First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Plectranthus amboinicus in Martinique

Plectranthus amboinicus, commonly known as Gwo ten in the French West Indies (Martinique), is a semi-succulent perennial plant of the Lamiaceae family. This aromatic plant wich is widespread naturally throughout the tropics is of economic importance because of the therapeutic and nutritional properties attributed to its natural phytochemical compounds wich are highly valued in the pharmaceutical industry. In March 2019, wilted P. amboinicus plants intercropped with tomato plants (cv. Heatmaster) in order to reduce the insect-pest damages on tomato, were observed in a field located at the CIRAD experimental station in Lamentin, Martinique (14.663194 N, -60.999167 W). Average disease incidence of 65.74% was recorded on P. amboinicus, in 3 plots with an area of 22.04 m2. The initial symptoms observed were irregular, black, necrotic lesions on leaves. After 10 days, plants wilted and black stripes were observed on stems. Within 4 weeks, more than 50% of plants were fully wilted. Longitudinal stem sections of the wilted plants showed brown vascular discoloration. The cut stems of the wilted plants released a whitish bacterial ooze in water. In all, 108 stem sections were collected, surface disinfected with 70% ethanol and each was crushed in 2 mL of Tris-buffer, then processed for bacterial isolation by plating on modified Semi-Selective Medium from South Africa SMSA (Engelbrecht 1994). Typical Ralstonia solanacearum colonies grew on SMSA medium for 100 of the 108 samples after incubation for 48h at 28°C and were identified as Ralstonia solanacearum using diagnostic PCR with 759/760 primers (Opina et al. 1997). A phylotype-specific multiplex PCR (Fegan and Prior 2005) classified all the strains in R. solanacearum Phylotype IIA. A subset of 11 strains was selected for sequevar identification. All the strains were identified as sequevar I-39 (100% nucleotide identity with strain ANT92 - Genbank accession EF371828), by partial egl sequencing (Fegan and Prior 2005) (GenBank Accession Nos. MT314067 to MT314077). This sequevar has been reported to be widespread in the Caribbean and tropical America on vegetable crops (particularly on tomato), but not on P. amboinicus (Deberdt et al. 2014; Ramsubhag et al. 2012; Wicker et al. 2007). To fulfil Koch’s postulates, a reference strain, isolated from diseased P. amboinicus (CFBP 8733, Phylotype IIA/sequevar 39), was inoculated on 30 healthy P. amboinicus plants. A common tomato cultivar grown in Martinique (cv. Heatmaster) was also inoculated on 30 plants with the same bacterial suspension. Three-weeks-old plants of both crops grown in sterilized field soil were inoculated by soil drenching with 20 ml of a calibrated suspension (108 CFU/mL). P. amboinicus and tomato plants drenched with sterile water served as a negative controls. Plants were grown in a fully controlled environment at day/night temperatures of 30–26°C ± 2°C under high relative humidity (80%). The P. amboinicus plants started wilting 9 days after inoculation, and within four weeks 60% of the P. amboinicus plants had wilted. The tomato plants started wilting 5 days after inoculation with 62% of wilted plants within four weeks. R. solanacearum was recovered from all symptomatic plants on modified SMSA medium. No symptoms were observed and no R. solanacearum strains were isolated from negative controls plants. To our knowledge, this is the first report of R. solanacearum causing bacterial wilt on Gwo ten (P. amboinicus) in Martinique. The importance of this discovery lies in the reporting of an additional host for R. solanacearum, which can be associated with other crops as tomato crop in order to reduce the abundance of insect-pests. Further studies need to be conducted to assess the precise distribution of bacterial wilt disease on P. amboinicus in Martinique and to develop a plan of action avoiding its association with R. solanacearum host crops as tomato for reducing epidemic risk.

