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

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.

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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 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.

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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.

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First Report of a Soft Rot of Philodendron ‘Con-go’ in China Caused by Dickeya dieffenbachiae

Plant Disease ◽

10.1094/pdis-09-11-0795 ◽

2012 ◽

Vol 96 (3) ◽

pp. 452-452 ◽

Cited By ~ 5

Author(s):

B. R. Lin ◽

H. F. Shen ◽

J. N. Zhou ◽

X. M. Pu ◽

Z. N. Chen ◽

...

Keyword(s):

16S Rdna ◽

Phylogenetic Trees ◽

Soft Rot ◽

Gyrb Gene ◽

Control Treatment ◽

Gene Sequences ◽

16S Rdna Gene ◽

First Report ◽

Rdna Gene ◽

Plastic Bags

Philodendron is a popular foliage plant cultivated in interiorscapes of homes, offices, and malls throughout China. A severe outbreak of a soft rot of Philodendron ‘Con-go’ occurred in Guangzhou, China from 2010 to 2011. The disease was characterized by leaf infections starting as pinpoint spots that are water soaked and yellow to pale brown. The lesions are sometimes surrounded by a diffuse yellow halo. When the humidity is high and temperatures are warm to hot, the spots expand rapidly, becoming slimy, irregular, and sunken with light tan centers, darker brown borders, and diffused yellow margins and may involve the entire leaf in a few days. An invasion of the midrib and larger veins by the causal bacterium often results in advancement into the petiole and stem. A survey of three areas of production of Philodendron ‘Con-go’ (5 ha) in Guangzhou revealed that 91% of the fields were affected at an incidence ranging from 15 to 30%. Of 41 bacterial isolates obtained from lesions, three were selected randomly for further characterization. All strains were gram negative, negative for oxidase and positive for catalase and tryptophanase (indole production), and utilized citrate, tartrate, malonate, glucose, sucrose, fructose, and maltose but not glucopyranoside, trehalose, or palatinose. Biolog analysis (version 4.20.05, Hayward, CA) identified the isolates as Pectobacterium chrysanthemi (SIM 0.804 to 0.914). According to Samson et al. (1), it was renamed as a Dickeya sp. PCR was performed on the 16S rDNA gene with primers 27f and 1495r (3) and 1,423 bp of the 16S rDNA gene (GenBank No. JN709491) showed 99% identity to P. chrysanthemi (GenBank No. AF373202), and 98% to Dickeya dieffenbachiae (GenBank No. JF311644). Additionally, the gyrB gene was amplified with primers gyrB-f1 (5′-atgtcgaattcttatgactcctc-3′) and gyrB-r1 (5′-tcaratatcratattcgcygctttc-3′) designed based on all the submitted gyrB gene sequences of Dickeya spp. The dnaX gene was amplified with primers dnaXf and dnaXr (2). The products were sequenced and phylogeny analyses were performed by means of MEGA 5.05. Results showed that the gyrB and the dnaX genes of the strains were 98% homologous to those of D. dieffenbachiae (GenBank Nos. JF311652 and GQ904757). Therefore, on the basis of phylogenetic trees of the 16S rDNA, gyrB, and dnaX gene sequences, the bacterial isolate named PC1 is related to D. dieffenbachiae (100% bootstrap values). Pathogenicity of each of the three strains on Philodendron ‘Con-go’ was confirmed by injecting 60 50-day-old seedlings each with 0.1 ml of the isolate suspension (108 CFU/ml) into the leaves. Another 60 were injected with sterile water to serve as the control treatment. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 32°C day and 28°C night temperatures with high humidity. After 72 h, all the injected plants started to show symptoms similar to those observed on field plants, but no symptoms appeared on the control plants. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of D. dieffenbachiae causing soft rot of Philodendron ‘Con-go' in China. References: (1) R. Samson et al. Evol. Microbiol. 55:1415, 2005. (2) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (3) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

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First Report of Bacterial Leaf Spot Caused by Pseudomonas syringae pv. aptata on Swiss Chard, Beta vulgaris subsp. vulgaris, in Arizona

Plant Disease ◽

10.1094/pdis-12-20-2554-pdn ◽

2021 ◽

Author(s):

Marilen Nampijja ◽

Mike Derie ◽

Lindsey J. du Toit

Keyword(s):

