First Report of Bacterial Leaf Blight of Carrot Caused by Xanthomonas hortorum pv. carotae in Korea.

In December 2012, symptoms of typical bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus) cultivated in commercial fields in Kujwa, Jeju, Korea. The disease was detected in 40% of 50 fields surveyed with an incidence of 10% on average. The bacterial leaf blight lesions on leaf blades were elongated, dark brown to black with water-soaked edges and chlorotic halos. Lesions were also crescent-shaped to V-shaped on leaflets. Four bacterial isolates were recovered on trypticase soy agar from leaf lesions that were surface-sterilized in 70% ethyl alcohol for 20 s. Identity of the isolates was confirmed by PCR product (1,266-bp) using a specific primer set for Xanthomonas hortorum pv. carotae (Kendrick 1934) Vauterin et al. 1995, XhcPP03 (1). All isolates were gram-negative, aerobic rods with a single polar flagellum. Isolates were positive for catalase and negative for oxidase. In phenotypic tests for differentiation of Xanthomonas (2), the isolates positive for mucoid growth on yeast extract-dextrose-calcium carbonate agar, growth at 35°C, hydrolysis of esculin, protein digestion, alkaline in litmus milk, acid production from arabitol, and utilization of glycerol and melibiose. The isolates were negative for growth on SX medium, hydrolysis of starch, and ice nucleation. The gyrB gene (863 bp) and the rpoD gene (870 bp) were sequenced to aid identification of the original isolates using published PCR primer sets, Xgyr1BF/Xgyr1BR and XrpoD1F/XrpoD1R (4), respectively. Sequences of the gyrB gene (GenBank accessions KC920729 to KC920732) from the carrot isolates shared 100% sequence identity with that of the X. hortorum pv. carotae strain NCPPB 425 (EU285243). In phylogenetic analyses based on the partial sequences of the gyrB and the rpoD genes for Xanthomonas spp. available at NCBI (4), and sequences of the carrot isolates (KC920734 to KC920737 for rpoD gene) using the Neighbor-joining method in MEGA Version 5.1 (3), the isolates were clustered in the X. hortorum-cynarae-garnderi group. Pathogenicity of the isolates was tested by spray inoculation with a bacterial suspension (106 CFU/ml) prepared in sterile distilled water at 6 to 7 true-leaf stage (three plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 15 hr, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 14 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through gyrB gene sequence analysis (4). Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Korea. The detection of this pathogen could have a significant economic impact due to yield losses from disease development. Consolidation of quarantine inspection on imported carrot seeds needs to control an outbreak of the disease. Crop rotation and plowing are recommended to reduce incidence of the disease in the infested fields. References: (1) J. A. Kimbrel et al. Mol. Plant Pathol. 12:580, 2011. (2) N. W. Schaad et al. Page 189 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

First Report of Bacterial Leaf Spot of Witloof, Caused by Pseudomonas cichorii in Korea.

