First Report of Bacterial Wilt on Chrysanthemum Caused by Dickeya chrysanthemi (syn. Erwinia chrysanthemi) in Hungary

Chrysanthemum (Chrysanthemum spp.) is a popular potted and cut plant ornamental in Hungary. In September 2012, chrysanthemum plants (Chrysanthemum morifolium Ramat. cv. Palisade) showing wilt symptoms were collected from different greenhouses in the cities of Budakalász and Pilis near Budapest. Affected plants had dark brown to black lesions on the leaves and stems. Spots on the leaves were first water soaked and then became necrotic, and the plants wilted. According to the growers, disease symptoms developed rapidly, resulting in losses of nearly 100%. The disease caused a loss of ~€2,000 for the growers in cities of Budakalász and Pilis in Hungary. Losses for growers and consumers could have reached half a million euros. Ten samples were used for disease diagnosis and bacteria were isolated according to the method of Schaad et al. (3). Briefly, diseased leaf and stem tissues were macerated and streaked onto King's medium B (KB). Colonies on KB were white and non-fluorescent. All 10 strains grew at 26°C, were gram negative, and induced a hypersensitive response on tobacco (Nicotiana tabacum L. ‘White Burley’) leaves (1). Biochemical tests were also used for identification, and the results of API 20E (Biomérieux, Marcy l'Etoile, France), demonstrated that the bacterium belonged to the Enterobacteriaceae. The strain was positive for β-galactosidase and citrate utilization, acetoin and indole production, gelatinase, and utilization of glucose, mannitol, saccharose, melibiose, and arabinose. For molecular identification of the pathogen, the 16S rDNA gene was amplified from strain DCBK-1H with a general primer pair (63f/1389r) (2). The PCR products were cloned into a pGEM T-Easy plasmid vector (Promega, Madison, WI) and transformed into Escherichia coli DH5α cells. A recombinant plasmid (2A2.5) was sequenced using the M13 forward and reverse primers. The sequence was deposited in NCBI GenBank (Accession No. HF913430) and showed 99 to 100% sequence identity with a number of Dickeya chrysanthemi strains found in the database, including type strain HM590189, GQ293897, GQ293898 with 99% similarity and 100% identity with sequence FM946179. On the basis of the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as D. chrysanthemi. Pathogenicity was tested by inoculating the recovered strains onto three 1-month-old, healthy potted chrysanthemum cuttings (C. morifolium cv. Palisade). Four leaves and stem each of three ‘Palisade’ cultivars were inoculated by injecting ~10 μl of a bacteria suspension containing 107 CFU/ml into each leaf and stem. As a negative control, one plant was inoculated with water in each of four leaves and stem. Plants were enclosed in plastic bags and incubated in a greenhouse under 80% shade at 26°C day and 17°C night temperatures. Within 24 h, water-soaked spots appeared on inoculated leaves and the plants were wilted. The water control appeared normal. D. chrysanthemi was re-isolated and identified as described above; thus, Koch's postulates were fulfilled. To our knowledge, this is the first report of bacterial wilt caused by D. chrysanthemi on chrysanthemum in Hungary. References: (1) Z. Klement. Nature 199:299, 1963. (2) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (3) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Bacterial Bark Canker of Walnut Caused by Brenneria nigrifluens in Hungary

Plant Disease ◽

10.1094/pdis-09-13-0949-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 988-988 ◽

Cited By ~ 2

Author(s):

A. Végh ◽

A. Tóth ◽

Á. Zámbó ◽

G. Borsos ◽

L. Palkovics

Keyword(s):