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First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Chard in Taiwan

Plant Disease ◽

10.1094/pdis-07-14-0715-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 282-282 ◽

Cited By ~ 3

Author(s):

C.-H. Lin ◽

M.-H. Chuang ◽

J.-F. Wang

Keyword(s):

Beta Vulgaris ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Close Relative ◽

Bacterial Suspension ◽

Tetrazolium Chloride ◽

First Report ◽

Initial Symptoms ◽

Field Samples ◽

Pepper Plants

Chard (Beta vulgaris var. cicla L.) is a biennial herbaceous plant in the Chenopodiaceae family. It is rich in vitamins and minerals and is one of the most popular traditional vegetables in Taiwan. Chard accessions VI048530 and VI050121 growing in fields at Shanhua, Tainan, showed wilting symptoms in March and April 2013. The initial symptoms of wilt were observed on young green leaves. These symptoms progressed over time to chlorosis, interveinal necrosis, and finally blight. Finally, the plants collapsed and died. Vascular and pith tissues were discolored, especially at the stem base. A whitish mass oozed from the cut end of diseased stems. A total of eight bacterial strains were isolated from stems and roots of wilted chard plants. On tetrazolium chloride (TZC) medium (4), colonies were round to oval, fluidal, and white with a pink or red center after incubation at 30°C for 48 h. A typical hypersensitive reaction was induced within 24 h when the strains were infiltrated into tobacco leaves. Koch's postulates on chard plants were confirmed using the eight strains within a greenhouse, under natural light, with temperature and humidity ranges from 25 to 34°C and 56 to 98%, respectively. Fifteen chard (VI048530) plants at the four- to six-leaf stage were inoculated by soil drenching with 30 ml of a ~108 CFU/ml bacterial suspension. Sterile water was used as negative control. After 4 to 6 days, the first symptoms of wilt were observed on the young chard leaves. The progression of symptom development was identical to that observed in the field. The colony morphology on TZC medium of isolates from the inoculated plants was identical to that previously described from field samples. Pathogenicity of the strains was also tested on tomato (VI005790), eggplant (VI046095), and pepper (PBC1367) plants using the previously described inoculation procedure. The mean disease incidences on tomato, eggplant, and pepper plants were 100% (120/120), 100% (120/120), and 79.2% (95/120) respectively. Latent infection was found in asymptomatic pepper plants (16/120) by a printing method. Polymerase chain reaction (PCR) amplification of total DNA from each strain using the Ralstonia solanacearum-specific primer pair AU759f and AU760r (5) produced the expected 282-bp amplicon. All the isolated strains were identified as biovar 3 based on their capacity to utilize three hexose alcohols (mannitol, sorbitol, and dulcitol) and three disaccharides (lactose, maltose, and cellobiose) (2) to produce acid. Based on the phylotype-specific multiplex PCR assay and the partial egl gene sequence (GenBank accession numbers KM100442 to KM100449) (1), all chard isolates were identified as R. solanacearum phylotype I, sequevar 34. Bacterial wilt symptoms have also been observed on beet (Beta vulgaris L.), a close relative of chard, but beet has not been confirmed as a host plant (3). To our knowledge, this is the first report of chard as a host of R. solanacearum worldwide. References: (1) M. Fegan and P. Prior. Page 449 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. APS Press, St. Paul, MN, 2005. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) A. Kelman. The Bacterial Wilt caused by Pseudomonas solanacearum. Tech. Bull. No. 99. N.C. Agric. Exp. Stn., 1953. (4) A. Kelman. Phytopathology 44:693, 1954. (5) 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 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 Ralstonia solanacearum Causing Bacterial Wilt of Yacon in China

Plant Disease ◽

10.1094/pdis-11-11-0940-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 904-904 ◽

Cited By ~ 1

Author(s):

P. Li ◽

X. X. Wu ◽

Z. Y. Wang ◽

H. H. Ho ◽

Y. X. Wu ◽

...