In August 2011, bacterial leaf spot was observed on witloof (Cichorium intybus L. var. foliosum) grown in a commercial field with 15% incidence in Injae, Korea. Symptoms on leaves included irregular brown to reddish brown spots in the center. Bacterial streaming from the lesions was observed microscopically. Bacterial isolates (BC3286, BC3287, and BC3308-BC3310) were recovered on Trypticase soy agar from lesions surface-sterilized in 70% ethyl alcohol for 30 s. The isolates were gram negative, urease negative, fluorescent on King's B agar, and had aerobic rods with 2 to 6 polar flagella. Pathogenicity tests were separately performed in different greenhouses located in Suwon (National Academy of Agricultural Science) and Chuncheon (Gangwondo Agricultural Research and Extension Services) in Korea. Pathogenicity was confirmed by spray inoculation of healthy, 10-day-old leaves of witloof plants (two plants/isolate) with a suspension of original field isolate (106 CFU/ml). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) overnight, then transferred to a greenhouse at 23 to 27°C and 60 to 70% RH. Characteristic leaf spot symptoms were observed on inoculated witloof plants 8 days after inoculation. No symptoms were observed on control plants. The bacterium reisolated from the inoculated leaves was confirmed by analyzing sequence of the gyrB gene with direct sequencing method of PCR products using primers gyr-F and gyr-R (2). The sequence of reisolated bacteria shared 100% similarity with inoculated ones. In LOPAT (1) tests, all isolates and the reference strain of Pseudomonas cichorii CFBP2101T (=BC2595) were levan negative, oxidase positive, potato rot negative, arginine dihydrolase negative, and tobacco hypersensitivity positive, indicative of group III (-, +, -, -, +) of fluorescent pseudomonads. The 16S rRNA (1,408 bp), and gyrB (676 bp) regions were sequenced to aid in identification of the original field isolates as well as P. cichorii CFBP 2101T (=BC2595) using reported sets of PCR primers, fD1/rP2 and gyr-F/gyr-R, respectively (2,4). Phylogenetic analyses based on partial sequences of the gyrB and the 16S rRNA of Psudomonas spp. available in GenBank, the reference strain of P. cichorii CFBP2101T (=BC2595), and the witloof field isolates were conducted using the neighbor-joining method with Juke-Cantor model of distance calculation in MEGA version 5.1 (3). The isolates and the reference strain of P. cichorii CFBP2101T (=BC2595) was clustered in one group with P. cichorii strains in both phylogenetic trees based on the two sequences. Sequences of the 16S rRNA region had a distance index value ranging from 0.000 to 0.001 between the reference strain of P. cichori CFBP2101T (GenBank JX913784) and the field isolates (JX913785 to JX913789), and ranged from 0.000 to 0.001 within the field isolates. Sequences of the gyrB region had a distance index value ranging 0.029 to 0.033 between the reference strain (JX913790) and the field isolates (JX913791 to JX913795), and ranged from 0.000 to 0.041 within the field isolates. To our knowledge, this is the first report of bacterial leaf spot of witloof caused by P. cihorii in Korea. P. cichorii has a wide host range, and an important economic impact on vegetables. The disease is expected to result in a significant economic impact on root production of witloof in Korea. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) H. Sawada et al. J. Mol. Evol. 49:627, 1999. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) W. G. Weinsburg et al. J. Bacteriol. 173, 697, 1991.

Bacterial Stripe of Hog Millet Caused by Acidovorax avenae subsp. avenae, a New Disease in Korea.

In July 2011, bacterial stripe was observed on a commercial field of hog millet (Panicum miliaceum L.) in Chuncheon, Korea, with a disease incidence of 37% in the field. Symptoms on leaves included reddish-brown, long, narrow stripes that varied in length and were sharply delineated by uninfected adjacent vascular bundles. Eleven bacterial isolates (BC3107, BC3214 to BC3223) were recovered on trypticase soy agar from lesions surface sterilized in 70% ethanol for 1 min. The isolates, all obtained from different plants, were gram negative, oxidase positive, aerobic rods with two to four flagella. The isolates produced circular, cream-colored, nonfluorescent, butyrous colonies with entire margins on King's B medium. Using the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), the isolates were identified as Acidovorax avenae subsp. avenae with Biolog similarity indices ranging from 0.52 to 0.72 after 24 hr. Characters for differentiating between Acidovorax spp. were tested according to Schaad et al. (2). The isolates were positive for gelatin liquefaction, nitrate reduction, lipase production, utilization of D-mannitol, sodium citrate, and alkaline in litmus milk. The isolates were negative for utilization of D-arabitol and did not amplify with PCR primer sets Aaaf5, Aaaf3/Aaar2, and Aacf2/Aacr2. Colonies were V-, V+, and V+ for utilization of D-fucose, maltose, and ethanol, respectively. Regions of the 16S rRNA (rrs) and the IGS were sequenced to aid in the identification of the isolates using reported PCR primer sets (1,4). A 1,426 bp fragment of the rrs region shared 100% similarity with all strains of A. avenae available in GenBank. Pathogenicity tests were separately performed for the 11 isolates in different greenhouses located in Suwon (National Academy of Agricultural Science), and Chuncheon (Gangwondo Agricultural Research and Extension Services) in Korea. Pathogenicity was confirmed by clip inoculation with sterilized scissors dipped into cell suspensions containing 105 CFU/ml on three 8-day-old leaves of hog millet (two plants per isolate), rice (Oryza sativa L. cv. Hopyeong), and sweet corn (Zea mays L. cv. Daehak) in a greenhouse maintained at 28 to 32°C and 90% relative humidity. The isolates induced similar symptoms as those originally observed on hog millet 5 days after inoculation. No symptoms were observed on the control plants (hog millet, rice, and sweet corn), which were clipped with scissors dipped in sterilized distilled water. The identity of bacteria reisolated from the stripes on inoculated leaves was confirmed by analyzing sequences of the 16S-23S rRNA intergenic spacer region (IGS) (1). On the basis of physiological, pathological, and sequence data, the isolates were identified as A. avenae subsp. avenae. To our knowledge, this is the first report of bacterial stripe of hog millet caused by A. avenae subsp. avenae in Korea. The spread of the bacterial disease is expected to have a significant economic impact on hog millet culture in the fields of Gangwon Province in Korea. Nucleotide sequence data reported are available under accession numbers JQ743877 to JQ743887 for rrs of BC 3207 and BC3214 to BC3223, and JQ743877 to JQ743887 for IGS of BC3207 and BC3214 to BC3223. References: (1) T. Barry et al. The PCR Methods Appl. 1:51, 1991. (2) N. W. Schaad et al. Syst, Appl. Microbiol. 31: 434, 2008. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) W. G. Weisburg et al. J. Bacteriol. 173: 697, 1991.

