Vol. 58, No. 12APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1992, p. 3932-39400099-2240/92/123932-09$02.00/0Copyright © 1992, American Society for Microbiology

Diversity among Streptomyces Strains Causing Potato ScabCHRISTIANE DOERING-SAAD,"2 PETER KAMPFER,3 SHULAMIT MANULIS,4 GIORA KRITZMAN,4

JORG SCHNEIDER,2 JOLANTA ZAKRZEWSKA-CZERWINSKA,2 HILDGUND SCHREMPF,2AND ISAAC BARASH"*

Department ofBotany, Tel Aviv University, Tel Aviv 69978, 1 and Department ofPlant Pathology, A. R 0.Volcani Center, Bet Dagan, Israel, and Fachbereich Biologie/Chemie, Universitat Osnabruck, D-4500

Osnabruck,2 and Fachgebiet Hygiene, Technische Universitat Berlin, D-1000 Berlin 65,3 Germany

Received 30 June 1992/Accepted 30 September 1992

Eighty Streptomyces isolates, including 35 potato scab-inducing strains and 12 reference strains ofStreptomyces scabies, were physiologically characterized by a total of329 miniaturized tests. Overall similaritiesof all strains were determined by numerical taxonomy, with the unweighted average linkage (UPGMA)algorithm and simple matching (Ssm) and Jaccard (SJ) coefficients used as measures for similarity. Three clustergroups (A to C) were defined at a similarity level of 80.1% (Ssm); these groups contained 14 clusters and 24unclustered strains defined at a similarity level of 86.5% (Ssm) Cluster group A contained strains phenotyp-ically related to S. griseus or S. exfoliatus, whereas cluster group B contained strains which were phenotypicallyrelated to S. violaceus or S. rochei. The majority of the pathogenic isolates and reference strains were assignedto S. violaceus (57%) and S. griseus (22%). A DNA probe derived from the rRNA operon of S. coelicolor IMET40271 was used to detect restriction fragment length polymorphisms (RFLPs) among 40 pathogenic andnonpathogenic Streptomyces isolates. Southern blots revealed a high degree of diversity among the pathogenicstrains tested. No significant correlation between numerical classification and RFLP grouping of Streptomycesstrains could be revealed. The results obtained suggest that RFLP data are of minor importance in classificationof Streptomyces species and that genes for pathogenicity determinants are spread among different Streptomycesspecies by mobilizable elements.

Common scab of potato (Solanum tuberosum L.) iscaused by Streptomyces strains and widely distributed inpotato-growing areas (10). Infection occurs through thelenticels of developing tubers and initially results in reddish-brown water-soaked lesions on the tuber periderm. As thepathogen continues to colonize the tuber, the host developswound periderm, resulting in slightly raised lesions com-posed of rough corky tissue (10, 15). Depending on theStreptomyces strain and the environment, shallow- or deep-pitted lesions occur (1).The causal organism of common scab was first described

in 1892 by Thaxter (32) as Oospora scabies and was charac-terized as a melanin producer with grey spores born in spiralchains. The species was renamed Actinomyces scabies byGussow (7) and later renamed Streptomyces scabies byWaksman and Henrici (35). However, S. scabies was in-cluded in the group "species incertae sedis" in the eighthedition of Bergey's Manual of Determinative Bacteriology(2) because the original strain was no longer extant andWaksman's "neo-type" strain IMRU 3018 (34) is not char-acteristic of the species. Therefore, S. scabies was notincluded in the Approved Lists of Bactenial Names (28) in1980. Revival of the name was proposed in 1989 by Lambertand Loria (17), who expanded the original description toinclude smooth spores and use of all International Strepto-myces Project sugars. S. scabies has been reported topredominate in dry neutral to alkaline soils (9, 10, 22).However, additional Streptomyces spp. have also beenreported to cause potato scab (1, 4, 10, 15, 31). A newspecies, S. acidiscabies, which causes potato scab symp-

* Corresponding author.

toms in soils with pH values below 5.2 has recently beendescribed (18).

Potato scab became a limiting factor in potato productionin southern Israel during the early 1960s (33). The causalagent, which was identified as S. scabies, was successfullycontrolled by improved irrigation techniques (19). However,deep-pitted scab became a problem after 1981, even inwell-irrigated fields. Streptomyces strains which differ fromS. scabies were shown to cause deep-pitted scab in highlymoist soils. In contrast, most scab-causing Streptomycesstrains in the United States were assigned to S. scabies (17,22).Grouping within the genus Streptomyces according to

numerical classification was carried out by Williams et al.(36). By miniaturized physiological tests using 329 unitcharacters, Kampfer et al. (14) have recently tested 821strains of Streptomyces and Streptoverticillium. Thus, 15major and 34 minor clusters (phena), as well as 40 single-member clusters, have been defined at the 81.5% similaritylevel. Restriction fragment length polymorphism (RFLP)analyses have also been successfully used for classificationof microbes, including phytopathogenic bacteria (3, 5, 21).The present study was undertaken to identify the scab-inducing Israeli Streptomyces strains and some Americanstrains according to numerical classification and RFLPs.

