of p. taxonomy of phytopathogenic pseudomonadst · taxonomy of phytopathogenic pseudomonadst d. c....

JOURNAL OF BACTERIOLOGY, Jan. 1970, p. 9-23 Vol. 101, No. 1Copyright 0 1970 American Society for Microbiology Printed In U.S.A.

Taxonomy of Phytopathogenic PseudomonadstD. C. SANDS, M. N. SCHROTH, AND D. C. HILDEBRAND

Department of Plant Pathology, University of California, Berkeley, California 94720

Received for publication 6 October 1969

Phytopathogenic pseudomonads were placed into four major groups on the basisof nutritional and physiological characteristics. Group I consists of 86 strains ofphytopathogens distinguishable from other fluorescent pseudomonads by lowgrowth rates, ability to induce hypersensitivity on tobacco, absence of arginine di-hydrolase, and relatively limited ranges of carbon sources. Most of these strainscannot utilize benzoate, 2-ketogluconate, spermine, ,B-alanine, L-isoleucine, L-valine,and L-lysine. Most of the organisms in group I clustered into a small number of sub-groups, each of which generally corresponded to a previously recognized nomen-species. These subgroups differ with respect to the number of substrates used. As arule, the organisms that utilize the fewest substrates have the most limited hostranges. The fluorescent pseudomonads of group II are arginine dihydrolase-positiveand utilize a considerably larger number of carbon sources. Most pathogens ofgroup II are similar to Pseudomonasfluorescens biotype A. Groups III and IV con-sist of nonfluorescent pseudomonads. These two groups can be distinguished by thenumber of carbon sources used and by pigmentation. An amended description of thefluorescent pseudomonads and their internal subdivision is presented.

The lack of a detailed characterization of thebacterial plant pathogens has seriously hinderedtheir identification. A large number of specieshave been described on the basis of rather in-complete characterization of host range, symp-tomatology, and morphological and physio-logical tests. For example, 62 nomenspecies ofphytopathogenic fluorescent pseudomonads aredescribed in the seventh edition of Bergey'sManual.

Since 1957, Rhodes (15, 16), Lysenko (12),Jessen (5), Klinge and Graff (9), Stanier et al.(19), and Stolp (21) have published results ofextensive studies of the pseudomonads and havegreatly reduced the number of species within thegenus Pseudomonas. Rhodes (16), using equalweighting of 45 characters, reported a continuumof phenotypes within the fluorescent pseudo-monads. Stanier et al. (19), who examined 147characters (principally nutritional ones), recog-nized seven biotypes of P. fluorescens, two ofP. putida, and 11 other species of aerobic pseudo-monads.The relationship of the fluorescent pathogens

to saprophytes has remained unclear (22). Onthe basis of an examination of phage host ranges,Stolp (21) concluded that the fluorescent patho-gens were closely related to P. fluorescens. Jessen

1 The material presented represents, in part, data included inthe dissertation submitted by the senior author in partial fulfill-ment of the requirements for the Ph.D. degree in Plant Pathology.

9

(6), however, showed that many of the fluorescentpathogens significantly differed from P. fluores-cens with respect to a number of phenotypiccharacters, particularly their low growth rates.Lelliott et al. (11) have shown that the fluorescentpseudomonads (pathogenic and nonpathogenic)can be divided into five groups on the basis oflevan production, the oxidase reaction, rottingof potato, arginine dihydrolase, and productionof hypersensitivity in tobacco. Their study pro-vided a useful, simplified operational key fordeterminative purposes.

This paper presents detailed comparativephysiological characterizations of a number ofplant pathogenic and nonpathogenic pseudo-monads. This study was based on the use of theseries of tests described by Stanier et al. (19).These were supplemented by over 40 additionaltests, including a number specifically chosen fortheir possible utility in characterization of phyto-pathogenic species [production of extracellularribonuclease (5) and polypectinase (20), andutilization of glucosides].

MATERIALS AND METHODS

Biological material. Strains (see Tables 1 and 6-9 forlisting) were received from the following culturecollections: CUPP-Department of Plant Pathology(J. W. Lorbeer), Cornell University, Ithaca, N.Y.;ICPB-International Collection of PhytopathogenicBacteria, Department of Bacteriology (M. P. Starr),

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SANDS, SCHROTH, AND HILDEBRAND

University of California, Davis; NCPPB-NationalCollection of Phytopathogenic Bacteria, PlantPathology Laboratory (R. A. Lelliott), Harpenden,Herts., England; NEAP-Estacao Agronomica Na-cional (Maria de Lourdes d'Oliveira and ManuelaPinto de Silva), Oerias, Portugal; UCBPP-Depart-ment of Plant Pathology, University of California,Berkeley; UCBB-Department of Bacteriology andImmunology (M. Doudoroff, N. J. Palleroni, andR. Y. Stanier), University of California, Davis;UFCo-United Fruit Company (I. Buddenhagen), LaLima, Honduras; and UMPP-Department of PlantPathology (B. W. Kennedy), University of Min-nesota, Minneapolis.

Nutritional tests. The replica plating techniques ofLederberg and Lederberg (10) were used to testorganisms for growth on many carbon sources. Themethod of nutritional screening was used as describedby Stanier et al. (19) with slight modifications. Thestandard mineral base was strongly buffered (0.05M phosphate, pH 6.8), and was used in all tests forgrowth on single carbon sources. The nitrogen sourcewas 0.1% (NH4)2SO4. Low levels of carbon sources(0.1%) other than carbohydrates were used to avoidproblems of toxicity and contamination. Carbohy-drates were tested at 0.2%. lonagar II (1%) was usedin all tests because of its purity. Replica plating in-volved patching 17 organisms onto a 0.9% yeastextract-mineral base master plate, incubating for 1day, and then replicating 10 submaster plates whichwere used for seeding 100 plates of the test media.

Four modifications of the procedure described byStanier et al. (19) were used: (i) the complete vitaminmixture of Ballard et al. (1) was added to the minimalmedium for all nutritional tests as a precaution againstpossible vitamin requirements; (ii) plates were in-cubated at 27 C rather than 30 C; (iii) growth readingswere made after 1 and 2 weeks rather than 2 and 4days; and (iv) stock cultures were maintained onYDCP slants (0.25% Difco yeast extract, 2% dextrose,1% calcium carbonate, 0.25% Bacto peptone, and 2%Difco agar).The carry-over of nutrients on the replica plating

cloth sometimes allowed a small amount of growth;therefore, questionable readings were recorded asnegative. Possible toxicity of each carbon source atthe concentration used was not investigated in ourstudy.

Several compounds tests-methylamine, naphtha-lene, oxalate, eicosanedioate, and methanol-werenot utilized by any of the pathogens or saprophytestested and therefore were excluded from the compara-tive analyses of organisms. Twenty-five carbonsources additional to those used by Stanier et al.(19) were employed to include some compounds com-monly found in plants. These were pectate, L-a-lecithin, choline, triacetin, triproprionin, tannate,uridine, urate, L-glutamine, DL-asparagine, DL-norvaline, 3-phosphoglycerate, L-ascorbate, isoascor-bate, a-hydroxy-a-methylbutyrate, lauryl sulfate, lino-lenate, linoleate, N-acetylglucosamine, raffinose,melibiose, ai-methyl-D-glucoside, f3-methyl-D-glucoside,chlorogenate, and L-hydroxyproline.