Bacterial Leaf Spot of Zinnia Caused by Xanthomonas campestris pv. zinniae, a New Disease in Korea.

In September 2011, bacterial leaf spot was observed on zinnia plants (Zinnia elegans L.) grown in a garden in Suwon, Korea. Leaf symptoms included angular lesions that were yellow or brown-to-reddish brown in the center. Bacterial isolates (BC3293 to BC3299) were recovered on trypticase soy agar from lesions surface-sterilized in 70% ethyl alcohol for 1 min. Pathogenicity of the isolates was confirmed by spray inoculation with a bacterial suspension (106 CFU/ml) prepared in sterile distilled water and applied to zinnia plants at the four- to five-leaf growth stage (two plants per isolate). Sterile distilled water was used as the negative control. The inoculated plants were incubated in a greenhouse at 26 to 30°C and 95% relative humidity. Characteristic leaf spot symptoms developed on inoculated zinnia plants 5 days after inoculation. No symptoms were observed on the negative control plants. The bacterium reisolated from the inoculated leaves was confirmed through gyrB gene sequence analysis (3). All isolates were gram-negative, aerobic rods, each with a single flagellum. Isolates were positive for catalase and negative for oxidase. The biochemical and physiological tests for differentiation of Xanthomonas were performed using methods described by Shaad et al. (2). The isolates were positive for mucoid growth on yeast extract-dextrose-calcium carbonate agar, growth at 35°C, hydrolysis of starch and esculin, protein digestion, acid production from arabitol, and utilization of glycerol and melibiose. Colonies were negative for ice nucleation, and alkaline in litmus milk. The gyrB gene (870 bp) and the 16S-23S rRNA internal transcribed spacer (ITS) regions (884 bp) were sequenced to aid in identification of the original field isolates using published PCR primer sets Xgyr1BF/Xgyr1BR (3) and A1/B1 (1), respectively. Sequence of the gyrB gene (GenBank Accession Nos. JQ665732 to JQ665738) from the zinnia field isolates shared 100% sequence identity with the reference strain of Xanthomonas campestris pv. zinniae (GenBank Accession No. EU285210), and the ITS sequences (GenBank Accession Nos. JQ665725 to JQ665731) had 99.9% sequence identity with X. campestris pv. zinnia XCZ-1 (GenBank Accession No. EF514223). On the basis of the pathogenicity assays, biochemical and physiological tests, and sequence analyses, the isolates were identified as X. campestris pv. zinniae. To our knowledge, this is the first report of bacterial leaf spot of zinnia caused by X. campestris pv. zinniae in Korea. The disease is expected to result in economic and aesthetic losses to plants in Korean landscapes. Thus, seed treatment with bactericides will be required to control the bacterial leaf spot of zinnia before planting. References: (1) T. Barry et al. The PCR Methods Appl. 1:51, 1991. (2) N. W. Schaad et al. Page 189 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Bacterial Leaf Spot of Onion Caused by Pseudomonas syringae pv. porri, a New Disease in Korea.

