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

Plant Disease ◽  
2010 ◽  
Vol 94 (9) ◽  
pp. 1164-1164 ◽  
Author(s):  
I.-S. Myung ◽  
Y.-K. Lee ◽  
S. W. Lee ◽  
W. G. Kim ◽  
H. S. Shim ◽  
...  
Keyword(s):  
South Korea ◽  
Ethyl Alcohol ◽  
Leaf Spot ◽  
Pcr Primers ◽  
Bacterial Suspension ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
Spray Inoculation ◽  
Pseudomonas Viridiflava ◽  
Detached Leaves

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.

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

Plant Disease ◽  
2010 ◽  
Vol 94 (6) ◽  
pp. 790-790 ◽  
Author(s):  
I.-S. Myung ◽  
S. Y. Moon ◽  
I. H. Jeong ◽  
S. W. Lee ◽  
Y. H. Lee ◽  
...  
Keyword(s):  
South Korea ◽  
Ethyl Alcohol ◽  
Leaf Spot ◽  
Xanthomonas Campestris ◽  
Control Treatment ◽  
Distilled Water ◽  
Type B ◽  
Bacterial Leaf Spot ◽  
Field Isolates ◽  
Iceberg Lettuce

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.

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

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1311-1311 ◽  
Author(s):  
I.-S. Myung ◽  
J. H. Joa ◽  
H. S. Shim
Keyword(s):  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Reference Strain ◽  
Leaf Surface ◽  
Bacterial Suspension ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
Rpod Gene ◽  
Allium Cepa L ◽  
Spray Inoculation

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.

scholarly journals First report of bacterial leaf spot disease on sunflower (Helianthus annuus) caused by Pseudomonas viridiflava in South Korea

Plant Disease ◽  
2021 ◽  
Author(s):  
Huan Luo ◽  
Yun Jung Lee ◽  
Jun Myoung Yu
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
South Korea ◽  
Leaf Spot ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
Plastic Bags ◽  
Pseudomonas Viridiflava ◽  
Rrna Sequences ◽  
16S Rrna Sequences

In September 2019, bacterial leaf spot symptoms were observed on sunflowers in an experimental field in Eumseong, South Korea. The leaves of infected plants initially showed irregular brown spots surrounded by haloes; as the disease progressed, the spots became enlarged and darkened (eXtra Fig. 1a). At the flowering stage, leaves became dry and showed signs of blight including defoliation; dark brown spots were also observed on sunflower stems and petioles but not on floral discs (eXtra Fig. 1b). Disease incidence ranged from 5% to 30% in three surveyed plots of the field. Symptomatic leaf tissue was surface-sterilized, macerated with sterile distilled water, and cultured on nutrient agar plates at 28°C for 48 h. After incubation, nine bacterial isolates, representing individually collected samples from each field, were selected for further study. All nine isolates were Gram-negative and fluorescent pigments produced under UV on King’s medium B. With the LOPAT test, the isolates were levan negative, oxidase negative, positive for pectinolytic activity, arginine dihydrolase negative, and positive for tobacco hypersensitivity. Based on 16s rRNA sequences, all isolates shared 100% identity with Pseudomonas viridiflava strain KNOX209.1 (GenBank accession no. AY604847). The 16s rRNA sequences of nine isolates were deposited in GeneBank (accession nos. MT393747, MW446479 to MW446486). Based on the phylogenetic analysis of the 16s rRNA, all isolates were grouped with P. viridiflava strains isolated from globe artichoke, Chinese cabbage, and rape (Myung et al. 2010, Sanver et al. 2019, and Liu et al. 2019). The isolates CPB 19362, CPB 19366 and CPB 19372, which represent each plot were selected for further phylogenetic analysis and pathogenicity assays. The identity of these isolates was confirmed by sequences of housekeeping genes of the gyrase B subunit (gyrB) and RNA polymerase σ70 factor (rpoD) (Yamamoto et al. 2000) (GenBank accession nos. MT409400, MW446487 and MW446494 for gyrB and MT409401, MW446495 and MW446502 for rpoD). Based on the phylogenetic analyses of gyrB and rpoD, the three isolates belong to the same clade as the P. viridiflava pathotypes and were distinguished from P. syringae complex (eXtra Fig. 2). These results indicated that the bacteria isolated from the spots on the sunflower plants were P. viridiflava strains. To confirm the pathogenicity, bacterial suspensions (approximately 108 CFU/mL) of three representative isolates sprayed onto 4-week-old sunflower (cv. Common) seedlings separately until runoff occurred. Sterile distilled water was used as a control and inoculated in the same manner. After inoculation, plants were covered with transparent plastic bags at room temperature for 24 h. Plastic bags were then removed and plants were grown on a plant growth shelf at 25°C in 50% relative humidity. The leaves of plants inoculated with the bacterial suspensions developed small brown spots after 24 h. After 3 days, brown spots surrounded by chlorotic or necrotic areas were observed on infected leaves (eXtra Fig. 1c). These spots gradually increased in size and formed brown lesions with haloes similar to those of infected field-grown plants (eXtra Fig. 1d), but not on the controls treated with sterile water. The pathogenicity test was repeated three times. Isolates recovered from infected leaves showed the same morphological, biochemical, and molecular characteristics as the original isolates from field samples. To our knowledge, this is the first report of bacterial leaf spot on sunflower caused by P. viridiflava in South Korea.