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Yunnan Province ◽

Capillary Tube ◽

Disease Spread ◽

Pathogenicity Test ◽

Carbohydrate Utilization ◽

Tetrazolium Chloride ◽

First Report ◽

Initial Symptoms

Yacon (Smallanthus sonchifolius) is an important cash crop in Yunnan Province, China. In 2003, yacon was introduced to Yunnan province as a novelty root crop and as an experimental source of natural sugars; now more than 15 provinces cultivate the crop. Yunnan is one of the major yacon producing areas of China, with up to 10,000 ha yielding up to 50,000 t of yacon, which is nearly half of the production in China. In April and May 2010, bacterial wilt of yacon was observed in the fields of Lion Mountain of Wuding County, Yunnan Province, China. In 2011, the disease occurred in approximately 1 ha of yacon, resulting in 10% crop loss in that area. The initial symptoms observed were irregular, black, necrotic lesions on leaf margins. After 4 to 7 days, leaves became totally necrotic, plants wilted, and black stripes were observed on plant stems. Within 2 to 3 weeks, more than 70% of leaves within the crop were wilted. Subsequently, the plants died and stems became brittle. When dead plants were pulled from the soil, tubers were found to have turned black. When diseased stems and/or petioles were cut with a sterile sharp knife or razor blade, bacterial ooze appeared on the cut ends. High populations of morphologically uniform bacteria were isolated from the diseased plants by conventional methods. When cultured on TZC (2,3,5-Triphenylte tetrazolium chloride) agar medium (3), colonies were large, elevated, fluidal, and entirely white with a pale red center. The isolated bacterium was gram-negative, grew aerobically, and did not form endospores. The cells were 0.5 to 0.7 × 1.5 to 2.0 μm and nonencapsulated. Ralstonia solanacearum was identified and confirmed as the pathogen on the basis of morphological and physiological characteristics, pathogenicity test, and 16S rDNA sequence analysis (1,4). The nucleotide sequence is available in GenBank (Accession No. HQ176322.1). The pathogenic strain belonged to race 1 and biovar 3 according to the pathogenicity and carbohydrate utilization tests (2). Koch's postulates were tested in the greenhouse, with 10 plants inoculated per species. Plants were inoculated with 15 μl of cell suspension containing 106 to 107 CFU ml–1 deposited into the third axilla with a capillary tube. The bacteria could infect tomato, pepper, tobacco, potato, common sage (Salvia dugesii Fernald), and patchouli, and caused typical symptoms of wilt and black lesions, but could not infect leaves of swamp mahogany (Eucalyptus robusta Smith), stramonium (Dature stramonium Datura L.), ginger, or maize. To our knowledge, this is the first report of yacon as a host of R. solanacearum. Since the pathogen has a wide host range, monitoring of the vegetation in and around yacon fields should be implemented as a mandatory management measure to prevent disease spread. References: (1) C. A. Boucher et al. J. Bacteriol. 169:5626, 1987. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) A. Kelman. Phytopathology 44:693, 1954. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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First Report of Bacterial Wilt Caused by Ralstonia solanacearum in Ghana, West Africa

Plant Disease ◽

10.1094/pdis-09-13-0963-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 840-840 ◽

Cited By ~ 2

Author(s):

N. Subedi ◽

R. L. Gilbertson ◽

M. K. Osei ◽

E. Cornelius ◽

S. A. Miller

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Reference Strain ◽

Disease Incidence ◽

Casamino Acid ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Phylotype I

Tomato and pepper plants exhibiting wilt symptoms were collected from fields in seven villages in Northern (Vea, Tono, Pwalugu), Ashanti (Agogo, Akumadan), and Brong Ahafo (Tanoso, Tuobodom) regions of western Ghana in November 2012. The plants were wilted without leaf yellowing or necrosis. Disease incidence was generally low, with less than 20% symptomatic plants observed. Most of the plants collected produced visible bacterial ooze in water in the field. Ooze was plated on 2,3,5-triphenyltetrazolium chloride-amended (TZC) medium. Isolated colonies were fluidal, irregularly round, white with pink centers, gram-negative, and oxidase positive. One strain from each of seven fields was selected for further study. All strains induced a hypersensitive reaction on tobacco. Randomly selected strains SM855-12 and SM857-12 tested positive in R. solanacearum ImmunoStrip assays (Agdia Inc., IN). An end-point PCR assay with primer set 759/760 (3) generated an R. solanacearum-specific 280-bp amplicon for all seven strains. Two of these strains were biovar I and the remaining five were biovar III based on utilization of cellobiose, lactose, maltose, dulcitol, mannitol, and sorbitol. A phylotype-specific multiplex PCR assay that recognizes four geographically linked monophyletic groups within R. solanacearum (1) indicated that one strain (SM855-12) was phylotype III (African origin), whereas the other six were phylotype I (Asian origin). All strains were subjected to repetitive sequence-based PCR (Rep-PCR) with BOXA1R and REP1R/REP2 primers (4). Strain SM855-12 was grouped with the phylotype III reference strain UW 368 and the remaining six strains were grouped with the phylotype I reference strain GMI 1000. A pathogenicity test was performed with bacterial wilt-susceptible tomato line OH7814. Inoculum was prepared from 48-h cultures of strains SM855-12, SM856-12, and SM858-12 grown on casamino acid peptone glucose (CPG) medium at 30°C. Roots of ten 4-week-old tomato plants per strain were drench-inoculated with 5 ml of a 108 CFU/ml bacterial suspension after wounding with a sterile scalpel. Non-inoculated control plants were drenched with 5 ml distilled water after root wounding. Plants were kept in a greenhouse at 25 to 30°C. By 12 days after inoculation, 80 to 100% of inoculated plants were wilted, whereas no symptoms appeared in non-inoculated plants. Bacteria re-isolated from wilted plants were confirmed to be R. solanacearum using techniques mentioned above. Although an association of bacterial wilt with tomato/pepper was mentioned previously (2), to our knowledge, this is the first documented report of bacterial wilt caused by R. solanacearum in Ghana. The presence of Asian strains (phylotype I) may be the result of one or more accidental introductions. Awareness of this disease in Ghana will lead to deployment of management strategies including use of resistant varieties and grafting desirable varieties onto disease-resistant rootstocks. References: (1) M. Fegan and P. Prior. Page 449 in Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. American Phytopathological Society, St. Paul, MN, 2005. (2) K. A. Oduro. Plant Protection and Regulatory Services Directorate of MOFA, Accra, Ghana, 2000. (3) N. Opina et al. Asia Pac. J. Mol. Biol. Biotechnol. 5:19, 1977. (4) J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.