16S Rdna ◽

Juglans Regia ◽

Negative Control ◽

Bacterial Suspension ◽

Lake Balaton ◽

Biochemical Tests ◽

First Report ◽

Potted Plants ◽

Sequence Identity ◽

Plastic Bags

During August 2012, vertical oozing cankers were sporadically observed on trunks and branches of walnut trees (Juglans regia) in the city of Zánka, near Lake Balaton and other parts of Hungary including Budapest, Győr, and Tatabánya cities. Cankers were observed on trunks and branches where brownish-black exudates staining the bark appeared mainly in the summer. Isolations were performed primarily from exudates but also from infected tissues using King's medium B (KB) (3) and EMB medium (2). Colonies similar in appearance to Brenneria nigrifluens (syn.: Erwinia nigrifluens) (1,5) were isolated. The bacterium, first reported in California, was also recorded in Iran, Spain, France, and several Italian locations, on walnut trees. The bacterial strain was gram negative and did not induce a hypersensitive response on tobacco (Nicotiana tabacum L. ‘White Burley’) leaves. The bacterium grew at 26°C. Colonies on KB were white and non-fluorescent, but on EMB medium were a typical dark purple with metallic green sheen. The results of substrate utilization profiling using the API 20E kit (Biomérieux, Marcy l'Etoile, France) showed that the bacterium belonged to the Enterobacteriaceae. The strain was positive for citrate utilization, H2S, and acetoin production and urease, glucose, inositol, saccharose, and arabinose reactions. Pathogenicity was tested by injecting five young healthy walnut branches on two separate 2-year-old grafted potted plants with a bacterial suspension containing 107 CFU/ml. Negative controls were walnut branches injected with sterile distilled water. Branches were enclosed in plastic bags and incubated in a greenhouse under 80% shade at 26°C day and 17°C night temperatures. Three months after inoculation, necrotic lesions were observed in the inner bark and dark lines were observed in internal wood, but no external cankers were observed on inoculated branches. The negative control appeared normal. B. nigrifluens was re-isolated from lesions on inoculated branches and identified as described above; thus, Koch's postulates were fulfilled. For molecular identification of the pathogen, 16S rDNA amplification was performed using genomic DNA from strain Bn-WalnutZa-Hun1 with a universal bacterial primer set (63f and 1389r) (4). The PCR products were cloned into a pGEM T-Easy vector (Promega, Madison, WI) and transformed into Escherichia coli DH5α cells. A recombinant plasmid (2A2.5) was sequenced using M13 forward and reverse primers. The sequence was deposited in NCBI GenBank (Accession No. HF936707) and showed 99% sequence identity with a number of B. nigrifluens strains, including type strains Z96095.1, AJ233415.1, JX484740.1, JX484739.1, JX484738.1, and FJ611884.1. On the basis of the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence identity, the pathogen was identified as Brenneria nigrifluens. To our knowledge, this is the first report of a natural outbreak of bacterial bark canker on walnut in Hungary and the presence of the pathogen may seriously influence in local orchards and garden production in the future. References: (1) L. Hauben et al. Appl Microbiol 21:384, 1998. (2) J. E. Holt-Harris and O. Teague. J. Infect. Dis. 18:596, 1916. (3) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954. (4) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (5) E. E. Wilson et al. Phytopathology 47:669, 1957.

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First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Mesona chinensis in China

Plant Disease ◽

10.1094/pdis-08-10-0603 ◽

2011 ◽

Vol 95 (2) ◽

pp. 222-222

Author(s):

Q. Liu ◽

Y. Li ◽

J. Chen

Keyword(s):

16S Rdna ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Vascular Tissue ◽

Negative Control ◽

Bacterial Suspension ◽

Herbaceous Plant ◽

Disease Etiology ◽

Tetrazolium Chloride ◽

First Report

Jellywort (Mesona chinensis Benth) is an herbaceous plant in the Lamiaceae (mint) family. The plant is referred to as ‘Xiancao’ (weed from angels) in Chinese and primarily used to make grass jelly, a popular refreshing drink. Currently, Xiancao cultivation is a fast-growing industry with a high profit margin in southern China. An estimated 7,000 ha is grown with a value of more than $50 million USD. In June, 2009, a wilting disease of Xiancao was observed in Guangdong and the neighboring Guangxi and Fujian provinces with incidence up to 50% in the severest case. Affected plants initially show withering symptoms on apical leaves during the daytime with recovery at night. As the disease develops, withering leaves spread downward, eventually encompassing the whole plant. Leaves lose vigor but remain green. After 3 to 4 days, wilting becomes irreversible. Roots and basal stem tissues blacken and rot, leading to plant death. Longitudinal sectioning of the basal stem shows browning of vascular tissues with whitish ooze visible when compressed. To investigate the disease etiology, 12 Xiancao plants from three cultivars showing typical wilting symptoms were collected from a production field in Zengcheng City of Guangdong Province in June 2010. A total of 27 bacterial isolates showing large, elevated, and fluidal colonies with a pale red center were isolated from vascular tissue on tripheny tetrazolium chloride medium (3) after incubation at 30° for 2 days. Fifteen 45-day-old Xiancao plants (cv. Zhengcheng 1) were inoculated by injection of 20 μl of bacterial suspension (1 × 108 CFU/ml) into the middle stem. Sterile water was used as a negative control. After 4 to 6 days of incubation in a greenhouse (28 to 30°), all (15 of 15) inoculated plants developed wilting symptoms as described above. The same bacterium was reisolated from inoculated plants. The five negative control plants did not show any wilting symptoms. With the same artificial inoculation procedure, this bacterium also caused similar wilting disease in tobacco, potato, tomato, pepper, and eggplant. An inoculation test with a tomato strain of Ralstonia solanacearum resulted in similar symptoms. On the basis of symptomatology and bacterial culture characteristics, R. solanacearum (formerly Pseudomonas solanacearum) was suspected as the causal agent. For confirmation, the universal bacterial 16S rDNA primer set E8F/E1115R (1) was used to amplify DNA from pure culture. A 1,027-bp DNA sequence was obtained and deposited in GenBank with Accession No. HQ159392. BLAST search against the current version of GenBank database showed 100% similarity with the 16S rDNA sequences of 26 R. solanacearum strains. Furthermore, primer set 759/760 (4) amplified a specific 280-bp fragment. Along with the result from multiplex PCR (2), the bacterium was identified as R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on M. chinensis. References: (1) G. Baker et al. J. Microbiol. Methods 55:541, 2003. (2) M. Fegan and P. Prior. Page 449 in Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. The American Phytopathological Society. St. Paul, MN, 2005. (3) A. Kelman, Phytopathology 44:693, 1954. (4) N. Opina et al. Asia Pac. J. Mol. Biol. Biotechnol. 5:19, 1997.