In April 2007, a bacterial leaf spot of onion (Allium cepa L.) was observed in fields of Namjeju, Jeju Province in Korea with incidence varying from 95 to 100%. Symptoms on leaves included leaf blight and white and brown spots on the leaf surface. Eight bacterial isolates were recovered on trypticase soy agar (TSA) from leaf spot and blight lesions that were surface sterilized in 70% ethanol for 1 min. The isolates were fluorescent on King's B agar and gram-negative, aerobic rods with one to three polar flagella. All isolates belonged to P. syringae (LOPAT) group Ia (+, -, -, -, +) (1). The gyrB, rpoD (2), and rpoB regions (4) of the isolates and reference strain Pseudomonas syringae pv. porri CFBP 1908PT (=BC2583) were partially sequenced using reported primers (2,4). The rpoB region (1,119 bp) of the isolates (GenBank Accession Nos. JF719311-JF719318 for rpoB) shared 100% identity with P. syringae pv. porri CFBP 1908PT (GenBank Accession No. JF719319). Phylogenetic analysis based on partial sequences of the gyrB (660 bp) and rpoD (590 bp) loci of Pseudomonas spp. available in the GenBank (2,4), the reference strain P. syringae pv. porri CFBP 1908PT, and the field isolates was conducted using Jukes-Cantor model in MEGA Version 4.1 (3). The isolates and reference strain P. syringae. pv. porri CFBP 1908PT clustered in one group (GenBank Accession Nos. JF719293-JF719300 for gyrB; JF719302-JF719309 for rpoD). On the basis of phenotypic and pathological characteristics and the sequences, the eight isolates were identified as P. syringae pv. porri. Pathogenicity was evaluated on 3-week-old onion plants (cv. Marushino 330) by spot and spray inoculation. Bacteria were grown on TSA for 24 h at 28°C. Five microliters of bacterial suspension in sterile distilled water (1 × 106 CFU/ml) were spot inoculated on pinpricked positions of five leaves for each isolate and incubated in humid plastic boxes at 27°C. Spot-inoculated surfaces turned white 2 days after inoculation, followed by brownish discoloration. A bacterial suspension in sterile distilled water (100 ml at 1 × 106 CFU/ml) was sprayed onto three plants for each isolate. Plants were maintained in a greenhouse at 18 to 27°C and 80% relative humidity. Isolates induced identical symptoms on all inoculated plants 2 weeks after spray inoculation as those originally observed on onion in the fields. Bacteria were reisolated 3 weeks after inoculation from diseased lesions surface sterilized in 70% ethanol for 1 min and the identity of the reisolated bacteria confirmed by analyzing the sequences of rpoD gene (2). No symptoms were noted on intact plants inoculated with sterilized distilled water. To our knowledge, this is the first report of bacterial leaf spot of onion caused by P. syringae pv. porri in Korea. The disease is expected to have a significant economic impact on onion culture in the fields of Jeju Province in Korea. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) H. Sawada et al. J. Mol. Evol. 49:627, 1999. (3) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (4) L. Tayeb et al. Res. Microbiol. 156:763, 2005.

A New Disease, Bacterial Leaf Spot of Rape, Caused by Atypical Pseudomonas viridiflava in South Korea.

