INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1983, p. 546-550 Vol. 33, No. 3 0020-7713/83/030546-05$02 .OO/O Copyright 0 1983, International Union of Microbiological Societies Pseudomonas Jicuserectae sp. nov., the Causal Agent of Bacterial Leaf Spot of Ficus erecta Thunb. MASAO GOT0 Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422, Japan Pseudomonas Jicuserectae, a new nonfluorescent , phytopathogenic pseudomo- nad species, is described. This bacterium produces dark brown, water-soaked spots on the leaves and stems of Ficus erecta Thunb., resulting either in defoliation or shoot blight on severely infected plants. The colonies on nutrient agar plates are white, circular, and 3 to 4 mm in diameter after 6 days. P. jicuserectae is similar to Pseudomonas amygdali in many properties. The differences between these two species include size of colonies on agar plates, hydrolysis of Tween 80, production of HzS, utilization of ribose, raffinose, mannitol, sorbitol, and malonate, and pathogenicity. The deoxyribonucleic acid base composition of the type strain of P. Jicuserectae, strain L7, is 59 mol% guanine plus cytosine. The specific epithet of this new species reflects the pathogenicity of the bacterium on F. erecta Thunb. __ Since 1973, a bacterial disease has been ob- stems with a needle through drops of bacterial suspen- served in Japan on the tree Ficus erecta Thunb. sion placed on the plant surface. The inoculated plants (Japanese common name, Inubiwa), which is were kept in a mist chamber for 24 h and then often planted in gardens as an ornamental plant. transferred to a greenhouse, where disease develop- ment was monitored. This disease is characterized by small, water- Bacteriological properties. Cell size was measured soaked spots on the leaves and shoot blight, and under a light microscope after staining with Ziehl a nonfluorescent pseudomonad has been consis- carbolfuchsin (12). Poly-P-hydroxy-butyrate accumu- tently isolated from diseased tissues. Therefore, lation was detected either by staining with Sudan I performed a taxonomic study comparing this black (9) or by observing under a phase-contrast organism with three nonfluorescent pseudomo- microscope a wet mount of cells grown on a synthetic nads, Pseudomonas andropogonis (Smith) medium (0.05% NH4H2P04, 0.02% MgS04 * 7H20, Stapp 1928 (14), Pseudomonas amygdali Psalli- 0.02% KCl, 5% glucose, pH 6.8) for 3 to 5 days. The das and Panagopoulos 1975 (8), and Pseudomo- modified Yamanaka method was used for flagellar subsp. Schaad et staining (11). Reactions for catalase, amino acid decar- nas pseudoalcaligenes citrulli boxylase, amylase, urease, and Kovacs oxidase, re- al. 1978 (lo), which are similar in some respects duction of KN03, and production of acetoin, 2-keto- to the bacterium isolated from diseased F. gluconate, and indole were tested by method 1, and erecta tissues. Pseudomonas syringae pv. pha- production of H2S was tested by method 3 described in seolicola (Burkholder) Young et al. 1978 (16) Cowan and Steel's Manual for the Identification of was also included as a reference bacterium. Medical Bacteria, 2nd ed. (2). Phenylalanine deami- nase was tested on 2-day-old phenylalanine agar slant MATERIALS AND METHODS cultures by method 2 of the same manual. Methods Bacteria. The bacterial strains used in this study are described in this manual were also used to determine listed in Table 1. Eleven cultures of the Ficus bacteri- the results for the litmus milk reaction, gelatin lique- um were isolated from new lesions formed on young faction, the methyl red test, growth inhibition by leaves and twigs by the conventional plating method KCN, the Hugh-Leifson test, and esculin hydrolysis. on yeast extract-peptone agar (0.5% yeast extract, The methods of Dye were used to test utilization of 1.0% peptone, 1.5% agar, pH 6.8). Small white colo- asparagine as a sole source of carbon and nitrogen and nies grew on the plates in 2 days at 28°C. Single tolerance to NaCl (4). The production of reducing colonies were selected and grown on yeast extract- substances from sucrose, arginine dihydrolase, and peptone agar slants. The cultures were maintained at levan formation were tested by methods described in 4°C for routine work. the Laboratory Guide for Identification of Plant Pathogenicity. Inoculation tests were made on F. Pathogenic Bacteria (9). Utilization of carbon sources erecta Thunb., Ficus carica L. (cultivar unknown), was tested by using the basal media developed by Morus alba L. (cultivar unknown), and Prunus armen- Ayers et al. (1) and by Starr (15). Denitrification was iaca L. cv. Wasesuimitsu; 2-year-old plants grown tested by the method of Stanier et al. (13). Pigmenta- from cuttings were inoculated either by spraying with tion was