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. 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. VOL. 28, 1978 P. PSEUDOALCALIGENES SUBSP. CITRULLI SUBSP. NOV. 119 The following plant cultivars were used in the path- ogenicity experiments: Citrullus lanatus (waterme- lon) “Charleston Gray” and “Florida Giant,” Cucumis melo (cantaloupe) “Edisto,” Cucumis satiuus (cucum- ber) “Marketer,”Cucurbita pep0 (squash) “Early Yel- low Summer Crookneck,” Vigna unguiculata (cow- peas) “Early Ramshorn,” Lycopersicum esculentum (tomato) “Rutgers,” Zea mays (corn) “Pioneer 3030,” Dianthus caryophyllus (carnation) “Improved White Sim,” and Setaria italica (foxtail millet, plant intro- duction 196293). Plants inoculated with sterile distilled water served as controls. Carnation plants, obtained from Yoder Brothers Nursery, Barberton, Ohio, were inoculated at an internode by using a needle containing cells of a 48-h-old slant culture (7). To determine the pathogenicity of other pseudomonads, watermelon seedlings were inoculated with suspensions of containing approximately lo6 and l@ viable cells per ml. Thirteen plants were inoculated with each suspen- sion, i.e., five for leaf inoculation and eight for stem inoculation. RESULTS Morphology. Cells of the bacterium isolated from watermelon were gram-negative rods with average dimensions of 0.5 by 1.7 pm, as deter- mined by light and electron microscopy. The cells had a single polar flagellum about 5.0 pm long (Fig. 1). FIG. 1. Electron micrograph of Pseudomonas Cultural characteristics. Growth on KB pseudoalcaligenes subsp. citrulli strain C-42. was slow and colonies were rarely visible before Shadowed preparation. xl1,oOO. 2 days. Colonies were nonfluorescent, round, transparent, smooth, slightly convex, and 2 to 3 pseudoalcaligenes C-146 were 10, 10,20,40, and mm in diameter after incubation for 5 days at 40, respectively. Titers of P. syringae C-7, P. 30°C. Colonies on yeast extract-dextrose-CaCOa aeruginosa C-91, P. fluorescens C-94, P. solan- were round, smooth, convex, tan, and 3 to 4 mm acearum C-34 and C-37, P. cepacia (2-92, P. in diameter after incubation for 5 days at 30°C. putida C-93, P. marginata C-133, and P. cary- On nutrient agar containing glucose, growth oc- ophylli C-87 were less than 5. Fluorescent-anti- curred only where masses of cells were deposited body stains were +2 or greater with the water- by streaking. melon strains. Except for a very slight f reaction Physiological and biochemical proper- with P. auenae C-11 and P. stizolobii C-134, ties. The characteristics of the 10 watermelon the other pseudomonads examined showed no strains are summarized in Tables 2 and 3. From visible fluorescence (-). the results obtained in this study, the waterme- Pathogenicity. Stems of watermelon seed- lon bacterium was easily differentiated from lings became water-soaked 3 days after being Pseudomonas syringae, P. lachrymans, and injected with a suspension containing 2 x lo6 several nonfluorescent, plant-pathogenic pseu- viable cells of the watermelon bacterium per ml. domonads (Table 2). However, it appeared to The water soaking often extended upwards into be similar to P. alcaligenes and P. pseudoalca- the cotyledons, true leaves, and apical meristem. ligenes (Table 3). At 5 days, stem collapse was observed (Fig. 2). DNA base composition. The overall DNA Seedlings inoculated with sterile water were base compositions of the strains examined were symptomless. Three other pyudomonads, P. ci- in the range of 65 to 67 mol% G+C. Thus, the chorii C-4, P. maculicola C-2, and P. aerugi- DNA base composition of these strains is re- nosa C-91, at concentrations of 4.3 X 108, 1.6 X ported as 66 f 1 mol% G+C. 108, and 4.2 X 108 cells per ml, respectively, Tube agglutination and fluorescent-an- produced water-soaked spots when brushed onto tibody staining. Agglutination titers of water- cotyledons and true leaves. However, when melon strains C-95, C-96, C-42, C-98, C-99, and fewer than lo7 cells per ml was used, none of C-130 were 20,20,40,40,80, and 80,respectively. the other pseudomonads, including those cited Titers of P. auenae C-11, P. alcaligenes C-145, above, caused symptoms. Pathogenicity results P. stizolobii C-134, P. setariae C-132, and P. with the watermelon strains were variable at TABLE 2. Characteristics that differentiate the watermelon pseudomonad from several other plant- pathogenic pseudomonads "