MATERIALS AND METHODS

Strains and growth conditions. The Streptomyces strainsused in this study and their original sources are listed inTable 1. The Israeli strains were isolated from either scabbypotatoes or soil in which potatoes were cultivated at the A.R. 0. Volcani Center. The American strains were obtainedfrom R. Loria of the Department of Plant Pathology, Cornell

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STREPTOMYCES STRAINS THAT CAUSE POTATO SCAB

TABLE 1. Strains used in this study, cluster assignment, origin, pathogenicity, and assignment to RFLP SGs

Cluster Cluster or Patho- RFLP SGfgroup' Cluster' Strainb Orginc subcluster genicity'groupa ~~~~~~~~~~~~~~~~assipnmentdgnctf BamHI BstEll PvuII SalII

1 RL 8101031 RL8301031 RL84012351 RL84012221 DSM 40230 (S. venezuelae)2 PBS12 202 422 RL 8501292 5112 262 22663 DSM 40078 ("S. scabies")'4 NP2154 RL 8601304 B14 DSM 40236T (S. griseus)5 NP2125 NPAct55 NPActll5 NPAct96 127 DSM 40611 ("S. scabies"r)8 DSM 40859 ("S. scabies"t)9 DSM 40996 ("S. scabies")10 RL 8401104 (S. acidiscabies)10 RL 83014010 RL 8401182 (S. acidiscabies)11 7011 260211 RL 87012211 2312 DSM 40994 ("S. scabies")13 RL 840142 (S. scabies)'13 RL 8401232 (S. scabies)14 2115 NP11315 NP21416 DSM 40231T (S. rochei)16 443316 4317 4417 417 B3018 1919 RL 840170 (S. scabies)'19 RL 840143 (S. scabies)'19 2719 3119 B29 (also called 29)19 NP11419 NP11220 NP11521 1121 221 321 P221 NP21121 DSM 40566 (S. pulcher)h21 NPActl21 NPAct2222 DSM 40232 (S. baamnensis)23 M313124 RL 84010525 RL 870150

USAUSAUSAUSA

ISRISRISRUSA

ISRISR

ISRUSAISR

ISRISRISRISRISR

USAUSAUSAISRUSAUSAISR

USAUSAISRISRISR

ISRISRISRISRISRISRUSAUSAISRISRISRISRISRISRISRISRISRISRISR

ISRISR

USAUSA

2229221-31-31-31-31-31-31-31-3B1-31-31-31-31-31-31-31-31_3B1-41-2299999B9999913999BSMC9B96666999999999999B9999B999B9999965996SMCSMCgSMCl

NPSPSNP

DPSDPSDPSDPS

SPSSPS

1117

1

NPDPS

NPNPNPNPSPS

DPSSPS

DPS

SPSSPSDPSNPNP

DPSDPSDPSDPSDPSDPSSPSSPSDPSDPSDPSNPSPSNPDPSDPSDPSDPSNP

66 1

-_ - 33 3 17 3 13 3 1

11

1 2

4

- - ~~~~3

5 1

11

5 4

3

7

466222

4

44

2

2

4 82 9

98

5 6 114 106 5 66 5 6

5

42 4

7 2 9

1 7

5 2

NPNP

3

NPSPS

Continued on followingpage

A

B

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3934 DOERING-SAAD ET AL.

TABLE 1-Continued

Cluster Cluster or Patho- RFLPSGfSgroup' Cluste& Straine OriginC subcluster geityassignmentd encte BamHI BstEII PvuII Sall

26 DSM 40960 627 NP111 ISR 9 SPS - 6 527 RL 8401151 (S. scabies)' CAN 9B DPS28 DSM 40998 ("S. scabies")' 929 DSM 40997 ("S. scabies"P) 930 DSM 40995 ("S. scabies")h 831 DSM 40999 ("S. scabies") 832 RL 840134T (S. scabies)' USA 9532 DSM 41000 ("S. scabies"t 9B33 DSM 40962 ("S. scabies")* 14