Beta Vulgaris ◽

Pseudomonas Syringae ◽

Leaf Spot ◽

Phosphate Buffer ◽

Negative Control ◽

Bacterial Suspension ◽

Bacterial Leaf Spot ◽

First Report ◽

Leaf Spots ◽

Swiss Chard

Arizona is an important region of the USA for winter production of baby leaf crops such as spinach (Spinacia oleracea), table beet (Beta vulgaris subsp. vulgaris Condivita Group), and Swiss chard (B. vulgaris subsp. vulgaris Cicla Group). In the winter of 2019, severe leaf spots were observed at 80% incidence and 40% severity per plant in a 1-ha baby leaf Swiss chard crop of an (unknown cultivar) in Arizona. The lesions were circular to irregular, necrotic, water-soaked, and 1 to 5 mm in diameter. Symptomatic leaf sections (1-cm2) were surface-sterilized with 0.6% NaOCl, rinsed, and macerated in sterilized, deionized water. An aliquot of each macerate was streaked onto King’s B (KB) agar medium. Cream-colored, non-fluorescent colonies typical of Pseudomonas were isolated consistently, and all were non-fluorescent. A dozen isolates selected randomly were all negative for potato soft rot, oxidase, and arginine dihydrolase, and positive for levan production and tobacco hypersensitivity, which is typical of fluorescent P. syringae isolates, but can also include non-fluorescent strains (Lelliot et al. 1966). Three isolates were tested for pathogenicity on the table beet cv. Red Ace and Swiss chard cv. Silverado. Strain Pap009 of P. syringae pv. aptata (Psa), demonstrated previously to be pathogenic on Swiss chard and table beet, served as a positive control strain (Derie et al. 2016; Safni et al. 2016). Each isolate was grown inoculated into medium 523 broth and incubated on a shaker at 175 rpm overnight at 25°C. Each bacterial suspension was adjusted to an optical density (OD) of 0.3 at 600 nm (108 CFU/ml), and diluted in 0.0125M phosphate buffer to 107 CFU/ml. Thirty-day-old seedlings grown in Redi-Earth Plug and Seedling Mix in a greenhouse at 22 to 26°C were inoculated by rubbing the abaxial and adaxial leaf surfaces of each plant with a cotton swab dipped in inoculum to which Carborundum had been added (0.06 g/10 ml). The negative control plants were treated similarly with phosphate buffer with Carborundum. The experiment was set up as a randomized complete block design with 4 replications per treatment and 6 seedlings per experimental unit. In both trials, leaf spots resembling those on the original plants developed on all table beet and Swiss chard plants inoculated with the Arizona isolates and Pap009, but not on negative control plants. Disease severity was greater on Swiss chard (average 39% leaf area with spots) than on table beet (14%). Re-isolates obtained from inoculated seedlings using the same method as the original isolations resembled Psa. Multilocus sequence analysis (MLSA) was carried out for the original three Arizona isolates and the re-isolates using DNA amplified from the housekeeping genes gyrB, rpoD, gapA, and gltA (Hwang et al. 2005; Sarkar and Guttman 2004). Sequence identities of these genes of the Arizona isolates (GenBank accession numbers MW291615 to MW291618 for strain Pap089; MW291619 to MW291622 for Pap095; and MW291623 to MW291626 for Pap096 for gltA, gyrB, rpoD, and gapA, respectively) and the re-isolates ranged from 98 to 100% with those of Psa pathotype strain CFBP 1617 in the PAMDB database (Almeida et al. 2010; Altschul et al. 1997). Based on Koch’s postulates, colony characteristics, and MLSA, Psa was the causal agent of leaf spots in the Arizona Swiss chard crop. To our knowledge, this is the first report of bacterial leaf spot on chard in Arizona. The pathogen could have been introduced on infected seed as Psa is readily seedborne and seed transmitted.

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First Report of a Soft Rot of Phalaenopsis aphrodita Caused by Dickeya dieffenbachiae in China

Plant Disease ◽

10.1094/pdis-11-11-0942 ◽

2012 ◽

Vol 96 (5) ◽

pp. 760-760 ◽

Cited By ~ 8

Author(s):

J. N. Zhou ◽

B. R. Lin ◽

H. F. Shen ◽

X. M. Pu ◽

Z. N. Chen ◽

...

Keyword(s):