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First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Ageratum conyzoides in China

Plant Disease ◽

10.1094/pdis-08-12-0780-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 418-418

Author(s):

X.-M. She ◽

Z.-F. He ◽

F.-F. Luo ◽

H.-P. Li

Keyword(s):

16S Rdna ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Vascular Tissue ◽

Negative Control ◽

Plant Diseases ◽

Bacterial Isolates ◽

Ageratum Conyzoides ◽

First Report ◽

Initial Symptoms

Ageratum conyzoides L. is believed to act as reservoir host for many plant diseases. In June 2011, a 30% incidence of bacterial wilt on A. conyzoides was observed in a field of Rhizoma kaempferiae in Yangchun city of Guangdong province. The initial symptoms were wilting of the apical leaves during the day, which recovered at night. After 4 to 6 days, the leaves became totally necrotic. The basal stems of the diseased plants were blackened and the vascular tissue turned brown. To investigate the disease etiology before understanding the disease link between A. conyzoides and R. kaempferiae, 10 plants with typical wilting symptoms were collected from the field. A total of 10 bacterial isolates were isolated from the vascular tissue of each diseased plant on tripheny tetrazolium chloride (TZC) medium. After incubation at 30°C for 2 days, the plates had large, irregular round, fluidal, white colonies with a pink center. Thirty healthy A. conyzoides plants at the four- to six-leaf growth stage were inoculated by injuring the roots and soaking them in a bacterial suspension (1 × 108 cfu/ml) for 20 min with the 10 bacterial isolates separately, and planted in 10-cm pots with sterile gardening soil in a glasshouse (28 to 35°C). Sterile water was used as a negative control. Five days after inoculation, a few leaves of the inoculated plants began to exhibit wilting. The inoculated plants eventually showed the same symptoms as those in the field. The same bacterium was reisolated from inoculated plants. The 30 negative control plants did not have wilt symptoms. With the same inoculation procedure, the bacterium also caused wilting on tomato (25 of 30), pepper (10 of 30), eggplant (2 of 30), ginger (11 of 15), and R. kaempferiae (8 of 15). Using the universal bacterial 16S rDNA primer set 27f/1541R (3), approximately 1,400 bp-fragments were amplified from the 10 isolates, respectively. The sequences for the 10 fragments (GenBank Accession Nos. JX294065 to JX294074) were identical and had 100% sequence identity with 16S rDNA of R. solanacearum GMI1000 (AL646052). The 10 isolates were able to oxidize disaccharides (lactose, maltose, and cellobiose) and hexose alcohols (mannitol, dulcitol, and sorbitol). According to Hayward's classification, all isolates were biovar 3 (2). Based on the pathogenicity tests, carbohydrate utilization, and near full-length 16S rDNA sequences, the bacterial isolates from the diseased A. conyzoides belonged to race 4 and biovar 3 of R. solanacearum. Furthermore, the specific 280-bp and 140-bp fragments were respectively amplified from all 10 isolates by using the multiplex PCR (1). In addition, specific 165-bp fragments were amplified from all the isolates using the specific primers AKIF/AKIR (3), which indicates the bacterium belongs to R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on A. conyzoides in China. References: (1) M. Fegan and P. Prior. Page 449 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. The American Phytopathological Society. St. Paul, MN, 2005. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) M. Horita et al. J. Gen. Plant Pathol. 70:278, 2004.