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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 Bacterial Canker of Pepper in Ohio

Plant Disease ◽

10.1094/pdis.2000.84.7.810c ◽

2000 ◽

Vol 84 (7) ◽

pp. 810-810 ◽

Cited By ~ 7

Author(s):

M. L. Lewis Ivey ◽

S. A. Miller

Keyword(s):

Positive Control ◽

Bell Pepper ◽

Bacterial Suspension ◽

Bacterial Canker ◽

Tomato Plants ◽

First Report ◽

Spray Inoculation ◽

Pure Cultures ◽

Negative Controls ◽

Pepper Plants

Light brown, raised lesions were observed on the leaves of bell pepper (Capsicum annuum L.) plants throughout a commercial field in northwest Ohio in 1999. Bacterial streaming from the lesions was observed microscopically. Five representative, pale yellow colonies isolated on yeast dextrose carbonate medium were selected and purified. All isolates induced a hypersensitive response in Mirabilis jalapa L. plants 24 h after inoculation with a 1 × 108 CFU/ml bacterial suspension. All five were identified as Clavibacter michiganensis subsp. michiganensis by fatty acid methyl ester analysis (mean similarity index [S.I.] = 0.76; MIDI, Newark, DE). Identity was confirmed at the University of Hawaii (W. Kaneshiro and A. Alvarez) by carbon substrate utilization pattern (mean S.I. = 0.75; Biolog, Hayward, CA) and positive reactions with C. michiganensis subsp. michiganensis-specific monoclonal antibodies (clones 103-142-1-1 and 103-148-2-1) in ELISA. DNA extracted from lesions and pure cultures was used as template in a polymerase chain reaction (PCR) assay with primers specific for C. michiganensis subsp. michiganensis (CMM-5 and CMM-6) (1). DNA from a known strain of C. michiganensis subsp. michiganensis served as a positive control, while water and DNA from healthy tomato plants were used as negative controls. A 0.6-kb PCR product was amplified from lesions, pure cultures of all five strains, and positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 5- to 6-week-old bell pepper (cvs. Collossal, Lafayette, King Arthur, Brigadier, and Commandant) and tomato (cv. Peto 696) plants. Pepper plants were inoculated with each strain by clipping the lowest petioles with scissors that had been dipped into a bacterial suspension (1 × 108 CFU/ml) or by spray inoculation (approximately 5 ml/plant). Tomato plants were inoculated by clipping. Both inoculation methods included a water control. All five strains caused water-soaked lesions on leaves of all pepper varieties within 7 days after spray inoculation. Pepper plants inoculated by clipping did not develop symptoms. DNA extracts from lesions of challenged pepper plants were positive in PCR. All inoculated tomato seedlings exhibited wilting, streaks, and cankers in the stems and necrosis of leaf margins within 15 days after inoculation. None of the control plants developed symptoms. All five strains were re-isolated from inoculated tomato and pepper plants. The original pepper strains and the strains re-isolated from tomatoes were compared using rep-PCR with ERIC primers (4). DNA fingerprints of the re-isolated strains were identical to those of the original strains and were characteristic of C. michiganensis subsp. michiganensis type C. Bacterial canker is a common disease of tomatoes worldwide and has occurred in Ohio for at least 70 years. However, this is the first report of C. michiganensis subsp. michiganensis infecting peppers in Ohio. While the pathogen does not appear to cause systemic disease in peppers, it may serve as a source of inoculum for tomatoes, which are highly susceptible to the disease and often produced in the same greenhouse as peppers or planted in adjacent fields. Bacterial canker has been reported previously from commercial pepper fields in California (2) and Indiana (3). References: (1) J. Dreier et al. Phytopathology 85:462, 1995. (2) M. Lai. Plant Dis. Rep. 60:339, 1976. (3) R. Latin et al. Plant Dis. 79:860, 1995. (4) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994.

