Bulletin OEPP/EPPO Bullerin 17, 237--240 (1987)

The taxonomic position of Xanthomonas ampelina'

by A. WILLEMS, M. GILLIS, K. KERSTERS, L. VAN DEN BROECKE and J. DE LEY

Laboratorium voor Microbiologie en Microbiele Genetica, Rijksuniversiteit, K. L. Ledeganckstaat 35, Gent (Belgium)

The internal taxonomic structure and the suprageneric relations of the causal agent of bacterial blight and canker of grapevine, the misnamed Xanthomonas ampelina, were examined. By studying more than 30 strains of different geographical origin by polyacryl- amide gel electrophoresis and DNA: DNA hybridization, we established the homogeneity of X . ampelina. ['4C]-labelled ribosomal RNA (rRNA) was prepared from the type strain of X. ampelina (NCPPB 22 17) and hybridized with DNA from different reprekntative strains from the Pseudomonas acidooorans rRNA complex in rRNA superfamily 111. X. ampelina was found to be a separate branch in rRNA superfamily 111, related at a suprageneric level to P. acidouorans, Alcaligenes paradoxus and the other organisms of the P. acidooorans rRNA complex.

Introduction

Xanthomonas ampelina, the causal agent of bacterial canker of grapevine, is a strictly aerobic, non-spore forming, Gram-negative rod, motile by one polar flagellum. It produces a yellow insoluble pigment and metabolizes sugars oxidatively. Therefore Panagopoulos (1 969) origi- nally placed this bacterium in the genus Xunthomonas as a new species: X . ampelina. Its DNA base composition (68.5 mol YO G + C) does not exclude X . ampelina from the genus Xanthomonas (63-71 mol Yo G + C , Bradbury, 1984). Other data, however, clearly prove that this bacterium is not a genuine Xanthomonas:

(1) hybridizations between DNA from different Xanthomonas species and rRNA from the type strain of the type species X . campestris clearly showed the absence of any relationship between X . ampelina and the genus Xanthomonas (De Vos & De Ley, 1983);

(2) with respect to inhibitors and cofactor specificity of the enzymes mediating the synthesis of tyrosine, tryptophan and phenylalanine, all genuine Xanthomonas species have been found to be similar. X . ampelina, however, was shown to have totally different enzymatic properties, not comparable to any other investigated organism (Byng et a/., 1980; Whitaker et al., 1981);

(3) the xanthomonadins, pigments unique to the genus Xanthomonas and occurring in all genuine Xanthomonas species, have not been found in X . ampelina. Instead, this bacterium contains pigments that are hard to purify and the structure ofwhich is still unknown (Starr et a/., 1977);

(4) the following phenotypic features also support the exclusion of X . ampelina from the genus Xanthomonas - the occasional production of filamentous cells, the presence of urease, the utilization of D,L-tartrate and the failure to produce acid from glucose (Bradbury, 1984).

' Paper presented at the EPPO Conference on Bacterial Plant Diseases, Angers (FR), 5-6 May, 1986

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238 A . Willems et al.

It was our aim to determine the exact taxonomic position of X . ampelina and to reveal its internal taxonomic structure and the possible geographic interstrain differences.

Materials and methods

We studied 33 X . ampelina strains, obtained from different culture collections and originating from Spain, Greece, Crete, France and South Africa. Most of the French strains were kindly provided by Dr M. Ride. During purity controls of the strains, fast and slow-growing colony types were sometimes isolated. The occurrence of these types was not correlated with geographical origin.

The cellular proteins of all strains were compared by sodium-dodecylsulphate polyacryl- amide-gel electrophoresis, using the method of Laemmli (1970) as modified by Kiredjian et al. (1986). DNA: DNA hybridizations were performed as described by De Ley et al. (1970). For studies of the suprageneric relations, 23s [i4C]-labelled rRNA of X . ampelina NCPPB 2217 was prepared and DNA:rRNA hybridizations were performed according to De Ley & De Smedt (1975). In this method, TmC,, (the temperature at which 50% of the hybrid was denatured) is the decisive parameter.