In March 2007, a bacterial leaf spot of rape (Brassica napus var. oleifera) was observed in fields near Seogwipo City, Jeju Province, South Korea. Symptoms on leaves included white and corky-brown spots and sometimes water-soaked spots on the lower leaf surface. Seven bacterial isolates (BC2495-BC2497 and BC2506-BC2509) were recovered on trypticase soy agar (TSA) from leaf spot lesions surface sterilized in 70% ethyl alcohol for 1 min. Isolates were gram-negative, aerobic rods with one to three flagella. Pathogenicity was evaluated on 2-week-old rape plants by spot and spray inoculation. Bacteria were grown on TSA for 48 h at 25°C. Five microliters of bacterial suspension in sterile distilled water (1 × 105 CFU/ml) were spot inoculated on pinpricked positions of five detached leaves for each isolate. The detached leaves were incubated in a plastic box with high humidity at 20°C. Spot-inoculated surfaces turned white 48 h after inoculation followed by a brownish discoloration. A bacterial suspension in sterile distilled water (100 ml at 1 × 105 CFU/ml) was sprayed onto three plants for each isolate. Plants were maintained in a growth chamber at 20°C and 90% relative humidity. Isolates induced identical symptoms 2 weeks after spray inoculation as those originally observed on rape in the fields. Bacteria were reisolated 18 days after inoculation from diseased lesions surface sterilized in 70% ethyl alcohol for 1 min. Pathogenicity of the reisolated bacteria was confirmed by spot inoculation as described above. No symptoms were noted on detached leaves and intact plants inoculated with sterilized distilled water. Using the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), the isolates were identified as Pseudomonas viridiflava with a Biolog similarity index range of 0.52 to 0.72 after 24 h. Results of LOPAT tests (2) of isolates were identical to that of atypical P. viridiflava reported by Gonzalez et al. (1). Levan production and pectolytic activity of the isolates were variable. All isolates were positive for tobacco hypersensitivity and negative for oxidase reaction and arginine dihydrolase production. The 16S rDNA region (1,442 bp) of the isolates (GenBank Accession Nos. HM190218-HM190224; P. viridiflava CFBP2107T = HM190229), amplified by using universal PCR primers, shared 100% sequence identity with atypical P. viridiflava (GenBank Accession No. AM182934) (1). The gyrB sequence (638 bp) from the isolates (GenBank Accession Nos. HM190232-HM190238; P. viridiflava CFBP2107T = HM190239), amplified by using previously reported PCR primers (3), had a distance index value range of 0.029 to 0.031 with that of the P. viridiflava CFBP2107T (=BC2597) as determined by Jukes-Cantor model using MEGA Version 4.1 (4). On the basis of phenotypic characteristics and the sequences, the seven isolates were identified as atypical P. viridiflava. The disease is named "bacterial leaf spot". To our knowledge, this is the first report of bacterial leaf spot of rape caused by atypical P. viridiflava. References: (1) A. J. Gonzalez et al. Appl. Environ. Microbiol. 69:2936, 2003. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (3) H. Sawada et al. J. Mol. Evol. 49:627, 1999. (4) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007.

A New Disease, Arboricola Leaf Spot of Bell Pepper, Caused by Xanthomonas arboricola.

In June 2007, a leaf spot disease was observed on seedlings of bell pepper (Capsicum annuum L. var. angulosum) in a commercial greenhouse in Iksan City, Korea. Symptoms on leaves included small, irregularly shaped, brown lesions with yellow halos and marginal necrosis. Four bacterial isolates, BC2526, BC2527, BC2528, and BC2529, were obtained from the diseased plants. The isolates were gram-negative aerobic rods with a single flagellum. On peptone sucrose agar, colonies were yellow and raised with smooth margins. Pathogenicity was confirmed by spraying cell suspensions containing 106 CFU/ml onto seedlings of bell pepper (cv. Fieste), tomato (Solanum lycopersicon cv. Seokwang), and hot pepper (Capsicum annuum cv. Daekwang) in a greenhouse maintained at 26 ± 3°C. The isolates induced symptoms, spots, and margin blights on leaves of bell pepper, tomato, and hot pepper 2 weeks after inoculation. No symptoms were noted on the control plants inoculated with sterilized distilled water. The identity of the bacteria was confirmed with the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA). The gyrB region was partially sequenced to aid in identification of four isolates using PCR primers reported by Parkinson et al. (1). A 701-bp fragment of the gyrB region from the isolates was compared with sequences of the reference strains of Xanthomonas available in the DDBL/EMBL/GenBank databases (4). The bacterial isolates clustered with Xanthomonas arboricola pathovars in a phylogenetic tree generated with the neighbor-joining method in MEGA (version 4.1) (3). The sequence of the gyrB from the isolates had distance indexes of 0.016, 0.014, 0.016, 0.013, 0.037, and 0.019 as determined by the Jukes-Cantor model (2) with sequences of the reference strains of X. arboricola pvs. pruni (EU498953), celebensis (EU498984), corylina (EU499002), juglandis (EU 498951), populi (EU 499035), and a X. arboricola strain from bell pepper (EU 499039) (4), respectively. To our knowledge, this is the first report of a leaf disease on bell pepper caused by X. arboricola. We propose the name arboricola leaf spot for the disease. Further studies are required for determining pathovar status of the strain. Nucleotide sequence data reported are available under Accession Nos. GQ502678, GQ502679, GQ502680, and GQ502681 for gyrB of BC2626, BC2527, BC2528, and BC2923, respectively. The disease is expected to have a significant economic impact on tomato and pepper production in Korea. References: (1) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (2) N. Saitou and M. Nei. Mol. Biol. Evol. 4:406, 1987. (3) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Bacterial Leaf Spot of Iceberg Lettuce Caused by Xanthomonas campestris pv. vitians Type B, a New Disease in South Korea.