~ Melon P. au- P. lachv- pseudo- Characteristic mans(n= enae I monad 2) (n=6) (n= 10) _._- -. Fluorescent-pigment production - - Nitrate reduction + - Tobacco hypersensi- tivity - - Lipid hydrolysis + + Litmus milk Alk Alk,(P) Growth at 41°C + + Gelatin hydrolysis v,1 (+) Oxidase production + + Utilization of D-Serine (+) + P-Alanine + + L-Leucine - (+) n-Propanol - + Ethanol - + Ethanolamine - + Citrate + + FiUCtose + + D-M~~~oM (+) - Sucrose - - Glucose + - "Symbols and abbreviations: n, number of strains tested; +, test was positive; -, test was negative; ( 1, weak or no response after 7 days, positive after 21 days; Alk, alkaline; C, coagulated (curd); N, neutral; ND, not determined; P, peptonized; V, variable, numerals indicate number of strains positive. lower concentrations when cotyledons were in- An independent test with strains C-42, C-58, oculated (Table 4). Nevertheless, results ob- and P. solanacearum C-39 and (2-37 was run tained with stem inoculation were consistent. by S. M. McCarter, Athens, Ga., who obtained AH melon strains were positive, even at lower similar results. The watermelon strains were concentrations (Table 4). None of the other found to cause only a slight internal discolora- pseudomonads caused stem infections regardless tion of the cortex, whereas P. solanacearum of cell concentration (Table 4). caused a wilting and collapse of the plant. Results obtained by injecting stems of eight Characteristics of the new strains. The different species of plants with cell suspensions phenotypic characteristics of the 10 new strains containing 106 viable cells of P. auenae C-1 1, P. are listed in Table 6. A total of 40 characters solanacearum C-34, P. lachrymans C-41 and was common to all strains (either all positive or C-49, or the watermelon bacterium (strains C- all negative), and there were two characteristics 42, C-43, C-44, (2-58, C-95, C-96, and (2-98) per in which one or more of the strains differed. ml are shown in Table 5. Symptoms produced by the eight watermelon strains were indistin- DISCUSSION guishable on cantaloupe, cucumber, squash, and The morphological and physiological charac- watermelon. Water-soaking and discoloration of teristics of the bacterium associated with the the stems were evident 4 days after inoculation. disease of watermelon seedlings indicate that After 10 days, the lesions were 4 to 5 cm long the bacterium can be classified as a member of and often extended into the cotyledons and ap- the family . The bacterium ical meristem. These symptoms were similar to is a gram-negative, aerobic, motile (by means those described by Webb and Goth (22). The of a single polar flagellum) rod possessing a cortex of stems of tomato plants was discolored DNA content of 66 mol% G+C. The 7th edition several centimeters above and below the inoc- of Bergey's Manual (2) lists 62 nomen species ulation site when inoculated with strains C-42 of plant-pathogenic pseudomonads. Only one and C-58, but no external water-soaking or wilt- species, Pseudomonas lachrymans, has been ing typical of that caused by P. solanacearum shown to infect plants in the family Cucurhita- was observed. ceae, and none is listed as being pathogenic to VOL. 28, 1978 P. PSEUDOALCALIGENES SUBSP. CITRULLI SUBSP. NOV. 121 watermelons. In the 8th edition of Bergey's and oxidase reactions, and a negative hypersen- Manual (3), all phytopathogenic pseudomonads sitive reaction; most strains reduced nitrate and have been lumped into eight nomen species as grew at 37°C. Group 111 contained nonpig- follows: P. syringae, P. cichorii, P. caryophylli, mented, nonfluorescent, oxidase-negative strains P. cepacia, P. solanacearum, P. marginata, P. that contained poly-P-hydroxybutyrate