora subsp. carotovora serogroups ma6es identification of this subspecies b" serolog" impractical. =ost E. carotovora subsp. atroseptica strains belong to serogroup 3 and se eral serogroup*specific monoclonal antibodies are useful for identification of the subspecies #%, /&(. Ho)e er, at least three other serogroups #?C333, ??, and ??33( occur among E. carotovora subsp. atroseptica strains, and although the" occur infre.uentl", it limits serological identification of all strains in the subspecies. Serolog" of the other E. carotovora subspecies has not been studied. 3t is estimated that about one third of the E. carotovora strains isolated from potato can be placed in one of fort" serogroups described to date. Some strains from other hosts also belong to these serogroups but the serological di ersit" of strains from non*potato hosts is un6no)n. Homologous strains representing the four E. carotovora subsp. atroseptica serogroups #3C=D 9-%&, 9-'8, 9-'%, and 9-''( and 0% E. carotovora subsp. carotovora serogroups #3C=D 9-%'*9/:%( are in the 3nternational Collection of =icro*organisms from Dlants, Landcare 2esearch, Auc6land, ,e) Fealand. Dol"clonal antisera produced against glutaraldeh"de*fixed cells of homologous strains normall" contain a high titre of antibodies to the lipopol"saccharide antigen and are useful for serot"ping. Strains of E. carotovora can be placed into serogroups b" the 1uchterlon" double diffusion test #$(. The test re.uires a dense a.ueous suspension of a pure culture, )hich after treatment )ith phenol #*/: @@-@ml(, is placed in a 0 mm )ell cut into an agar double diffusion plate #-$ ml of :.'K agar, :.'$K ,aCl, /:: ppm ,a,0 in a -:: x -$ mm plastic Detri dish(. An ad!acent )ell is loaded in the same )a" )ith the phenol*treated cells of the homologous strain of the serogroup being tested. Serogroup*specific antibod" is placed in a )ell e.uidistant #8 mm( from the t)o sample )ells. Drecipitin bands that form in -/*/8 h bet)een the sample and antibod" )ells indicate a serological reaction, but the serogroup can onl" be identified to that of the homologous strain and antibod" used if the band formed b" the test strain fuses )ith that formed b" the homologous strain.
64

Serological techni.ues using +1+ antigens #--,8%,8&(, purified membrane protein complex #$9(, or fimbrial*specific monoclonal antibodies #89( are er" useful for rapid presumpti e identification of suspected cultures of E. chrysanthemi. Se eral serological t"ping schemes of three or four sero ars or serogroups ha e been suggested but these ha e not been reconciled )ith one another or adopted for further studies #--, /&(. Dol"clonal and monoclonal antibodies produced to the lipopol"saccharide of E. chrysanthemi do react )ith man" strains of the species but their use for identification and detection is greatl" hampered b" their cross*reacti it" )ith a lipopol"saccharide epitope of some common soil pseudomonads #$&(. A monoclonal antibod" to a fimbrial epitope of E. chrysanthemi has potential for identification and perhaps detection 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( l Ea( ons

The commerciall" a ailable Biolog s"stem #Biolog, 3nc., Ha")ard, CA( for substrate utili4ation is helpful for rapid identification of those species and subspecies included in the database. These currentl" include7 E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. chrysanthemi, and E. cypripedii. See Appendix C for description and details. "+ !<# rRNA -ene se?uen& n-

The =icroSe. =icrobial 3dentification s"stem is produced b" D; Applied Bios"stems. 3dentification ser ice using the =icroSe. -%S r2,A <ene Hit is pro ided b" =3D3 Labs #,e)ar6, D; -9&-0(. The commercial database currentl" contains -%S r2,A se.uences for E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. cypripedii, and E. rhapontici of the soft rot er)iniae group. &. CULTURE 9RE#ERVATION

=embers of the soft rot Erwinia group ar" considerabl" in iabilit" )hen stored under arious conditions. Se eral methods are a ailable for the storage of Erwinia cultures. These include agar plate cultures, )ater cultures, gl"cerol cultures and l"ophili4ed cultures. The method of choice depends on the duration of storage, the species or subspecies in ol ed and the a ailable e.uipment and@or cost. Agar plate or slant cultures are a common )a" of storing soft rot Erwinia bacteria for a short time. Cultures can be stored at 8AC for, at least for one month, on IDC agar slants #see b, p. 8(. <ro) strain for -*/ da"s at /0*/&AC, then refrigerate.

