Pseudomonas Pseudoalcaligenes Subsp. Citruzzi Subsp. Nov
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OO20-7713/78/OO28-0 I 17$02.OO/O INTERNATIONALJOLJRNAL OF SYSTEMATIC BACTERIOLOGY,Jan. 1978, p. 117-125 Vol. 28, No. I Copyright 0 1978 International Association of Microbiological Societies Printed in U.S. A. Pseudomonas pseudoalcaligenes subsp. citruZZi subsp. nov. N. W. SCHAAD,' G. SOWELL, JR.,' R. W. GOTH,3 R. R. COLWELL? AND R. E. WEBB3 Department of Plant Pathology, University of Georgia, Athens, Georgia .30602l; and Agricultural Research Seruice, Georgia Experiment Station, Experiment, Georgia 30212; Agricultural Research Seruice, P G G I, Beltsuille, Maryland 29705'; and Department of Microbiology, University of Maryland, College Park, Maryland 20747L Ten nonfluorescent Pseudomonas strains isolated from water-soaked lesions on cotyledons of plants of five Citrullus lanatus (watermelon) plant introductions were characterized and compared phenotypically with 22 other pseudomonads. The strains were distinguished phenotypically from other known plant pathogenic pseudomonads. The watermelon bacterium was aerobic. Cells were rod-shaped, gram negative, and motile by means of a single polar flagellum. They were nonfluorescent and grew at 41°C but not at 4°C. Oxidase production and the 2- ketogluconate reaction were positive. The 10 strains utilized p-alanine, rA-leucine, D-serine, n-propanal, ethanol, ethanolamine, citrate, and fructose for growth. No growth occurred with sucrose or glucose. Their deoxyribonucleic acid base com- position was 66 1mol% guanine plus cytosine. The bacterium is phenotypically similar to P. pseudoalcaligenes but differs from it in being pathogenic to water- melon, Cucumis melo (cantaloupe), Cucumis sativus (cucumber), and Cucurbita pep0 (squash). The name P. pseudoalcaligenes subsp. citrulli is proposed for the new subspecies, of which strain C-42 (= ATCC 29625) is the type strain. For several years a bacterial disease of water- yledons and the first true leaves showing greasy, dark melons, characteAed by water-soaked lesions green lesions were collected, and the surfaces were of cotyledons, has been observed on certain plant disinfested in a freshly prepared 0.5% solution of so- introductions at the Regional Plant Introduction dium hypochlorite for 3 min. After rinsing for 2 min Station, Experiment, Ga. A disease with similar in sterile distilled water, the tissue from the lesions symptoms on watermelon seedlings was re- was removed and cut into smaller pieces in 0.1 ml of sterile distilled water, using a sterile scalpel. The sus- ported to be caused by Pseudomonas lachry- pension was streaked onto King medium B (KB; 11) mans (9), but we could not isolate a pathogenic and incubated for 5 days at 27°C (colonies of nonpath- fluorescent pseudomonad. Our preliminary in- ogenic pseudomonads were usually present after 2 vestigations showed that the causal bacterium days). Colonies of the watermelon bacterium were was similar to the unidentified nonfluorescent removed from the agar with a transfer loop and puri- pseudomonad isolated by Webb and Goth (22) fied by streaking onto plates of KB. Stock cultures from plants of watermelon plant introductions were maintained on KB slants. The media used for 174103 and 174104. The purpose of this investi- determining cultural characteristics were KB, yeast gation was to identify the causal bacterium iso- extract-dextrose-CaC03 (22),and Difco nutrient agar. All media were sterilized for 20 min at 121°C and at lated from diseased watermelon seedlings and a presure of 1.1 kg/cm2. to compare it with several other fluorescent and Morphological properties. Cell morphology was nonfluorescent pseudomonads, including the recorded from light microscope observations of gram- strain of P. lachrymans isolated from waterme- stained smear preparations and from electron micro- lon (9). scope observations. Flagella were observed by light microscopy by the method of Blenden and Goldberg MATERIALS AND METHODS (1) and, using shadowed preparations, by electron Bacterial strains. Ten strains of the nonfluores- microscopy. cent pseudomonad isolated from water-soaked lesions Physiological and biochemical properties. The of watermelon seedlings were studied. Strains C-43, methods of Stanier et al. (20) were used to test for C-44, C-58, (3-95,C-96, C-97, C-98, C-99,and C-130 the following characters: starch hydrolysis, oxide,2- originated from plant introductions 174103, 181744, ketogluconate production, gelatin liquefaction, deni- 164475,279462,164475,164748,164748,164475,164475,trification, and poly-P-hydroxybutyrate production. and 164475, respectively. Other pseudomonads in- For carbon source utilization, the methods of Palleroni cluded irl the study as reference strains are listed in and Doudoroff were employed (16).Arginine dihydro- Table 1. lase activity was determined by the method of Misaghi Isolation and cultural properties. Seeds from and Grogan (15). Hypersensitivity on tobacco was