C 34 RL 870160 USA SMCW NP35 RL 870161 USA SMV NP36 DSM 40961 ("S. scabies',)h SMC 45937 Clpg 110 SMCY38 RL 830139 USA SMCY NP 4 - 10

a For cluster group and cluster composition, see Fig. 1 (the sequence of strains in this table corresponds to the phenogram in Fig. 1).b Strains with the prefix RL were obtained from R. Loria. DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen.c CAN, Canada; ISR, Israel; USA, United States of America.d Clusters and subclusters according to the study of KYampfer et al. (14): subcluster 1-2 (S. halstedii), subcluster 1-3 (S. griseus), subcluster 1-4 (S. olivaceus),

cluster 2 (S. exfoliatus), cluster 6 (S. rochei), cluster 8 (Streptomyces sp.), cluster 9 (S. violaceus), cluster 13 (S. niveoruber), cluster 14 (Streptomyces sp.), cluster45 (S. scabies DSM 40961), and SMC, single-member cluster.

I NP, nonpathogenic; SPS, shallow-pitted scab; DPS, deep-pitted scab.f For classification of RFLP SGs, see Table 4. -, strain could not be assigned to any RFLP SG.g Identification and assignment to phena by using the probability matrix of Kampfer and Kroppenstedt (13), which is composed of 50 characters (tests are listed

in Table 2), could not be made unambiguously (Willcox probability <0.99 and/or taxonomic distance >0.4).h Strain included in the numerical study of Kampfer et al. (14); quotation marks indicate strains tentatively assigned to S. scabies.Strain included in the DNA-DNA hybridization study of Healy and Lambert (8).

University, Ithaca, N.Y., and the reference strains wereprovided by R. M. Kroppenstedt of the Deutsche Sammlungvon Mikroorganismen und Zellkulturen, Braunschweig, Ger-many.

Strains were grown at 28°C on R2 agar medium (11)without sucrose until spores were formed. The spores were

kept in glycerol at -20°C. For pathogenicity tests or DNAextraction, the cells were grown in tryptic soy broth (11).Before analysis for numerical classification, the strains werecultivated on glucose-yeast extract-malt extract agar con-taining the following ingredients (in grams per liter): yeastextract, 4; malt extract, 10; glucose, 4; CaCO3, 2; and agar,15. The plates were incubated aerobically at 28°C. Thestandard procedures used to determine spore chain micro-morphology were those described by Shirling and Gottlieb(27). Scanning electron microscopy was used to determinespore ornamentation.

Pathogenicity test. Each Streptomyces strain was grown inan Erlenmeyer flask containing 200 ml of tryptic soy broth.The flasks were incubated for 3 to 4 days on a rotary shakerat 28°C. The cultures were used to inoculate sterile perliteno. 1 crumbs in plastic bags supplemented with a mediumcontaining the following nutrients (in grams per liter): yeastextract, 4; soluble starch, 15; K2HPO4 3H20, 1.3;MgSO4. 7H20, 0.5; Bacto Peptone, 0.6; Bacto ProteosePeptone, 0.6; nalidixic acid, 0.01; polymyxin B, 0.001;penicillin G, 0.002; and cycloheximide, 0.15. Five liters ofperlite crumbs was aseptically inoculated with 400 ml ofculture of each strain, mixed, and incubated at room tem-perature under moist conditions. After 5 days, the cultureswere mixed with steam-sterilized sandy soil (1:1, vol/vol).Potato seed tubers were planted in pots containing thismixture and grown under greenhouse conditions. Symptomdevelopment was recorded after 120 days.

Biochemical tests and analyses for numerical classification.

Each strain was examined for 329 unit characters, includingcarbon source utilization, sugar fermentation, and qualita-tive enzyme activity, by using chromogenic substrates.Tests were performed with standard microtitration plates(Greiner, Nurtingen, Germany). Details of test media andconditions were as described by Kampfer et al. (14). Char-acters were scored + or - and then clustered with theunweighted average linkage (UPGMA) algorithm (30) byusing the simple matching coefficient (Ssm) and the Jaccardcoefficient (S.). The cophenetic correlation coefficient (Lr),as a measure of agreement between similarity values impliedby a phenogram and those of the original similarity matrix,was calculated for both classifications by the method ofSneath and Sokal (30). The characters providing the bestdifferentiation of the obtained phena have been described byKiampfer and Kroppenstedt (13). Each analyzed strain wastested for these 50 characters (Table 2), and the identificationscores were calculated by the MATIDEN program (29).DNA manipulations. Genomic DNA was isolated from