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First Report of Erwinia amylovora Causing Fire Blight on Plum (Prunus domestica) in Hungary

Plant Disease ◽

10.1094/pdis-12-11-1013-pdn ◽

2012 ◽

Vol 96 (5) ◽

pp. 759-759 ◽

Cited By ~ 3

Author(s):

A. Végh ◽

Zs. Némethy ◽

L. Hajagos ◽

L. Palkovics

Keyword(s):

16S Rdna ◽

Sequence Homology ◽

Fire Blight ◽

Plasmid Vector ◽

Fruit Production ◽

Hypersensitive Reaction ◽

Bacterial Suspension ◽

Prunus Domestica ◽

Biochemical Tests ◽

First Report

During July 2011, a severe, unusual disease symptom was observed on young shoots on a 10-year old plum tree (Prunus domestica L. ‘d'Agen’) in the city of Budaörs, near Budapest. The naturally infected shoots showed typical symptoms of fire blight including terminal shoots with brown-to-black necrotic lesions and later, shepherd's crook deformation. Symptoms were the same as fire blight, symptoms reported from other hosts and locations. The first occurrence of fire blight on European plum was recorded in Germany in 2002 (4). Shoots containing regions of dead and healthy tissue were surface sterilized with ethanol (50-mg sample homogenized with 500 μl of sterile water and 50 μl of the homogenate streaked to King's B agar medium). After 48 h of incubation at 26°C, the medium contained pure cultures of a bacterium with white mucoid colonies, which is morphologically consistent with E. amylovora (1). Isolates were gram negative and induced a hypersensitive reaction in tobacco (Nicotiana tabacum L. ‘White Burley’) leaves (2). Biochemical tests were also used for identification, and the results of API 20E and API 50 CH kits (Biomérieux, Marcy l'Etoile, France), demonstrated that the bacterium belongs to Enterobacteriaceae. Pathogenicity was tested by injecting five healthy young plum shoots from the same tree with a 10-μl bacterial suspension of 107 CFU/ml. Controls were injected with sterile distilled water. Shoots were kept at 26°C and 80 to 100% relative humidity. Five days after inoculation, dark brown-to-black lesions and shepherd's crook symptoms were observed only on inoculated shoots. The bacterium was reisolated from lesions on inoculated shoots, fulfilling Koch's postulates. No lesions were observed on controls. For molecular identification of the pathogen, the 16S rDNA region was amplified from isolate EA-PlumBo1 with a general bacterial primer pair (63f forward and 1389r reverse) (3). The PCR products were cloned into a pGEM T-Easy plasmid vector (Promega, Madison, WI) and were transformed into Escherichia coli DH5α cells. A recombinant plasmid (2A2.5) was sequenced by M13 forward and reverse primers. The sequence was deposited in GenBank (Accession No. HE610678) and showed 99 to 100% sequence homology with a number of E. amylovora isolates, including type strain AJ233410 with 99% similarity and 100% homology with sequences FN434113 and FN666575, where the complete genomes are known. On the basis of the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as E. amylovora. To our knowledge, this is the first report of a natural outbreak of fire blight on plum in Hungary and the presence of the pathogen may seriously influence local stone fruit production in the future. References: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954. (2) Z. Klement. Nature 199:299, 1963. (3) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (4) J. L. Vanneste et al. Acta Hortic. 590:89, 2002.

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First Report of Candidatus Phytoplasma solani Associated with Potato Plants in Greece

Plant Disease ◽

10.1094/pdis-05-14-0488-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1739-1739 ◽

Cited By ~ 3

Author(s):

M. C. Holeva ◽

P. E. Glynos ◽

C. D. Karafla ◽

E. M. Koutsioumari ◽

K. B. Simoglou ◽

...

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Pathogenic Bacteria ◽

Related Strain ◽

Northern Greece ◽

First Report ◽

Potato Plants ◽

Pcr Product ◽

16S Rdna Pcr ◽

Stolbur Phytoplasma

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated. References: (1) U. Ahrens and E. Seemuller. Phytopathology 82:828, 1992. (2) A. S. Alivizatos. Pages 945-950 in: Proceedings of the 7th International Conference of Plant Pathogenic Bacteria. Academiai Kiado, Budapest, Hungary, 1989. (3) J. Jović et al. Bull. Insectol. 64:S83, 2011. (4) L. Lotos et al. J. Plant Pathol. 95:447, 2013. (5) E. Vellios and F. Lioliopoulou. Bull. Insectol. 60:157, 2007.