16S Rdna ◽

Large Scale ◽

Soft Rot ◽

The Philippines ◽

Gyrb Gene ◽

Bacterial Suspension ◽

Sterile Water ◽

Gene Sequences ◽

First Report ◽

Tropical Asia

Phalaenopsis orchids, originally from tropical Asia, are mainly planted in Thailand, Singapore, Malaysia, the Philippines, and Taiwan and have gained popularity from consumers all over the world. The cultivation area of Phalaenopsis orchids has been rising and large-scale bases have been established in mainland China, especially South China because of suitable environmental conditions. In September 2011, a soft rot of Phalaenopsis aphrodita was found in a Phalaenopsis planting base in Guangzhou with an incidence of ~15%. Infected plants initially showed water-soaked, pale-to-dark brown pinpoint spots on leaves that were sometimes surrounded by a yellow halo. Spots expanded rapidly with rising humidity and temperatures, and in a few days, severely extended over the blade with a light tan color and darker brown border. Lesions decayed with odorous fumes and tissues collapsed with inclusions exuding. The bacterium advanced to the stem and pedicle. Finally, leaves became papery dry and the pedicles lodged. Six diseased samples were collected, and bacteria were isolated from the edge of symptomatic tissues after sterilization in 0.3% NaOCl for 10 min, rinsing in sterile water three times, and placing on nutrient agar for culture. Twelve representative isolates were selected for further characterization. All strains were gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Biolog identification (version 4.20.05, Hayward, CA) was performed and Pectobacterium chrysanthemi (SIM 0.868) was confirmed for the tested isolates (transfer to genus Dickeya). PCR was used to amplify the 16S rDNAgene with primers 27f and 1492r, dnaX gene with primers dnaXf and dnaXr (3), and gyrB gene with primers gyrBf (5′-GAAGGYAAAVTKCATCGTCAGG-3′) and gyrB-r1 (5′-TCARATATCRATATTCGCYGCTTTC-3′) designed on the basis of the published gyrB gene sequences of genus Dickeya. BLASTn was performed online, and phylogeny trees (100% bootstrap values) were created by means of MEGA 5.05 for these gene sequences, respectively. Results commonly showed that the representative tested strain, PA1, was most homologous to Dickeya dieffenbachiae with 98% identity for 16S rDNA(JN940859), 97% for dnaX (JN989971), and 96% for gyrB (JN971031). Thus, we recommend calling this isolate D. dieffenbachiae PA1. Pathogenicity tests were conducted by injecting 10 P. aphrodita seedlings with 100 μl of the bacterial suspension (1 × 108 CFU/ml) and another 10 were injected with 100 μl of sterile water as controls. Plants were inoculated in a greenhouse at 28 to 32°C and 90% relative humidity. Soft rot symptoms were observed after 2 days on the inoculated plants, but not on the control ones. The bacterium was isolated from the lesions and demonstrated identity to the inoculated plant by the 16S rDNA sequence comparison. Previously, similar diseases of P. amabilis were reported in Tangshan, Jiangsu, Zhejiang, and Wuhan and causal agents were identified as Erwinia spp. (2), Pseudomonas grimontii (1), E. chrysanthemi, and E. carotovora subsp. carovora (4). To our knowledge, this is the first report of D. dieffenbachiae causing soft rot disease on P. aphrodita in China. References: (1) X. L. Chu and B. Yang. Acta Phytopathol. Sin. 40:90, 2010. (2) Y. M. Li et al. J. Beijing Agric. Coll. 19:41, 2004. (3) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) Z. Y. Wu et al. J. Zhejiang For. Coll. 27:635, 2010.

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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 Bacterial Blight of Carrot in Indiana Caused by Xanthomonas hortorum pv. carotae

Plant Disease ◽

10.1094/pdis-10-13-1083-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 685-685 ◽

Cited By ~ 2

Author(s):

L. J. du Toit ◽

M. L. Derie ◽

C. E. Christianson ◽

L. Hoagland ◽

P. Simon

Keyword(s):