mycelia grown for 3 to 4 days in tryptic soy broth by the 2xKirby lytic mix procedure as described previously (11).DNA (5 to 10 ,ug) from each strain was used for digestionwith the restriction enzyme BamHI, BstEII, PvuII, or Sall.The procedures for agarose gel electrophoresis (TBE buffer)and Southern blotting were described previously (23).A 7.2-kb DNA fragment containing the 16S, 23S, and 5S

portions of the rRNA operon from S. coelicolor IMET 40271(38) cloned into the Sall site of pUC18(p64) and propagatedin Escherichia coli DH5at was used as a probe. It was labeledwith digoxigenin II-dUTP by the random priming procedure,according to the manufacturer's instructions, with a kitpurchased from Boehringer GmbH (Mannheim, Germany).Southern hybridization and subsequent washes were con-ducted with Biodyne membranes (Pall Bio Support, EastHills, N.Y.), as instructed by the kit manufacturer.

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STREPTOMYCES STRAINS THAT CAUSE POTATO SCAB 3935

38-

33-32r31-

28-C

26-

21[20-

15

17 [

CE12-~

10

9-

. 5

64 j ]

A 3

2

100 90 80 70

% SIMILARITYFIG. 1. Simplified phenogram showing relationship between

phena recovered in the UPGMA-Ssm analysis.

Similarity coefficients for RFLPs. Similarity coefficients (F)for all pairwise combinations of RFLP groups were calcu-lated by using the following estimator of DNA fragmenthomology (5): F = (nxy + nyx)/(nx + ny), where nx and nyare the number of major fragments (not total fragments) instrains X and Y, respectively, nxy is the number of majorfragments in strain X that match any fragment (major or

minor) in strain Y, and nyx is the number of major fragmentsin strain Y that match any fragment in strain X. An RFLPsimilarity group (SG) was arbitrarily defined as the assem-

blage of isolates with an F of greater than 0.85 for all possiblepairwise combinations within the group.

RESULTS

Micromorphology. The micromorphological characteris-tics of the Israeli pathogenic strains were diverse. Sporechain morphology ranged from spiral (e.g., strains 12, 23, 31,and B30) to flexous (e.g., strain PBS1) to retinaculum-apertum (e.g., strains 2, 11, and P2) to rectus (e.g., strain21). Spore ornamentation ranged from smooth (e.g., strains12, 21, 23, and PBS1) to warty (e.g., strains 31 and B30) tospiny (e.g., strains 2, 11, and P2). Spore color on yeast-maltextract agar (27) ranged from grey (e.g., strains 11, 23, 31,P2, and B30) to white (e.g., strains 2, 12, 21, and PBS1).These results clearly indicate that potato scab-causingstrains in Israel did not necessarily match the traditionaldescription of S. scabies, namely, smooth grey spores bornein spiral chains (1, 9, 15, 17, 32).

Numerical classification. A total of 80 Streptomyces iso-lates consisting of 40 strains from Israel, 22 strains from theUnited States and Canada, and 18 reference strains from theDeutsche Sammlung von Mikroorganismen und Zellkulturenwere subjected to numerical classification. A phenogramderived from UPGMA-SSm analysis is shown in Fig. 1. Thecophenetic correlation coefficient Lr, a measure betweencorresponding pairs from the similarity matrix and thecophenetic matrix, was 0.79, which has been found to bereasonably good (12). A total of 14 clusters (containing twoor more strains) and 24 unclustered strains were obtained bytaking a similarity level of 86.5% (Ssm). They were numberedin descending order of similarity. Three cluster groups (A toC) could be defined at a similarity level of 80.1% (Ssm).Exactly the same clusters were recognized with the Sicoefficient (Lr = 0.67) by taking similarity levels rangingfrom 66 to 73% (Sj). Because of the similarity of the twoclassifications, only the UPGMA-Ssm analysis is discussed indetail in comparison with the results of the numerical studyof Kampfer et al. (14).

Cluster group A (clusters 1 to 9) contained strains pheno-typically similar to subclusters 1-2 (S. halstedii), 1-3 (S.gnseus), 1-4 (S. olivaceus), and 2 (S. exfoliatus) describedby KYampfer et al. (14) (Fig. 1 and Table 1). Cluster 1, whichcontains S. venezuelae DSM 40230 and four Americanstrains, and the unclustered strain S. scabies DSM 40996(cluster 9) were found to be similar to S. e4oliatus (cluster 2described by Kampfer et al. [14]). The remaining strains(clusters 2 to 8) were similar to subclusters 1-2 through 1-4 ofreference 14. Cluster 4 contained S. griseus DSM 40236T.

