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

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

First Report of a Leaf Spot of Radermachera sinica in China Caused by Bacillus megaterium

Plant Disease ◽

10.1094/pdis-05-14-0469-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1425-1425 ◽

Cited By ~ 3

Author(s):

Y. L. Li ◽

Z. Zhou ◽

Y. C. Yuan ◽

J. R. Ye

Keyword(s):

16S Rdna ◽

Bacillus Megaterium ◽

Leaf Spot ◽

Disease Incidence ◽

Bacterial Suspension ◽

Distilled Water ◽

Biochemical Tests ◽

16S Rdna Gene ◽

First Report ◽

Rdna Gene

Radermachera sinica is widely planted as an ornamental plant in homes, offices, and malls in China. A leaf spot of R. sinica occurred in Luoyang, China, from 2013 to 2014. Lesions mostly occurred in wounds and were irregular with light brown centers and purple borders. One or more lesions on a leaf sometimes covered the entire blade. Eighty plants were surveyed in Luoyang, with disease incidence of 17%. Five millimeter pieces from the borders of lesions were surface-disinfected with 75% ethanol for 30 s, 1% sodium hypochlorite for 5 min, washed three times in sterilized distilled water, placed on nutrient agar (NA) medium at 25°C in darkness, and incubated for 24 to 48 h. Four white, round, smooth, and shiny colonies were selected for further identification. All strains were gram-positive, aerobic rods with many peritrichous flagella, and could grow in medium containing 5% NaCl. The strains were positive for catalase, starch hydrolysis, liquefaction of gelatin, reduction of nitrate, acid production from glucose, mannitol, maltose, lactose, xylose, and pectinose. The strains were positive for phenylalanine deaminase, decomposition of tyrosine, and utilization of citrate. The strains were identified by biochemical tests as Bacillus megaterium (1). To confirm pathogenicity, the strains were grown on NA for 48 h and suspended in sterile distilled water to produce a suspension with a final concentration of 108 CFU/ml. Healthy leaves of biennial R. sinica plants were sterilized with 75% ethanol and washed three times with sterilized distilled water. Fresh wounds were made with a sterile needle on the healthy leaves. Each of four strains was tested by spray inoculation with a bacterial suspension on three leaves. Sterile distilled water was used as negative control. Plants were enclosed in plastic bags and placed in a growth chamber at 28°C with 80% relative humidity. After 5 days, water-soaked lesions were observed. Two weeks later, lesions 4 mm in diameter turned light brown with purple borders, and most of lesions occurred in puncture wounds. Symptoms similar to those observed on field plants developed on all inoculated leaves, while no symptoms appeared on the control leaves. B. megaterium was re-isolated from the lesions of inoculated leaves, but not from the control leaves. To confirm the bacterial identification, PCR was performed on the 16S rDNA gene with P1/P2 (P1: CAGAGTTTGATCCTGGCT, P2: AGGAGGTGATCCAGCCGCA) (2) and 1,463 bp of the 16S rDNA gene (GenBank Accession No. KJ789369) showed 100% sequence identity to B. megaterium DSM 319 (NC_014103.1). To our knowledge, this is the first report of a leaf spot of R. sinica caused by B. megaterium in China as well as anywhere in the world. References: (1) P. Vos et al. Bergey's Manual of Systematic Bacteriology. Vol 3: The Firmicutes. Springer, 2009. (2) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

Download Full-text

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.

Download Full-text

First Report of Bacterial Blight of Guar Caused by Xanthomonas axonopodis pv. cyamopsidis in China

Plant Disease ◽

10.1094/pdis-09-13-0983-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 840-840 ◽

Cited By ~ 2

Author(s):

Y. Z. Ren ◽

Y. L. Yue ◽

G. X. Jin ◽

Q. Du

Keyword(s):