65

Cell suspensions of some soft rot Erwinia also preser e )ell in )ater at room temperature. Ho)e er, some species, notabl" E. chrysanthemi, lose iabilit" almost immediatel" )hen stored in )ater. <ro) the culture on an appropriate agar medium and use a loop to scrape the cells into a scre)*capped ial filled )ith sterile )ater. Co er the ial, label and store at room temperature. Bacterial cell suspensions can also be stored at *&: AC in Luria*Bertani #LB( #see -, p. 8-( *gl"cerol broth #LB broth containing /:K gl"cerol, mixed after autocla ing(. Cultures are gro)n as described abo e for )ater cultures and cells are resuspended in the LB*gl"cerol medium in cr"o ials. These cells )ill remain iable for a er" long time. Ehen re i ing a gl"cerol culture, it is ad isable not to let the fro4en culture tha) before strea6ing out. 3t is best to simpl" remo e samples of fro4en cells )ith a loop and strea6 onto the medium. A iable culture should start gro)ing during o ernight incubation. This method of culture preser ation has the ad antage that culture re i al is .uic6 but re.uires a *&: AC free4er. A commercial product, +Drotect+, consisting of medium and beads in cr"o ials is also a ailable as a bacterial preser ation s"stem #Drotect, STC Technical Ser ice Consultants Ltd, He")ood Lanes, LH(. Bacterial cells are suspended in the medium, mixed )ith the beads in the ial and then excess medium remo ed. The ials are stored at *&: AC and bacteria are reco ered b" remo ing indi idual beads )hich are strea6ed directl" on a culture medium. An alternate method for long*term culture preser ation is l"ophili4ation. Cells are suspended in -:K s6im mil6 and free4e dried under acuum. Drepare -:K s6im mil6, autocla e, and ali.uot at about 8:: ul per sterile tube. Suspend bacterial cells from an o ernight agar plate culture in the mil6, ortex and use a sterile Dasteur pipette to transfer about -:: ul of the suspension into a sterile l"ophili4ation tube. Cap each tube )ith a cotton plug and free4e o ernight. Lse a free4e*dr"ing machine to l"ophili4e the cultures and then seal the ials b" melting the top )ith a torch. L"ophili4ed cultures can be stored at room temperature for man" "ears. '. L3T;2ATL2; C3T;D

-. Alcorn, S. =, T. C. 1rum, A. <. Steiger)alt, J. L. =. 5oster, J. C. 5ogleman and D. J. Brenner. -99-. Taxonom" and pathogenicit" of Erwinia cacticida subsp. no . 3nt. J. S"st. Bacteriol. 8-7-9&*/-/. /. Burr, T. J., and =. ,. Schroth. -9&&. 1ccurrence of soft*rot Erwinia spp. in soil and plant material. Dh"topatholog" %&7-0'/*-0'&. 0. Cother, ;. J, andH. Si asithamparam. -9'0. Erwinia. TheGcaroto oraGgroup. Dages '&*-:% in7 D. C. 5ah" and <. J. Dersle", eds., Dlant Bacterial Diseases7 A Diagnostic <uide. Academic Dress, Australia. 8. Cuppels, D., and A. Helman. -9&8. ; aluation of selecti e media for isolation of soft* %%