Cluster group B (clusters 10 to 33) was composed ofstrains which were mainly assigned by Kampfer et al. (14) tocluster 6 (S. rochei) and cluster 9 (S. violaceus). Cluster 10contained two strains of S. acidiscabies which were found tobe phenotypically similar to S. violaceus, i.e., cluster 9 (141.It is noteworthy that cluster 32 contained strain RL 840134 ,which has been designated the type strain of S. scabies byLambert and Loria (17). This strain had the highest degree ofphenotypic similarity to S. scabies DSM 41000, which wasgrouped into the S. violaceus cluster (14). Clusters 16, 22,and 26 were assigned to S. rochei (cluster 6 [14]). Cluster 16contained the type strain of S. rochei, DSM 40231T (Fig. 1).Other S. scabies strains, i.e., DSM 40994 (cluster 12) andDSM 40962 (cluster 33), remained unclustered (Fig. 1).

Cluster group C (single-member clusters 34 to 38) con-tained additional nonpathogenic strains and S. scabies DSM40961, which could not be assigned to any cluster of Ka-mpfer et al. (14).

Results of physiological characterizations of all of thestrains according to characters found to be useful for differ-

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3936 DOERING-SAAD ET AL.

a b c d e *f g-

a a a a ah i k I m n o p q r s t u v w x y z a b c d e

!_-- w

*e23.1 kb

;E* ttt:t;-0:',!t0-t;i b 4 v4 *|" lE *Xt -94kb

EsI Es 9i46*0 6.6kb

;f: 4.4 kb

FIG. 2. RFLP pattern of genomic digest of Streptomyces strains after endonuclease restriction by BamHI. Electrophoresis was carried outin 1% agarose gels (20 by 20 cm, 3 V/cm) for approximately 20 h. Southern hybridization was performed with the rRNA operon from S.coelicolor. Lanes: a, strain PBS1; b, 20; c, 26; d, 42; e, 29; f, 31; g, 27; h, 41; i, RL 860130; k, 11; 1, P2; m, 3; n, 19; o, 21; p, 12; q, 23; r,RL 840170; s, RL 840143; t, RL 850129; u, B30; v, 43; w, 44; x, NP112; y, 4; z, 33; aa, 2; ab, NP111; ac, 8401151; ad, 70; ae, X-HindIII. RFLPSGs are composed of strains in the following lanes: 1, a to d; 2, e to g; 3, h and i; 4, k to n; 5, o to q; 6, r and s; and 7, t and u.

entiating Streptomyces spp. (13) indicate a high diversity ofthe Streptomyces isolates causing potato scab (Tables 2 and3). The majority of isolates was assigned to different phena(14) with Willcox probabilities higher than 0.99 (Table 1).For successful identification, Willcox probabilities greaterthan 0.90, combined with low scores for taxonomic distanceand its standard error, are required (13). However, some ofthe strains did not meet these criteria. The low Willcoxprobabilities with high taxonomic distances obtained forseveral strains (Table 1) reflect the difficulties involved instreptomycete taxonomy.RFLP analyses. RFLPs of cleaved DNA derived from the

various isolates were analyzed in order to test their suitabil-ity for a rapid identification of pathogenic strains. Theisolated DNAs were digested by different restriction en-zymes and hybridized to the SalI fragment containing therRNA operon of S. coelicolor IMET 40271 (38). RFLPpatterns observed in a Southern blot of BamHI-digestedDNA from various Streptomyces strains are presented inFig. 2. Seven RFLP SGs could be observed among the 29strains analyzed with BamHI, but some strains could not beincluded in any of these RFLP SGs. Similar RFLP analyseswere performed with BstEII, PvuII, and SalI, and the resultsare summarized in Tables 1 and 4. The calculated similaritycoefficients for strains restricted with BamHI, as well asthose for strains restricted with the other three enzymes,ranged from 0 to 100%.

Table 4 summarizes the RFLP SGs obtained from RFLPpatterns of each of the restriction endonuclease tests. De-pending on the restriction enzyme used, different RFLP SGscould be distinguished. For BamHI, BstEII, PvuII, and Sail,7, 7, 6, and 11 RFLP SGs, respectively, were characterized.Generally, strains grouped within the same RFLP SG for onerestriction enzyme fell into different RFLP SGs if tested withother endonucleases. There were, however, a few excep-tions. For example, the American strains RL 840143 and RL840170 were in identical RFLP SGs for all four enzymestested, whereas strains PBS1 and 42 were in the same RFLP

SGs for three restriction endonucleases. Pathogenic as wellas nonpathogenic strains, and American as well as Israelistrains, fell within the same RFLP SGs. For example, PvuIIRFLP SG is composed of strain 70 (an isolate from Israel)and strain 2602 (an isolate from the United States), whichinduce deep- and shallow-pitted scabs, respectively, and thenonpathogenic strain RL 8401235 (an isolate from the UnitedStates) (Table 4).

