Panagopoulos 1969 to a New Genus, Xylophilus Gen

Home , Xylophilus (bacterium), Xylophilus ampelinus

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1987, p. 422430 Vol. 37, No. 4 0020-7713/87/040422-09$02.00/0 Copyright 0 1987, International Union of Microbiological Societies

Transfer of Xanthomonas ampelina Panagopoulos 1969 to a New Genus, Xylophilus gen. nov., as Xylophilus ampelinus (Panagopoulos 1969) comb. nov.

A. WILLEMS, M. GILLIS, K. KERSTERS, L. VAN DEN BROECKE, AND J. DE LEY* Laboratorium voor Microbiologie en Microbiele Genetica, Rijksuniversiteit, B-9000 Ghent, Belgium

Thirty-four strains of Xanthomonas ampelina, the causal agent of bacterial necrosis of grape vines, were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their cellular proteins and by numerical analysis of 106 enzymatic features (API systems). These organisms formed a very homogeneous taxon. Generic and suprageneric relationships were determined by hybridizations between 23s 14C-labeled ribosomal ribonucleic acid from Xanthomonas ampelina NCPPB 2217T (T = type strain) and deoxyribonucleic acids from eight Xanthomonas ampelina strains, Xanthomonas campestris NCPPB 52€lT, and the type strains of 16 possibly related species. Xanthomonas ampelina was found to be a totally separate subbranch in ribosomal ribonucleic acid superfamily 111, without any relatives at the generic level. It is not related to the genus Xanthomonas. Genetically its closest relatives are, among others, Pseudomonas acidovorans, Alcaligenes paradoxus, and Comamonas terrigena. We propose the transfer of Xanthomonas ampelina to a new genus, Xylophilus. The only species and thus the type species is Xylophilus ampelinus. The type strain is NCPPB 2217.

The causal agent of bacterial necrosis and canker of grape to a new genus, Xylophilus, as Xylophilus ampelinus (Pan- vines in the Mediterranean region and South Africa was agopoulos 1969) comb. nov. Strain NCPPB 2217 is main- originally described as Xanthomonas ampelina (16, 26). This tained as the type strain. In the rest of this paper we use the organism possesses the following features of the genus name Xylophilus ampelinus for this organism. Xanthomonas. It is an aerobic, nonsporeforming, gram-negative, rod-shaped organism with one polar flagellum; it has oxidative carbohydrate metabolism; it produces a yellow MATERIALS AND METHODS insoluble pigment; and it has a mean deoxyribonucleic acid Bacterial strains. The strains which we used are listed in (DNA) base composition similar to that of the genus Table 1. They were checked for purity by plating and by Xanthomonas. However, other data clearly prove that it examination of living and gram-stained cells. In all experi- does not belong to the genus Xanthomonas. Hybridizations ments Xanthomonas and Xylophilus strains were grown on between ribosomal ribonucleic acid (rRNA) from Xantho- GYCA medium (1% [wt/vol] glucose, 0.5% [wt/vol] yeast monas campestris NCPPB 52gT (T = type strain) and DNAs extract, 3% [wthol] CaC03, 2% [wt/vol] agar); Rhodocyclus from different Xanthomonas species showed that Xantho- gelatinosus was grown on DSM medium 27 (4), and nutrient monas ampelina is definitely not related to the authentic agar (0.1% beef extract, 0.2% yeast extract, 0.5% NaC1, Xanthomonas species (11). This conclusion is supported by 0.5% peptone, 2% agar, pH 7.4) was used for all other research on the regulation of the biosynthesis of aromatic strains. All strains were grown at 28"C, except the amino acids in Pseudomonas strains and related organisms Xylophilus strains, which were grown at 24°C. In some (3, 32, 33). Additional features differentiating Xanthomonas cultures of Xylophilus ampelinus we isolated two colony ampelina from the other Xanthomonas species are the types, tl and t2, which showed similar colony morphology absence of xanthomonadins (30), very slow growth, a max- but differed in growth rate. The average colony diameters imum growth temperature of 30"C, the presence of urease, after 2 weeks on GYCA medium were 1.4 k 0.6 mm for tl utilization of meso-tartrate, and no production of acid from and 0.7 k 0.3 mm for t2. glucose or sucrose (2, 26). It is now well accepted that Enzymatic features. A total of 31 Xylophilus ampelinus Xanthomonas ampelina is not a Xanthomonas species; strains and a number of reference strains (Table 1) were however, despite its unknown taxonomic position, it has tested at 30°C in the commercially available API ZYM been maintained up to now as a separate species in this gallery and the experimental API galleries Osidases, genus (2). Esterases, and Aminopeptidases AP1, AP2, AP3, AP4, AP5, In this paper we present the results of extensive research and AP6 (API Systems S.A., La Balme-les-Grottes, on (i) the internal taxonomic structure of Xanthomonas Montalieu-Vercieu, France). The experimental procedure ampelina and (ii) its generic and suprageneric relationships. and the scoring of the results have been described previously We examined 34 strains from various geographic origins, (24). In total, 106 enzyme activities were recorded per strain. together with a number of possibly related strains and some Canberra metric coefficients (dcANB) were calculated (22) authentic Xanthomonas strains, by means of protein elec- and transformed into similarity coefficients (%S) by using the trophoresis, numerical analysis of enzymatic features, and following equation: %S = 100 x (1 - dCANB). Cluster DNA-DNA and DNA-rRNA hybridizations. Based on our analysis by the unweighted average pair group method (29) results, we propose the transfer of Xanthomonas ampelina was performed by using the Clustan 2.1 program of Wishart (34) and the Siemens model 7551 (BS2000) computer of the * Corresponding author. Centraal Digitaal Rekencentrum, Rijksuniversiteit, Ghent,