rot bacteria from soil and plant tissue. Dh"topatholog" %878%'*8&$. $. De Boer, S. H, 2 J. Copeman, and H. Cruggin6. -9&9. Serogroups of Erwinia carotovora potato strains determined )ith diffusible somatic antigens. Dh"topatholog" %970-%*0-9. %. De Boer, S. H. and =. ;. =c,aughton. -9'&. =onoclonal antibodies to the lipopol"saccharide of Erwinia carotovora subsp. atroseptica serogroup 3. Dh"topatholog" &&7'/'*'0/. &. De Boer, S. H. and L. J. Eard. -99$. DC2 detection of Erwinia carotovora subsp. atroseptica associated )ith potato tissue. Dh"topatholog" '$7'$8*'$'. '. Dic6e", 2. S. -9&9. Erwinia chrysanthemi# A comparati e stud" of phenot"pic properties of strains from se eral hosts and other Erwinia species. Dh"topatholog" %97 0/8*0/9. 9. Dic6e", 2S. -9'-. Erwinia chrysanthemi. 2eaction of eight plant species to strains from se eral hosts and to strains of other Erwinia species. Dh"topatholog" &-7/0*/9. -:. Dic6e", 2. S., and J. 3. Cictoria. -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. H. L"emoto. -9'8. Erwinia chrysanthemi# serological relationships among strains from se eral hosts. Dh"topatholog" &87-0''* -098. D"e, D. E. -9%9. A taxonomic stud" of the genus Erwinia. 33. The +caroto ora+ group. ,. F. J. Sci. -/7'-*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", =. <oto, A. C. Ha")ard, 2. A. Lelliott, and =. ,. Schroth. -9':. 3nternational standards for naming patho ars of ph"topathogenic bacteria and a list of patho ar names and pathot"pe strains. 2e . Dlant Dathol. $97-$0*-%'. ;rcolani, <. L. -9'8. 3nfecti it" titration )ith bacterial plant pathogens. Annu. 2e . Dh"topathol. //70$*98. 5rechon, D., D. ;xbra"at, 1. <allet, ;. <uillot, C. Le Clerc, ,. Da"et and I. Bertheau. -99$. Se.uences nuceotidi.ues pour la detection des Erwinia carotovora subsp. %&

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atroseptica. 3nstitut ,ational de la 2echerche Agronomi.ue, 3nstitut ,ational Agronomi.ue Daris*<rignon, Sanofi Diagnostics Dasteur. Bre et 9$7-/*':0. -&. <allois, A., 2. Samson, ;. Agaron, D. A. D. <rimont. -99/. Erwinia carotovora subsp. odorifera subsp. no ., associated )ith odorous soft rot of chicor" (&hichorium intybus L(. 3nt. J. S"st. Bacteriol. 8/7$'/*$''. <erhardt, D. #;d.(. -990. =ethods for <eneral and =olecular Bacteriolog". Am. Soc. =icrobiol. Eashington, D. C. <oto, =., and H. =atsumoto. -9'&. Erwinia carotovora subsp. wasabiae subsp. no . isolated from diseased rhi4omes and fibrous roots of Japanese horseradish (Eutrema wasabi =axim(. 3nt. J. S"st. Bacteriol. 0&7-0:*-0$. <raham, D.C. -9&/. 3dentification of soft rot coliform bacteria. Dages /&0*/&9 in7 H. D. =aas <eesteranus, ed., Droc. 0rd 3nt. Conf. Dlant Dathogenic Bacteria, Eageningen, the ,etherlands. <raham, D. C. and =. D. Harisson, eds. -9'$. 2eport of the 3nternational Conference on Dotato Blac6leg Disease. Dotato =ar6eting Board, 1xford. Hampton, 2, ;. Ball and S. De Boer, eds. -99:. Serological =ethods for Detection and 3dentification of Ciral and Bacterial Dlant Dathogens7 A Laborator" =anual. ADS Dress, =,. Holt, J. <., ,. 2. Hrieg, D. H. A. Sneath, J. T. Stale" and S. T. Eilliams, eds. -998. Berge"Gs =anual of Determinati e Bacteriolog". ,inth edition. Eilliams R Eil6ins, Baltimore, =D. Hugh, 2, and ;. Leifson. -9$0. The taxonomic significance of fermentati e ersus oxidati e metabolism of carboh"drates b" arious <ram*negati e bacteria. J. Bacteriol. %%7/8*/%. H"man, L. J., 3. H. Toth and =. C. =. Derombelon. -99'. 3solation and identification. Dages %:*&- in7 =. C. =. Derombelon and J. =. an der Eolf, eds., =ethods for the detection and .uantification of Erwinia carotovora subsp. atroseptica on potatoes7 Laborator" =anual. Scottish Crop 2esearch 3nstitute. 1ccasional Dublication ,o. -:. 3 ergo)rie, Dundee, DD/ $DA, Scotland, LH. H"man, L. J., A. Eallace, =. =. Lope4, =. Cambra, = .T. <orris and =. C. =. Derombelon. -99$. Characteri4ation of monoclonal antibodies against Erwinia carotovora subsp. atroseptica serogoup 37 specificit" and epitope anal"sis. J. Appl. Bacteriol. &'780&* 888. %'