scholarly journals First Report of Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Lei Li ◽  
Yishuo Huang ◽  
Yanxia Shi ◽  
A LI CHAI ◽  
Xuewen Xie ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Citrate Synthase ◽  
Morphological Characteristics ◽  
Likelihood Method ◽  
Bacterial Suspension ◽  
Leaf Spot Disease ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
First Report

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander fields in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King’s B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer’s recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml−1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

scholarly journals First Report of Bacterial Leaf Spot of Basil Caused by Pseudomonas viridiflava in Hungary

Plant Disease ◽  
2012 ◽  
Vol 96 (1) ◽  
pp. 141-141 ◽  
Author(s):  
A. Végh ◽  
M. Hevesi ◽  
Zs. Némethy ◽  
L. Palkovics
Keyword(s):  
Leaf Spot ◽  
Sequence Similarity ◽  
Soft Rot ◽  
Hypersensitive Reaction ◽  
Bacterial Suspension ◽  
Bacterial Leaf Spot ◽  
Biochemical Tests ◽  
First Report ◽  
Arginine Dihydrolase ◽  
Pseudomonas Viridiflava

In April 2011, typical bacterial spot symptoms were observed on sweet basil plantlets (Ocimum basilicum L.) in a supermarket in Budapest, Hungary. Affected plants had dark brown-to-black lesions on the cotyledons. Spots on the leaves were first water soaked and then became necrotic and progressed inward from the margins. Symptoms were similar to those reported by Little et al. (3) on basil affected by Pseudomonas viridiflava. Bacteria consistently isolated from leaf lesions formed mucoid colonies with a green fluorescent pigment on King's B medium. Strains were gram negative. In LOPAT (levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity) tests (2), all induced a hypersensitive reaction (HR) in tobacco (Nicotiana tabacum L. cv. White Burley) leaves (1), caused soft rot of potato tuber slices, and were negative for levan, oxidase, and arginine dihydrolase. Biochemical tests, API 20NE and API 50 CH (Biomérieux, Marcy l'Etoile, France), were also used for identification. The pathogenicity of three isolates was tested twice by injecting 20-day-old healthy basil plants with a bacterial suspension (107 CFU/ml). Controls were injected with sterile distilled water. Plants were kept at 25 to 28°C and 80 to 100% relative humidity. Forty-eight hours after inoculation, dark brown-to-black lesions were observed only on inoculated plants. The bacterium was reisolated from lesions of all plants tested, fulfilling Koch's postulates. No lesions were observed on controls. To identify the pathogen, a PCR technique was used. The 16SrDNA region was amplified with general bacterial primer pair (63f forward and 1389r reverse) (4) then the PCR products were cloned into Escherichia coli DH5α cells and a recombinant plasmid was sequenced by M13 forward and reverse primers. The sequence was deposited in GenBank (Accession No. HE585219). On the basis of the symptoms, biochemical tests, and 16SrDNA sequence homology (99% sequence similarity with a number of P. viridiflava isolates), the pathogen was identified as P. viridiflava. To our knowledge, this is the first report of bacterial leaf spot of basil in Hungary, which can seriously affect the basil production. References: (1) Z. Klement. Nature 199:299, 1963. (2) R. A. Lelliot et al. Appl. Bacteriol. 29:470, 1966. (3) E. L. Little et al. Plant Dis. 78:831, 1994. (4) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000.