gran- aeruginosa ( P. polycolor), and P. fluorescens ules; the group I11 strains grew slowly at 27°C biotype B (P.marginalis). but fast at 37°C. Group IV comprised the yellow- Sands et al. (18) divided the plant-pathogenic pigmented, nonfluorescent, oxidase-positive pseudomonads into the following four groups on strains with poly-/3-hydroxybutyrate granules; the basis of nutritional and physiological char- the group IV strains were fast-growing at 27°C acteristics. Group I comprised strains demon- and slow-growing at 37°C. strating a green fluorescent pigment, a hypersen- On the basis of the characteristics given by sitive reaction on tobacco, but negative arginine Sands et al. (18), the watermelon bacterium dihydrolase and oxidase tests; group I strains described here fits most closely into group 111, did not reduce nitrates or grow at 37°C. Group which includes P. ru brilineans, P. ru brisu bal- I1 consisted of strains demonstrating green flu- bicans, P. setariae, P. caryophylli, and P. solan- orescent pigments, positive arginine dihydrolase acearum. However, the watermelon bacterium

TARLE3. Phenotypic characters of the watermelon pseudomonad, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes" Watermelon bacte Characteristic P. pseudoalcali- P. alcaligenesb rium (n= 10) genesb -~ ~ ~ . ~ - ~ ~ ~ ____ Mol% G+C ...... 65-67 62-64 66-68 Flagella ...... Single polar Single polar Single polar Fluorescent-pigment production ...... - Oxidase production ...... + + + Lipase (Tween 80) ...... + - V 2-Ketogluconate production ...... + V Utilization of: P-Alanine ...... + Citrate ...... - Fructose ...... n-Propand ...... Ethanol ...... Ethanolamine ...... Lactate ...... y- Aminobutyrate ...... Glucose ...... Inositol ...... Sucrose ...... Cellobiose ...... Arabinose ...... D-Mannose ...... Maltose ...... Rhamnose ...... Gelatin liquefaction ...... f f Growth at 41°C ...... + + Growth at4"C ...... - d Hypersensitivity" ...... - Nitrate reduction ...... V Arginine dihydrolase ...... V Poly-/?-hydroxybutyrate production ...... v Starch hydrolysis ...... - Growth on: Salmonella-shigella agar ...... ND ND HEagar ...... - ND ND MacConkey agar ...... + ND ND

a Symbols and abbreviations: n, number of strains tested; +, all strains positive; -, all strains negative; f, sli ht or delayed; ND, not determined; V, variable reaction depending upon the strain. 'Data from Ralston-Barrett et al. (17). Determined on tobacco (13). This paper. 122 SCHAAD ET AL. INT. J. SYST.BACTEHIOL. was oxidase positive. Of the above-mentioned on the biochemical characteristics evaluated in species, the watermelon bacterium appeared to ths study, the watermelon bacterium also ap- be most closely related to P. caryophylli. How- peared to be similar to P. avenae (19). In our ever, the latter bacterium is polarly multi- opinion, P. avenue reduces nitrate, utilizes glu- tichous, produces a yellow-green, nonfluores- cose and mannose but not L-leucine, n-propanol, cent, diffusible pigment, utilizes glucose and su- ethanol, or ethanolamine, and does not infect crose, and does not infect watermelon. Based any of the cucurbits tested. Finally, the DNA of P. avenae is in the range 70 to 75 mol% G+C (18), whereas the DNA of the watermelon bac- terium is 66 f 1 mol% G+C. The description of the organism from water- melon failed to correlate with that of any previ- ously described plant-pathogenic species. The ability of the watermelon bacterium to cause an infection of stems and leaves after injection of suspensions containing fewer than lo6 viable cells per ml into the stems of healthy waterme- Ion seedlings is strong evidence that it is patho- genic. On the other hand, the interaction be- tween P. cichorii, P. aeruginosa, and P. macu- Licola at cell concentrations greater than 108/ml FIG. 2. Watermelon seedlings 7 days after inoc- ulation by injection with 0.1 ml of a suspension of and young leaves of watermelon fits the descrip- sterile distilled water (left) and 2 x I@ viable cells tion of a nonpathogen (hypersensitive reaction) of P. pseudoalcalgenes subsp. citrulli strain C-42 host combination (12). Its description most (right)per ml. closely approximates that by Stanier et al. (20)