DISCUSSION

The results of the present study demonstrate that potatoscab in Israel is caused by a phenotypically and geneticallydiverse population of Streptomyces strains. The Israelistrains, as well as the few American strains examined, fellinto different phena when analyzed according to the numer-ical classification of Kampfer et al. (14). The majority of thepathogenic strains were assigned to S. violaceus (57%) andS. gnseus (22%), and some were allocated to S. exfoliatus,S. rochei, and to single-member clusters.Numerical taxonomy has been extensively used for clas-

sification of the genus Streptomyces and has resulted inidentification of several hundred species (14, 36). However,DNA-DNA hybridization studies revealed a high diversityamong the strains tested, and intracluster pairing valueswere as low as those found between members of differentclusters defined by numerical taxonomy (16, 24, 25, 37).Similarly, a high diversity in DNA-DNA pairing values hasrecently been demonstrated among Streptomyces isolatescausing potato scab (8).

Six strains of S. scabies exhibiting zreater than 70% DNArelatedness to S. scabies RL 840134 were included in ournumerical study. They were grouped in cluster group B(Table 1), which contained the majority of strains phenotyp-ically similar to S. violaceus (cluster 9 [14]). As indicated byHealy and Lambert (8), phenotypically S. scabies mostclosely resembles the S. diastaticus or S. cyaneus groupdefined earlier by Williams et al. (36), and genotypically it

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TABLE 2. Characteristics useful for differentiating Streptomyces spp. for clusters containing two or more strains

% of strains positive in indicated cluster' (no. of strains in cluster)

1 (5) 2 (7) 4 (4) 5 (4) 10 (3) 11 (4) 13 (2) 15 (2) 16 (3) 17 (3) 19 (7) 21 (8) 27 (2) 32 (2)

Utilization of:N-Acetyl-D-galactosamineL-ArabinoseL-FucoseD-GlucarateD-GlucuronateLactoseLactuloseL-Lyxosea-D-Melibiosea-Methyl-D-mannosideMethyl-ot-D-glucopyranosideca-D-RaffinoseL-RhamnoseD-RiboseD-SucroseD-TuranoseAdonitolD-Arabitoli-InositolMaltitolD-MannitolCaprateIsobutyrateSorbinic acidAdipateAzelateGlycerophosphate2-HydroxyvalerateDL-LactateMalonateSuberateAcetamidocaprateD-AlanineL-CitrullineD-GlutamateL-GlycineL-HydroxyprolineDL-IsoleucineL-LeucineL-LysineL-OrnithineSpermineL-LeucinamideL-ValinamidePhenyllactate

Hydrolysis of:pNP-N-acetyl-a-D-glucosaminidepNP-a-L-arabinopyranosidepNP-P-D-lactosidepNP-a-L-rhamnopyranosideL-Valine-pNA

100 100100 7160 140 0

100 5780 4320 140 14

40 00 00 140 0

40 8640 1000 0

20 00 570 1000 0

20 140 100

100 10080 4320 2920 060 2920 080 0100 8620 2980 430 00 00 0

60 0100 14100 71100 2980 14100 020 1460 080 1460 8620 14

0 0100 2960 2940 8620 0

100 100 3325 100 10050 25 1000 0 67

50 0 3350 25 1000 25 1000 0 1000 0 1000 0 100

75 75 670 0 33

25 0 100100 75 1000 50 100

50 100 6775 0 33100 100 100

0 0 10050 100 67100 100 100100 100 100100 100 10050 75 3350 75 10075 0 10025 50 10050 75 100100 100 3375 100 67

100 100 10075 75 10025 0 00 0 670 0 67

100 100 100100 25 100100 0 10050 75 100100 50 025 25 1000 0 0

75 75 100100 25 10025 75 100

0 0 025 100 1000 100 100

25 0 10025 0 0

50 100 100100 100 100100 100 10050 0 050 100 50100 100 100100 100 0100 100 0100 100 1000 50 100

25 50 50100 100 100100 100 100100 100 100100 100 1000 50 50

25 0 50100 100 50100 100 10025 50 5075 50 0100 100 100100 100 1000 50 0

100 50 10050 100 5075 50 025 50 1000 100 100

75 0 075 100 100100 0 100100 50 5075 100 10075 100 0100 100 100100 50 50100 100 0100 100 10025 0 100100 100 100