N-hexadecane, n-dodecane, triglycolate, and tri-caproin were mixed with 0.01% dimethyl sulfoxide(DMSO) to aid solubilization in water. Control plateswith DMSO and DMSO plus glucose or succinatewere made to determine whether the solvent wastoxic or could serve as an energy source.

Oxidase test. The oxidase test was performed asdescribed by Stainer et al. (19). Cultures grown for 24hr on King B medium (KB) were used (8).

Urease test. Cells grown for 18 hr on YDCP plateswere examined with Patho-Tec-U indicator sticks(Warner-Chilcott Laboratories, Morris Plains, N.J.)for constitutive or adaptive production of urease.

Hypersensitivity test. A suspension of cells (5 X108 to 5 X 109/ml) from a 24-hr-old culture grown onKB was injected into the intercellular spaces ofNicotiana tabacum leaves. The plants were maintainedat 24 C, with the exception of those injected with P.solantacearum which were incubated at 32 C. Deathof the infiltrated portion of the leaf within 24 hr wasconsidered a hypersensitive response (8).

Growth rate. Growth rates were determined formost of the organisms in the standard basal mediumwith the addition of 9.5% Difco yeast extract and0.4% glucose (autoclaved separately). The mediumwas dispensed in 5-ml amounts in 25-ml shakerflasks, and in 25-ml amounts in 250-ml Erlenmeyerflasks with side arms that fit into a Klett-Summersoncolorimeter with a red filter. The organisms werebrought into log phase at 27 C in the small flasks,inoculated into the large precalibrated Klett flasks,shaken (rotary action) at 90 cycles/min, and readhourly until the stationary phase was attained.

Antibiotic sensitivity. Antibiotic sensitivity discs(Multidiscs, no. 11-1608, Colab Laboratories, Inc.,Chicago Heights, Ill.) were used to determine sensi-tivity to: albamycin, 30 4g/disc; chloramphenicol,30,g; erythromycin, 15 ug; penicillin G, 10 units;dihydrostreptomycin, 10,ug; and tetracycline, 30,Ag.The bacteria were seeded onto a plate containingNutrient Agar (Difco) plus 1% glucose. Inhibitionzones around the discs after 48-hr incubation at 28 Cdenoted sensitivity.

Temperature. Growth in broth at 4, 37, 39, and41 C was measured by the methods of Stainer et al.(19), except that readings were made after 10 days.

Salt tolerance. Sodium chloride at concentrationsof 2, 3.5, and 5% was added with 0.5% glucose to 1%Ionagar in the standard mineral base which contained0.3% Na+ and 0.006% Cl-. The plates were inoculatedwith replica plating techniques.

Deoxyribonuclease and ribonuclease. The occurrenceof deoxyribonucleic acid (DNA) hydrolysis (DifcoDNase Test Agar, code 0632) was examined after 4days. Only large zones which could not be accountedfor by cell lysis were considered positive. Hydrolysis ofribonucleic acid (Torula RNA, from Sigma ChemicalCo., St. Louis, Mo.) was determined by the methodsof Jeffries et al. (5).

Polypectinase. Hydrolysis of polypectate wastested by the methods of Starr (20), except that theamount of polypectate was reduced to 0.3 g/100 ml.

Lipase. The Tween 80 test of Sierra (17) was used.

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PHYTOPATHOGENIC PSEUDOMONADS

Denitrification. Tests for denitrification were thoseused by Stanier et al. (19).

Orthocleavage of protocatechuate. The methodused for examining the orthocleavage of protocatechu-ate was described by Wheelis et al. (24).

Arginine dihydrolase. The production of the enzymearginine dihydrolase was measured by the methods ofThomley (23), with the use of her medium 2A.

Numerical taxonomic methods. Results of all testswere recorded as "plus" or "minus," or occasionally as"no test made". Organisms were compared in termsof matching characters. Two types of comparisonswere used: SM, a simple-matching similarity coefficientobtained by dividing all matching test results by alltests (18), and PO, a positive-only similarity coefficient(positive matches/all tests less negative matches).The SM coefficient was useful when examining the

entire assemblage of organisms and to divide thisassemblage into several large groups through cluster-ing of the coefficients. In this context, there were noinvariant characters among the 188 characters onwhich the study was based. However, the organismsfailed to utilize a large proportion of the substrates intwo of the groups delineated by clustering the SMcoefficients. Consequently, the SM coefficients ofthese organisms were high, primarily because thelarge number of negative tests in essence wouldconstitute invariant characters for these groups.Thus, if two organisms have 70 negative tests incommon, 10 positive tests in common, and 20 variabletests, the SM coefficient is (70 plus 10)/100 or 80%,and the organisms would appear to be similar. ThePO coefficient tends to minimize this imbalance byeliminating all negative matches between two orga-nisms. Thus, the PO coefficient of the same twoorganisms would be 10/30 or 33%, and the orga-nisms would appear dissirnilar.The SM and PO coefficients were clustered by the

weighted pair group method of Sokal and Sneath(18). The programs were written by L. Niel Bell ofthe Department of Entomology, University of Cali-fornia, Berkeley.

RESULTS

Biological material selected for analysis. Theorganisms selected for study included 113 strainsof plant-pathogenic and nonpathogenic fluores-cent pseudomonads representing 31 nomen-species. Nonfluorescent pseudomonads consistedof 18 strains from 10 nomenspecies. In addition,nine strains from seven nomenspecies of threeother genera of phytopathogens (Agrobacterium,Erwinia, and Xanthomonas) were included forcomparative purposes, as were three strainsrepresenting three genera of Hydrogenomonas, agenus closely related to Pseudomonas.The collection of pseudomonads examined

could be differentiated into four major groupson the basis of SM similarity coefficients (Fig. 1).Much the same groupings emerged from the PO

,, I ,,II I I I I I. r- P MARGINALIS 139

P CORONAFACIENS 53E AMYLOVORA 34

- P SP from SQUASH 134P SAVASTANOI 171

-P IAVAITAN81 206- P SAVASTANOI 194- P SAVASTANOI 203- P SAVASTANOI 174- P SAVASTANOI 200

- P PHASEOLICOLA 111

- P PHASEOLICOLA 104- P PHASEOLICOLA 109- P PHASEOLICOLA 103- P PHASEOLICOLA 108P PHAEOLICOLA 90

-P PHASEOLICOLA 92- P PHASEOLICOLA 101P GLYCINEA 71

- P GLYCINEA 66

-P MORI var. HUSIP MOR var HUSZIl 82Pp PHASEOLICOLA 110-PMORI 84P GLYCINEA 69

MORSPRUNORUM 81*P MORI 85

P LACHRYMANS 167P PASSIFLORAE 166P CORONAFACIENS 55P GARCAE 61P DYSOXYLI 58p CORONAFACIENS 56

-P GELPCINEAP CORONAFACIENS 54P ATROFACIENS 48P HELIANTHI 73P MELLEA 80P HELIANTHI 74P TOMATO 133P TOMATO 132P TOMATO 128

P CICHORII 136P CICHORII 137

P SYRINGAE 127P$YRINGAE 122P SYRINGAE 123P SYRINGAE 121P SYRINGAE 120P PISI 113P ANGULATA 45P $YRINGAE 118P SYRINGAE 118P APTATA 47P PANICIS 88