422 VOL. 37, 1987 XYLOPHILUS AMPELINUS 423

TABLE 1. Organisms used, their origins, and the tests in which they were used Species Strain" Source, place, and year of isolation Tests"

Xylophilus ampelinus NCPPB 2217T (= LMG 5856T = PDDCC Vitis vinifera var. sultana, Crete, 1966 4298T = ATCC 33914T = CNBP 1192T) Xylophilus ampelinus CNBP 1192T (= LMG 5949T) Vitis viniferu var. sultana, Crete, 1966 Xylophilus ampelinus Ride AA2 (= LMG 500 = CNBP 1800) Vitis viniferu, Aude, France, 1976 Xylophilus ampelinus CNBP 1802 (= LMG 501 = Ride A1.03) Vitis vinifera, France, 1968 Xylophilus ampelinus Ride AC1 (= LMG 502 = CNBP 1803) Vitis vinifera, Aude, France, 1976 Xylophilus ampelinus CNBP 1833t1 and CNBP 1833t2 (= LMG Vitis vinifera, France, 1975 503 = Rid6 A14)" Xylophilus ampelinus CNBP 1834 (= LMG 504 = Ride A7) Vitis vinifera, France, 1975 Xylophilus ampelinus CNBP 1837t1 and CNBP 1837t2 (= LMG Vitis vinifera, France, 1975 505 = Ride C12)" Xylophilus ampelinus Ride AR1 (= LMG 506 = CNBP 1841) Vitis vinifera var. ugniblanc, Ardeche, France, 1977 Xylophilus ampelinus CNBP 1926t1 and CNBP 1926t2 (= LMG Vitis vinifera, Spain 507)' Xy lop hilus amp elin us CNBP 1927t1 and CNBP 1927t2 (= LMG Vitis vinifera, Spain 508)' Xylophilus ampelinus CNBP 1928 (= LMG 509) Vitis vinifera, Spain Xylophilus ampelinus CNBP 1938t1 and CNBP 1938t2 (= LMG Vitis vinifera, Spain 510)' Xylophilus ampelinus CNBP 2059 (= LMG 511) Vitis vinifera Xylophilus ampelinus CNBP 2060tl and CNBP 2060t2 (= LMG Vitis viniferu 512)' Xylophilus ampelinus CNBP 2061 (= LMG 513) Vitis vinifera Xylophilus ampelinus CNBP 2098 (= LMG 514) Vitis vinifera Xylophilus ampelinus NCPPB 2218 (= LMG 516 = ATCC 29074) Vitis vinifera, Greece, 1966 Xylophilus ampelinus NCPPB 2219 (= LMG 517 = ATCC 29075) Vitis vinifera, Greece, 1966 Xylophilus ampelinus NCPPB 2220 (= LMG 518 = PDDCC 4299) Vitis vinifera cv. sultana, Crete, 1966 Xylophilus ampelinus NCPPB 2221t1 and NCPPB 2221t2 (= LMG Vitis vinifera cv. sultana, Crete, 1966 519 = PDDCC 4300)' Xylophilus ampelinus NCPPB 2590 (= LMG 520) Vitis vinifera, South Africa, 1972 Xylophilus ampelinus NCPPB 2591t1 and NCPPB 2591t2 (= LMG Vitis vinifera, South Africa, 1972 521)" Xylophilus ampelinus PDDCC 4299 (= LMG 522 = NCPPB 2220) Vitis vinifera cv. sultana, Crete, 1966 Xylophilus ampelinus PDDCC 4300tl and PDDCC 4300t2 (= LMG Vitis vinifera cv. sultanu, Crete, 1969 523 = NCPPB 2221)' Xylophilus ampelinus RidC C2' (= LMG 524 = CNBP 1796) Vitis vinifera var. ugni, Charente Maritime, France, 1975 Xylophilus ampelinus RidC C13 (= LMG 525) Vitis vinifera var. ugni, Charente Maritime, France, 1975 Xylophilus ampelinus RidC 011 (= LMG 526)d Vitis vinifera var. ugni, Ile d'OlCron, France, 1969 Xylophilus ampelinus Ride 012 (= LMG 527)" Vitis vinifera var. ugni, Ile d'OlCron, France, 1969 Xylophilus ampelinus Ride P5 ( = LMG 528) Vitis vinifera var. grenache, Pyrenees Orientales, France, 1975 Xylophilus ampelinus Ride P6 (= LMG 529) Vitis vinifera, var. grenache, Pyrenees Orientales, France, 1975 Xylophilus ampelinus Ride P7 (= LMG 530 = CNBP 1799) Vitis vinifera var. macabeu, PyrCnCes Orientales, France, 1975 Xylophilus ampelinus CNBP 1800 (= LMG 544 = Ride AA2) Vitis vinifera, Aude, France, 1976 Xylophilus ampelinus CNBP 1841 (= LMG 545 = Rid6 AR1) Vitis vinifera var. ugniblunc, Ardkche, France, 1977 Xanthomonas campestris pv. NCPPB 528T (= LMG 568T = ATCC Brassica olerucea cv. gemmiferu, England, campestris 33913T) 1957 Xanthomonas campestris pv. NCPPB 1149 (= LMG 939)< Vitis carnosu, India vitiscarnosae Xanthomonas campestris pv. NCPPB 2475 (= LMG 965)' Vitis vinifera, India, 1969 viticola Xanthomonas campestris pv. NCPPB 1451 (= LMG 940)'' Vitis trifolium, India, 1961 vitistrifoliae Xanthomonas campestris pv. NCPPB 1014 (= LMG 954)' Vitis woodrowii. India vitis woodrow ii Xanthomonas albilineans NCPPB 2969T (= LMG 494T) Succharum oficinarum, Fiji, Japan, 1961 Xanthomonas albilineans NCPPB 1050 (= LMG 487) Sacchurum oficinarum, Guyana Xanthomonas axonopodis NCPPB 457T (= LMG 538T) Axonopus scoparius, Columbia, 1949 Xanthomonas axonopodis NCPPB 2376 (= LMG 540) Axonopus scoparius, Columbia, 1949 Xanthomonas fragariae NCPPB 1469T (= LMG 708T) Fragaria chiloensis var. unanassa, United States, 1966 Continued on following page 424 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TAB LE 1-Con t in ued Species Strain“ Source, place. and year of isolation Tests’