TABLE4. Reaction of watermelon seedlings to inoculations with the watermelon pseudomonad and several other pseudomonads No. of infected leavesh of water- Inoculum (viable melon seedlings inoculated by: Strain Organism cells/ml") Leaf brushing' Stem injection" . Pseudomonaa svringue B-3 1.2 x 10'' 0 0 P. lachrymans 11921 1 3.2 x 10; 0 0 P. lachrymans 64-3 8.3 x 10" 0 0 P. solanacearum K-fjg 2.1 x 10'' 0 0 P. margina Iis 10844 I.i x 10'; 0 0 P. maculicola 11781 1.6 x 10'' 0 0 P. aerugin.osa 11-5759 4.2 x l@' 0 0 P. fluorewens KC 678 1.2 x loti 0 0 P. putida KC 1074 5.0 x 10" 0 0 Y.caryophylli B- 1 6.3 x 10" 0 0 P. cepacia KC 1372 3.4 x 10'' 0 0 P. maltophilia 5015 2.5 x 10; 0 0 P. setariae 19882 2.5 x loh 0 0 P. stizolobii 19309 4.7 x 1oh 0 0 P. avenae c-11 2.1 x 10" 0 0 P. ulcaligenes 14908 1.2 x 10'' 0 0 P. pseudoalcalqenes 17440 2.6 x lo6 0 0 Watermelon pseudomonad c-42 1.6 x 10" 0 7 Watermelon pseudomonad c-96 1.5 X 10' 3 7 Watermelon pseudomonad c-99 1.0 x 10" :1 8 Watermelon pseudomonad c-97 3.0 x 10;' 2 a

" Actual number determined by plating 0.1 ml of a 10-" dilution of a 50 Klett suspension onto five plates of KR. " Symptoms recorded after plants were inoculated, placed overnight in a dew chamber at '27°C. and incubated 6 da.vs in a Percival PCW environmental chamber at 2i"C, 704 relative humidity, and 100-hlx light intensity. ' Inoculum brushed onto two leaves of each of five plants, using a sterile camel hair brush. ,' A (1. I-ml volume of inoculum was injected into the stem of each of eight plants, using a syringe and 25- gauge needle. VOL. 28, 1978 P. PSEUDOALCALIGENES SUBSP. CITRULLI SUBSP. NOV. 123

TABLE5. Susceptibility of eight plant species to the watermelon pseudomonad and selected plant pathogenic Pseudomonas species” Susceptibility of - - - .. - - 2. Water- Canta- Squash Cucum- Tomato Cow- Carna- Organism melon loupe ber (Lyco- pea Corn ( Citrui- ( ~ucu- (~ucu- persicurn ( Vigna (zea tested lus mis bitu rnis escu- ungui- mays) lhus ianatus) meio) Pep0) satruus) lenturn) culator) caryo- phyllus) - _. - .------P. caryophylli 1 -h ------+ P. auenae 1 ------+ NT P. solanacearum 1 - - - - + - - NT P. lachrymans 2 ------NT Watermelon pseudo- monad 8 + + + + -< - - - “Stems of plants (first to second true-leaf stage of growth) were inoculated with a bacterial suspension containing approximately lo6 viable cells per ml. Four plants were inoculated with each bacterial species. Carnation plants were inoculated by needle with cells from a 48-h-old slant culture (7). ’Symbols and abbreviation: +, susceptible; -, not susceptible; NT, not tested. ‘Strains C-42 and C-58caused dark streaks in the pith; no external symptoms were observed. for the “alcaligenes group,” which consists of P. 17440 with respect to the utilization of glucose, alcaligenes and P. pseudoalcaligenes. These sucrose, arabinose, mannose, fructose, n-pro- species were distinctive in their inability to uti- panol, ethanol, ethanolamine, lactate, aminobu- lize sugars (except for fructose) as carbon tyrate and growth at 4 and 41°C are in agree- sources. Furthermore, they were described as ment with those of Ralston-Barrett et d. (17). being monotrichous, growing at 41°C but not at Because of its somewhat wider nutritional ver- 4”C, and demonstrating a restricted nutritional satility (utilization of n-propanol, ethanol, etha- spectrum. Stanier et al. (20) separated the two nolamine, and fructose), the watermelon bacte- species on the somewhat greater nutritional ver- rium is concluded to be more similar to P. pseu- satility and DNA base composition of P. pseu- doalcaligenes than to P. alcaligenes. However, doalcaligenes. RaLston-Barrett et al. (17) char- the watermelon bacterium differed from P.pseu- acterized 18 strains of bacteria assigned to the doalcaligenes in two important characteristics: “Pseudomonas alcaligenes” group by pheno- cells of P.pseudoalcaligenes failed to stain with typic