0 0 075 100 10050 100 100100 100 100

75 0 100100 100 100100 100 50100 100 0

0 0 0

33 100 29 100 100100 100 100 100 1000 33 71 62 1000 0 0 0 100

100 67 71 100 100100 100 100 100 100100 100 100 100 10033 33 86 88 100100 33 43 50 100100 33 0 38 0100 33 29 88 0

0 0 100 0 100100 100 100 100 100100 100 100 100 10067 33 100 50 100100 33 57 100 00 0 14 12 50

100 100 71 100 100100 100 100 100 100100 33 57 100 0100 100 43 100 100100 100 43 100 10067 100 100 88 10067 100 14 62 00 67 71 75 0

33 33 71 88 500 0 14 25 0

33 100 86 75 10033 100 71 88 0100 33 57 75 100100 100 86 88 50100 0 86 100 1000 0 43 0 100

100 0 86 100 100100 100 71 62 50100 0 100 62 10067 0 71 88 1000 33 86 75 100

100 67 71 75 5067 33 43 88 50100 67 100 100 100

0 0 0 0 0100 67 71 88 10067 100 71 75 50100 100 100 100 100

0 0 0 0 0100 100 100 100 100100 100 100 100 100

0 100 86 100 500 0 0 0 0

I Tests selected according to the report of KYimpfer and Kroppenstedt (13). pNP, para-nitrophenyl; pNA, para-nitroanilide.b For cluster numbers, see Fig. 1.

most closely resembles S. diastatochromogenes ATCC12309T and S. bottropensis ATCC 25435T, which belong tothe S. violaceus cluster according to Kampfer et al. (14). Thetwo strains of S. acidiscabies which have less than 20%DNA relatedness to S. scabies RL 840134T (8) were alsofound in our study to belong to cluster group B, albeit in thedistinct cluster 10 (Fig. 1). American strains which are

assigned to cluster group A (Table 1) have not been investi-gated for DNA-DNA relatedness by Healy and Lambert (8).

However, other strains of the S. albidoflavus group (36)tested by these authors, such as S. scabies ATCC 3352, S.setonii ATCC 25497T, S. sampsonii ATCC 25495T, and S.griseus ATCC 10317 and ATCC 23345T (DSM 40236T)showed uniformly low DNA-DNA relatedness and were notrelated to S. scabies and S. acidiscabies (sensu Healy andLambert [8]). These strains were also tested by Kampfer etal. (14) and grouped into subclusters 1-2 to 1-4.RFLP analyses obtained in the present studies also imply

Charactera

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TABLE 3. Characteristics useful for differentiating Streptomyces spp. for unclustered strains

Test result for strainb:Characteristie

3 6 7 8 9 12 14 18 20 22 23 24 25 26 28 29 30 31 33 34 35 36 37 38

Utilization of:N-Acetyl-D-galactosamineL-ArabinoseL-FucoseD-GlucarateD-GlucuronateLactoseLactuloseL-Lyxosea-D-Melibiosea-Methyl-D-mannosideMethyl-a-D-glucopyranosidea-D-RaffinoseL-RhamnoseD-RiboseD-SucroseD-TuranoseAdonitolD-Arabitoli-InositolMaltitolD-MannitolCaprateIsobutyrateSorbinic acidAdipateAzelateGlycerophosphate2-HydroxyvalerateDL-LactateMalonateSuberateAcetamidocaprateD-AlanineL-CitrullineD-GlutamateL-GlycineL-HydroxyprolineDL-IsoleucineL-LeucineL-LysineL-OrnithineSpermineL-LeucinamideL-ValinamidePhenyllactate

Hydrolysis of:pNP-N-acetyl-CL-D-glucosaminidepNP-a-L-arabinopyranosidepNP-13-D-lactosidepNP-a-L-rhamnopyranosideL-Valine-pNA

-++++++_-+_-+++_----+__- +_

+- ++ + ++ ++ + ++ + +++ ++ + + + + ++ +_ +_ ++ +_---- +

-- + -+++ - +

+

+ -+ -- ++ +++ ++ ++ +- + -- - + + +- ++ -++ ++ + ++ + +++ + ++++ ++ + + - +- ++ +- ++ +- + ++ + +++ + + + -- +- +

+ -- -+ + ++- ++ + + +++++-+++-+-+---__+ +-+ +-__+ +++. ++++__

-+---+_+ ++ +_+-+ ++ -+ +++__ + -+ -_-+---_-_++_+ + + + + .+_.__+ +- -_ __+-+ ++_- +.__++++++_+ + +