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First Report of Bacterial Wilt of Tomato (Solanum lycopersicum) Caused by Ralstonia solanacearum in Benin

Plant Disease ◽

10.1094/pdis-93-5-0549b ◽

2009 ◽

Vol 93 (5) ◽

pp. 549-549 ◽

Cited By ~ 2

Author(s):

R. Sikirou ◽

F. Beed ◽

V. Ezin ◽

G. Gbèhounou ◽

S. A. Miller ◽

...

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Rural Areas ◽

Reference Strain ◽

Plant Protection ◽

Tomato Production ◽

Tetrazolium Chloride ◽

Tomato Plants ◽

First Report ◽

Ecological Zones

In June 2004, wilted tomatoes with no foliar yellowing were observed in Ouègbo, Atlantique District, Benin. The cut tomato stems released whitish bacterial ooze. Longitudinal sections of most stems showed brown vascular discoloration. Twenty symptomatic tomato plants were collected from 10 fields and exported to the Institute of Plant Disease and Plant Protection, Leibniz Universität Hannover, Germany. Bacteria were isolated on triphenyl tetrazolium chloride (TTC) medium (2) and three of the nine bacterial isolates that resembled Ralstonia solanacearum (colonies with red center and whitish periphery) and reference strain ToUdk (race 1 biovar 3; N. Thaveechai, Kasetsart University, Bangkok, Thailand) were used for pathogenicity tests. Five 4-week-old tomato plants cv. Tohounvi, grown in individual plastic pots (14 × 16 cm) containing sterilized field soil, were inoculated with each of the four isolates individually by soil drenching with 30 ml of the test cultures at 108 CFU/ml. Control plants were treated with 30 ml of sterile water. All plants were incubated in a glasshouse at 30°C. All plants inoculated with the isolates from Benin wilted 4 days after inoculation with symptoms similar to those observed in the field. Plants inoculated with the reference strain wilted 7 to 11 days after inoculation. Control plants treated with water remained healthy. R. solanacearum was recovered from the 20 symptomatic plants on TTC medium. The identity of the strains in comparison with the reference strain was confirmed by PCR with species-specific primers 759/760, which produced a single 281-bp fragment (3). Because similar symptoms were being increasingly reported by farmers across Benin and linked with reduced tomato yields, a disease survey was undertaken by IITA in 2006 and 2007. Wilted tomato plants were found across all agro-ecological zones of Benin (3 to 72% of plants per field). Isolates were recovered from the southeastern districts of Adja-Ouèrè, Sakété, Adjohoun, and Dangbo, the southwestern districts of Klouékanmè and Athiémé, the southern districts of Toffo and Bohicon, the central districts of Dassa and Savè, and the northern districts of Malanville and Karimama. Identification of R. solanacearum was confirmed following inoculation of tomato, production of characteristic wilting symptoms, recovery of the pathogen on TTC medium, and positive identification with ELISA kits (Pathoscreen Rs; Agdia Inc., Elkhart, IN). To our knowledge, this is the first report of R. solanacearum infecting tomato in Benin. Tomato is the most cultivated vegetable crop in Benin and important to the livelihood of many people in peri-urban and rural areas. Understanding that the cause of the observed crop losses is R. solanacearum may lead to implementation of management strategies such as deployment of disease-resistant cultivars or grafting tomatoes onto bacterial wilt-resistant rootstocks (1). References: (1) P. Aggarwal et al. Indian J. Agric. Sci. 78:379, 2008. (2) A. Kelman. Phytopathology 44:693, 1954. (3) N. Opina et al. Asian Pac. J. Mol. Biol. Biotechnol. 5:19, 1997.