P VIRIDIFLAVA 169P SYRINGAE 125P SYRINGAE 124

A TUMEFAC IENS 5A TUMEFA IEN 16A TUMEFACIENS 23X PHASEOLI 38E CAROTOVORA 36A. RHIZOGENES 41

GROUPS

IAAjIB

JID

]IE

]IH--

IF

_IG

P PUTIDA B 231P FLUORESCENS 214P FLUORESCENS 215P PRIMULAE 116

~~~~~PFLUORE ENS A._ < = ~~~PFLUORREtESCNS G 3P ANGULATA 46P MARGINALIS 142P SP from LETTUCE 78P AERUGINOSA 25P PUTIDA A. 29P ALCALIGENES 33P PSEUDOALCALIGENES 26

V

P TESTOSTERONI 28-P ACIDIVORANS 24H EUTROPHA 42H PANTOTROPHA 164P RUBRISUBALBICANS 151P FACILIS 212

pP iULTANEA EA3RUM 157P SOLANACEARUM 159P SETARIAE 117P RUBRILINEANS 149

P CEPACIA 162P MULTIVORANS 32

I I I I I I I Ii II I I I I I

60 70 80 90 100SM Similarity CoefficientFIG. 1. Dendrogram of SM similarity coefficients

showing relationships among various pseudomonadspecies and some reference strains representing othergenera (dotted lines indicate isolates with low POcoefficients to group I).

similarity coefficients (Fig. 2), except that thedispersion was considerably greater. The nu-merical analysis produced a fifth group contain-ing a heterogeneous collection of plant patho-gens belonging to other genera.

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GROUPSP CORONAFACIENS 53E AMYLOVORA 34P SP from SQUASH 134

P PHASEOLICOLA 109P PHASEOLICOLA 104P PHASEOLICOLA 111

P PHASEOLICOLA I0 IBP PHASEOICOLA 108P PHASEOLICOLA 90P PH LICLA 92PPHASEOLICOLA 1I1

P GLYCINEA 71R GLYCINEA 66P GLYCINEA 85P SAVASTANOI 171P SAVASTANOI 177P SAVASTANOI 206P SAVASTANQ2 14 A

P SAVASTANOI 203P SAVASTANOI 174

P LACHRYMANS 167P PASSIFLORAE 166P ATROFACIENS 48

P CORONAFACIENS 54

p CORONAPACIENS 55

P GARCAE 61DYoSOXYLI 58 IDJ

P COROALFACIENS 56

P DELPHINII 57P GLYCINEA 62

P MORI var. HUSZI 82P MORI var HUSZI 83P PHASEOLICOLA 110 ICP MORI 84P GLYCINEA 69P MORSPRUNORUM 81

P SYRINGAE 127P SYRINGAE 122P SYRINGAE 123P SYRINGAE 121P SYRINGAE 120P PISI 113 IFP ANGULATA 45

YNYt"A 119P APTATA 47P PANICIS 88

P CICHORI I 136P CICHORI I 137 IH

P TOMATO 128 IEP TOMATO 132

P HELIANTHI 74P TOMATO 133P HELIANTHI 73P MELLEA 80

P VIRIDIFLAVA 169P SYRINGAE 125 IGP SYRINGAE 124

X. PHASEOLI 38

E. CAROTOVORA36_A. RHIZOGENES 16 \A, TUMEFACIENS 23A. TUMEFACIENS 16A, TUMEFACIENS 5

H FACILIS 212P ACIDOVORANS 24H EUTROPHA 42H. PANTOTROPHA 164P TESTOSTERONI 28 mP RUBRISUBALBICANS 151P STUTZERI 31

P SOLANACEARUM 157P SETARIAE 117P RUBRILINEANS 149

P PSEUDOALCALIGENES 26P ALCALIGENES 33P AERUGINOSA 25P FLUORESCENS A 30P PRIMULAE 116P FLUORESCENS G 224P ANGULATA 46P MARGINALIS 142P SP from LETTUCE 78P PUTIDA A. 29P FLUORESCENS E 215P FLUORESCENS D214P PUTIDA B 231

P CEPACIA 162P MULTIVORANS 32

10 0 30 40 50 60 70 80 90 100P0. Similarity Coefficient

FIG. 2. Dendrogram of PO similarity coefficientsshowing relationships among various pseudomonadspecies and some reference strains representing othergenera (dotted lines indicate isolates with low POcoefficienits to grouip 1).

Fluorescent pseudomonads. The fluorescentpseudomonads fell into two of the major groups.Group I contained the majority of phytopatho-gens, and group II consisted principally ofsaprophytes.

Group I: arginine dihydrolase-negative, hyper-sensitive positive, green-fluorescent pseudomonads(Table 1). All organisms in group I were phyto-pathogens. These organisms have a low growthrate (generation times from 68 to 135 min) rela-tive to the strains of group II (45 to 86 min/generation). With the exception of P. cichorii,they are oxidase-negative. They do not denitrifynor grow at 37 C. The organisms are nutritionallyfastidious, utilizing from 19 to 43% of the 165carbon sources tested, far fewer than the 55%or more utilized by other fluorescent pseudo-monads. Fifty-two carbon sources are not utilized(Table 2). Five of these-j3-alanine, L-iSoleucine,L-valine, L-lysine, and spermine-are utilizedby nearly all other fluorescent pseudomonads.The inability of an organism to grow with anyof these differentiates it from the members ofgroup II. An additional 39 substrates were usedby 10% or fewer of the strains (Table 2).No single carbon source tested was utilized by

all 86 strains in group I. Ten carbon sources,however, were nearly universally used (Table 3).All strains that grew on p-hydroxybenzoate andthat were tested on protocatechuate utilized the,3-keto-adipate pathway and therefore contain1,2-protocatechuate dioxygenase (ortho cleavageof protocatechuate).Most of the organisms in group I clustered

into eight subgroups, each of which generallycorresponded to a previously recognized nomen-species (Fig. 1 and 2). Eight of the nine strainsreceived as P. phaseolicola clustered together.Also assembled in separate clusters were eight ofthe P. syringae strains, four P. mori strains, thethree isolates of P. tomato, and three of four P.coronafaciens strains.From the dendrograms (SM and PO) for this

group, we arbitrarily designated a number ofclusters as subgroups I-A to I-H. These sub-groups (and the strains comprising them) werelisted (Table 1) in order of increasing nutritionalversatility. It is apparent from the dendrograms(Fig. 1 and 2) that the subgroups are not neces-sarily discrete groups, but noteworthy areas ofrelative strain homogeneity. The most usefultests for distinguishing subgroups are listed inTable 4. Additional test results are listed inTable 5.

Subgroup I-A. P. savastanoi comprises sub-group I-A. The 34 strains had a limited nutri-tional spectrum, utilizing from 19 to 30% of the165 carbon sources examined. Strains of thisgroup clustered at 84% (SM) and 53% (PO).Many of the P. savastanoi cultures received

were probably different from their originallyvirulent phenotypes. Two strains (no. 175 and

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VOL. 101, 1970 PHYTOPATHOGENIC PSEUDOMONADS

TABLE 1. Group I strainis of arginine dihydrolase-negative, hypersensitive-positive, green-fluorescentpseudomonads ranged in order of increasing nutritional versatility

Pathogen No. Source Strain Pathogen No. Source Strain

Subgroup I-A (the savastanol subgroup)P. savastanoi 170 UCDPP HCS-2 (olive)

171 UCDPP S-00 (olive)172 UCDPP 100 (olive)173 UCDPP 200 (oleander)174 UCDPP 1007 (olive)175 UCDPP 2019 (oleander)176 UCDPP 1017 (olive)177 UCDPP HCS-3 (olive)178 UCDPP 2024 (oleander)179 UCDPP 2023 (oleander180 UCDPP 2018 (oleander)181 UCDPP 2021 (oleander)183 UCDPP 1023 (olive)184 UCDPP 1006 (olive)185 UCDPP 100 (olive)186 UCDPP 2008 (oleander)187 UCDPP 1021 (olive)189 UCDPP 1024 (olive)190 UCDPP HCM-4 (olive)191 UCDPP 1026 (olive)192 UCDPP 2016 (oleander)194 UCDPP SC-3 (olive)195 UCDPP S-0-13 (olive)196 UCDPP S-0-14 (olive)197 UCDPP MC-3 (olive)198 UCDPP 1010 (olive)199 UCDPP 1025 (olive)200 NCPPB 1481 (olive)201 NCPPB 1464 (Fraxinus)203 NEAP N-4 (oleander)204 NEAP B-2 (olive)206 NEAP E-4 (olive)207 NEAP Elvas-I (olive)208 NEAP Elvas-4 (olive)

Subgroup I-B (the phaseolicola subgroup)P. phaseolicola 90 UCDPP G-2

92 UCDPP 6-16101 UCDPP G-55103 UCDPP G-71104 UCDPP G-72108 UCDPP G-124109 UCDPP HB-29II1 NCPPB 1341