different watermelon plant introductions were sown determined by injecting leaves of Nicotina glutinosa in trays (30 by 50 cm) of soil in a greenhouse with a with a bacterial suspension containing approximately day/night .ternpertwe of approximately 30/21 "C. Cot- 108 cells/ml as described by Klement et al. (13). All 117 118 SCHAAD ET AL. INT. J. SYST.BACTERIOL. TABLE1. Pseudomonas strains included in this study as reference strains Received as: Iaboratory no. Sourcen Habitat Name Strain - c-7 Pseudomonas syringae B-3 1 Peach C-41 P. lachrymans 64-3 2 Watermelon c-49 P. lachrymans ATCC 1192 1 3 Squash c-11 P. avenae ATCC 119860 3 Corn c-14 P. eriobotryae 4083 4 Loquat C-15 P. panci ATCC19875 3 C-34 P. solanacearum K-60- 1 5 Tomato c-37 P. solanacearum K- 132 5 Pepper C-90 P. andropogonis 934 6 c-3 P. marginalis ATCC10844 7 Endive c-2 P. maculicola ATCCll781 7 (2-133 P. rnarginata ATCC10247 3 Gladiolus c-91 P. aeruginosa D-5759 8 C-94 P. fluorescens KC-678 8 c-93 P. putida KC- 1074 8 C-87 P. caryophyli B- 1 9 Carnation c -4 P. cichorii a-3 7 Cabbage C-92 P. cepacia KC- 1372 8 C-88 P. maltophilia NCH-5015 6 C- 132 P. setariae ATCC19882 3 Oryra sativa c-134 P. stizolobii ATCC 19309 3 Stizolo bium C-145 P. alcaligenes ATCC 14909 3 Water C-146 P. pseudoalcaligenes ATCC17440 3 Sinus drainage a 1, H. English, [Jniversity of California, Davis; 2, D. Hopkins, University of Florida, Leesburg; 3, American Type Culture Collection, Rockville, Md.; 4, M. hi. State Department of Agriculture, Sacramento, Calif.; 5, A. Kelman, University of Wisconsin, Madison; 6, A. Vidaver, Lincoln, Neb.; 7, D. Sumner, University of Georgia, Tifton; 8. R. Weaver, Center for Disease Control, Atlanta, Ga.; 9, R. Dickey, Cornell University, Ithaca, N.Y. other tests were performed as described previously fixative (60% ethanol, 30% chloroform, 10% Formalin), (9). Tween 80 and 1-propanol were obtained from J. stained with the globulin, and counter-stained with a T. Baker Co.; ethanolamine was from Calbiochem; fluorescent anti-rabbit globulin (GIBCO Diagnostics, and N,N-dimethyl-p-phenylenediaminewas from Grand Island, New York), as described by Goldman Eastman Kodak Co. 2-Ketoglutamate, m-P-hydroxy- (8). After rinsing in phosphate-buffered saline (pH butyric acid, m-lactic acid, y-aminobutyrate, and p- 8.0) and distilled water, the smears were air-dried and D-(-)-fhctose were purchased from Sigma Chemical mounted to 0.5 M carbonate-buffered (pH 9.0) glycerin co. (21). They were observed with a Zeiss fluorescent G+C content of DNA. The guanine-plus-cytosine microscope (X40objective) equipped with BG 12 ex- (G+C) contents of purified deoxyribonucleic acids citation and 50-barrier filters. The intensity of the (DNAs) extracted from selected strains in this study fluorescence was classified in the range - to +4, with were determined by thermal denaturation techniques - representing no visible fluorescence and +4 indicat- (5, 14). ing intense fluorescence. Serological study. Strain (2-42 antiserum was pro- Pathogenicity. For pathogenicity tests, cultures duced in New Zealand White rabbits. Cells of strain grown in medium 523.(10) were adjusted to 50 Klett C-42 from 48-h-old KB slant cultures were suspended units (green filter) and diluted with sterile distilled in 0.85%NaCl (saline)and adjusted to 100 (f10) Klett water to provide suspensions containing the desired units (Klett-Summerson colorimeter). Formalin was number of viable cells per milliliter. The number of added to a final concentration of 296, and 1 ml of the viable cells per milliliter was determined by plating suspension was emulsified with 1 ml of Difco incom- 0.1 ml of a dilution onto five plates of KB. For plete adjuvant, using a high-speed stirrer. Rabbits spray and brush inoculations, a cell suspension was were given four intramuscular injections at 10-day atomized by employing 0.7-kg/cm2 pressure or was intervals. The animals were bled 7 days after the last brushed with a sterilized camel hair brush, respec- injection, and the sera were stored at -20°C. The tively. For stem inoculations, approximately 0.1 m'l of immunoglobulin was prepared from the sera by col- inoculum was injected into the stem with a syringe umn chromatography on diethylaminoethyl-Sephadex fitted with a 25-gauge needle. All plants were in the A-50 columns (6). Tube agglutination tests, using so- first to second true-leaf stage of growth. The plants matic antigens, were performed by the method of were allowed to stand overnight in a dew chamber at Carpenter (4). Smears for fluorescent-antibody stain- 27"C, after which they were placed in a Percival ing were made from a suspension of cells in saline. (Percival Manufacturing Co., Boone, Iowa) PGW en- The smears were made on multiwell slides (Cell-Line vironmental chamber adjusted to 27 * l0C, 70 * 2% AKwciates, Inc., Minotola, N.J.) with a 0.001-ml loop. relative humidity, and 100 hlx light intensity. Symp- When dry. the smears were fued with Kukpatrick toms were recorded after incubation for 5 and 7 days.