studies and DNA hybridization data and the fluorescent-antibody stain of the watermelon found that all strains examined were accommo- bacterium or to infect watermelon. Although dated within the two species, with a close rela- the latter could have been due to a loss of tionship to P. mendocina and P. stutzeri and a pathogenicity during storage, it appears more distant relationship to the fluorescent pseudo- judicious to class@ the organism as a new sub- monads. The following phenotypic characters of species of P. pseudoalcaligenes. The name P. P. alcaligenes and P. pseudoalcaligenes were pseudoalcaligenes subsp. citrulli subsp. nov. given as useful in distinguishing them from other (ci’trul,li. M.L. mas.n. Citrullus generic name of Pseudomonas species: motile by means of a watermelon; M.L. mas.gen.n. citrulli of the wa- single polar flagellum, soluble pigments not pro- termelon) is proposed. Strain C-42 is designated duced, positive oxidase reaction, organic growth as the type strain of this new subspecies, and a factors not required, no hydrolysis of starch, no culture of it has been deposited in the American autotrophic growth with the oxidation of HPas Type Culture Collection (ATCC), Rockville, energy source, growth at 41°C but not at 4”C, Md., under the number 29625. A condensed de- and utilization of y-aminobutyrate, caprate, and scription of this subspecies follows (additional lactate but not sucrose, glucose, cellobiose, mal- characteristics are listed in Tables 3 and 6): onate, sebacate, glycolate, L-isoleucine, and L- asparate. The only consistent phenotypic differ- Pseudomonas pseudoalcaligenes subsp. ci- ence between strains of P. pseudoalcaligenes trulli subsp. nov. and P. alcaligenes was utilization of fructose Gram-negative rods, 0.5 by 1.7 pm, which are by P. pseudoalcaligenes. motile by means of a single polar flagellum 5.0 The results of this study of P. alcaligenes pm in length. ATCC 14908 and P. pseudoalcaligenes ATCC Colonies on KB are round, transparent, 124 SCHAAD ET AL. INT. J. SYST.BACTEHIOL.

TABLE6. Phenotypic characteristics of Pathogenicity: Infects plants of the family Cu- Pseudomonas pseudoalcaligenes subsp. citrulli curbitaceae. subsp. nor,!. DNA base composition: 66 f 1 mol% G+C.

All strains (10) positive ACKNOWLEDGMENTS Hydrolysis of lipid We thank R. C. Donaldson and C. A. Walker, Jr., for Alkaline reaction in litmus [Jtilization of ethanolamine technical assistance, S. S. Hearon for preparation of the elec- milk tromicrograph, S. M.McCarter for running the pathogenicity Growth at 41" C Utilization of citrate tests with tomatoes, and R. E. Weaver for verification of the Liquefaction of gelatin Iftilization of fructose characteristics of Pseudomonas pseudoalcaligenes subsp. ci- Cytochrome oxidaw produc- Utilization of lactate trulli subsp. nov. strain ATCC 29625. tion Utilization of u-serine Utilization of y-aminobutyr- REPRINT REQUESTS ate Address reprint requests to: N. W. Schaad, Department of Utilization of 8-alanine Production of Z-ketoglucon- ate Plant Pathology, Georgia Experiment Station, Experiment, GA 30212. Utilization of 1.-leucine Growth on MacConkey agar Utilization of n-propanol lnfection of watermelon LITERATURE CITED Infection of cantaloupe Utilization of ethanol Infection of squash 1. Blenden, P. C., and H. S. Goldberg. 1965. Silver im- Infection of cucumber pregnation stain for Leptospira flagella. J. Bacteriol. _. 89:899-900. 2. Breed, R. S., E. G. D. Murray, and N. R. Smith (ed.). A11 strains (10) negative 1957. Bergey's manual of determinative bacteriology, Fluorescence on KB Utilization of glucose 7th ed. The Williams & Wilkins Co., Baltimore. Nitrate reduction Utilization of inositol 3. Buchanan, R. E., and N. E. Gibbons (4.).1974. Ber- Tobacco hypersensitivity Utilization of sucrose gey's manual of determinative bacteriology, 8th ed. The Growth at 4" C Utilization of cellobiose Williams & Wilkins Co., Baltimore. Arginine dihydrolase Utilization of arabinose 4. Carpenter, P. L. 1975. Immunology and serology, 3rd Starch hydrolysis Utilization of D-mannose ed. W. G. Saunders Co., Philadelphia. Growth on salmonella-shi- Utilization of maltose 5. Citarella, R. V., and R. R. Colwell. 