scholarly journals First Report of Pseudomonas syringae pv. coriandricola Causing Bacterial Leaf Spot on Carrot, Parsley, and Parsnip in Serbia

Plant Disease ◽  
2015 ◽  
Vol 99 (3) ◽  
pp. 416-416 ◽  
Author(s):  
T. Popović ◽  
Ž. Ivanović ◽  
M. Ignjatov ◽  
D. Milošević
Keyword(s):  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Bacterial Suspension ◽  
Pathogenicity Test ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
Koch’S Postulates ◽  
First Report ◽  
Vegetable Farm ◽  
Koch's Postulates

During the spring of 2014, a severe leaf spot disease was observed on carrot (Daucus carota), parsley (Petroselinum crispum), and parsnip (Pastinaca sativa) on a 0.5-ha vegetable farm in Vojvodina Province, Serbia. The disease appeared under wet and cool conditions with 5 to 25% of plants infected for each of the three crops. Symptoms were characterized as brown angular leaf spots, ~2 mm in diameter, often limited by veins. Collected symptomatic leaves were rinsed and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in sterile phosphate buffer and streaked onto nutrient agar with 5% (w/v) sucrose (NAS). After isolation, whitish, circular, dome-shaped, Levan-positive colonies consistently formed. Five strains from each host (carrot, parsley, and parsnip) were used for further study. Strains were gram-negative, aerobic, and positive for catalase and tobacco hypersensitive reaction but negative for oxidase, rot of potato slices, and arginine dihydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (3). Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the REP, ERIC, and BOX primers (4) were identical for all strains. Sequence typing of the housekeeping genes gyrB and rpoD (1) was performed for three representative strains (one from each host). Sequences were deposited in the NCBI GenBank database as accessions KM979434 to KM979436 (strains from carrot, parsnip, and parsley, respectively) for the gyrB gene and KM979437 to KM979439 (strains from parsnip, parsley and carrot, respectively) for the rpoD gene. Sequences were compared with pathotype strain Pseudomonas syringae pv. coriandricola ICMP12471 deposited in the Plant Associated and Environmental Microbes Database ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ). BLAST analysis revealed 100% homology for gyrB and 99% homology for rpoD. Pathogenicity was tested with five representative strains from each host on four-week-old plants of carrot (cv. Nantes), parsley (cv. NS Molski), and parsnip (cv. Dugi beli glatki) using two methods: spraying the bacterial suspension (108 CFU ml−1) on the leaves until runoff (5) and injecting the bacterial suspension into leaves with a hypodermic syringe (2). Four plants were used per strain and method. Sterile distilled water was applied as a negative control treatment for each plant species. All plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse at 25°C and 80% relative humidity and examined for symptom development over a period of three weeks. For all strains, inoculated leaves first developed water-soaked lesions on the leaves 5 to 7 days after inoculation (DAI); 14 DAI lesions became dark brown, often surrounded by haloes. No symptoms were observed on control plants inoculated with sterile distilled water. For fulfillment of Koch's postulates, re-isolations were done onto NAS. Re-isolated bacteria were obtained from each inoculated host and confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR fingerprinting profiles. Based on the pathogenicity test accompanied by completion of Koch's postulates, sequence analysis, and bacteriological tests, the strains were identified as P. s. pv. coriandricola. To our knowledge, this is the first report of bacterial leaf spot of carrot, parsley, and parsnip in Serbia. It may present a threat to production due to quality requirements for fresh market. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) M. Gupta et al. Plant Dis. 97:418, 2013. (3) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (4) F. J. Louws et al. Appl. Environ. Microb. 60:2286, 1994. (5) X. Xu and S. A. Miller. Plant Dis. 97:988, 2013.