16S Rdna ◽

Bacterial Blight ◽

Negative Control ◽

Bacterial Suspension ◽

Cyamopsis Tetragonoloba ◽

Sterile Water ◽

Bacterial Strains ◽

Xanthomonas Axonopodis ◽

First Report ◽

Pathogenicity Tests

Bacterial blight was observed on field-grown guar (Cyamopsis tetragonoloba L.) for the first time in China. The disease outbreak occurred in the Xinjiang Uyghur Autonomous Region after several weeks of unusually heavy rains during late summer 2013. The disease incidence was generally 40 to 50%, although values as high as 80% were observed in several fields. Initial field symptoms included water-soaked spots on leaves, pods, petioles, and stems. During later stages of infection, the color of the spots became dark. We also observed large, angular, necrotic lesions at leaf tips, black streaks on petioles and stems, split stems, defoliation, wilting or top withering, vascular necrosis, and dieback. Samples of diseased leaves, stems, petioles, pods, and seeds were surface sterilized, ground, and then plated onto King's B medium. Plates were incubated at 28°C for 72 h. Fifteen bacterial strains with yellow-pigmented, opaque, and round colonies were isolated. These strains were aerobic, gram-negative rods with a single, polar flagellum. They were positive for H2S, esculin, oxidase, tobacco hypersensitivity, indole production from tryptophan, nitrate reduction to nitrite, and the utilization of glucose, mannose, trehalose, galactose, and starch. The maximum salt tolerance of the strains was 2 to 3%. Pathogenicity tests using eight strains were conducted in July 2013. A bacterial culture was suspended in sterile water with a final concentration of 108 CFU/ml. Eight 4-week-old guar plants were inoculated by (i) spraying the bacterial suspension on the leaves until runoff, or (ii) puncturing the stems with a needle that had been dipped into the bacterial suspension. Sterile water was used as a negative control. Plants were kept in a mist room with 100% relative humidity for 24 h. Stem and leaf symptoms similar to those of the original plants were observed on the inoculated guar plants within 10 days of inoculation. No symptoms developed on the negative control plants. Yellow bacterial colonies re-isolated from inoculated plant tissues were morphologically identical to the original. 16S rDNA was amplified using universal primers (Pa 5′-AGTTTGATCCTGGCTCAG-3′ and Ph 5′-TACCTTGTTACGACTTCGTCCCA-3′) and sequenced. A BLAST search of the NCBI GenBank database indicated that the 16S rDNA sequences of three strains (accession nos. KF563926, KF563927, and KF563928) had 99.9% identity to Xanthomonas axonopodis strain XV938 (AF123091). Under greenhouse conditions, bacterial strains wilted asparagus bean and pea but rarely infected bean, kidney bean, faba bean, mung bean, soybean, red bean, pea, garbanzo bean, and peanut. Based on morphology, pathogenicity tests, 16S rDNA sequencing, and host plant specificity, the pathogen was confirmed as X. axonopodis pv. cyamopsidis (synonym: X. campestris pv. cyamopsidis [Patel et al., 1953]). To our knowledge, this is the first report of bacterial blight of guar caused by X. axonopodis pv. cyamopsidis in China. Guar has recently been introduced in Xinjiang Province. Our findings indicate that bacterial blight may pose a threat to the economic sustainability of guar production in the region. References: (1) I. A. Milyutina et a1. FEMS Microbiol. Lett. 239:17, 2004. (2) I. M. G. Almeida et al. Summa Phytopathol. 18:255, 1992. (3) J. D. Mihail et al. Plant Dis. 69:811, 1985.

Download Full-text

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

Plant Disease ◽

10.1094/pdis-09-13-0948-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 988-988 ◽

Cited By ~ 5

Author(s):

A. Végh ◽

Zs. Némethy ◽

P. Salamon ◽

Z. Mándoki ◽

L. Palkovics

Keyword(s):

16S Rdna ◽

Bacterial Wilt ◽

Pathogenic Bacteria ◽

Plasmid Vector ◽

Disease Diagnosis ◽

Negative Control ◽

Chrysanthemum Morifolium ◽

Water Control ◽

Biochemical Tests ◽

First Report

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.

Download Full-text

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.