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First Report of Xanthomonas hortorum pv. hederae Causing Bacterial Leaf Spot of Hedera hibernica in Slovenia

Plant Disease ◽

10.1094/pdis-06-11-0498 ◽

2012 ◽

Vol 96 (1) ◽

pp. 141-141 ◽

Cited By ~ 2

Author(s):

M. Pirc ◽

T. Dreo ◽

M. Šuštaršič ◽

J. Erjavec ◽

M. Ravnikar

Keyword(s):

Type Strain ◽

Negative Control ◽

The European Union ◽

Bacterial Strains ◽

Biochemical Tests ◽

Adaxial Side ◽

Banding Patterns ◽

First Report ◽

Dna Fragment ◽

Pale Green

In October 2008, water-soaked spots, each 5 to 10 mm in diameter and surrounded by a pale green halo, were observed on leaves of a single Atlantic ivy plant (Hedera hibernica Kirchn.) out of 89 plants imported from the European Union into a commercial greenhouse in Slovenia. Leaves were surface disinfected, and spots were cut and suspended in 10 mM phosphate buffered saline. From this extract, yellow-pigmented, Xanthomonas-like bacterial colonies were isolated onto nutrient agar, and two colonies (NIB Z 1310 and NIB Z 1312) were each identified as a Xanthomonas sp. based on biochemical tests (oxidase negative; positive for hydrolysis of H2S, starch, and tributyrin; and positive for acid production from sucrose). Both isolates caused a hypersensitive reaction (1) on leaves of tomato cv. Moneymaker. A repetitive extragenic palindromic sequence (REP)-PCR assay using the BOXA1R primer (3) resulted in highly similar DNA fragment banding patterns (Pearson's correlation: 95% identity) between the two isolates (NIB Z 1310 and NIB Z 1312) and the type strain of Xanthomonas hortorum pv. hederae CFBP 4925 (ICMP 453). Partial sequences of the gyrB gene (DNA gyrase, subunit B) (2) from isolates NIB Z 1310 (Accession No. JF794785; 599 bp) and NIB Z 1312 (Accession No. JF794784; 544 bp), showed identical sequences (100% identity with 100% coverage) to type strain ICMP 453 (Accession No. EU498975.1). The pathogenicity of the two isolates from H. hibernica was confirmed on three plants of H. helix ‘Evita’ (each 6 months old) for each isolate. Plants were sprayed on the abaxial and adaxial side of leaves with 10 ml of a 48-h suspension of the appropriate isolate with approximately 106 CFU/ml (1), covered individually with plastic bags for 24 h, and incubated under high relative humidity (>80%) with 16 h of daylight at 25°C by day and 20°C by night. Three positive and three negative control plants were inoculated with the type strain of X. hortorum pv. hederae CFBP 4925 and 0.01 M magnesium sulfate buffer, respectively. After 21 days, water-soaked spots with a pale green halo were observed on all plants inoculated with the bacterial strains, including the positive control plants. Colonies isolated from these lesions were identical in morphology and BOX-PCR DNA fragment banding patterns to the original isolates. Negative control plants did not develop symptoms, and colonies similar to X. hortorum pv. hederae were not isolated from these plants. To our knowledge, this is the first report of X. hortorum pv. hederae on H. hibernica in greenhouse production in Slovenia. If the disease spreads, it could reduce quality and marketability of this popular ground cover plant. References: (1) Z. Klement et al. Inoculation of Plant Tissues. In: Methods in Phytobacteriology. Akadémiai Kiadó, Budapest, 1990. (2) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (3) J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.

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First Report of Brenneria nigrifluens as the Causal Agent of Shallow-Bark Canker on Walnut Trees (Juglans regia) in Serbia

Plant Disease ◽

10.1094/pdis-03-13-0267-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1504-1504 ◽

Cited By ~ 4

Author(s):

T. Popović ◽

Ž. Ivanović ◽

S. Živković ◽

N. Trkulja ◽

M. Ignjatov

Keyword(s):