scholarly journals First Report of Bacterial Leaf Spot of Lettuce (Lactuca sativa) Caused by Xanthomonas campestris pv. vitians in Saudi Arabia

Plant Disease ◽  
2009 ◽  
Vol 93 (1) ◽  
pp. 107-107 ◽  
Author(s):  
M. Al-Saleh ◽  
Y. Ibrahim
Keyword(s):  
Saudi Arabia ◽  
Lactuca Sativa ◽  
Leaf Spot ◽  
Xanthomonas Campestris ◽  
Bacterial Suspension ◽  
Strictly Aerobic ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
Biochemical Tests ◽  
First Report

In April of 2008, lettuce (Lactuca sativa L. cv. Darkland) plants grown in the Al-Ouunia Region of Saudi Arabia were observed with numerous lesions typical of bacterial leaf spot. Leaf lesions were irregular, small, pale green to black, and 2 to 5 mm in diameter. Bacteria were isolated from diseased leaf tissues by cutting leaves into small pieces (0.5 mm) and soaking them in 2 ml of sterile distilled water. The resulting suspension was streaked onto yeast dextrose calcium carbonate agar (YDC) (1) and plates were incubated at 28°C. Large, round, butyrus, bright yellow colonies typical of Xanthomonas spp. formed after 48 h and five strains were selected for further tests. A yellow, mucoid bacterium was consistently isolated from lettuce samples with typical bacterial leaf spot symptoms. All five strains tested in this study were gram negative, oxidase negative, nitrate reduction negative, catalase and esculin hydrolysis positive, motile, and strictly aerobic. All were slightly pectolytic but not amylolytic. All were identified as Xanthomonas campestris pv. vitians. The bacterium was identified with specific oligonucleotide primers (2). This primer pair directed the amplification of an approximately 700-bp DNA fragment from total genomic DNA of all X. campestris pv. vitians strains tested. Pathogenicity tests were performed by using bacterial cultures grown on YDC for 48 h at 28°C. Each strain was suspended in sterile distilled water and the bacterial concentration was adjusted to 106 CFU/ml. Leaves of 5-week-old lettuce plants (cv. Darkland) were sprayed with the bacterial suspension. The inoculated and sterile-water-sprayed control plants were covered with polyethylene bags for 48 h at 25°C, after which the bags were removed and plants were transferred to a greenhouse at 25 to 28°C (1). All strains were pathogenic on the lettuce cv. Darkland, causing typical bacterial leaf spot symptoms by 2 weeks after inoculation. All inoculated plants showed typical symptoms of bacterial leaf spot and symptoms similar to those observed on the samples collected. No symptoms developed on the control plants. The bacterium was reisolated from inoculated plants and identified as X. campestris pv. vitians by morphological, physiological, and biochemical tests as described above. To our knowledge, this is the first report of bacterial leaf spot of lettuce by X. campestris pv. vitians in Saudi Arabia. References: (1) F. Sahin and A. Miller. Plant Dis.81:1443, 1997. (2) J. D. Barak. Plant Dis.85:169, 2001.

scholarly journals First Report of Pseudomonas syringae pv. aptata Causing Bacterial Leaf Spot on Sugar Beet in Serbia

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 281-281 ◽  
Author(s):  
V. Stojšin ◽  
J. Balaž ◽  
D. Budakov ◽  
Slaviša Stanković ◽  
I. Nikolić ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Leaf Surface ◽  
Negative Control ◽  
Gyrb Gene ◽  
Bacterial Suspension ◽  
Bacterial Strains ◽  
Gene Sequences ◽  
Bacterial Leaf Spot