Download Full-text

First Report of Xanthomonas axonopodis pv. poinsettiicola Causing Bacterial Leaf Spot of Euphorbia pulcherrima in Slovenia

Plant Disease ◽

10.1094/pdis-09-10-0630 ◽

2011 ◽

Vol 95 (1) ◽

pp. 70-70 ◽

Cited By ~ 2

Author(s):

T. Dreo ◽

M. Pirc ◽

J. Erjavec ◽

M. Ravnikar ◽

I. Miklič-Lautar

Keyword(s):

Hypersensitive Reaction ◽

Negative Control ◽

Leaf Blight ◽

Economic Damage ◽

Bacterial Suspension ◽

Euphorbia Pulcherrima ◽

Biochemical Tests ◽

Xanthomonas Axonopodis ◽

First Report ◽

Leaf Spots

In September 2009, water-soaked spots, 2 mm in diameter, surrounded by a pale yellow halo were observed on leaves of pot-grown poinsettia plants (Euphorbia pulcherrima L.) cv. Christmas Feeling in a commercial greenhouse in Slovenia. Several spots per leaf developed on 10% of 84 plants used for propagation and slowly progressed to necrotic brown spots. While all plants were watered by overhead irrigation until mid-September, and afterward by flooding, no symptoms were observed on parent plants of four other separately grown cultivars. Propagated cuttings were all grown together, and in addition to cv. Christmas Feeling, an estimated 90% of 315, 35% of 29, 10% of 240, and 5% of 840 plants of cvs. Crazy Marble Star, Crazy Christmas, Lemon Snow, and Cortez Red, respectively, developed leaf spots. Yellow, smooth, and butyrous colonies with entire margins were isolated from symptomatic leaves of poinsettia parent plants of cv. Christmas Feeling on yeast peptone glucose agar (YPGA). They were identified as a Xanthomonas sp. based on biochemical tests (oxidase negative, positive for hydrolysis of H2S, starch and tributiryn and acid production from sucrose) and the isolates caused a hypersensitive reaction in leaves of tomato cv. Moneymaker. Partial sequences of gyrase subunit B-like (gyrB) gene (2) from an isolate (Accession No. HQ215596, 676 bp) showed highest similarity to Xanthomonas axonopodis pv. poinsettiicola strain LMG 5401 (Accession No. GU144264.1, 99% identity, 98% coverage) and 98% identity with gyrB sequence of X. axonopodis pv. poinsettiicola pathotype strain LMG 849 (Accession No. GU144273.1, 99% coverage, 3 gaps). Repetitive BOX-PCR (3) revealed high similarity of our isolate to pathotype strain LMG 849 with one additional band of approximate size of 1,500 bp present in our isolate. The pathogenicity of two isolates from parent plants of cv. Christmas Feeling was confirmed on four young poinsettia plants each. Plants were inoculated with a bacterial suspension of approximate concentration of 106 CFU/ml by spraying on the under side and upper side of the leaves, some of which were pricked with a sterile needle (1). Plants were then incubated under high relative air humidity (minimum 85%), 12 h of daylight, and 25°C day and 20°C night temperature regimens. After 10 days, all inoculated plants developed faint leaf spots, consistent with mild symptoms observed in the greenhouse. Colonies isolated from the developed spots had identical morphology and BOX-PCR profile to original isolates. Mock inoculated, negative control plants did not develop characteristic symptoms and no colonies similar to X. axonopodis pv. poinsettiicola were isolated from them. Bacteria isolated from leaf spots of other poinsettia cultivars had the same biochemical characteristics and BOX-PCR profiles as the first isolate. Since no leaf blight was observed on poinsettias in the greenhouse in the previous season and no host plants were kept between the seasons, imported parent plants are the most likely source of infection. To our knowledge, this is the first report of X. axonopodis pv. poinsettiicola on poinsettia in Slovenia, providing further data on the occurrence and potential economic damage of leaf blight of poinsettia in Europe. References: (1) R. A. Lelliott and D. E. Stead. Host tests. In: Methods in Plant Pathology. Vol 2. Blackwell, Oxford, 1987. (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.