Xanthornonas fragariae NCPPB 1822 (= LMG 709) Fragaria chiloensis var. ananassa, United States, 1966 “Xanthornonas populi” CNBP 1817 (= LMG 5743)’ Populus euramericana, 1966 “Xanthornonas populi” CNBP 2226 (= LMG 5755) Popidus euramaricana, Belgium Pseudornonas acidovorans Stanier 14T (= LMG 1226T = ATCC 1566tIT) Acetamide-enriched soil, The Netherlands, 1926 Pseudornonas palleronii Stanier 362tlT (= LMG 2366T)g Water, United States Pseudornonas testosteroni NCTC 10698T (= LMG 1786T = Stanier 7gT) Soil, United States, 1952 Pseudornonas saccharophila ATCC 15946T (= LMG 2256T = NCIB Bay of San Francisco, United States, 1940 8570T) Pseudornonas flava DSM 619T (= LMG 218ST) Mud from ditch, 1942 Pseudornonas delafieldii CCUG 1779T (= LMG 5943T = Stanier 133T Soil = ATCC 17505T) Pseudornonas facilis ATCC 11228T (= LMG 2193T) Lawn soil Pseudornonas avenue NCPPB lollT (= LMG 2117T = ATCC Zea mays, United States, 1958 19860T) Pseudornonas rubrilineans NCPPB 920T (= LMG 2281T = ATCC Saccharum oficinarurn, Riunion, 1960 19307T) “Pseudornonas setariae” NCPPB 1392T (= LMG 1806T = ATCC Otyza sativa, Japan, 1955 19882T) Pseudornonas cattleyae NCPPB 961T (= LMG 5286T) Unknown Pseudornonas PDDCC 7500T (= LMG 5376T = ATCC Citrullus lanatus, United States, 1977 pseudoalcaligenes subsp. 29625T) citrulli Alcaligenes paradoxus ATCC 17713tlT and ATCC 17713t2T (= Soil, United States LMG 1797T)“ Alcaligenes paradoxus ATCC 17712 (= LMG 3572) Soil Alcaligenes paradoxus ATCC 17719t1 and ATCC 17719t2 (= LMG Panthotenate-enriched soil 3599y Alcaligenes latus ATCC 29712T (= LMG 3321T) Soil Cornarnonas terrigena NCIB 8193T (= LMG 1253T) Hay infusion, United States Cornarnonas terrigena NCIB 2581 (= LMG 1249) Soil Rhodocyclus gelatinosus NCIB 8290T (= LMG 4311T = ATCC 1944 17011T) ’ ATCC, American Type Culture Collection, Rockville, Md; CCUG, Culture Collection of the University of Goteborg, Department of Clinical Bacteriology, University of Goteborg, Goteborg, Sweden; DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany; LMG, Culture Collection, Laboratorium Microbiologie, Ghent, Belgium; NCIB, National Collection of Industrial Bacteria, Aberdeen, Scotland; NCPPB, National Collection of Plant Pathogenic Bacteria, Hatching Green, England; NCTC, National Collection of Type Cultures. Central Public Health Laboratory, London, United Kingdom; PDDCC, Culture Collection of the Plant Disease Division, New Zealand Department of Scientific and Industrial Research, Auckland, New Zealand. ’ A, Enzymatic API galleries; P, polyacrylamide gel electrophoresis; D, DNA-DNA hybridization; R, DNA-rRNA hybridization. While subculturing, we found two colony variants in this strain, which we labeled then tl and t2. When, for strains other than Xylophilus wnpelinus strains, both types yielded an identical protein electrophoretic pattern, only tl was used. Strians Rid6 011 and Rid6 012 are yellow and white subcultures of strain Ride 01, respectively. ‘ Pathotype strain (15). Strain CNBP 1817 was proposed as the type strain for “Xanthornonas populi” by Rid6 and Ride (27). This stain was received from Stanier as one of two colony variants of strain Stanier 362. Strains Stanier 362t1 and Stanier 362t2 correspond to strain ATCC 17724.