Phosphate Buffer ◽

Bacterial Blight ◽

Agar Medium ◽

Negative Control ◽

Control Treatment ◽

Bacterial Suspension ◽

Pcr Assay ◽

First Report ◽

Seed Testing ◽

Symptomatic Leaf

In summer 2012, bacterial blight symptoms (2) were observed on leaves of carrot plants in 7 out of 70 plots of carrot breeding lines at the Purdue University Meig Horticulture Research Farm, Lafayette, IN. Symptoms included small to large, variably shaped, water-soaked to dry, necrotic lesions, with or without chlorosis, at <5% incidence. Microscopic examination of symptomatic leaf sections revealed bacterial streaming from the cut ends of each leaf piece. For each of the seven plots, symptomatic leaf sections (each 5 to 10 mm2) were surface-sterilized in 1.2% NaOCl for 60 s, triple-rinsed in sterilized, deionized water, dried on sterilized blotter paper, macerated in sterilized water, and a loopful of the suspension was streaked onto yeast dextrose carbonate (YDC) agar medium (1). Colonies with morphology similar to that of strain Car001 of Xanthomonas hortorum pv. carotae from California (3) were obtained consistently from all seven plots, and serial dilutions streaked onto YDC agar medium to obtain pure cultures. One bacterial strain/plot was then subjected to a PCR assay for X. hortorum pv. carotae using the protocol of Meng et al. in (5), except for an annealing temperature of 60°C. All seven Indiana strains and Car001 produced a 355-bp DNA fragment indicative of X. hortorum pv. carotae. The Indiana strains and Car001 were each tested for pathogenicity on five 11-week-old carrot plants of a proprietary Nantes inbred line grown from a seed lot that tested negative for X. hortorum pv. carotae (1,3). Each strain was grown for 16 h in 523 broth (4) on a shaker (200 rpm) at 28°C, and diluted in 0.0125M phosphate buffer to 108 CFU/ml. Approximately 24 h prior to inoculation, the five plants for each strain were enclosed in a large plastic bag to create a moist chamber. The plants were inoculated by atomizing 30 ml of the appropriate bacterial suspension onto the foliage using an airbrush. Five plants inoculated with sterilized phosphate buffer served as a negative control treatment. The plants were re-sealed in plastic bags for 72 h, and placed in a randomized complete block design in a greenhouse set at 25 to 28°C. Symptoms of bacterial blight were first observed 14 days after inoculation, and developed on all inoculated plants by 21 to 28 days after inoculation, with slight variation in severity of symptoms among strains. Symptoms did not develop on negative control plants. Re-isolations were done 32 days after inoculation from symptomatic leaves of three replicate plants/strain and from three plants of the negative control treatment, using the protocol described for the original samples. Bacterial colonies typical of X. hortorum pv. carotae were obtained from symptomatic leaves for all seven Indiana strains and the control strain, but not from the negative control plants. Identity of the re-isolated strains as X. hortorum pv. carotae was confirmed by PCR assay. To our knowledge, this is the first report of bacterial blight of carrot in Indiana. References: (1) M. Asma. Detection of Xanthomonas hortorum pv. carotae on Daucus carota. 7-020. International Rules for Seed Testing, Annex to Chapter 7: Seed Health Testing Methods. Internat. Seed Testing Assoc., Bassersdorf, Switzerland, 2006. (2) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (3) L. J. du Toit et al. Plant Dis. 89:896, 2005. (4) E. I. Kado and M. G. Heskett. Phytopathology 60:969, 1970. (5) X. Q. Meng et al. Plant Dis. 88:1226, 2004.

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First Report of Crown Gall, Caused by Agrobacterium tumefaciens on Soapberry (Sapindus delavayi) in China

Plant Disease ◽

10.1094/pdis-10-12-0988-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 685-685

Author(s):

Y. J. Wang ◽

Y. Y. He ◽

Z. Xie ◽

L. Q. Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

16S Rdna ◽

Crown Gall ◽

Maximum Parsimony Analysis ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Identification ◽

Poor Growth ◽

First Report

Soapberry (Sapindus delavayi (Franch.) Radlk.,) plants are widely grown as shade trees in the subtropical to tropical regions of China. In July 2011, large, aerial galls were observed on the above-ground trunks of 5-year-old soapberry plants in two commercial nursery gardens located in Zhejiang Province. Disease incidence was estimated to be 75%. The galls varied in weight from 2 to 24 g and in texture from soft and spongy to hard, and in some cases, the galls completely girdled the trunk. The trees with galls exhibited poor growth compared with healthy trees. Isolations from the grinded and macerated galls yielded nearly pure white, circular, and glistening bacterial colonies on Roy Sauer medium (2). Six random colonies from different galls were selected for bacterial identification, and showed the same morphological, physiological, and biochemical characters and 16S rDNA sequences. All six isolates (isolate SD01 to SD06) were gram negative, rod-shaped bacteria. Carbon source utilization testing with the Biolog GN Bacterial Identification System (version 3.50) confirmed the bacteria as Agrobacterium tumefaciens with a similarity of 0.90. The most-parsimonious tree from the maximum parsimony analysis (PHYLIP package, version 3.68, 500 replicates) of bacterial 16S rDNA gene sequences showed that A. tumefaciens SD01 (GenBank Accession No. JX997939) clustered phylogenetically most closely (99.5% similarity) with A. tumefaciens C58 (AE007870.2). Pathogenicity was confirmed by injecting 3- to 5-week old tomato and sunflower plants and 2-year-old soapberry with approximately 5 μl of the bacterial suspension (108 CFU/ml) in sterile, distilled water. Sterile distilled water was used as a negative control. Ten plants of each treatment were inoculated. Inoculated plants were then transferred to a greenhouse at 25°C. Typical tumors developed at the inoculation sites on tomatoes and sunflower plants 3 weeks after inoculation and on soapberry 6 weeks after inoculation. No symptoms were observed on the control plants. The bacteria that were readily reisolated from the inoculated plants exhibited the same morphological, physiological characters and 16S rDNA sequence as the original culture and were confirmed as A. tumefaciens, fulfilling Koch's postulates. A. tumefaciens is endemic to China and has a very wide host range (1). However, crown gall of soapberry has never been found in China and other countries. To our knowledge, this is the first report of A. tumefaciens on soapberry plants in China. References: (1) M. A. Escobar and A. M. Dandekar. Trends Plant Sci. 8:380, 2003. (2) L. W. Moore et al. Page 17 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.

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