Download Full-text

First Report of Enterobacter cowanii Causing Bacterial Spot on Mabea fistulifera, a Native Forest Species in Brazil

Plant Disease ◽

10.1094/pdis-02-12-0160-pdn ◽

2012 ◽

Vol 96 (10) ◽

pp. 1576-1576 ◽

Cited By ~ 5

Author(s):

G. Q. Furtado ◽

L. M. S. Guimarães ◽

D. O. Lisboa ◽

G. P. Cavalcante ◽

D. A. A. Arriel ◽

...

Keyword(s):

16S Rdna ◽

Phylogenetic Trees ◽

Rpob Gene ◽

Negative Control ◽

Experimental Unit ◽

Bacterial Suspension ◽

Native Forest ◽

Forest Species ◽

First Report ◽

Molecular Tests

In the summer of 2011, in a nursery located in Viçosa City, Minas Gerais State, brownish, necrotic, irregular spots were observed on leaves of Mabea fistulifera Mart. (Euphorbiaceae), an indigenous forest species commonly found in Brazil. Around 6,300 seedlings were evaluated and as many as 60% of them showed disease symptoms, including severe defoliation and plant death. Leaves with coalescing lesions turned papery in texture and had a blighted appearance. Bacterial colonies were isolated from these symptomatic leaves on King B's medium and identified based on biochemical and molecular analysis, as a member of the Enterobacteriaceae family. Like other members of the Enterobacteriaceae family, the bacteria were facultative anaerobic, gram-negative, cream-colored on YDC medium, urease and oxidase negative, as well as catalase and asparagine positive. Bacterial DNA was extracted from pure culture grown overnight in liquid 523 medium at 28°C using the Wizard Genomic DNA Purification kit (Promega) and conserved sequences in 16S rDNA (3) and rpoB (1) were amplified by PCR. The sequence of the 1,300-bp 16S rDNA fragment and the 750-bp rpoB gene were analyzed by NCBI BLAST. Related sequences were aligned and analyzed by ClustalW in MEGA 5 software. Phylogenetic analysis by maximum likelihood, using PAUP version 4.0 and TBR algorithm with 1,000 bootstrap replications, grouped the isolate in a clade with Enterobacter cowanii and the result showed 99% and 98% identity to the 16s rDNA and rpoB, respectively. The isolate clustered closely with the type strain of E. cowanii in both phylogenetic trees constructed. Pathogenicity tests were carried out by inoculating leaves of healthy seedlings either by spraying or cutting with a scissor previously dipped into a 108 CFU/ml bacterial suspension. The experiment was in a completely randomized design, with six replications. A pot with one plant was considered one experimental unit. Control seedlings were sprayed or cut with a scissor treated with saline solution. Prior to and after inoculation, plants were kept in a humid chamber for 24 h at 26°C in the dark and at room temperature. Subsequently, plants were transferred to growth chamber at 26°C, under a 12-h photoperiod (40 μmol/s/m2). Consistent with the symptoms observed originally, 7 days after inoculation, all seedlings developed leaf spots. No characteristic symptoms could be observed in the negative control. Furthermore, Koch's postulates were confirmed by reisolation of the bacterium from symptomatic tissues. In summary, the phenotypic, biochemical, and molecular tests identified the pathogen as E. cowanii. Recently, E. cowanii was isolated from Eucalyptus trees with symptoms of bacterial blight, although its pathogenicity was not demonstrated (2). To the best of our knowledge, this is the first report of a member of the Enterobacteriaceae family causing disease in M. fistulifera. The result has a great importance to better understand the role of E. cowanii as a pathogen-causing disease on a forest species. References: (1) C. L. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. L. Brady et al. Lett. Appl. Microbiol. 49:461, 2009. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Download Full-text

Severe Outbreak of Bacterial Blight Caused by Xanthomonas arboricola pv. corylina on Hazelnut cv. Tonda di Giffoni in Central Italy