Subgroup I-C (the mori subgroup)P. mori 84 NCPPB 1415

85 NCPPB 1445P. mori var. 82 NCPPB 1037

huszi 83 NCPPB 1413P. phaseolicola 110 UCBPP HB-67P. glycinea 69 UMPP R7P. morsprunorum 81 NCPPB 560

Subgroup I-D (the coronafaclens subgroup)P. corona- 54 NCPPB 1351faclens 55 NCPPB 1355

56 NCPPB 600P. delphinit 57 NCPPB 650P. dysoxyUli 58 NCPPB 225P. garcae 61 NCPPB 1399P. atrofaciens 48 NCPPB 117P. glyclnea 62 UMPP RI

Subgroup I-E (the tomato subgroup)P. tomato 128 UCBPP P. tom 3

132 UCBPP P. tom 2133 UCBPP P. tom 9

Subgroup I-F (the syringae subgroup)P. syringae 105 UCDPP G-10l

118 UCBPP S-3 (pear)119 UCBPP S-4 (pear)120 UCBPP S-5 (pear)121 UCBPP S-6 (pear)122 UCDPP B-IS (sorghum)123 UCDPP B-3 (stonefruit)127 NCPPB 649

P. pisi 113 NCBPP 1652P. aptata 47 ICPB PA-122P. panacis 88 NCPPB 1498P. angulata 45 UCBPP 1238

Subgroup I-G (the viridtflava subgroup)P. viridtflava 169 NCPPB 1249P. syringae 124 UCBPP S-9 (plum)

125 UCDPP G-28Subgroup I-H (the cichorit subgroup)

P. cichorii 136 NCPPB 1512137 NCPPB 907

Strains that did not appear in subgroups I-A to I-H, butwere intermediate between subgroups

P. glycinea 66 UMPP R-3 (race 3)71 NCPPB 1245

P. helianthi 73 NCPPB 122974 NCPPB 1054

P. lachrymans 167 NCPPB 277P. mellea 80 NCPPB 280P. passlflorae 166 TCPB PP-1P. corona- 53 NCPPB 1348

faclensPseudomonas 134 UCBPP SQ-1 (squash)

sp.

178) did not induce hypersensitivity on tobacco fore one of the least nutritionally versatile sub-and were avirulent. Eight other strains were groups. They clustered at 88 and 65% PO.avirulent, and 10 strains were nonfluorescent. These halo blight organisms are host specific,These pathogens are specific to oleander and infecting only species of beans and soybeans (2).olive and produce knots or tumors, not leaf- Subgroup I-C. This subgroup consists of fourspotting (2). strains of P. mori and one each of P. phaseolicola,Subgroup I-B. Eight strains of P. phaseolicola P. glycinea, and P. morsprunorum. This subgroup

comprise subgroup I-B. These strains utilized was designated as the P. mori subgroup as a22 to 31 % of the carbon sources and were there- matter of convenience. The inclusion of strains

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TABLE 2. Substrates which are utilized by few or none of, and tests negative for, the oxidase-negative,dihydrolase-negative fluorescent pseudomonads (group 1)

Utilization of Utilization of Utilization of

CarbohydratesD-ArabinoseMaltose"Trehalose"CellobioseaLactose"Starch"InulinMelibioseD-FucoseaL-Rhamnose"2-KetogluconateuKelzana,bSalicinaa-Methyl-D-glucosideft-Methyl-D-glucoside

Fatty acids and derivativesIsobutyrateIsovalerateLinoleateLauryl sulfate3-Phosphoglycerate"

Dicarboxylic acidsAzelateaPimelateaSebacateaMaleateAdipateSuberateaOxalatec

Hydroxy acidsGlycollateaPoly-j-hydroxybutyrateaHydroxy-a-methylbutyrateaTriglycolateaTannate

Miscellanzeous organic acidsPectateCitraconateaItaconateaMesaconateaLaevulinateEicosanedoatec

Polyalcohols and glycolsAdonitolEthyleneglycol"Propyleneglycol"2, 3-Butyleneglycol"

AlcoholsEthyl alcoholn-Propanoln-Butyl alcoholIsobutanol"MethanolcGeraniol"Non-nitrogenous aromatic and

other cyclic compoundsD-Mandelate"L-Mandelate"BenzoylformateBenzoateo-Hydroxybenzoatem-HydroxybenzoatePhthalate"Phenylacetate"PhenylethanediolaPhenol"Testosterone"Naphthalenec

Aliphatic amino acidsGlycinea,j-Alanine"L-LeucineDL-CitrullineL-ThreonineaL-IsoleucineaL-ValineaDL-NorvalineaL-LysineaDL-NorleucineaL-Ornithine

Amino acids and related compoundscontaining a ring structure

D-TryptophanaL-TryptophanaDL-KynerenineaL-KnurenateaL-PhenylalanineL-HydroxyprolineAnthranilate

AminesSpermineaButylamine"EthanolamineMethylaminecBenzylamineHistamineTryptaminen-Acetyl-glucosaminea-Amylamine

Miscellaneous nitrogenouscompounds

Creatine"AcetamideIndoleacetate"PantothenateaUrateNicotinate

Paraffin hydrocarbonsN-dodecane"N-hexadecane"

Miscellaneous tests

OxidasedDenitrificationaArginine dihydrolaseaGelatine hydrolysisPolypectate hydrolysisaDNA hydrolysis5% NaCl + glucoseGrowth at 39 CaGrowth at 41 Ca

a Substrates not utilized by, or tests which are negative for, all group I organisms.6 Polysaccharide from Xanthomonas (Kelco Co.).c Substrates not utilized by any organisms tested.d Oxidase test negative for all group I organisms except for P. cichorii.

of P. phaseolicola in this group reflects the factthat subgroups I-B and I-C are very similar. Afew errors or misinterpretations could easilyplace a strain into the other group. The onlyclear differences between subgroups I-B and I-Cwere that the strains in the P. mori subgroupgrew on D-xylose and meso-inositol. Also, or-ganisms in this subgroup utilized a slightly higher

percentage of carbon sources (25 to 35%) thanthose in subgroup I-B (22 to 31%); P. mori re-portedly is specific to the genus Morus (2).Subgroup I-D. Subgroup I-D consists of three

strains of P. coronafaciens and one of P. del-phinii, P. dysoxylii, P. garcae, and P. atrofaciens.This subgroup was designated the P. corona-faciens subgroup. These grew on 28 to 35% of

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the carbon substrates. They clustered at 86%SM and 68% PO. Some of these pathogens (P.coronafaciens and P. atrofaciens) infect plants infour genera; others are reportedly more host-specific (2). Little is known about the host rangesof the strains labeled P. garcae and P. dysoxylii.Subgroup I-E. The three strains of P. tomato

which comprise subgroup I-E were positive in 31to 39% of their nutritional tests and clustered at88% SM and 66% PO. These strains infect onlytomatoes.Subgroup I-F. This group (designated the P.

syringae subgroup for convenience) includeseight strains of P. syringae, and one each of P.pisi, P. aptata, P. panicis, and P. angulata. Theseorganisms utilized 34 to 43% of the carbonsources; they clustered at 87% SM and 73%PO. The nomenspecies in this subgroup gen-erally exhibit wide host ranges: 43 genera of hostplants for P. syringae, 6 for P. aptata, 5 for P.pisi, 2 for P. angulata, and one for P. panicis (2).Subgroup I-G. This subgroup (designated the

P. viridiflava subgroup) consists of three strains,P. syringae 124 and 125, and P. viridiflava 169.They utilized 43, 42, and 38% of the carbonsources, respectively, and clustered at 83% SMand 63% PO. P. viridiflava has been reported toinfect plants in six genera (2).Subgroup I-H. Subgroup I-H consists of two

strains of P. cichorii. The principal characteristicsthat distinguish it from other group I strains isits positive oxidase reaction. The two strainswere 43 and 36% positive for nutritional tests.They grew on most of the compounds utilized byorganisms in the other subgroups. The host rangeis not well defined.