Belgium. The centrotype strain was calculated by the 16s or 23s rRNA and filter-fixed, single-stranded, high- method of Rogers and Tanimoto (28). molecular-weight DNA were performed as described by De Polyacrylamide gel electrophoresis of proteins. Xylophilus Ley and De Smedt (8). Each DNA-rRNA hybrid was char- ampelinus strains were grown on Roux flasks at 24°C for 72 acterized by the following two parameters: the midpoint (in h on GYCA medium. The other reference strains were degrees centigrade) of the thermal denaturation curve [ T,(,)] grown on the same medium at 28°C for 40 h. Whole-cell and the percent rRNA binding (i.e., the amount [in micro- protein extracts were prepared, and sodium dodecyl sulfate- grams] of labeled rRNA duplexed to 100 pg of filter-fixed polyacrylamide gel electrophoresis was performed by using DNA after ribonuclease treatment). small modifications of the procedure of Laemmli (21), as DNA base composition. The average mol% guanine-plus- described previously (20). cytosine (G+C) content was determined by thermal dena- DNA-rRNA hybridization. Preparation of high-molecular- turation (10) and was calculated by using the equation of weight DNA, fixation of DNA on cellulose nitrate filters, and Marmur and Doty (23), as modified by De Ley (5). chemical determination of the amount of DNA on the filters DNA-DNA hybridization. The degree of binding (D) was were carried out as reported previously (8, 11). Purified 23s determined spectrophotometrically from the initial renatur- [14C]rRNA from Xylophilus ampelinus NCPPB 2217T was ation rates by the method of De Ley et al. (7). Values of 20% prepared as described previously (8). Purified 23s [3H]rRNA D and less do not represent any significant DNA homology. from Pseudomonas acidovorans Stanier 14T and 16s [3H] The total DNA concentration was ca. 50 pg/ml, and the rRNA from Alcaligenes paradoxus ATCC 17713tlT were optimal renaturation temperature in 2 x SSC (sodium saline prepared previously by M. Goor and M. Gillis (12) and by P. citrate; lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate, Segers (13), respectively. Hybridizations between labeled pH 7.0) was 82.5”C. A Gilford model 2600 spectrophotome- VOL. 37, 1987 XYLOPHILUS AMPELINUS 425

ampelinus, and representatives of the acidovorans rRNA complex. Enzymatic features, The test reproducibility of the enzymatic API systems was good, as 91 to 95% S was found between duplicates of tests for five Xylophilus ampelinus strains. As expected from the protein electropherograms, the results obtained for strains with two colony types were similar (95% S between strains CNBP 1833t1 and CNBP 1833t2 and 90% S between strains CNBP 1838t1 and CNBP 1838t2). Consequently, only colony type tl strains were included in the enzyme tests of the other six Xylophilus ampelinus strains displaying two colony types. The dendrogram obtained after numerical analysis of our data is shown in Fig. 3. All Xylophilus ampelinus strains clustered above 87% S. The centrotype strain is CNBP 1833t1. The Xylophilus ampelinus cluster (Fig. 3) is clearly distinct from the genuine Xanthomonas species and is more closely related (Fig. 3) to Pseudomonas acidovorans, Pseu- domonas Java, Pseudomonas avenue, Pseudomonas facilis, Pseudomonas delafieldii, Pseudomonas testosteroni, Pseu- domonas palleronii, Alcaligenes paradoxus, and Coma- monas terrigena, which are all members of the acidovorans rRNA complex. DNA-DNA hybridizations. Hybridizations of Xylophilus ampelinus CNBP 1926t1 with either CNBP 2098 or NCPPB 2591t1 gave 100% D. Hybridizations of Xylophilus ampe- FIG. 1. Normalized protein electrophoregrams of some Xylo- linus NCPPB 2217T with Xylophilus ampelinus CNBP 2098 philus ampelinus strains. and CNBP 2061 yielded 100 and 96% D, respectively. Pseudomonas avenue NCPPB 101lT, Pseudomonas flava ter equipped with a thermostatically controlled cuvette DSM 619T, Pseudomonas palleronii Stanier 362tlT, and chamber and a Hewlett-Packard model 7225A plotter was Alcaligenes paradoxus ATCC 17713tlT gave less than 20% D used. with Xylophilus ampelinus NCPPB 2217T. RESULTS DNA base composition. The average G+C values of the strains studied are listed in Table 2. Protein electrophoresis. All Xylophilus ampelinus strains DNA-rRNA hybridizations. The specific activities of the displayed very similar protein patterns (Fig. 1). Four strains 23s [14C]rRNA fraction from Xylophilus ampeZinus NCPPB (NCPPB 2219, NCPPB 2220, Ride ACl, and CNBP 1802) 2217T, the 23s [3H]rRNA fraction from Pseudomonas lacked a heavy band near the top of the gel. This slight acidovorans Stanier 14T, and the 16s [14C]rRNA fraction aberration was reproducible. Highly similar protein patterns from Alcaligenes paradoxus ATCC 17713tlT were 3,662, were found for both colony types isolated in 8 of the 32 12,432, and 6,618 cpm/kg of rRNA, respectively. The results strains tested. Figure 2 shows the protein electropherograms of the DNA-rRNA hybridizations are summarized in Table 2 of the type strains of Xanthomonas campestris, Xylophilus and are presented in Fig. 4 as a dendrogram based on Tm(c)

FIG. 2. Normalized protein electropherograrns of the type strains of Xylophilus ampelinus, Xanthomonas campeWs, and some taxa from the acidovorans rRNA complex. Abbreviations: A., Alcaligenes; C.,Comamonas; P., Pseudomonas; X., Xanthomonas; Xy., Xylophilus. 426 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