16S Rdna ◽

Juglans Regia ◽

Causal Agent ◽

Nutrient Agar ◽

Gene Sequences ◽

Biochemical Tests ◽

First Report ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Plant Pathol

In late summer 2011, shallow, irregular cankers were observed on trunks and branches of non-chemically-treated walnut trees (Juglans regia L.) on a 30-year-old orchard in the region of Fruška Gora (Vojvodina, Serbia). Disease incidence was ~80% and yield loss was ~50%. For pathogen isolation, small pieces (~5 mm diameter) of wood tissue collected at the edge of the cankers were macerated in sterile distilled water and streaked onto nutrient agar with 5% sucrose. Plates were then incubated at 28°C for 2 days. The prevalent bacterial colonies and those similar in appearance to Brenneria nigrifluens (Wilson et al.) Hauben et al. were purified on nutrient agar (NA). Eight gram-negative, oxidasenegative, catalase-positive strains, showing oxidative and fermentative metabolism, were selected for further characterization. To identify the bacteria on a molecular basis, we analyzed the 16S rDNA and gyr B gene sequences. The 16S rDNA partial sequences of analyzed strains were amplified using the primers P0 (5′-GAGAGTTTGATCCTGGCTCAG-3′) and P6 (5′-CTACGGCTACCTTGTTACGA-3′) (3). Additionally, the gyr B gene sequences were generated with primers GyrB-F (5′-MGGCGGYAAGTTCGATGACAAYTC-3′) and GyrB-R (5′-TRATBKCAGTCARACCTTCRCGSGC-3′) (2). All amplicons were purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions and sequenced by Macrogen Inc. (Seoul, South Korea) using the same primers used for amplification. The sequences were edited using FinchTV v.1.4.0, assembled using the Clustal W program integrated into MEGA5 software (4), and deposited in NCBI GenBank under accessions JX484738 to 40 for the 16S rDNA gene and KC571240 to 47 for the gyr B gene. The 1,359-bp 16S rDNA sequences obtained for the eight strains were compared to the reference 16S rDNA sequences retrieved from GenBank. BLAST analysis revealed 100% homology of Serbian strains with sequences of B. nigrifluens (Z96095 and FJ611884). The gyr B gene sequences of our strains were 100% homologous to the sequences of B. nigrifluens deposited in GenBank (JF311612 to 15). Pathogenicity of all strains was confirmed on young fruits by infiltration of bacterial suspensions (108 CFU ml–1 from a 48 h NA culture) with syringe into the mesocarp of walnut fruits and by stem infiltration with syringes without needles into branch wounds (1). Inoculated fruits were incubated in plastic boxes for 8 days at 20°C, 80 to 100% RH, with a 12-h photoperiod. Inoculated plants were maintained for 3 months at 22 to 28°C with continuous light and at 70 to 80% RH in plastic tunnels. Inoculated fruits developed bark canker symptoms at the inoculation sites, which became necrotic and released a reddish brown exudate. Necrotic lesions were observed on inoculated branches. B. nigrifluens was reisolated from the margins of necrotic fruit and stem tissue. Physiological and biochemical tests showed that strains grew at 36°C and did not produce arginine dihydrolase, H2S, indole, nitrate, nor a fluorescent pigment on King's B medium. They did not induce a hypersensitive reaction on tobacco leaves and did not hydrolyse gelatin and starch. They produced acid without gas from glucose, inositol, sorbitol, arabinose, and sucrose, but not from maltose and lactose (1). Results of pathogenicity and biochemical tests were also the same for reisolated strains. This is the first report of B. nigrifluens as the causal agent of shallow-bark canker on walnut trees in Serbia. References: (1) E. G. Biosca and M. M. López. J. Plant Pathol. 94:105, 2012. (2) P. Ferrente and M. Scotrichini. Plant Pathol. 59:954, 2010. (3) A. Grifoni et al. FEMS Microbiol. Lett. 127:85, 1995. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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First Report of Bacterial Spot Caused by Xanthomonas campestris pv. vesicatoria on Sweet Pepper (Capsicum annuum L.) in Saudi Arabia

Plant Disease ◽

10.1094/pdis-04-12-0354-pdn ◽

2012 ◽

Vol 96 (11) ◽

pp. 1690-1690 ◽

Cited By ~ 1

Author(s):

Y. Ibrahim ◽

M. Al-Saleh

Keyword(s):