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First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Roselle in Taiwan

Plant Disease ◽

10.1094/pdis-02-13-0186-pdn ◽

2013 ◽

Vol 97 (10) ◽

pp. 1375-1375 ◽

Cited By ~ 3

Author(s):

Y.-F. Wu ◽

A.-S. Cheng ◽

C.-H. Lin ◽

C.-Y. Chen

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Wilt Disease ◽

Bacterial Suspension ◽

Herbaceous Plant ◽

Infested Soil ◽

Single Plant ◽

Bacterial Strains ◽

Tetrazolium Chloride ◽

First Report

Roselle (Hibiscus sabdariffa L.) is a herbaceous plant belonging to the Malvaceae family. Its calyxes are rich in vitamin C and anthocyanins and are used to make roselle drink and hibiscus tea. Roselles are grown in counties of Taitung, Pingtung, and Chiayi in Taiwan. In addition to a few local cultivars, the major cultivar currently grown in Taiwan is Roselle cv. Victor. In April of 2012, a wilt disease appeared on seedlings of a cultivar, Chiada 1, at the Chungpu Township of Chiayi County. Mature plants were free from this disease. Leaves appeared weak and drooping when they were still green, followed by collapse of the whole plant a few days later. Browning of vascular and pith tissues was evident, especially at the base of the stem. A whitish mass of bacteria oozed from the cut end of diseased stems, suggesting that bacteria might be the cause of this disease. A total of 15 bacterial strains were collected. Colonies on tetrazolium chloride medium (3) were round to oval and fluidal, each with a pink or red center after incubation at 30°C for 48 h. When tobacco leaves were infiltrated with these strains, a hypersensitive reaction (HR) typical of phytopathogenic bacteria was induced. All strains produced the expected amplicon (282 bp) after PCR with the Ralstonia solanacearum-specific primer pair, AU759f and AU760r (4). Three hexose alcohols (mannitol, sorbitol, and dulcitol), rather than three disaccharides (lactose, maltose, and cellobiose), were utilized, which suggests R. solanacearum biovar 4 (2). R. solanacearum phylotype I was determined by phylotype-specific multiplex PCR (1). Pathogenicity of the strains was tested on roselle, tomato, pepper, and eggplant. Young plants of the various species were inoculated at the four- to six-leaf stage by soil drenching with 30 ml of bacterial suspension (about 108 CFU/ml). Control plants were inoculated with sterile water. Each treatment comprised eight plants with a single plant in each pot. Plants were incubated in a greenhouse at 25 to 31°C and 56 to 93% humidity. Wilting was observed 4 to 6 days after inoculation, while the control did not wilt. To find the correlation between plant growth stage and resistance to the pathogen, 2-, 3-, 4-, and 5-week-old roselle plants cv. Chiada 1 were transplanted into artificially infested soil. Eight plants in each treatment were planted with a single plant in each pot. The disease incidences for plants of different ages were 75%, 62.5%, 50%, and 12.5%, respectively. This study showed that resistance increases with plant age. Hence, if older seedlings are transplanted, the risk of bacterial wilt of roselle can be reduced. To our knowledge, this is the first report of R. solanacearum on roselle in Taiwan. References: (1) M. Fegan and P. Prior. Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex, page 449. C. Allen et al., eds. The American Phytopathological Society. St. Paul, MN, 2005. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (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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Ralstonia solanacearum Needs Motility for Invasive Virulence on Tomato

Journal of Bacteriology ◽

10.1128/jb.183.12.3597-3605.2001 ◽

2001 ◽

Vol 183 (12) ◽

pp. 3597-3605 ◽

Cited By ~ 201

Author(s):

Julie Tans-Kersten ◽

Huayu Huang ◽

Caitilyn Allen

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Soft Agar ◽

Cell Protein ◽

In Planta ◽

Tomato Plants ◽

Bacterial Wilt Disease ◽

Virulence Assay ◽

Flagellar Filament

ABSTRACT Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found thatR. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lackingfliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphAcassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

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