DSM 6191 P FLAVA NCPPB10111 P AVENAE ATCC 11228l I? FAClLlS STANIER 1LT P ACIDOVORANS STANIER 362 tlT P PALLERONII ATCC 17713111 A. PARA DOX U S NClB 81937 C TERRIGENA NCTC 106981 P TESTOSTERONI CCUG 17791 f? DELAFlELDll CNBP 2098 RID^ c2 RIDE ~7 CNBP 1831 CNBP 2059 CNBP 1803 RID$ P6 CNBP 1938 11 NCPPB 2591 t1 NCPPB 2218 RIDE 011 CNBP 1800 7 RIDE P5 CNBP 183311 PDDCC 4299 CNBP 2061 XY. AMPELINUS CNBP 1926 11 RID& ci3 CNBP 1811 PDDCC 13M)tl CNBP 192711 NCPPB 2221 11 CNBP 206011 NCPPB 2590 CNBP 1802 NCPPB 22177 CNBP 1928 CNBP 1192 CNBP1837 tl

NCPPB 1822 X. FRACARIAE NCPPB 11691 NCPPB 1050 X.AL8ILINEANS NCPPB 29691 CNBP 2226 “X.POPULI .’ CNBP 1817 NCPPB 2376 X. AXONOPODIS NCPPB 1571 NCPPB 528l NCPPB 1016 NCPPB 1651 X. CAMPESTRIS NCPPB 1119 NCPPB 2175

FIG. 3. Dendrogram obtained by unweighted average pair group clustering of similarity coefficients. Each of the 53 strains (Table 1) was characterized by 106 enzymatic features. For abbreviations see the legend to Fig. 2. values, the most valuable taxonomic parameter of the hybrid value of about 60°C when it is hybridized with rRNA from (8, 9, 11). Xanthomonas campestris NCPPB 52gT (rRNA superfamily 11) and about 76°C when it is hybridized with rRNA from DISCUSSION Pseudomonas acidovorans Stanier 14T (rRNA superfamily 111). Xylophilus ampelinus belongs in rRNA superfamily 111, Our results indicate that Xylophilus ampelinus is a very in which it is related at an equidistant Tm(ellevel of 76.2”C to homogeneous taxon; all of its strains, even those from Pseudomonas acidovorans, Alcaligenes paradoxus, and different geographic origins, show very similar protein pat- many other organisms in the acidovorans rRNA complex terns (Fig. l),as well as very similar characteristics in the (Fig. 4). The numerical analysis of the enzymatic API tests enzymatic API systems (Fig. 3). The latter were preferred likewise points toward a closer relationship of Xylophilus over classical phenotypic tests, because of the slow growth ampelinus to the members of the acidovorans rRNA com- of Xylophilus ampelinus. Strains with almost identical pro- plex than to the genuine Xanthomonas species, including tein electropherograms possess a high level of genome Xanthomonas campestris strains pathogenic for grape vines similarity (18). Indeed, DNA-DNA hybridizations between (Table 1 and Fig. 3). some pairs of selected representative Xylophilus ampelinus The organisms of the acidovorans rRNA complex show strains result in a D of almost 100%. great diversity, and many of them (including Pseudomonas In previous papers from this laboratory, the impact and acidovorans) are generically misnamed. Since no definite significance of DNA-rRNA hybridizations in detecting intra- nomenclatural proposals have yet been made (11, 25), we and intergeneric relationships within the gram-negative bac- have to retain the inappropriate names of these organisms in teria have been shown extensively (12, 17, 31). Based on this paper. The following taxa belong to the acidovorans Tm(e)values, one can divide the gram-negative bacteria into rRNA complex: the Hz oxidizers Pseudomonas facilis, at least five groups, called rRNA superfamilies, which are Pseudornonas palleronii, Pseudomonas JEava, Pseudomonas related only very remotely and certainly beyond the family saccharophila, Alcaligenes paradoxus, and Alcaligenes level (6, 11). Xylophilus ampelinus is definitely not related to latus;the plant pathogens Pseudomonas avenue, Pseudomo- the genus Xanthomonas since its DNA has a mean T,(,, nus rubrilineans, and Pseudomonas pseudoalcaligenes VOL. 37, 1987 XYLOPHILUS AMPELINUS 427

TABLE 2. DNA base compositions of strains and parameters of hybrids of their DNAs with 14C-labeled23s rRNA from Xylophilus ampelinus, 'H-labeled 235 rRNA form Pseudoinonas acidovorans, and 'H-labeled 16s rRNA from Alcaligenes paradoxus Hybridized with [14C]rRNA Hybridized with [3H]RNA Hybridized with [3H]RNA G+C from Xylophilus ampe- from Pseudomonas from Alcaligenes para- DNA from strain: content linus NCPPB 2217T ucidovorans Stanier 14* doxus ATCC 17713tlT