In 2008 and 2009, a leaf spot of iceberg lettuce (Lactuca sativa var. capitata) was observed in two fields of Pyeongchang District and Jecheon City in South Korea, respectively. Disease incidence averaged 3.5% in the two fields. Symptoms on leaves included black, water-soaked, angular lesions with halos. Two bacterial isolates, BC2932 and BC3095, were recovered on trypticase soy agar (TSA) from lesions surface sterilized in 70% ethyl alcohol for 1 min. Both isolates had gram-negative, aerobic rods each with a single flagellum. Colonies on peptone sucrose agar were yellow and raised with smooth margins. Pathogenicity was evaluated on 3-week-old lettuce plants (cv. Avi). Bacteria were grown on TSA for 48 h at 28°C. A bacterial suspension in sterile distilled water (100 ml at 1 × 105 CFU/ml) was sprayed onto three plants for each isolate. Plants were maintained in a growth chamber at 28°C and 90% relative humidity. Isolates induced identical symptoms 3 days after inoculation as those originally observed in the fields. Pathogenicity of bacteria reisolated 10 days after inoculation from lesions surface sterilized in 70% ethyl alcohol was confirmed by inoculation as described above. No symptoms were observed on two control plants treated with sterile distilled water. Identity of bacteria reisolated from inoculated leaves was confirmed by PCR with specific primer set B162 (1). DNA of the original two isolates and 12 reisolates (two per inoculated plant) was amplified by PCR assay using Xanthomonas campestris pv. vitians Type B LMG938 (= BC2575) as a positive control treatment and X. axonopodis pv. vitians strain CFBP2538 (= BC2610) as a negative control treatment. The PCR amplicon for each of the 14 test isolates was identical in size to that of X. campestris pv. vitians Type B LMG938. No fragment of X. axonopodis pv. vitians CFBP2538 was amplified. Patterns of metabolic fingerprinting of the original two isolates were more similar to those of X. campestris pv. vitians Type B LMG938 than X. axonopodis pv. vitians CFBP2538 using Biolog Microbial Identification System Version 4.2 (Biolog Inc., Hayward, CA). X. campestris pv. vitians Type B LMG938, BC2932, and BC3095 were identified as X. campestris pv. pelargonii with a Biolog similarity index of 0.68, 0.45, and 0.78, respectively. Strain X. axonopodis pv. vitians CFBP2538 was identified as X. campestris pv. juglandis with an index of 0.48. The dnaK (958 bp), gyrB (859 bp), and rpoD (884 bp) regions were partially sequenced to aid in identification of the two original field isolates as well as X. campestris pv. vitians Type B LMG 938 and X. axonopodis pv. vitians CFBP2538 using reported PCR primers (3). Sequences were compared with those of reference strains of Xanthomonas in GenBank. Sequences of the three genes from the two lettuce field isolates shared 100% similarity to those of the genes of X. campestris pv. vitians Type B LMG938 and had a distance index value of 0.040, 0.099, and 0.046, respectively, with the reference strain of X. axonopodis pv. vitians CFBP2538 determined by p-distance modeling using MEGA Version 4.1 (2). Based on the pathogenicity test and sequence analyses, the isolates were identified as X. campestris pv. vitians Type B. To our knowledge, this is the first report of bacterial leaf spot of iceberg lettuce caused by X. campestris pv. vitians Type B in South Korea. References: (1) J. D. Barak et al. Plant Dis. 85:169, 2001. (2) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (3) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Wildfire of Soybean Caused by Pseudomonas syringae pv. tabaci, a New Disease in Korea.