Intermediate strains. Of the 86 strains in groupI, 12 were not placed in a subgroup, as theyappear to be intermediate between subgroups.Thus, P. helianthi (no. 73 and 74) cannot beclearly separated from subgroups I-E and I-F,and P. glycinea strains 66 and 71 are intermediatebetween subgroups I-B and I-C. These strainsmay constitute additional valid subgroups, or maybelong to one of the described subgroups. Theorganisms in group I utilized so few substratesin our tests (19 to 43%) that a few differencesin the interpretation of nutritional spectra wouldbe sufficient to blur the boundaries of the sub-groups.

Numerical analysis of P. savastanoi, a testgroup. A detailed numerical analysis of manystrains of each nomenspecies of plant pathogenwas not within the scope of this project. However,it was considered useful to determine the pat-terns and limits of variation for a single well-known nomenspecies. The olive knot pathogen,

TABLE 3. Pseudomonias group 1: substrates utilizedby, or tests positive for, 90% or

more strainis

Utilization of Utilization of

Carbohydrates and sugar. Aliphatic amino acidsderivatives DL-Aspartate

D-Glucose y-AminobutyrateMucate L-Glutamate

Fatty acids AminesHeptanoate L-Glutamine

Dicarboxylic acidsSuccinate Miscellaneous testsFumarate

Miscellanieous organiic Chloramphenicolacids Urease-

Pyruvate HypersensitivityOrtho cleavage

P. savastanoi (subgroup I-A), was selected be-cause it caused a distinct set of symptoms on asmall number of hosts and could be easily iden-tified. Thirty-four strains were obtained fromseveral areas of the world where the disease isprevalent.The results of the analysis indicated that the

distribution frequency of SM coefficients withinthis group assume a symmetrical distributionfrom 80 to 98% with the mode at 89%. The POcoefficients ranged from 43 to 94% in a sym-metrical distribution with a mode at 67%. Theentire set of 34 strains clustered at 84% SM and53% PO.Group II: arginine dihydrolase-positive, oxidase-

positive, hypersensitive-negative, green-fluores-cent pseudomonads (Table 6). These organismsincluded 9 strains, representing six nomenspeciesof pathogens, and 16 strains representing threesaprophytic fluorescent species. These strainsclustered at 68% SM and 56% PO. The POcoefficients with some strains of this group wereas low as 50% (Fig. 3). The strains of group IIcould be differentiated from those of group I bytheir higher growth rate (45 to 86 min/genera-tion), and broader nutritional spectrum (utiliza-ation of 55 to 60% of the 165 substrates tested).The saprophytes of the P. putida biotypes

clustered (Fig. 1) at 74% SM and 67% PO.P. aeruginosa and the plant pathogen P. poly-color were highly similar (94% SM; 76% PO).Despite these high similarity coefficients, P.polycolor differed in some tests. It deviated fromthe ideal phenotype (see Table 11) of P. aeru-ginosa in failure to grow with geraniol, in produc-tion of a positive egg yolk reaction, and ingrowth at 4 C.Most of the arginine dihydrolase-positive or-

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TABLE 4. Useful tests for distinguishing subgroups within group I

Savas- Phase- Mor Corona- Tom- Syrin- Viridi- Cich-Test tanoi olicola ic faciens ato gae flava orii

IA lB ID IIE IF IGIH.

Number of strains...............................Utilization tests

Carbohydrates and sugar derivativesD-Xylose....................D-Gluconate.................................

Fatty acids and derivativesAcetate........................................Propionate..Linolenate...................Triacetin ......................................Triproprionin..................

iTricaproin.Dicarboxylic acidsGlutarate.......

Hydroxy acidsL-Malate ......................................L-(+)-Tartrate....D-(-)-Tartrate.meso-Tartrate.................................DL-jl-HydroxybutyrateDL-Lactate...................................DL-Glycerate.DL-Glycerat ...............................

Miscellaneous organic acidsL-Ascorbate..Isoascorbate..a-Ketoglutarate....

Amino acids and related compounds containing aring structure

L-Histidine ....................................L-Tyrosine......

Miscellaneous nitrogenous compoundsBetaine........................................

Polyalcohols and glycolsErythritol.....................................Sorbitol.......................................meso-Inositol................................

Non-nitrogenous aromatic and other cyclic com-pounds

Quinate.......................................PhospholipidsL-a-Lecithin ...................................

Aliphatic amino acidsDL-Asparagine...............................

Miscellaneouts testsOxidase.. ..................................Egg yolk reaction...............................Ribonuclease ....................................Growth at 37 C..................................

34

la32

1200000

23

2924020020

01632

110

26

03018

8

08

040883

0

5000300

006

00

0

000

0 17

2

26

022533

5

0

0180

7

77

620775

3

5114101

5/67

31//6

4

047

2

0

Y60

70

a Numbers represent strains positive for tests. NT = not tested.

8

80

2NT0865

5

0015322

8

0

1

5

764

NT

0

5

0450

3 12 3 2

33

112320

1

2023201

033

03

3

033

3

3

3

0020

1212

708

1189

12

1210129812

31212

81

12

101212

11

5

10

08101

11

223333

3

2032233

030

22

221221

2

2202222

002

3 12 2

3

333

3

2

3

0230

2

002

2

2

2

2202

ganisms which were sent to us as plant pathogens their nutritional spectrum. Each organism utilizedwere not clearly distinguishable from P. fluores- a few carbon sources not commonly utilized bycens biotype A. These strains were P. primulae P. fluorescens biotype A (19).117, and strains 112, 116, and 143 labeled as Nonfluorescent pseudomonads. The nonfluo-P. pisi, P. syringae, and P. marginata, respec- rescent pseudomonads fall into two groups.tively. The latter strains were certainly incorrectly Group III contains both phytopathogens andlabeled, as shown by their nonpathogenicity and saprophytes. The Hydrogenonomas strains also

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TABLE 5. Additional test resdlts among subgroups I-A to I-H ofgroup I

Savas- Phase- Mor Corona- Tom- Syrin- Viridi- Cich-Test tanoi olicola I-c faciens ato gae flava orii

I-A I-B - I-D I-E I-F I-G I-H

Number of strains................................Utilization Tests

Carbohydrates and sugar derivativesSucrose........................................D-Mannose .....................................D-Raffinose...................................D-Galactose .. .................................

D-Fructose .....................................L-Arabinose ....................................D-Ribose .......................................Saccharate.....................................

Fatty acidsValerate.......................................Pelargonate ....................................Caprylate......................................Caprate.Caproate .......................................

Dicarboxylic acidsFumarate......................................Malonate......................................

HydroxyacidsB-Hydroxymethylglutarate ......................D-Malate ..... ..............................