Saudi Arabia ◽

Xanthomonas Campestris ◽

Sweet Pepper ◽

Negative Control ◽

Bacterial Suspension ◽

Distilled Water ◽

Bacterial Spot ◽

Biochemical Tests ◽

First Report ◽

Pepper Fruit

In the summer of 2009 and 2010, 18 sweet pepper fruit with blister-like, raised, rough lesions were collected from four greenhouses (total of 0.1 ha) in the Al-Kharj region of Saudi Arabia. All samples were collected from commercial crops of the sweet pepper cv. California Wonder. Disease incidence was ≤5%. Isolations were made from all diseased fruits. A small piece (3 mm2) of symptomatic tissue from pepper fruit was placed in a sterile mortar and macerated in sterile distilled water with a pestle. A loopful of bacterial suspension from each sample was streaked onto Tween B agar medium (3). Plates were incubated at 28°C for 48 h. Single yellow, circular, butyrous, shiny colonies were picked from the plates and transferred to nutrient agar plates containing 5% D+ glucose agar (NGA). Gram-negative, rod-shaped bacteria were consistently isolated from the fruit and 10 of the isolates were identified as Xanthomonas campestris pv. vesicatoria on the basis of morphological, physiological, and biochemical tests (1,2). The isolates were oxidase positive and levan negative, arginine-dihydrolase positive, and did not macerate potato discs. The isolates were also non-fluorescent, grew at 37 and 4°C but not at 40°C, did not liquefy gelatine or starch, but did produce H2S. The identity of the 10 bacterial strains was confirmed by PCR assay using primers RST65 and RST69 (4). Four-week old pepper plants (cv. California Wonder) were inoculated by spraying five potted plants with each isolate using a bacterial suspension (108 CFU/ml). Sterile distilled water was sprayed on an additional five plants as a negative control treatment. The bacterial isolates caused necrotic lesions, each with a yellow halo, on leaves of inoculated plants. Bacteria reisolated from the necrotic lesions using the technique previously described were identical to the original strains according to the morphological, cultural, and biochemical tests described above. Negative control plants inoculated with sterile distilled water did not show symptoms and no bacterial colonies were recovered from them. To our knowledge, this is the first report of bacterial spot on pepper fruits in Saudi Arabia. References: (2) R. F. Bradbury. Genus II Xanthomonas Dowson 1939. In: Bergey's Manual of Systematic Bacteriology, Vol. 1, Krieg, R., Holt, J. G. (Eds.), Williams & Wilkins Co., Baltimore, MD, 1987. (3) R. A. Lelliott and D. E. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications, Oxford, UK. (1) R. G. McGuire et al. Plant Dis 70:887, 1986. (4) A. Obradovic et al. Eur. J. Plant Pathol. 110:285, 2004.

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First Report of the Purple Variant of Curtobacterium flaccumfaciens pv. flaccumfaciens, Causal Agent of Bacterial Wilt of Bean, in Canada

Plant Disease ◽

10.1094/pd-90-1262a ◽

2006 ◽

Vol 90 (9) ◽

pp. 1262-1262 ◽

Cited By ~ 9

Author(s):

H. C. Huang ◽

R. S. Erickson ◽

L. J. Yanke ◽

C. D. Chelle ◽

H.-H. Mündel

Keyword(s):

Bacterial Wilt ◽

Blue Pigment ◽

Bacterial Suspension ◽

Water Control ◽

First Report ◽

Randomized Design ◽

Conventional Tests ◽

Completely Randomized Design ◽

Hydrolysis Of ◽

Pooled Sample

Bacterial wilt of bean (Phaseolus vulgaris L.) caused by the yellow and orange variants of Curtobacterium flaccumfaciens pv. Flaccumfaciens (Hedges) Collins & Jones was found in western Canada in 2002 (1). A purple variant was found in a pooled sample of discolored cull seeds of great northern bean (cv. US1140) from a crop grown near Bow Island, Alberta, Canada in 2005. Bacterial colonies isolated from purple seed using modified Burkholder's agar (MBA) (3) were convex, glistening, and smooth edged with blue pigment diffusing into the medium. Three isolates (V154, V155, and V254) were identified with conventional tests (2), carbohydrate oxidation (GP Microplates, Biolog Inc., Hayward, CA), and cellular fatty acids (CFA) (MIDI, Inc., Newark, DE). All were grampositive, motile, aerobic rods with yellow colonies producing extracellular blue pigment on MBA when grown at 20 ± 2°C. Bacterial isolates grew at 27°C but grew weakly at 37°C. They were positive for catalase and hydrolysis of hippurate and indoxyl acetate and negative for urease, gelatin liquification, and oxidase. CFA profiles were approximately 48% 15:0 anteiso, 40% 17:0 anteiso, 7% 16:0 iso, and 3% 15:0 iso; with 17:1 anteiso A variable but <1%. Many carbohydrates were oxidized in the Biolog microplates with little acid production. The results match C. flaccumfaciens (2) and the MIDI and Biolog databases, as well as the purple variant of C. flaccumfaciens found in Nebraska, the only previous report of this variant (4). The pathogenicity of the three isolates was tested. Seeds of great northern (cv. US1140) and navy (cv. Morden003) beans were soaked in a bacterial suspension (1 × 108 CFU/ml) or distilled water (control) for 1 h, planted in Cornell mix in root trainers, incubated at 28/22°C (16-h day/8-h night) in a growth cabinet for 14 days, and examined for seedling wilt. The test had three replicates per treatment and 20 seeds per replicate in a completely randomized design. All three isolates were pathogenic to both bean cultivars. The wilt incidences were 51, 57, and 56% on US1140 and 64, 76, and 69% on Morden003 for isolates V154, V155, and V254, respectively. The purple variant of C. flaccumfaciens was reisolated from hypocotyls of wilted seedlings but not from healthy controls. The experiment was repeated using the reisolated bacteria and the results were similar to the first experiment, thus fulfilling Koch's postulates. To our knowledge, this is the first report of the purple variant of C. flaccumfaciens pv. flaccumfaciens in Canada. References: (1) T. F. Hsieh et al. Plant Dis. 86:1275, 2002. (2) K. Komagata et al. Page 1313 in: Bergey's Manual of Systematic Bacteriology. Vol. 2. Williams and Wilkens, Baltimore, MD, 1986. (3) G. A. Nelson and G. Semeniuk. Phytopathology 54:330, 1964. (4) M. L. Schuster et al. Can. J. Microbiol. 14:423, 1968.