A severe bacterial leaf spot was observed during June and July 2013 on commercial cultivars of sugar beet (Beta vulgaris var. saccharifera) in the Vojvodina Province of Serbia. Serbia is a major sugar beet production area in southeastern Europe, with 62,895 ha and 3 million tons of sugar beet yield in 2013. A foliar leaf spot observed in 25 commercial sugar beet fields surveyed ranged from 0.1 to 40% severity. Symptoms were characterized as circular or irregular, 5- to 20-mm diameter, white to light brown necrotic spots, each with a dark margin. Diseased leaves were rinsed in sterilized, distilled water (SDW) and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in SDW. Isolations from 48 symptomatic leaves onto nutrient agar with 5% (w/v) sucrose (NAS) produced bacterial colonies that were whitish, circular, dome-shaped, and Levan-positive. Representative isolates (n = 105) were Gram negative; aerobic; positive for catalase, fluorescence on King's medium B, and tobacco hypersensitivity; and negative for oxidase, potato rot, and arginine dehydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (2). Repetitive extragenic palindromic sequence (rep)-PCR was used for genetic fingerprinting the isolates using the REP, ERIC, and BOX primers. Twenty-five different profiles were obtained among the strains. From each profile group, one representative strain was sequenced for the gyrB gene (1). Four heterogenic groups were observed, and representative gyrB gene sequences of each group were deposited in the NCBI GenBank (Accession Nos. KJ950024 to KJ950027). The sequences were compared with those of pathotype strain P. syringae pv. aptata CFBP 1617 deposited in the PAMDB database; one strain was 100% homologous, and the other three were 99% homologous. To fulfill identification of the Serbian sugar beet isolates, gltA and rpoD partial gene sequences were determined (1), and the sequences were deposited as Accession Nos. KM386838 to KM386841 for gltA and KM386830 to KM38683033 for rpoD. The sequences were 100% homologous with those of pathotype strain CFBP 1617. Pathogenicity of each of four representative bacterial strains was tested on 3-week-old plants of the sugar beet cultivars Marinela, Serenada, and Jasmina (KWS, Belgrade, Serbia) and Lara (NS Seme, Novi Sad, Serbia) by atomizing a bacterial suspension of ~106 CFU/ml of the appropriate isolate onto the abaxial leaf surface of three plants per cultivar until water-soaking of the leaf surface was observed. Three plants of each cultivar atomized similarly with P. syringae pv. aptata CFBP 2473 and SDW served as positive and negative control treatments, respectively. Inoculated plants were kept in a clear plastic box at 80 to 100% RH and 17 ± 1°C and examined for symptom development over 3 weeks. For all test isolates and the control strain, inoculated leaves first developed water-soaked lesions 7 days after inoculation (DAI). By 10 to 14 DAI, lesions were necrotic and infection had spread to the petioles. By 21 DAI, wilting was observed on more than 50% of inoculated plants. Negative control plants were symptomless. Bacteria re-isolated onto NAS from inoculated leaves had the same colony morphology, LOPAT results, and gyrB partial gene sequences as described for the test strains. No bacteria were re-isolated from negative control plants. Based on these tests, the pathogen causing leaf spot on sugar beet in Serbia was identified as P. syringae pv. aptata. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.

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

Plant Disease ◽  
2012 ◽  
Vol 96 (7) ◽  
pp. 1064-1064 ◽  
Author(s):  
I.-S. Myung ◽  
J. Y. Lee ◽  
H. L. Yoo ◽  
J. M. Wu ◽  
H.-S. Shim
Keyword(s):  
Leaf Spot ◽  
Xanthomonas Campestris ◽  
Negative Control ◽  
Gyrb Gene ◽  
23S Rrna ◽  
Distilled Water ◽  
Bacterial Leaf Spot ◽  
Field Isolates ◽  
Sequence Identity ◽  
Physiological Tests

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.

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