+-+- -+ ++ ++ + + + ++ +++ + +-++ +++ -+

+++ +--+ ++ +- + + + + ++ + + - + + ++-+_-+-+_--+++ +_++ ++++++++-- - + +--+

_-++__+_+ + +++ + + ++- +++ +

+ -+-+-+ +++ + + ++ + + +-+-+_ + +-+ + +_-_-+_++ ++ ++-+ ++-+ +.++++--+ -+_+-+__++_+++_+++___++__

+-++ - -++ + -++++ +++++ ++_ - -++ + -_++++_+__+ + ++ + +++++- + ---+ ++ + -+

_ +_ _+ ++- ++-++-+++++ - -+-+ +_+ ___++_ +_+++ + ++-++---+-+--++-+ ++ -- +-+ +- ++_+ +++__ -_+-_+ -+ -++ +-++ + + ++ ++-+-+- +-+ - +++

_ ~ ~ -_++++++ -++ _- ++_ - +__+ +

+++-+++-+-+-++ +__++__-_++_-_-+ ++ ++ +++ +-++ + ++ + ++_ + + + +_

+-_++-_+ ++ ++ ++_+ + +++++ +++- - + -+

+--+ --+++-+ + +++ + ++ ++-+ ++- - + - +

+-+-++ +-+++++++-+++-++-++++ ++++ +++ +.+-+++---+ + -

a Tests selected according to the report of KAmpfer and Kroppenstedt (13). pNP, para-nitrophenyl; pNA, para-nitroanilide.b For cluster numbers, see Fig. 1.

that the pathogenic traits cannot be assigned to any Strep-tomyces species. Furthermore, although DNA homologyamong these strains has not been determined, the highdegree of the observed polymorphism suggests that in con-trast to the case for other bacteria (e.g., see references 3 and5) RFLPs may not be useful for rapid species identificationof Streptomyces strains. The latter assumption is also sup-ported by the high genetic variability characteristic of Strep-

tomyces spp. (26). The lack of correlation between thenumerical classification of the investigated strains and theirassemblage according to RFLP SGs (Table 1) indicates thatthe phenetic clustering of Streptomyces can hardly reflectthe genomic relationship. Similar results were also shown byGladek et al. (6), who used DNA-rRNA pairing to establishphylogenetic relationships. As pointed out by Witt andStackebrandt (37), at present it is not possible to determine

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STREPTOMYCES STRAINS THAT CAUSE POTATO SCAB 3939

TABLE 4. Summary of RFLP SGs obtained withrestriction endonucleases

Endonuclease Streptomyces strains in groupand SG'

BamHI1. PBS1, 20, 16, 422. 29, 31, 273. 41, RL 8601304. 11, P2, 3, 195. 21, 12, 236. RL 840170, RL 8401437. B30

BstEII1.12, 26

2.NP113, 40231

3.42, PBS1

4.RL 830139, 19, NP2155.P2, 40015, B30

6.RL 840143, RL840170, RL 8401234, RL 830103

7.20, NP115

PvuII1.RL 8401235, 70, 2602

2.NP114, NP115, NP113

3.42, 20, PBS1

4 23, 2

5.RL 840143, RL 840170, 31

6.NP111, B30

SaIlI1 42, 20

2 P2, 26

3.40236, 40232, 40230

4.NP114, 29

5.27, NP1l1, 40015

6.RL 840143, RL 840170

7 2, 41

8 4, 21

9.NP113, 44

10 RL 830139, 1911 B30, RL 850129

a An SG is composed of Streptomyces strains which exhibit a similaritycoefficient (F) of >0.85 with each other.

the phylogenetic depth of the phenetic clusters solely byDNA-DNA hybridization, and the question about their phy-logenetic coherency is difficult to answer.

Since isolates causing shallow- and deep-pitted scabsrepresent neither phenotypically nor genetically homoge-neous groups, it appears likely that genes required forpathogenicity are spread by mobilization elements amongdifferent Streptomyces species within their natural habitat.Thus, only if these pathogenic determinants are character-ized will it be feasible to develop a rapid screening procedurefor strains inciting potato scab. Recently, Lawrence et al.(20) have isolated and chemically characterized vivotoxins(thaxtomins) from scabby potato tissue. These toxins inducescab development on aseptically cultured minitubers. Ifthaxtomins are proven to be indispensable for scab forma-tion, then genes encoding their biosynthesis might serve as

tools for investigating gene mobilization among pathogenicStreptomyces spp. and for development of diagnostic DNAprobes.

ACKNOWLEDGMENTSWe thank R. Loria and R. M. Kroppenstedt for providing Strep-

tomyces strains for this study.

This study was partially supported by grant I-1418-88 from theUS-Israel Binational Agricultural Research and Development Fundand MWK Hannover, Germany.

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