(mol%) T,,IIP) % rRNA T,,lC,,, % rRNA T"W % rRNA ("C) binding ("C) binding ("0 binding Xylophilus ampelinus NCPPB 2217T 68" 80.0 0.11 76.0 0.09 76.0 0.13 Xylophilus ampelinus CNBP 1938t1 68 81.0 0.14 76.5 0.11 Xylophilus ampelinus CNBP 2218 68 80.5 0.12 76.5 0.08 Xylophilus ampelinus CNBP 1926t1 69 80.0 0.12 76.5 0.08 Xylophilus ampelinus NCPPB 2220 68 79.5 0.11 76.0 0.10 Xylophilus ampelinus CNBP 2098 68 79.0 0.11 76.5 0.10 Xylophilus ampelinus CNBP 2061 69 78.5 0.14 76.5 0.11 Xylophilus ampelinus NCPPB 2591t1 68 80.0 0.09 76.5 0.10 Pseudomonas acidovorans Stanier 14T 67' 76.0 0.07 80Sh 0.12h 77.0' 0.10'' Pseudomonas testosteroni NCTC 1069gT 62' 76.0 0.19 76.5' 0.17' 76.5' 0.18'' Pseudomonas palleronii Stanier 362tlT 66' 77.0 0.04 76.0' 0.05h 77.5" 0.06" Pseudomonas facilis ATCC 1122gT 65' 77.5 0.08 77.0' 0.09' 77.5" 0.07" Pseudomonas delajieldii CCUG 1779T 66 77.5 0.10 77.0 0.11 76.5 0.02 Pseudomonas Java DSM 619= 67' 76.5 0.05 75.9 0.02h 76.0" 0.04" Pseudomonas saccharophila ATCC 67' 73.0 0.05 72.5 0.05 73.4 0.05 15946T Pseudomonas avenue NCPPB lollT 71' 76.5 0.05 76.5 0.12 76.5 0.19 Pseudomonas cattleyae NCPPB 961T 69" 77.0 0.07 77.d 0.05 Pseudomonas rubrilineans NCPPB 920T 69' 78.0 0.07 75.5' 0.06' "Pseudomonas setariae" NCPPB 1392T 69' 77.5 0.08 78.0' 0.08' Pseudomonas pseudoalcaligenes subsp. 68 77.0 0.08 76.5 0.24 citrulli PDDCC 7500T Comamonas terrigena NCIB 8193T 64" 76.0 0.14 76.0" 0.19'' 75.5" 0.16" Comamonas terrigena NCIB 2581 66" 76.5 0.19 75.5" 0.16" 76.0" 0.20" Alcaligenes paradoxus ATCC 17713tlT 67h 78.0 0.02 76Sh 0.03' 81.0'' 0.06" Alcaligenes paradoxus ATCC 17712 68 77.0 0.04 76.0' 0.03h 805d 0.05" Alcaligenes paradoxus ATCC 17719t1 67' 76.5 0.03 76.5' 0.03h 81.0d 0.07" Alcaligenes latus ATCC 29712T 7OX 72.0 0.10 74.5 0.12 70.0 0.11 Rhodocyclus gelatinosus NCIB 8290T 70' 71.5 0.07 74.5 0.05 71.0 0.02 Xanthomonas campestris NCPPB 528T 65 59.5 0.05

All of the results reported for the Xylophilus ampelinus type strain were obtained with strain NCPPB 2217= of lyophilization batch I. The type strain previously reported to have G+C content of 70.8 mol% (11) was not a genuine Xylophilus ompelinus strain. Data from reference 11. Data from reference 13. P. Segers and J. De Ley, unpublished data. ' Data from reference 12. fSeveral hybridizations confirmed that the previously reported T,,,,,, value of 733°C (12) should be corrected to 77.0"C (M.Goor et al., manuscript in preparation). Data from reference 19. subsp. citrulli; the photoautotrophic and CO-oxidizing or- acidovorans rRNA complex have Tm(e)values above 78.0"C ganism Rhodocyclus gelatinosus; and, from soil and clinical when their DNAs are hybridized with rRNA from Xylophilus origin, Pseudomonas testosteroni, Pseudomonas delajieldii, ampelinus NCPPB 2217T. Similar values are found versus and Comamonas terrigena (11-13, 19). Some of the taxa rRNAs from Pseudomonas acidovorans Stanier 14T and related at a mean Tm(e)of 76.2"C to Pseudomonas Alcaligenes paradoxus ATCC 17713tlT. The average level at acidovorans and Alcaligenes paradoxus could be related to which the different subbranches in the acidovorans rRNA each other more closely. The obvious step to determine the complex ramify is 76.2 5 0.7"C. Since the DNAs of many position of Xylophilus ampelinus within this rRNA complex genera in the Enterobacteriaceae have comparable Tm(e) was to prepare a labeled [14C]rRNA from its type strain and values versus rRNA from Escherichia coli (R. Tytgat and J. to hybridize this preparation with DNAs from the different De Ley, unpublished data), we can postulate that each members of the rRNA complex. The Tm(e)values which we subbranch in the acidovorans rRNA complex deserves ge- obtained for these hybrids clearly demonstrate that neric rank, provided sufficient phenotypic and genotypic Xylophilus ampelinus constitutes a separate rRNA sub- arguments are available. We have the following reasons to branch in the acidovorans rRNA complex (Table 2 and Fig. propose creation of a new genus for all of the strains known 4). Indeed, the DNAs of seven of the eight Xylophilus up to now as Xanthomonas ampelina. (i) They constitute a ampelinus strains investigated have a mean Tm(e)value of separate subbranch in the acidovorans rRNA complex. (ii) 80.1"C (standard deviation, 03°C) when they are hybridized They form a homogeneous taxon and can be differentiated with rRNA from Xylophilus ampelinus NCPPB 2217T. Strain from the other members of the acidovorans rRNA complex CNBP 2061 has a slightly lower Tm(e)value of 78.5"C; it is by their unique protein electropherograms (Fig. 2) by their nevertheless indistinguishable from the other Xylophilus enzymatic characteristics (Fig. 3) and by the lack of DNA ampelinus strains in its protein electropherogram and in its binding. (iii) They utilize only few carbohydrates, organic enzymatic features, None of the other strains from the acids, and amino acids for growth (M Van den Mooter et al., 428 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