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Janse, J. D. and =. A. 2uissen. -9''. Characteri4ation and classification of Erwinia chrysanthemi strains from se eral hosts in the ,etherlands. Dh"topatholog" &'7'::*':'. Hado, C. 3., and =. <. Hes6ett. -9&:. Selecti e media for isolation of 0!robacterium, &orynebacterium, Erwinia, Pseudomonas and 1anthomonas. Dh"topatholog" %:79%9*9&%. Helman, A. -9$8. The relationship of pathogenicit" in Pseudomonas solanacearum to colon" appearance on a tetra4olium medium. Dh"topatholog" 887%90*%9$. Helman, A, and 2. S Dic6e". -9'9. Detection of Erwinia carotovora and E. chrysanthemi in potato. Dages &%*9- in7 A. E. Saetler, ,. E. Schaad and D. A. 2oth, eds. Detection of Bacteria in Seed and 1ther Dlanting =aterial. ADS Dress, St. Daul, =,. Hlement. F., H. 2udolph and D. C. Sands. -99:. =ethods in Dh"tobacteriolog". A6ademiai, Budapest, Hungr". Lelliott, 2. A, and2. S. Dic6e". -9'8. <enus C33. Erwinia Einslo), Broadhurst, Buchanan, Hrum)eide, 2ogers, and Smith -9/:. Dages 8%9*8&% in7 ,. 2. Hrieg and John <. Holt, eds., Berge"Gs =anual of S"stematic Bacteriolog". Col. -. Eilliams and Eil6ins, Baltimore, =D. Lum, H. I., and A. Helman. -9'-. 3nfecti it" titrations of Pseudomonas solanacearum on tomato. Dh"topatholog" &-7'9-. #Abstr.( =enele", J. C, and =. ;. Stanghellini. -9&%. 3solation of soft*rot Erwinia spp. from agricultural soils using an enrichment techni.ue. Dh"topatholog" %%70%&*0&:. =ergaert, J., L. Cerdonc6, H. Hersters, J. S)ings, J.*=. Boeufgras, and J. De Le". -9'8. ,umerical taxonom" of Erwinia species using AD3 s"stems. J. <en. =icrobiol. -0:7-'90* -9-:. =iller, J. =. and J. E. Eright. -9'/. Spot indole test7 e aluation of four methods. J. Clin. Dathol. -$7$'9*$9/. =iller, T., and =. ,. Schroth. -9&/. =onitoring the epiph"tic population of Erwinia amylovora on pear )ith selecti e medium. Dh"topatholog" %/7--&$*--'/. ,assar, A., A. Darrasse, =. Lemattre, A. Hotou!ans6", C. Der in, 2. Cedel and I. Bertheau. -99%. Characteri4ation of Erwinia chrysanthemi b" pectinol"tic iso4"me pol"morphism and restriction fragment length pol"morphism anal"sis of DC2*amplified fragments of pel genes. Appl. ;n iron. =icrobiol. %/7///'*//0$. 1G,eill, 2., and C. Logan. -9&$. A comparison of arious selecti e isolation media for their %9