Miscellaneous organic acidsCitrate .........................................Aconitate......................................

Polyalcohols and glycolsGlycerol.......................................Mannitol .......................................


Chlorogenate.................................p-Hydroxybenzoate ...........................

Aliphatic amino acidsD-a-Alanine ....................................c Aminovalerate..............................L-Serine ........................................L-a-Alanine...................................DL-Arginine.....................................

Amino acids and related compounds containing aring structure

L-Proline .......................................AmineCholine ........................................Putrescine.....................................

Miscellaneous nitrogenous compoundsSarcosine......................................Trigonelline ....................................Uridine.

Miscellanieous testsErythromycin...Streptomycin .....................................Tetracycline ......................................Ortho cleavage..................................Growth at 4 C....................................Halo on Tween 80................................Fluorescent pigment on King B medium...........

- Number of positive isolates. NT = no test.

34 8

22280163138

25

1525303221

3223

020

2634

3430

224

240

273226

30

3210

91

16

28343430341424

88063857

28S54

73

16

87

81

32

8078

4

27

367

8888738

7

77577475

47775

77

07

71

77

34

85555

7

4

140

8

88188875

38888

NT8

2

88

NT6

08

61881/

8

73

422

8888888

3 I 12 I 3 2

33032333

23332

33

02

33

23

22

30333

3

32

122

2333333

12121

1212111212

1112121212

1211

711

1111

1212

511

12812116

12

116

4124

1212121291212

33033222

23333

32

23

22

33

22

22222

1

2

212

22

02

22

22

1 03 2

32323

3

22

032

3333213

11222

2

10

121

2222112

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TABLE 6. Group 11: strains of arginine dihydrolase-positive, oxidase-positive,green-fluorescent pseudomonadsa

hypersensitive-negative,

Species No. Source Strain Species No. Source Strain Biotype

P. marginalis 139 NCPPB 247 P. fluorescens 30 UCBB 192 A141 NCPPB 667 221 UCBB 12 A142 NCPPB 1689 227 UCBB 126 A

P. "marginata" 143 NCPPB 316 222 UCBB 2 B(mislabeled) 215 UCBB 210 C

P. polycolor 146 NCPPB 1224 214 UCBB 31 DP. primulae 116 NCPPB 1619 230 UCBB 30 DPseudomonas sp. 78 UCBPP L-3 (lettuce) 223 UCBB 143 FP. "pisi" (mis- 112 NCPPB 1434 224 UCBB 33 G

labeled)P. "syringae" (mis- 126 UCDPP G22 P. putida 29 UCBB 90 A

labeled) 229 UCBB 6 AP. aeruginosa 25 UCBB 45 (ATCC 163 UCBB 110 B

17423) 217 UCBB 53 B220 UCBB 167 B221 UCBB 107 B

a The following two strains were somewhat similar to group II organisms: no. 33, P. alcaligenes UCBBstrain 417, and no. 26, P. pseudoalcaligenes UCBB strain 63.

-Ii} POSITIVE ONLY

.±.1.Ji..WJI. I hIItIlIIrb[~~~~~~~~~~~~~~~~~~~~~~~~~~~..[......14.50 60 70 80 90 0

PERCE N T SI M ILA RI TY

FiG. 3. Distribution of PO coefficieiitsgroup II organisms.

withini

are included in this group on the basis of theirSM and PO similarity coefficients (Fig. 1 and 2).Group V contains two nomenspecies which ap-pear synonymous. One of these nomenspecies(P. cepacia) is a plant pathogen.Group III: nonpigmented, slow-growing, non-

fluorescent pseudomonads (Table 7). All isolates(except P. stutzeri) contained poly-,B-hydroxy-butyrate granules in the cells. The strains of thisgroup were distinguished from those of group IVby their slow growth at 27 C (generation times of90 to 140 min as compared with 76 to 87 min) andtheir utilization of fewer carbon sources (19 to52% compared with 61 to 71 %). They grew at37 C, and usually at higher temperatures.The group consists of pathogens and sapro-

phytes. The saprophytes in the group are P. tes-tosteroni, P. acidovorans and P. stutzeri. Thepathogens in this group include strains of P. sola-nacearum, P. setariae, P. rubrilineans, and P.rubrisubalbicans. Three. species of Hydrogeno-monas also were included in this group.

TABLE 7. Group III: strains of nonpigmented,slow-growing, nonfluorescent pseudomonads

Species No. Source Strain

PathogensPseudomonas 51 NCPPB 349caryophylii

P. rubrilineans 147 NCPPB 359148 NCPPB 1118149 NCPPB 920

P. rubrisubalbi- 151 NCPPB 520cans 152 NCPPB 1027

P. setariae 117 NCPPB 1392P. solanacearum 157 UFCo 19 6, race T

(tomato)158 UFCo SFR (ba-

nana)159 UFCo 139, race B

(banana)160 UFCo 100, race D

SaprophytesP. acidovorans 24 UCBB 14P. stutzeri 31 UCBB 14P. testosteroni 28 UCBB 78Hydrogenomonas 42 UCBB 373eutropha

H. facilis 212 UCBB 332H. pantotropha 164 UCBB 350

P. solanacearum, P. setariae, and P. rubrilineanscaused hypersensitivity on tobacco, whereas Pcaryophylli and P. rubrisubalbicans did not.Group IV: yellow-pigmented, rapid-growing,

nonfluorescent pseudomonads that contain poly-,B-hydroxybutyrate granules in the cells (Table 8).The new saprophytic species described by Stanier

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et al. (19), P. multivorans, is remarkably similar tothe onion pathogen P. cepacia in growth rates(76 to 86 min/generation) and nutritional spectra.The strains were 61 to 69% positive for P. cepaciaand 71% positive for P. multivorans. The or-ganisms produced a nonfluorescent yellow,diffusible pigment. On the basis of this compari-son of three strains of P. cepacia with one of P.multivorans, it appears these nomenspecies aresynonymous.

Reference bacteria. The strains in group V werefrom other genera of pathogens (Table 9). Thesewere included to determine how successfullynutritional tests of the type used for Pseudomonascould be applied in differentiating other groupsof bacteria. Most group V strains were clearlyseparated from the pseudomonads (Fig. 1 and 2).One strain of Erwinia amylovora fell within thegroup I cluster of pseudomonads on the basis ofSM similarity (Fig. 1). This can be attributed tothe fact that this strain, like the group I pseudo-monads, utilized very few substrates. The POanalysis (Fig. 2) separates this strain much moresharply from the group I pseudomonads, sincenegative matches (which essentially are invariantcharacters for strains which utilize few sub-strates) are eliminated.

DISCUSSIONNumerical taxonomic studies of pseudomonads

have been attempted several times (12, 14, 16).Although several plant pathogens have been in-cluded, the analyses did not clearly differentiatethe arginine dihydrolase-negative pathogens(group I) as a distinct group of organisms separa-ble from other fluorescent saprophytes and patho-gens. Inability to obtain differentiation presum-ably occurred because too few pathogens wereincluded or because the tests employed were in-adequate. Our study has shown that the argininedihydrolase-negative phytopathogenic pseudo-monads can be clearly differentiated from otherpseudomonads, by use of the 147 tests of Stanieret al. (19) and 41 additional tests.