tested on King B medium (5). bacterial suspensions (concentration, 10' cells per ml) G+C content of DNA. Deoxyribonucleic acid (DNA) over young shoots or by puncturing mature leaves and was isolated and purified by the method of Marmur 546 VOL.33, 1983 PSEUDOMONAS FICUSERECTAE SP. NOV. 547 TABLE 1. Pseudomonas strains included in this study Taxon Strain(s) History" Habitat -____________ Pseudomonas ficuserectae L2, L3, L5, L6, L7, L8, L9, Goto F. erecta L10, L11, s1, S2h Pseudomonas pseudoalcaligenes PDDCC 6521, PDDCC 6522 Schaad Watermelon subsp. citrulli (PDDCC) Pseudomonas amygdali PDDCC 3918T, PDDCC 3920 Panagopoulos Almond (PDDCC) Pseudomonas andropogonis PA1 ' Goto Sorghum Pseudomonas andropogonis PA21' Goto Clover Pseudomonas andropogonis PA31' Goto Strelitzia Pseudomonas syringae pv. PP1' Goto Kidney phaseo licola bean a Person who isolated the strain(s). PDDCC, Plant Diseases Division Culture Collection, Auckland, New Zealand. ' Strains L2, L3, L5, L6, L7, L8, L9, L10, and L11 were isolated from leaves, and strains S1 and S2 were isolated from stems. ' Culture Collection of Phytopathogenic Bacteria, at the Laboratory of Plant Pathology, Shizuoka University, Shizuoka, Japan. (6). The guanine-plus-cytosine (G+C) content of the ectae sp. nov. is proposed for the new strains. DNA was calculated by using the equation of Marmur Pseudomonas Jicuserectae sp. nov. Pseudomo- and Doty (7) and the thermal denaturation tempera- nus Jicuserectae (fi.cus.e.reCtae. Ficus erecta ture, which was measured with a Gilford model 250 name of host species; L. n. ficuserectae of Ficus spectrophotometer. DNA of Micrococcus luteus cells are gram-negative, non-encapsulat- (Schroeter) Cohn strain IAM 1056 was used as a erecta) reference; the base composition of this DNA was 72 ed, nonsporeforming rods with an average size mol% G+C. of 2.0 by 0.5 km and are motile by means of one to five polar flagella (Fig. 1). Poly-P-hydroxybu- tyrate granules accumulate. Colonies on yeast RESULTS AND DISCUSSION extract-peptone agar plates at 28°C are white, The 11 strains of the Ficus bacterium were transparent, circular, convex, and 0.1 to 0.3 mm identical in their morphological, physiological, in diameter after 2 days and 2 to 3 mm in and biochemical properties. In addition, as diameter after 6 days. Growth on yeast extract- shown below, they were distinct from strains of peptone agar slants becomes very viscid after three other phytopathogenic nonfluorescent spe- several days. cies (Pseudomonas amygdali, Pseudomonas Pseudomonas ficuserectae was identical to pseudoalcaligenes subsp. citrulli, Pseudomonas the other four species studied in being positive andropogonis) and Pseudomonas syringae pv. for the following tests: motility, aerobic growth, phaseolicola. The name Pseudomonas ficuser- catalase reaction, oxidative metabolism of fruc- FIG. 1. Pseudomonas jkuserectac cells. 548 GOT0 INT.J. SYST.BACTERIOL. TABLE 2. Phenotypic characteristics of Pseudomonas jicuserectae, Pseudomonas amygdali, Pseudomonas pseudoalcaligenes subsp. citrulli, Pseudomonas andropogonis, and Pseudomonas syringae pv. phaseolicola Pseudo- Pseudo- Pseudomonus Pseudotnonns Pseudomonas Character monus pseridoulcciligene~ syritigne pv. monas andropogonis jicuserectae arnygduli subsp. citrulli phaseolicolu Flagella 1-3 (polar) Poly- p-hydroxybut yrate - accumulation Oxidase Growth at 41°C Growth at 4°C Utilization of asparagine as C and N sources Nitrate reduction Levan formation Gelatin liquefaction Tween 80 hydrolysis H2S production Oxidation of gluconate Reducing substances from sucrose Urease Arginine decarboxylase Growth in 4% NaCl G+C content Utilization of L- Arabinose D- Arabinose Xylose Ribose Glucose Mannose Rhamnose Cellobiose Lactose Saccharose Melibiose Raffinose Melezitose Ethanol Glycerol Inositol Mannitol Adonitol Sorbitol Formate Acetate Lactate Maleate Malonate Propionate Caprate Tartrate p-Alanine Serine Leucine y-Aminobutyric acid Valine Ethylene glycol +, Positive reaction; -, negative reaction; *, weak reaction; (d), delayed reaction. ’ From reference 10. ND, Not determined. Strain S2 was positive. VOL. 33, 1983 PSEUDOMONAS FICUSERECTAE SP. NOV. 549 TABLE 3. Main differences between Pseindornonas jicuserectae and Pseudornonas arnygdali Size of colonies Hydrol- Utilization of G+C on nutrient agar ysis of Produc- Species plates after 7 Tween Of Ribose Raffi- Glyc- Manni- Sorbi- 'Ontent days (mm) go H2S nose erol to1 to1 (mol%) Pseudornonus Jicuserectae 3-4 - - - ++ - - 59 Pseudomonas arnygdali 0.3-1 .O + + + - - + + 61 Strain S2 was positive. tose, alkaline reaction of litmus milk, growth in spots are angular, 1 to 3 mm across, and dark 3% NaCl, utilization of L-galactose, fructose, green. to dark brown, with a water-soaked ap- malate, fumarate, succinate,