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First Report of Bacterial Fruit Blotch of Watermelon Caused by Acidovorax avenae subsp. citrulli in Hungary

Plant Disease ◽

10.1094/pdis-92-5-0834c ◽

2008 ◽

Vol 92 (5) ◽

pp. 834-834 ◽

Cited By ~ 6

Author(s):

L. Palkovics ◽

M. Petróczy ◽

B. Kertész ◽

J. Németh ◽

Cs. Bársony ◽

...

Keyword(s):

Sequence Similarity ◽

Citrullus Lanatus ◽

Acid Methyl Ester ◽

Negative Control ◽

Sterile Water ◽

Biochemical Tests ◽

Cucumis Sativus L ◽

Green Pepper ◽

First Report ◽

Bacterial Fruit Blotch

Typical bacterial fruit blotch (BFB) symptoms were observed on cvs. Crisby, Suzy, Top Gun, and Lady watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai.) fruits in southeastern Hungary (Medgyesegyháza) in July 2007 when the mean maximum daytime temperature was greater than 32°C. Approximately 20 to 30 ha of watermelons were observed to be affected by the disease. Source of the infection was not determined, however, grafted watermelon transplants that were grown in this area had been imported from Turkey where the pathogen is present (2). Disease symptoms started with irregularly shaped, water-soaked lesions on the surface of the fruits. The lesions enlarged and the epidermis became brown and cracked. BFB symptoms were not readily visible on mature foliage. Colonies of the BFB pathogen were creamy white on nutrient agar (Difco, Detroit, MI). Strains were gram negative, oxidase positive, and produced acid from glucose aerobically. A cell suspension (50 μl of ~1 × 107 CFU/ml) from a 24-h nutrient plate culture was infiltrated with a hypodermic syringe into the intercellular spaces of fully developed intact tobacco (Nicotiana tabacum L. cv. White Burley) leaves to determine the hypersensitive reaction (HR) (1). A typical HR developed 20 h after leaf infiltration. Bacterial pathogenicity was tested on surface-sterilized, mature fruits of different plant species by injecting cell suspensions into the fruit tissues as previously described (each fruit was injected in five places; the negative control (sterile water) as well). Fruits were incubated for 7 days at 25°C and then observed for symptom development. Necrosis was observed at each point of inoculation with the pathogen for watermelon and green pepper (Capsicum annuum L.). Necrosis was also observed for cucumber (Cucumis sativus L.), zucchini (Cucurbita pepo L. convar. giromontiina Greb.), squash (C. pepo L.), and patisson (C. pepo L. convar. patissoniana Greb.). Necrosis was not observed when the pathogen was inoculated onto fruit of melon (Cucumis melo L.), tomato (Lycopersicon esculentum Mill.), and eggplant (Solanum melongena L.). Additionally, symptoms were not observed at the points inoculated with sterile water (negative control) for any of the fruits tested. To identify the pathogen, PCR was used with Acidovorax avenae subsp. citrulli-specific primers WFB1/2 (4). The 16s rDNA region amplified with a general bacterial primer pair (63f forward and 1389r reverse) (3) was cloned into a pBSK+ vector (Stratagene, La Jolla, CA) and sequenced by M13 forward and reverse primers (GenBank Accession No. AM850114). On the basis of the symptoms, biochemical tests (API 20NE; Biomérieux, Marcy l'Etoile, France), fatty acid methyl ester analysis (74.5 to 83.6% similarity), and 16SrDNA sequence homology (100% sequence similarity with AAC00-1), the pathogen was identified as A. avenae subsp. citrulli. To our knowledge, this is the first report of BFB of watermelon in Hungary. References: (1) Z. Klement. Nature 199:299, 1963. (2) M. Mirik et al. Plant Dis. 6:829, 2006. (3) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (4) R. R. Walcott and R. D. Gitaitis. Plant Dis. 84:470, 2000.

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