1970. Polyphasic gella agar of the genus Vibrio: polynucleotide sequence Growth on HE agar [Jtilization of rhamnose relationships among selected Vibrio species. J. Bacte- No infection of cowpea No infection of corn rial. 104:434-442. No infection of camation 6. Dedman, R E., A. W. Holmes, and F. Dienhardt. - 1W.Preparation of fluorescein isothiocyanate-labeled Refer- globulin by dialysis, gel filtration, and ion-exchange ence chromatography. J. Bacteriol. 89:734-738. No . Results no. of 7. Dickey, R. S., and P. E. Nelson. 1970. Pseudomonas of of strains Strain caryophylli in carnation. IV. Unidentified bacteria iso- difference strains type that lated from carnation. Phytopathology 60:647-653. Dosi- strain .,gave 8. Coldman, M. 1968. Fluorescent antibody methods, p. tive (C-42) less 157-158. Academic Press Inc., New York. common 9. Hopkins, D. L., and N. C. Schenck. 1972. Bacterial leaf result - spot of watermelon caused by Pseudomonas lachry- Production of poly-8-hy- 4/10 - c-58, c-95, mans. Phytopathology 62:542-515. droxybu trate c-98, c-130 10. Kado, C. I., and M. G. Heskett. 1970. Selective media Production of dark 2/10 + C-42,C-58 for isolation of Agrobacterium, Corynebarteriurn, Er- streaks in tomato pith winia, Pseudomonas, and Xanthornonas. Phytopath- 010gy 60:969-976. 11. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two smooth, slightly convex, and 2 to 3 mm in di- simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44901-307. ameter after 5 days at 30°C. Colonies on yeast 12. Klemenf Z. 1968. Pathogenicity factors in regards to extract-dextrose-CaCOs are 3 to 4 mm and tan relationships of phytopathogenic bacteria. Phytopath- in color. 0100 58: 1218-1221. Pigment production: None. 13. Klement, Z., C. L. Karkas, and L. Lourekovich. 1964. Hypersensitive reaction induced by phytopathogenic Strictly aerobic. bacteria in the tobacco leaf. Phytopathology. Nitrates are not reduced. 54 :474-477. Temperature relationships: Grows at 41°C but 14. Marmu, J., and P. Doty. 1962. Determination of the not at 4°C. base composition of deoxyribonucleic acid from its ther- mal denaturation temperature. J. Mol. Biol. 5: 109-1 18. Oxidase production and the 2-ketogluconate 15. Misaghi, I., and R. G. Grogan. 1969. Nutritional and reaction are positive. biochemical comparisons of plant-pathogenic and sap- Utilizes p-alanine, L-leucine, D-Whe, n-pro- rophytic fluorescent pseudomonads. Phytopathology panol, ethanol, ethanolamine, citrate, and fruc- 59: 1436-1450. 16. Palleroni, N. J., and M. Doudoroff. 1972. Some prop- tose as sole carbon sources for growth. No erties and taxonomic subdivisions of the genus Pseu- growth occurs with sucrose or glucose. domonas, p. 77-78. In K. F. Baker (ed.), Annual review Tobacco hypersensitivity: Negative. of phytopathology, vol. 10. VOL. 28, 1978 P. PSEUDOALCALIGENES SUBSP. CITRULLI SUBSP. NOV. 125

17. Raleton-Barrett, E, N. J. Palleroni, and M. Doudo- 1966. The aerobic peeudomonadtx a taxonomic study. roff. 1976. Phenotypic characterization and deoxyribo- J. Gen. MicrobioL 41:159-271. nucleic acid homologies of the “Pseudomnas akdi- 21. Thomason, B. M., and J. G. Wells. 1971. Preparation genes” group. Ink J. Syst. Bacterial. 26:4!21426. and tea% of polyvalent conjugates for fluomcent- 18. Sands, D., M. N. Schmth, and D. C. Hildebrand. 1970. antibody detection of salmonellae. Appl. Microbiol. Taxonomy of phytopathogenic pseudomonada J. Bac- a2:mi~atu. teriol. 101:9-23. 22. Webb, R E, and R W. Goth. 1965. A &-borne 19. Schaad, N. W., C. L Kado, and D. R Sumner. 1975. bacterium isolated from watermelon. Plant Dis. Reptr. Synonymy of Pseudomonacl auenae Manns 1906 and 49:818-821. Pseudomonae albopmipituns Roeen 1922. Int. J. Syst. 23. Wileon, E. E., M Zeitoun, and D. L Fredriclreon. Bacteriol. 26:133-137. 1967. Bacterial phloem canker, a new disease of Persian 20. Stanier, R Y., N. J. Palleroni, and M. Doudoroff. walnut trees. Phytopathology 67:618-621.