In 2006 and 2007, a new bacterial disease was observed in field-cultivated soybeans in Boeun District and Munkyung City of Korea. The disease caused severe blighting of soybean (Glycine max) leaves. Soybean leaves in fields showed yellowish spots with brown centers. Brown and dead areas of variable size and shape were surrounded by wide, yellow haloes with distinct margins. Spots might coalesce and affected leaves fell readily. Seven bacterial strains were isolated from chlorotic areas of soybean leaves and all produced white colonies on trypticase soy agar. With the Biolog Microbial Identification System, version 4.2, (Biolog Inc., Hayward, CA) all strains and Pseudomonas syringae pv. tabaci CFBP2106T were identified as P. syringae pv. tabaci with a Biolog similarity index of 0.28 to 0.52 and 0.48 after 24 h. Pathogenicity of the strains (three plants per strain) on soybean leaves at the V5 stage (cv. Hwanggeum) was confirmed by rub inoculation with bacterial suspensions (1 × 108 CFU/ml) in sterile distilled water on the lesions cut 1 cm long on the upper side of the leaves with razor blades and by pinprick on 3-week-old leaves of tobacco (Nicotiana tabacum cv. Samsun) in the greenhouse. Wildfire symptoms on the soybean leaves and faint halos on tobacco leaves were observed 4 days after inoculation. The identification of reisolated bacterial strains was confirmed with the metabolic fingerprintings on Biolog. LOPAT tests (1) and phenotypic characteristics (3) of the strains were similar to those of the CFBP2106T. Colonies were levan positive, oxidase negative, potato soft rot negative, arginine dihydrase negative, and tobacco hypersensitivity negative. All strains were gram-negative, aerobic rods with a polar flagellum. Strains were negative for esculin hydrolysis, gelatin liquefaction, urea production, accumulation of poly-ß-hydroxy butyrate, starch hydrolysis, ornithine dihydrolase, lysine dihydrolase, growth at 37°C, utilization of geraniol, benzoate, cellobiose, sorbitol, trehalose, l-rhamnose, and adonitol. Positive reactions were catalase and arbutin hydrolysis, utilization of sorbitol, d-arabinose, and dl-serine. The strains were variable in utilization of mannitol, sucrose, and d-arabinose. The 1,472-bp PCR fragments of strains, BC2366 (GenBank Accession No. FJ755788) and BC2367 (No. FJ755789) was sequenced using 16S rDNA universal primers (2). The sequences shared 100% identity with the analogous sequences of P. syringae pv. glycenea (GenBank Accession No. AB001443) available in NCBI databases. Based on the phenotypic, genetic, and pathological characteristics, all strains were identified as P. syringae pv. tabaci. To our knowledge, this is the first report of P. syringae pv. tabaci causing wildfire on soybean in Korea. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) I.-S. Myung et al. Plant Dis. 92:1472, 2008. (3) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

Bacterial Spot of Tomato Caused by Xanthomonas perforans, a New Disease in Korea.

In July 2007, a leaf spot was observed on seedlings of tomato (Solanum lycopersicum) in a commercial greenhouse in Sungju County, Korea. Symptoms were dark, circular-to-irregular, water-soaked spots surrounded by chlorotic halos. Affected leaves turned yellow and readily detached. Two bacterial isolates, BC2642 and BC2923, were obtained from leaf lesions. The isolates were gram-negative, aerobic rods with a single flagellum. On peptone sucrose agar, colonies were yellow and raised with smooth margins. Starch and pectate hydrolysis tests were positive. Pathogenicity was confirmed by spraying cell suspensions containing 108 CFU/ml on seedlings of tomato (cv. Seokwang) and hot pepper (Capsicum annuum cv. Daekwang) in a greenhouse maintained at 28 ± 2°C. The isolates induced similar symptoms as those originally observed on tomato and also caused spots and a marginal blight of leaves of pepper 2 weeks after inoculation. No symptoms were noted on the control plants sprayed with sterilized distilled water. The identity of bacteria reisolated from spots on leaves of both plants were confirmed by comparison of patterns of metabolite fingerprints with those from preliminary identification of the isolates using the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), and reinoculation of the seedlings as above. The 16S rRNA gene (rrs) and the intergenic spacer (IGS) located between the rrs and the 23S rRNA gene, and partial sequences of gyrB were sequenced to aid in the identification of the isolates (1-3). A 2,134-bp fragment of the rrs and IGS regions and 701-bp fragment of the gyrB region from isolates BC2642 and BC2923 were compared with sequences in GenBank. Sequences from both isolates shared 100% similarity to sequences of Xanthomonas perforans (Genbank Accession No. AF123091). On the basis of the sequences and other assays, the two isolates were identified as X. perforans. To our knowledge, this is the first report of bacterial spot of tomato caused by X. perforans in Korea. Nucleotide sequence data reported are available under Accession Nos. GQ461739 and GQ461740 for rrs and IGS of BC2642 and BC2923, respectively, and GQ368187 and GQ380567 for gyrB of BC2642 and BC2923, respectively. An outbreak of this disease in the greenhouse may be due to the use of tomato seeds harvested in foreign countries where spot is known to occur. The disease is expected to have a significant economic impact on tomato culture in Korea. References: (1) J. B. Jones et al. Int. J. Syst. Evol. Microbiol. 50:1211, 2000. (2) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (3) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