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efficienc" in the diagnosis and enumeration of soft rot coliform bacteria. J. Appl. Bacteriol. 097-09*-8%. 8:. 8-. 8/. Derombelon, =. C. =. -9&-. A semi*selecti e medium for estimating population densities of pectol"tic Erwinia spp. in soil and in plant material. Dotato 2es. -87-$'*-%: Derombelon, =. C. =, and A. Helman. -9':. ;colog" of the soft rot er)inias. Annu. 2e . Dh"topathol. -'70%-*0'&. Derombelon, =. C. =. and J. =. Can Der Eolf. -99'. =ethods for the detection and .uantification of Erwinia carotovoa subsp. atroseptica on potatoes. Scottish Crop 2esearch 3nst. 1ccasional Dublication ,o. -:. 3 ergo)rie, Dundee, DD/ $DA, Scotland, LH. Dhillips, J. A, and A. Helman. -9'/. Direct fluorescent antibod" stain procedure applied to insect transmission of Erwinia carotovora. Dh"topatholog" &/7'9'*9:-. Dierce, L. and A. H. =ccain. -99/. Selecti e medium for isolation of pectol"tic Erwinia sp. Dlant Dis. &%70'/*0'8 2"u, ;. -90&. A simple method of staining bacterial flagella. Hitasato Arch. ;xp. =ed. -87/-'*/-9. Samson, 2. -9&0. Les Erwinia pectinol"ti.es. 33. 2echerches sur les antigenes somati.ues tfErwinia carotovora ar. chrysanthemi #Bur6holder( D"e -9%9. Ann. Dh"topathol. $70&&* 0''. Samson, 2., 2. Doutier, =. Saill" and B. Jouan. -9'9. Caracterisation des Erwinia chrysanthemi isolees de 2olanum tuberosum et dGautres plantes*hotes selon les bio ars et serogroupes. ;DD1 Bull. -&7--*-%. Sell)ood, J., and 2. A. Lelliott. -9&'. 3nternal bro)ning of h"acinth caused b" Erwinia rhapontici. Dlant Dathol. /&7-/:*-/8. Singh, L., C. =. Tre ors, S. H. DeBoer, and J. D. Janse. -999. A fimbrial*specific monoclonal antibod"*based ;L3SA test for ;uropean potato strains of Erwinia chrysanthemi and comparison to DC2. Dlant Dis. '87880*88'. S6erman, C. B. D., C. =c<o)n, and D. H. A. Sneath, eds. -9':. Appro ed list of bacterial names. 3nt. J. S"st Bacteriol. 0:7//$*8/:. Smid, ;. J., A. H. J. Jansen, and L. <. =. <orris. -99$. Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using pol"merase chain reaction. Dlant Dathol. 887-:$'*-:%9
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Stanghellini, =. ;. -9'/. Soft rotting bacteria in the rhi4osphere. Dages /89*/%0 in7 =.S. =ount and <. H. Lace", eds., Dh"topathogenic Dro6ar"otes. Academic Dress, ,e) Ior6. Starr, =. D. -9'0. The genus Erwinia. Dages -/%:*-/&- in7 Dh"topathogenic Bacteria. =. D. Starr, ed. Chapter -:/. Springer Cerlag, ,e) Ior6. Starr, =. D., and A. H. Chatter!ee. -9&/. The genus Erwinia# ;nterobacteria pathogenic to plants and animals. Annu. 2e . =icrobiol. /$70'9*8/%. Thomson, S. C., D. C. Hildebrand, and =. ,. Schroth. -9'-. 3dentification and nutritional differentiation of the Erwinia sugar beet pathogen from members of Erwinia carotovora and Erwinia chrysanthemi. Dh"topatholog" &-7-:0&*-:8/. Tsu"ama, H., and =. Sa6amoto. -9$/. 3solationmethodsof soft*rot causing bacteria from the soil. Sci. 2ep. 2es. 3nst. Toho6u Lni ., Ser. D., Biol. 07/9*08. Can Der Eolf, J. =., J. 2. C. =. Can Bec6ho en, ;. De Boef, and H. J. =. 2oo4en. -990. Serological characteri4ation of fluorescent Pseudomonas strains cross*reacting )ith antibodies against Erwinia chrysanthemi. ,eth. J. Dlant Dathol. 997$-*%:. Ealdee, ;. L. -98$. Comparati e studies of some peritrichous ph"topathogenic bacteria. 3o)a State College J. Sci. -9780$*8'8. Iar6us, =. and ,. E. Schaad. -9&9. Serological relationships among strains of Erwinia chrysanthemi. Dh"topatholog" %97$-&*$//. C@e) &al L s( #our&e

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Lnless stated other)ise, all chemicals in this list )ere obtained from Sigma Chemical Co., D. 1. Box -8$:', St. Louis, =1 %0-&'. Agar Ammonia Arabitol Bromcresol purple CaCl/*/H/: Casamino acid Citrate Cr"stal iolet CuSC"$H/: ;r"throm"cin &-

H"drochloric acid H/HD:8 HH/D:8 Heto*meth"l glucoside Lactose =gS:8*&H/: =elibiose ,a/C:0H/: ,a,:0 2affinose Sorbitol ,a1H ,H8C#,H8(/S:8 D*dimeth"laminocinnamaldeh"de Deptone Dhenolphthalein diphosphate sodium Sodium citrate Sodium h"pochlorite Sodium pol"pectate Dr. =. Burger, ;ton 2idge 2oad, =adison, E3. LSA Sodium dodec"l sulfate Sucrose Tetrameth"l*p*phen"lenediaminedih"drochloride /,0,$ triphen"l tetra4olium chloride Tr"ptone Tr"ptophan

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