It was apparent that most numerical taxono-mists, although professing to weigh charactersequally, often use a heavily imbalanced series oftests. They frequently bias their character selec-tion in favor of carbohydrate metabolism andmorphological characters at the expense of themetabolism of other compounds, the formationof specific metabolites, or the formation and rateof formation of certain enzymes.The hazard of relying too heavily on one or two

groups of characters instead of a wide range isapparent if the approach is applied to the fluores-cent arginine dihydrolase-negative group I orga-nisms. These organisms appear to be very similar

TABLE 8. Group IV: strains of yellow-pigmented,rapid-growing, nonfluorescent pseudomonads

Species No. Source Strain

PathogensP. cepacia 134 CUPP 61-52

161 CUPP 63-87162 CUPP 63-53

SaprophytesP. multivorans 32 UCBB 382

TABLE 9. Group V: reference strains of plantpathogens from other genera

Species No. Straina

Agrobacterium rhizogenes 41 A. rhiz 1A. tumefaciens 5 CG 5A. tumefaciens 16 CG 18A. tumefaciens 23 CG 63Erwinia carotovora 36 ECE. amylovora 34 FB-1-AE. quercina 35 AcCXanthomonas incanae 39 XIX. phaseoli 38 X phas

aAll UCBPP strains.

to fluorescent saprophytes if a comparison isbased primarily on sugar utilization; however,they are very unlike the saprophytes if the com-parison includes their ability to utilize many otherclasses of compounds. Our work and that ofStanier et al. (19) and Misaghi and Grogan(Phytopathology, in press) still is biased in that itis based primarily on the nutritional capacity oforganisms and, as such, may not provide theseparation within the major groups that othertests would.The taxonomy of the phytopathogenic Pseudo-

monas species is far from resolved (22). Lelliottet al. (11) developed a determinative scheme thatwas of considerable value for classifying fluores-cent pathogens into major groups. They wereunable, however, to find physiological evidenceof the speciation within these groups because ofthe small number of definitive characters that wereemployed. Our study verifies their conclusion thatthe arginine dihydrolase-negative fluorescentpathogens are distinct, and provides additionalinformation concerning phenotypic properties ofthese organisms. An ideal phenotype has beenconstructed by Stanier et al. (19) for the fluores-cent pseudomonads. Since fluorescent phyto-pathogens (our group I) were not included in thatstudy, we have amended the description of theideal phenotype to include data for this group oforganisms (Table 10).

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Stanier et al. (19) further presented a set ofcharacters useful for differentiating the internalsubdivisions of the fluorescent pseudomonads.That set of characters also has been amended toprovide useful characters for distinguishing thephytopathogen group (I) from the other fluores-cent pseudomonads (Table 11).The present nomenspecies in group I cannot

justifiably be maintained. The question is whetherthere should be one large collective species, or agrouping of present nomenspecies into a smallernumber of species distinguishable by reliable char-acters. There are two reasons for not consideringgroup I as a single species. First, analysis of datafrom the characterization studies reveals that theorganisms are clustered in groups or subgroups.These clusters tend to correspond to currentlyrecognized nomenspecies, such as P. phaseolicolaand P. savastanoi, which have been proven to bedistinct pathological groups on the basis of hostspecificity and type of symptoms evoked. To a

TABLE 10. Group characters ofgreatest differentialvalue in the recognitioni of the fluorescent

pseudomonads as amended fromStanier et al.

No. of positivestrains

IdealCharacter group I Group pheno

(86 (1 typstrainsstanused) used)

1. Fluorescent pigment.. 76 155 +2. Poly-3-hydroxybutyrate. 0 0 -

Utilization of3. Poly-,3-hydroxybutyrate. 0 0 -

4. D-Fucose................ 0 0 -

5. D-Glucose............... 84 174 +6. Salicin .................. 0 0 -

7. Cellobiose............... 0 0 -

8. Starch0 0 -

9. 2-Ketogluconate......... 0 167 NUb10. Pelargonate. 74 175 +11. Benzoate and/or p-hy-

droxybenzoate ........... 72c 172 +12. m-Hydroxybenzoate ... 1 7 -

13. O-Alanine...............0 174 NU14. Norleucine. 0 0 -

15. Arginine ................ 60/79 173 NU16. Putrescine and/or sperm-

ine.................. 36d 173 NU17. Betaine and/or sarcosine. 65 175 NU

a Data from Stanier et al. (19).bTest listed by Stanier et al. (19) but not useful

for recognizing fluorescent pseudomonads inthe amended description.

r Benzoate utilized by two strains.d Spermine not utilized by any strains.

plant pathologist who used cross-inoculation ofplants as a major criterion for identification, theseare very distinct organisms which he recognizesas separate species. Second, most of the tests usedin this study were developed for study of sapro-phytes, and probably do not reveal a sizableportion of the genotypic characters of plantpathogens. We believe that the taxonomic resolu-tion of the phytopathogens could be greatly in-creased by using more tests that reflect their specialcapabilities. It is probable that clustering would bemore evident if the battery of tests in our studyhad included such additional tests as indole-acetic acid synthesis, specific toxin production,,3-glucosidase synthesis, casein hydrolysis, andtyrosinase activity. These tests appear to be veryspecific for certain nomenspecies. For example,P. savastanoi, in contrast to P. syringae, does notproduce ,B-glucosidase (3), does convert trypto-phan to indole-acetamide and indole acetic acid(13), and produces indole-3-acetyl-E-L-lysine (4).Many nomenspecies also produce supposedlycharacteristic phytotoxins, some of which havebeen chemically defined.The reasons in favor of lumping and against

retaining the large number of nomenspeciespresently found in the literature are as follows.(i) The resolution of the clusters representingvarious nomenspecies is insufficiently clear todesignate species. There appears to be a series ofphenotypes with relative areas of homogeneity.P. savastanoi and P. syringae, organisms at theextremities of variation in this series, appear asdistinct species; however, other groups of namedorganisms show various combinations of the char-acters which distinguish these two species. Withmore isolates from different environments, it ispossible that a continuum of intermediate pheno-types would be found. (ii) Not enough standard-ized cross-inoculations have been made to verifythat host ranges of the nomenspecies are as re-stricted as their definitions imply. Our laboratory,for example, has shown that P. phaseolicola andP. glycinea have similar host ranges.