ACIDOVORANS rRNA COMPLEX I I

F! TESTOSTERONI J "I? SETARIAE" P FAClLlS F! PSEUDOALCALIGENES ,I? SACCHAROPHILA SUBSF! ClTRULLl -A. LATUS P PALLERONII R. GELATINOSUS m 65 I 60L Liz FIG. 4. rRNA cistron similarity dendrogram showing the position of Xylophilus urnpelinus as a separate subbranch in the acidovorans rRNA complex. Data are from Table 2 and reference 11. Solid bars represent the T,n(e)ranges within the individual rRNA branches; the roman numerals are the rRNA superfamily designations sensu De Ley (6). For abbreviations see the legend to Fig. 2; R., Rhodocyclus. manuscript in preparation). (iv) They can easily be differen- enzymatic features of 31 strains; all studies included the type tiated from the other members of the acidovorans rRNA strain. complex by their much slower and less abundant growth and On nutrient agar, colonies are circular, semitranslucent, by their lower optimal growth temperature of 24°C. We slightly raised, glistening, and pale yellow with entire mar- propose a new genus, Xylophilus, for these organisms. gins. They attain diameters of 0.2 to 0.3 and 0.6 to 0.8 mm Description of Xylophilus gen. nov. Xylophilus (Xy.10. after 6 and 15 days, respectively. Better growth is obtained phi'lus. Gr. n. xylon wood; Gr. n. philos friend; M.L. masc. on GYCA medium, and the best growth occurs on a medium Xylophilus friend of wood) cells are straight to slightly containing 1% yeast extract, 2% D-galactose, 2% CaC03, curved rods, 0.4 to 0.8 by 0.6 to 3.3 km. Filamentous cells and 2% agar at 24°C. On this medium, colonies are yellow, (length, 230 pm) may occur is older cultures. Cells occur and a brown diffusible pigment is produced. Some strains singly, in pairs, or in short chains; they are motile by a single may produce two colony types on GYCA medium. Average polar flagellum. Gram negative, oxidase negative, cataiase colony diameters of 1.4 k 0.6 and 0.7 ? 0.3 mm are attained positive, strictly aerobic, chemoorganotrophic. Oxidative after 15 days at 24°C by the fast- and slow-growing types, carbohydrate metabolism. Even at the optimal temperature respectively. Minimal and maximal growth temperatures are of 24"C, growth is generally very slow and poor. The G+C 6 and 30°C, respectively. The maximal NaCl concentration content of the DNA ranges from 68 to 69 mol%. In DNA- tolerated is 1%.Growth is very slow in the presence of 0.01 rRNA hybridizations TmC,,values of 78.0 to 81.5"C are to 0.02% 2,3,5-triphenyltetrazolium chloride. L-Glutamate obtained versus rRNA from Xylophilus ampelinus NCPPB (0.1%) is required for growth; L-methionine is not required. 2217T. Xylophilus belongs to the acidovorans rRNA complex Acid is produced from L-arabinose and D-galactose but not in rRNA superfamily I11 and is equidistantly related to the from D-xylose, D-ribose, L-rhamnose, D-glucose, D-man- other taxa in this complex, Comamonas terrigena and the nose, D-fructose, sucrose, trehalose, cellobiose, lactose, generically misnamed organisms (11, 12, 19) Pseudomonas maltose, D-melibiose, raffinose, glycogen, indin, dextrin, acidovorans, Pseudomonas testosteroni, Pseudomonas adonitol, dulcitol, D-mannitol, D-sorbitol, L-sorbose, salicin, facilis, Pseudomonas delajieldii, Pseudomonas palleronii, a-methylglycoside, arbutin, and meso-inositol. Grows on Pseudomonas Java, Pseudomonas avenae, Pseudomonas acetate (0.2% but not 0.5%), citrate, DL-malate, succinate, rubrilineans, "Pseudomonas setariae," Pseudomonas cat- DL-tartrate, and fumarate; does not grow on formate, tleyae, Pseudomonas pseudoalcaligenes subsp. citrulli, and propionate, malonate, maleate, oxalate, benzoate, or cal- Alcaligenes paradoxus. The type species is Xylophilus cium gluconate. Grows on Dye asparagine medium (14). No ampelinus. Xylophilus strains can grow on L-glutamine but hydrolysis of gelatin, esculin, starch, casein, arbutin, and not on calcium lactate, whereas the reverse is true for sodium hippurate; lipolysis of Tween 80. Potato soft rot test Xanthomonas strains (Van den Mooter et al., manuscript in negative. H2S formed from cysteine and weakly from preparation). thiosulfate. Urease positive. The following features are Xylophilus ampelinus (Panagopoulos 1969) comb. nov. The lacking in all strains that have been studied: nitrate reduc- description of Xylophilus ampelinus (am.pe.li'nus. Gr. n. tion, production of indole and ammonia, Voges-Proskauer ampelos grape vine; Gr. adj. ampelinos and M.L. masc. adj. test, arginine dihydrolase, lysine and ornithine decarbox- ampelinus of the vine) is the same as for the genus. The ylase, lecithinase, and acid from glucose. All Xylophilus following description is based on previously published data ampelinus strains hydrolyze the following substrates in API for 15 strains (1, 2, 26) and on our own research on the systems (NA is an abbreviation for 2-naphthylamide): 2- VOL. 37, 1987 XYLOPHILUS AMPELINUS 429

TABLE 3. Enzymatic features in which the 31 Xylophilr~sumpelinus strains differ from each other