First Report of Bacterial Canker of Tomato Caused by Clavibacter michiganensis subsp. michiganensis in Korea.

In 2007, a new bacterial disease was observed in greenhouse-cultivated cherry tomatoes in Cheorwon and Iksan provinces, Korea. The disease caused severe wilt of tomatoes (Solanum lycopersicum cv. Koko). Infected young petioles were curled downward. Margins of the leaves rolled upward and whole leaves were distorted. Stem cankers had reddish or dark brown cavities. Vascular tissues in stems cut longitudinally were brown to deep brown, but no bird's eye lesions were observed. Eight bacterial strains recovered from the stems of wilted tomatoes produced yellow colonies on nutrient broth-yeast extract agar and pink colonies on triphenyl tetrazolium chloride. Pathogenicity of the strains (three plants per strain) on 18-day-old tomatoes (cv. Koko) was confirmed by clip inoculation of petioles of second leaves and spray inoculation with bacterial suspensions (1 × 108 CFU/ml) in sterile distilled water. Wilt and canker symptoms were observed 2 weeks after inoculation. Symptoms produced by both inoculation methods were systemic and localized. Clip inoculation of tomatoes resulted in wilt, defoliation, and open stem cankers, whereas small, white spots (2 to 3 mm in diameter) and sometimes water-soaked, dark brown-to-black lesions on the leaf margins were observed with spray inoculation. Bacteria were reisolated from stems and leaves of the inoculated plants and their identities confirmed by direct PCR using specific primer set CMM5/CMM6 (1). No symptoms were observed on negative control plants inoculated with sterile water. All strains were gram-positive aerobic rods with no polar flagella. Strains were positive for esculin hydrolysis, gelatin liquefaction, H2S production from peptone, utilization of citrate and succinate, and acid from d(+)mannose and negative for starch hydrolysis, casein hydrolysis, methyl red reaction, acid from inulin, mannitol, d(+)-melezitose and d(-)sobitol, and utilization of acetate, formate, lactate, propionate, and ribose. Identification as C. michiganensis subsp. michiganensis was confirmed using 16S rDNA universal primers fD1 and rP2 (4) and internal primers (3). The 1,439-bp PCR fragment of strain BC2643 was sequenced (GenBank Accession No. EU685335) and compared with reference C. michiganensis subspecies strains in GenBank: AM410696 (C. michiganensis subsp. michiganensis), AM410693 (C. michiganensis subsp. tessellarius), AM410697 (C. michiganensis subsp. nebraskensis), AM410694 (C. michiganensis subsp. sepedonicus), and AM410695 (C. michiganensis subsp. insidiosus). The sequence had a similarity index of 0.999 calculated by Juke-Cantor model (2) with the 16S rRNA sequence of C. michiganensis subsp. michiganensis (AM410696). The fragment size of eight strains amplified by PCR using CMM5/CMM6 (1) was identical to that of the C. michiganensis subsp. michiganensis reference strain KACC20122. On the basis of the physiological, genetic, and pathological characteristics, all strains were identified as C. michiganensis subsp. michiganenesis. To our knowledge, this is the first report of C. michiganensis subsp. michiganenesis causing bacterial canker on tomato in Korea. References: (1) J. A. Dreier et al. Phytopathology 85:464, 1995. (2) S. Kumar et al. Brief. Bioinform. 5:50, 2004. (3) S. W. Kwon et al. Int. J. Syst. Bacteriol. 47:1061, 1997. (4) W. G. Weinsburg et al. J. Bacteriol. 173, 697, 1991.

Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction.

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two-step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X. fastidiosa. A total of 347 leafhoppers representing 16 species were captured and sampled from American elm (Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta, regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa. Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.