Regardless of their taxonomic status, severalfacts concerning the organisms of group I emergefrom the present study: (i) there are clusters ofgroup I organisms according to the substancesthey utilize, (ii) in most cases these clusters fit cur-rent nomenspecies, (iii) these clusters (subgroupsof group I) also can be arranged in a series on thebasis of the number of substrates utilized, and (iv)the position of a subgroup in this series generallyrelates quite closely to the host specificity of theorganisms within this subgroup.We suggest that the resolution of these sub-

groups will be markedly improved as more or-ganisms are intensively studied and a greater as-

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VOL. 101,1970 PHYTOPATHOGENIC PSEUDOMONADS 21

TABLE 1 1. Characters of differel1tial value for the internal subdivision of the fluorescent pseudomonads asamended from Stanier et al.

No. of positive strains

Characters P. aeru-| f |

re|

Group I gi: saer P.foenS- P. pusida(86 strains) (2~9strains) (94 strains) (32 strains)

Extracellular enzyme production(a) Proteases (gelatin liquefaction). 7c 29 94 0(b) Lecithinases (egg-yolk reaction). 19 6 77 0(c) Lipases (hydrolysis of Tween 80) ................ .5Y8 29 58 2

Temperature relations(a) Growth at 4 C....... 78 0 93 11(b) Growth at 41 C ................................. 0 29 0 0

Oxidase ........................................... 2d 29 94 32Denitrification ........................................ 0 29 46 0Production of pyocyanine............................. 0 25 0 0Hypersensitivity in tobacco ........................... 76 0 0 0Nutritional propertiesCarbohydrates and sugar derivativesD-Xylose ......................................... 35 0 51 5L-Arabinose ...................................... 47 0 64 6D-Mannose ....................................... 74 0 91 6D-Galactose ...................................... 66 0 83 0Sucrose........................................... 75 0 66 3Trehalose......................................... 0 0 62 0Saccharate........................................ 64 0 75 32Mucate........................................... 84 0 84 322-Ketogluconate ................ .................. 0 29 91 32

Dicarboxylic acidsAdipate .......................................... l 29 13 0Pimelate.......................................... 0 25 9 0Suberate.......................................... 0 19 10 0Azelate........................................... 0 28 11 0Subacate ......................................... 0 29 13 0

HydroxyacidsL-(+)-Tartrate.................................... 32 0 9 23Hydroxymethylglutarate ......................... 14 0 43 0

Miscellaneous organic acidsItaconate......................................... 0 29 68 6Laevulinate....................................... 0 29 21 8Mesaconate....................................... 0 29 65 5Citraconate....................................... 0 0 36

Polyalcohols and glycolsErythritol ........................................ 26 0 61 1Sorbitol ......................................... 21 0 53 3meso-Inositol ..................................... 57 0 85 0Adonitol ......................................... 1 0 47 1Propyleneglycol.................. 0 28 38 262,3-Butyleneglycol ............... ................. 0 27 53 23

AlcoholsGeraniol ......................................... 0 29 0 0Isobutanol. 0 29 17 32


L-Mandelate...................................... 0 29 3 2Phenylacetate.... 0 0 15 29Benzoate ......................................... 2 29 71 32

Aliphatic amino acidsGlycine..... 0 22 0 26DL-a-Aminovalerate............................... 24 0 2 22j8-Alanine...... ... .............................0 29 94 32

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TABLE 11-Continued

No. of positive strains

CharactersGroup I P. aeru- P. .lores- p3 pstidai.giosd" censa(86 strains) (29 strains) (94 strains) (32 strains)

L-Isoleucine.. 15 94 32L-Lysine......................................... 0 26 71 32L-Valine. 0 25 90 31

AminesBenzylamine...................................... 1 0 4 32Histamine ........................................ 1 29 32 32Butylamine....................................... 0 0 17 29ar-Amylamine .................................... 0O 27 29Spermine......................................... 0 29 92 31

Miscellaneous nitrogenous compoundsCreatine......................................... 0 0 2 28Hippurate ........................................ 1 0 2 23Acetamide........................................ 0 29 0 10Trigonelline......... 38 0 34 32

Paraffin hydrocarbonsn-Hexadecane ..............................0.... 32 0 0

a Data from Stanier et al. (19).b Data from Stanier et al. (19). Excludes data for P. putida biotype B which is no longer recognized as

P. putida (N. J. Palleroni, personal communication).c Number of positive strains.d P. cichorii exception.

semblage of characters is made available fortaxonomic usage. The designation and ranking ofthese subgroups of group I will depend upon thefindings of such studies.Any decision concerning taxonomic treatment

of the organisms of group I is at present extremelyarbitrary. It is not yet feasible to make subdivi-sions within group I, as too few representatives oftoo few nomenspecies have been examined by usand by Misaghi and Grogan (in press). Our onlyalternative, therefore, is to recommend that theorganisms of this group (the arginine dihydrolase-negative, hypersensitive-positive, fluorescent pseu-domonads) be divided into two species. Theoxidase-positive strains are designated as P.cichorii and the oxidase-negative strains as P.syringae. However, in recognition that manyphysiological and pathological differences (nomatter how ill-defined) do exist within the oxidase-negative phytopathogens, we recommend that thenomenspecies representing these organisms listedin the seventh edition of Bergey's Manual bedesignated as pathotypes until such a time as theirstatus can be fully determined.

ACKNOWLEDGMENTS

This investigation was supported by a Public Health ServicePredoctoral Fellowship to D.C.S. (5-FL-FM-28,242), a PublicHealth Service research career development award to D.C.H.(K3-AI-34,942) from the National Institute of Allergy and In-fectious Diseases, and by U.S. Department of Agriculture grant(12-14-100-9199-34).

We also thank M. Doudoroff, N. J. Palleroni, and R. Y. Stanierfor contributions of cultures and chemicals, and for their ableassistance and advice, and Manuela Pinto da Silva for her tech-nical help.

LITERATURE CITED

1. Ballard, R. W., M. Doudoroff, R. Y. Stanier, and M. Mandel.1968. Taxonomy of the aerobic Pseudomonads: Pseudo-monas diminuta and P. veskculare. J. Gen. Microbiol. 53:349-361.

2. Elliott, C. 1951. Manual of bacterial plant pathogens, 2nd ed.Chronica Botanica Co., Waltham, Mass.

3. Hildebrand, D. C., and M. N. Schroth. 1964. ,-Glucosidaseactivity in phytopathogenic bacteria. AppI. Microbiol. 12:487-491.

4. Hutzinger, O., and T. Kosuge. 1968. Microbial synthesis anddegradation of indol-3-acetic acid. III. The isolation andcharacterization of indol-3-acetyl-e-L-lysine. Biochemistry7:601-605.

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