Results and discussion

Comparison of electrophoretic protein patterns is a very fine technique, giving information mainly at the species or intraspecific level. Strains with identical or very similar protein patterns are genotypically highly related (Kersters & De Ley, 1980). All X . ampelina strains investigated showed very similar protein electrophoregrams. From a limited number of strains, slightly aberrant protein electrophoretic patterns were obtained. Usually the fast and slow-growing types produced identical protein patterns, although sometimes very small differences were observed. It is our experience that such minor differences in protein patterns reflect only small, almost indetectable genotypic differences. Indeed, 100% DNA: DNA binding was found in control hybridizations of total DNA from an electrophoretically aberrant strain with DNA from a typical strain.

The intergeneric relationships of the X . ampelina cluster were determined by DNA: rRNA hybridizations. The application, for over 10 years, of the DNA: rRNA hybridization technique in the study of the evolutionary relations between various taxa of Gram-negative bacteria has proved to be very useful and has led to a classification of the Gram-negatives into at least five large, clearly distinct groups, which we call rRNA superfamilies (De Ley, 1978; De Vos & De Ley, 1983; De Ley, 1984; De Vos et al., 1985). There is little relation between the different rRNA superfamilies, and within one rRNA superfamily different branches are related at levels ranging from order to species (De Vos & De Ley, 1983). The genus Xanthomonas belongs to rRNA superfamily 11, whereas X . ampelina has proved to be a member of rRNA superfamily 1II:it belongs to the Pseudomonas acidovorans - Alcaligenes paradoxus rRNA branch at about the same level as, for example, Pseudomonas facilis, P. dela$eldii, P. palleronii, P. j a v a , P. testosteroni, Alcaligenes latus, Comamonas terrigena and the plant pathogen P. avenae. We have united all these organisms in what we call the P. acidovorans rRNA complex. In our classification scheme the type species of the genera Pseudomonas and Alcaligenes are only distantly related to the P. acidovorans rRNA complex. Therefore most of the taxa from this complex are generically misnamed.

In order to find out which other organisms of the P. acidovorans rRNA complex are more closely related to X . ampelina, hybridizations were performed between their DNAs and 23s [I4C]- labelled rRNA from the type strain of X . ampelina, NCPPB 2217. None of the known members of the P. acidovorans rRNA complex was found to be closely related to X . ampelina, which consequently constitutes a separate rRNA branch next to A . paradoxus and P. acidovorans.

Taxonomy of Xanthomonas ampelina 239

These three branches are related at the same level Tm(e) = 76.2 & 0.7”C). A comparable level of relatedness is found between different genera of the Enterobacteriaceae (R. Tytgat & J. De Ley, unpubl.). Just as in this family, the organisms of the P. acidovorans rRNA complex are very heterogeneous; therefore the different rRNA branches of this complex may be considered to be related at the generic level.

Conclusions

From our research using protein electrophoresis, DNA: DNA and DNA: rRNA hybridizations, we can thus conclude that X . ampelina is a homogeneous taxon which has been generically misnamed. It belongs to the third rRNA superfamily in which it forms a totally separate branch, without any relatives a t the generic level. Consequently a new genus name will be proposed.

Situation taxonomique de Xanthomonas ampelina

Nous avons examine la structure taxonomique interne de l’agent de la maladie d’Ol6ron de la vigne, appele a tort Xanthomonas ampelina, ainsi que ses relations suprageneriques. L’Ctude de plus de 30 souches d’origine geographique diffirente par electrophorese sur gel polyacrylamide et par hybridation d’ADN a permis d’etablir I’homogeneite de l’esp2ce. Nous avons prepare un A R N ribosomique marque au 14C (rARN) a partir de la souche type de X . ampelina (NCPPB 221 7) et l’avons hybride avec I’ADN de difftrentes souches representatives du complexe rARN Pseudomonas acidovorans de la superfamille rARN 111. X . ampelina apparait comme un element distinct de celle-ci, se rapprochant au niveau supragenerique de P. acidovorans, Alcaligenes paradoxus et des autres organismes du complexe rARN P. acidovorans.

TaKcoHoMwiecKoe nonoxenne Xanthomonas ampelina

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