No' Of Result for strain Hydrolysis of strains Strains that gave the less common result NCPPB 2217T positive" L-Serine-N A 30 + CNBP 1926t1 L-Phenylalanine-N A 30 + CNBP 1841 L-Leucyl-glycine-N A 30 + Ride AA2 L-Ornithine-NA 29 + CNBP 1926t1, CNBP 2098 L-Pro1 yl-L-arginine-N A 29 + CNBP 2098, CNBP 2059 2-Naphthyl-nonanoate 29 + CNBP 1837t1, CNBP 2059 L-Histid yl-L-phenylalanine-N A 28 + CNBP 1803, CNBP 1834, Ride P7 L-Phen ylalan yl-L-proline-N A 4 + NCPPB 2217T, CNBP 1834. Ride P7, CNBP 1192T L-Lysyl-L-l ysine-N A 3 - NCPPB 2219, NCPPB 2220, CNBP 1837t1 Naphthol-AS-BI-phosphodiamideh 3 - Ride C13, Ride C2', CNBP 2098 L-Valine-N A 1 - NCPPB 2219 y-L-Glutamic acid-NA 1 - CNBP 1928 a+-Aspart yl-L-alanine-N A 1 - PDDCC 4299 L-Alanyl-L-phenylalanine-L-prolyl-L-alanine-NA 1 - Ride C2' (' For hydrolysis of the following substrates. various results were obtained (the results obtained with the type strain are given in parentheses): naphthylphosphate at pH 8.5 (+), 2-naphthyl-caprate (+), L-pyrrolidonyl-NA (+), S-benzyl-L-cysteine-NA ( + ), L-arginyl-L-arginine-NA (+ ), L-lysyl-L-alanine- NA (+), L-phenylalanine-L-prolyl-L-alanine-NA(-), and glycyl-L-tryptophan-NA (variable). ' 6-Bromo-2-phosphodiamide-3-naphthoicacid-2-met hoxyanilide. naphthyl-butyrate, 2-naphthyl-caprylate, 2-naphthyl- G+C content of the DNA is 68 to 69 mol%. The type strain valerate, 2-naphthyl-caproate, 2-naphthyl-phosphate (at pH is NCPPB 2217 (= LMG 5856 = PDDCC 4298 = CNBP 1192 5.4), L-leucine-NA, L-tyrosine-NA, L-lysine-NA, glycine- [= LMG 59491 = ATCC 33914), which was isolated from NA, L-aspartic acid-NA, L-arginine-NA, L-alanine-NA, DL- Cretan vines in 1966 (26). Its characteristics are the same as methionine-NA, L-tryptophan-NA, L-glutamine-NA hydro- those listed for the species. The G+C content of its DNA is chloride, a-L-glutamic acid-NA, glycyl-glycine-NA 68 mol%. hydrobromide, glycyl-L-phenylalanine-NA, L-seryl-L-tyrosine-NA, L-alanyl-L-arginine-NA, glycyl-L-alanine-NA, glycyl-L-arginine-NA, L-leucyl-L-alanine-NA, L-phenyl- ACKNOWLEDGMENTS alanyl-L-arginine-NA, and L-seryl-L-methionine-NA. None J.D.L. is indebted to the Fonds voor Geneeskundig Wetenschap- of the Xylophilus ampelinus strains hydrolyzes the following pelijk Onderzoek for research and personnel grants, and K.K. is substrates in API systems: 2-naphthyl-a-~-glucopyranoside, indebted to the Nationaal Fonds voor Wetenschappelijk Onderzoek 2-naphthyl-P-~-galactopyranoside,2-naphthyl-a-~-fuco- for a research grant. A.W. is indebted to the Instituut tot pyranoside, 6-Br-2-naphthyl-a-~-galactopyranoside,6-Br-2- Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid naph t h y 1- P- D-glucop y ranosi de , 6-B r-2- naph t h y 1-a- D- en Landbouw for a scholarship. mannopyranoside, 1-naphthyl-N-acetyl-p-D-glucosaminide, We are grateful to API Systems S.A. for supplying galleries for the enzyme tests. We thank C. G. Panagopoulos, J. Veremans, and naphthol-AS-BI-P-D-glucuronic acid (6-bromo-2-hydroxy-3- M. Ride for valuable discussion on selecting a new genus name. naphthoic acid-2-methoxyanilide-~-~-glucuronate),2- naphthyl-myristate, 2-naphthyl-laurate, 2-naphthyl-palm- LITERATURE CITED itate, 2-naphthyl-stearate, L-cystine-NA, P-alanine-NA, L- 1. Bradbury, J. F. 1973. Xanthomonas ampelina. Commonwealth histidine-NA, L-hydroxyproline-NA, L-isoleucine-NA, L- Mycological Institute descriptions of pathogenic fungi and bac- threonine-NA, L-proline-NA hydrochloride, N-benzoyl- teria no. 378. Commonwealth Mycological Institute, Kew, En- L-leucine-NA, N-benzoyl-DL-arginine-NA, N-benzoyl-L- gland. a1 anine-4-met hox y -NA, N-car bobenz y lox y - L-argi nine-4- 2. Bradbury, J. F. 1984. Genus 11. Xanthornonas Dowson 1939, p. methoxy-NA, N-glutaryl-DL-phenylalanine-NA, glycyl-L- 199-210. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual proline-NA, aspartyl-L-arginine-NA, a-L-glutamyl-a-L- of systematic bacteriology, vol. 1. 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