Plant Disease ◽

10.1094/pdis-04-12-0375-pdn ◽

2012 ◽

Vol 96 (10) ◽

pp. 1577-1577 ◽

Cited By ~ 6

Author(s):

J. R. Lamichhane ◽

A. Fabi ◽

L. Varvaro

Keyword(s):

16S Rdna ◽

Bacterial Blight ◽

Central Italy ◽

Positive Control ◽

Negative Control ◽

Early Summer ◽

Nutrient Broth ◽

Universal Primers ◽

Bacterial Suspension ◽

Xanthomonas Arboricola

Hazelnut (Corylus avellana L.) is one of the most economically important tree crops in Italy. Xanthomonas arboricola pv. corylina (Xac) causes bacterial blight of hazelnut (4). During early summer 2010, a survey of three orchards (5 ha total) containing 4-year-old hazelnut trees (cv. Tonda di Giffoni) in Viterbo Province, Latium region, Italy, showed an 80 to 100% incidence of bacterial blight. Initially, water-soaked, necrotic spots were visible on leaves, fruit involucres, and shells, followed by lateral shoot dieback and development of cankers as longitudinal bark cracks on twigs, branches, and main trunks. Brown necrosis of the cambium was observed when bark tissue was removed. By late summer, necrosis had extended down main branches to the trunk, causing complete girdling of branches. Symptomatic tissues were collected from leaves, branches, and trunks, sections were surface-sterilized in 1% NaOCl for 1 min followed by two rinses in sterile distilled water (SDW, each for 1 min), and each section was then crushed in SDW. A loopful of the suspension was streaked onto yeast extract-dextrose-calcium carbonate agar medium (YDCA). Thirty six (12 from each type of tissue) yellow-mucoid, shiny, round bacterial colonies, each approximately 2 mm in diameter, were subcultured on YDCA. All strains were gram-negative and aerobic; negative for indole, lecithinase, urease, tyrosinase, and nitrate reduction; and positive for catalase, growth in 2% NaCl in nutrient broth, and growth at 35°C. All strains produced dark green pigment on succinate-quinate (SQ) medium. Inoculum of each of 15 isolates was prepared in nutrient broth, and washed cells from late log-phase cultures used to prepare a bacterial suspension of each isolate for inoculation of 2-year-old potted hazelnut plants cv. Tonda di Giffoni. A suspension of 106 CFU/ml for each isolate was sprayed onto leaves (10 ml/plant), and drops of inoculum were placed on wounds made on twigs with a sterile scalpel (0.10 μl/wound). For each isolate, three plants were inoculated per inoculation method. Inoculations with two reference strains of Xac (Xaco 1 from central Italy (3) and NCPPB 2896 from England) and SDW were performed on the same number of plants for positive and negative control treatments, respectively. Inoculated plants were maintained at 26 ± 1°C in a greenhouse. After 21 days, all inoculated plants had developed symptoms on leaves, while cankers developed on twigs after 40 days. Positive control plants developed the same symptoms, while negative control plants remained asymptomatic. Bacteria recovered from lesions on plants inoculated with the test strains or positive control strains had the same morphological and physiological characteristics as the original strains. No bacteria were recovered from negative control plants. Total DNA was extracted from bacterial suspensions and 16S rDNA amplified using universal primers (2). Sequences (GenBank Accession Nos. JQ861273, JQ861274, and JQ861275 for strains Xaco VT3 to VT5) had 99 to 100% identity with 16S rDNA sequences of Xac strains in GenBank. In Italy, Xac was reported by Petri in 1932 in Latium, and later in other regions on several hazelnut cultivars (1). However, to our knowledge, this is the first report of the disease causing severe damage in Italy. References: (1) M. Fiori et al. Petria 16:71, 2006. (2) J. R. Lamichhane et al. Plant Dis. 95:221, 2011. (3) J. R. Lamichhane et al. Acta Horticol.:In press. 2012. (4) OEPP/EPPO Bull. 179:179, 2004.

Download Full-text