First Report of Pectobacterium carotovorum subsp. carotovorum Causing Soft Rot of Orostachys japonica in Korea

Orostachys japonica (Maxim) A. Berger is an important traditional medicine in Korea. The extract of this plant has antioxidant activity and suppresses cancer cell proliferation (1). From summer through fall of 2012 and 2013, a high incidence (~10% to 30%) of disease outbreaks of all plants characterized by water-soaked lesions and soft rot with a stinky odor was observed in cultivated O. japonica around Uljin (36°59′35.04″N, 126°24′1.51″E), Korea. Water-soaked lesions were first observed on the stem base of plants. Subsequently, the plants collapsed, although the upper portion remained asymptomatic. Thereafter, the lesions expanded rapidly over the entire plant. To isolate potential pathogens from infected leaves, small sections (5 to 10 mm2) were excised from the margins of lesions. Ten bacteria were isolated from ten symptomatic plants. Three representative isolates from different symptomatic plants were used for identification and pathogenicity tests. Isolated bacteria were gram negative, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All isolates caused soft rot of potato tubers. These isolates also differed from isolates of Erwinia chrysanthemi (Ech) that they were insensitive to erythromycin and did not produce phosphatase. These isolates differed from known strains of E. carotovora subsp. atroseptica in that they did not produce reducing substances from sucrose (2). Use of the Biolog GN microplate and the Release 4.0 system identified the isolate as Pectobacterium carotovorum subsp. carotovorum with 81.2% similarity. The 16S rRNA of the isolated bacteria was amplified by PCR and sequenced as described by Weisburg et al. (3). A BLAST analysis for sequence similarity of the 16S rRNA region revealed 99% similarity with nucleotide sequences for P. carotovorum subsp. carotovorum isolates (KC790305, KC790280, JF926758, JX196705, and AB680074). The pathogenicity of three bacterial isolates was examined on three 2-year-old O. japonica plants by adding 50 μl of a bacterial suspension containing 108 CFU/ml when wounding the leaves with sterile needles. Ten control plants were inoculated with sterilized water. After inoculation, plants were maintained in a growth chamber at 25°C with relative humidity ranging from 80 to 90%. After 2 to 3 days, tissue discoloration, water-soaked lesions, and soft rot developed around the inoculation point. Severe symptoms of soft rot and darkening developed on leaves of inoculated plants within 3 to 5 days after inoculation. All controls remained healthy during these experiments. The bacterial strains re-isolated from the parts of the leaf showing the symptoms and identified as P. carotovorum subsp. carotovorum on the basis of the biochemical and physiological tests, as well as Biolog system. The results obtained for pathogenicity, Biolog analysis, and molecular data corresponded with those for P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of the presence of P. carotovorum on O. japonica in Korea. References: (1) C.-H. Kim et al. Kor. J. Med. Crop Sci. 11:31, 2003. (2) 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. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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First Report of Pectobacterium carotovorum Causing Soft Rot of Opium Poppy in Spain

Plant Disease ◽

10.1094/pdis-92-2-0317a ◽

2008 ◽

Vol 92 (2) ◽

pp. 317-317 ◽

Cited By ~ 5

Author(s):

S. Aranda ◽

M. Montes-Borrego ◽

F. J. Muñoz-Ledesma ◽

R. M. Jiménez-Díaz ◽

B. B. Landa

Keyword(s):

Pathogenic Bacteria ◽

Soft Rot ◽

Opium Poppy ◽

Bacterial Suspension ◽

Fluorescent Light ◽

Pectobacterium Carotovorum ◽

Bacterial Strains ◽

First Report ◽

Biolog System ◽

Carotovorum Subsp

Opium poppy (Papaver somniferum L.) is an economically important pharmaceutical crop in Spain. Approximately 8,000 ha are cultivated annually in southern and central Spain. To improve yields, opium poppy cultivation is expanding to more humid or irrigated areas of Spain. In the springs of 2005 and 2007, we observed poppy plants with wilt and stem rot symptoms in irrigated, commercial opium poppy (cv. Nigrum) at Carmona and Écija, which are in Seville Province in southern Spain. Closer observations of affected plants revealed darkening and water soaking of the leaves and stem at the soil level, wilting of the lower leaves or the entire plant, and dark brown discoloration of stem vascular tissues and pith of the plant. Severely affected plants became completely rotten and collapsed. Isolations from symptomatic tissues on nutrient agar consistently yielded bacterial colonies. Pure cultures of four representative bacterial strains (two per each of affected field and year of isolation) were used in triplicate for a comparative analysis of biochemical and physiological traits in the ‘carotovora’ group of Erwinia (1) with known isolates of Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. atrosepticum, and Dickeya chrysanthemi. The isolates from opium poppy were gram negative, facultatively anaerobic, oxidase negative, catalase positive, grew at 37°C, and did not produce gas from D-glucose. Acid was produced from D(+)-arabinose, lactose, and D(+)-trehalose, but not from α-D-methylglucoside. In addition, the opium poppy bacterial isolates caused soft rot on potato slices within 24 h at 25°C and did not induce a hypersensitive reaction on tobacco leaves. Use of the Biolog GN microplates and the OmniLog ID 1.2 system identified the four poppy isolates as P. carotovorum (showing a 66.7% similarity with the subsp. carotovorum). Pathogenicity of poppy isolates was tested on three 6-week-old opium poppy plants (cv. Nigrum) by injecting 100 μl of a bacterial suspension containing 108 CFU/ml in the basal stem. Plants that served as controls were injected with sterile water. Plants were incubated in a growth chamber adjusted to 28°C, 90% relative humidity, and a 14-h photoperiod of fluorescent light of 360 μE·m-2·s-1. Severe symptoms of soft rot and darkening developed on stems of inoculated plants within 3 to 5 days after inoculation. No symptoms developed on control plants. Bacterial strains reisolated from inoculated plants were identified as P. carotovorum on the basis of the Biolog system, as well as biochemical and physiological characters. To our knowledge, this is the first report of P. carotovorum causing soft rot of commercial opium poppy crops in Spain and elsewhere. The presence of this bacterial pathogen to irrigated crops and humid areas may pose an important constraint on the yield of opium poppy crops in Spain. References: (1) R. S. Dickey and A. Kelman. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. The American Phytopathological Society, St. Paul, MN, 1988.

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First Report of Bacterial Soft Rot of Potato Caused by Pectobacterium carotovorum subsp. carotovorum in Guangdong Province of China

Plant Disease ◽

10.1094/pdis-07-12-0620-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1819-1819 ◽

Cited By ~ 1

Author(s):

J. X. Zhang ◽

B. R. Lin ◽

H. F. Shen ◽

X. M. Pu ◽

Z. N. Chen ◽

...

Keyword(s):

Guangdong Province ◽

Housekeeping Genes ◽

Soft Rot ◽

Bacterial Suspension ◽

Pectobacterium Carotovorum ◽

Bacterial Soft Rot ◽

First Report ◽

Wide Range ◽

Carotovorum Subsp ◽

Agar Media

Potato (Solanum tuberosum L.) is a major crop in China, with 80.0 million tons being produced in 2010 on 3.3 million ha. Pectobacterium carotovorum subsp. carotovorum Jones 1901; Hauben et al. 1999 causes soft rot worldwide on a wide range of hosts including potato, carrot, and cabbage. During spring 2010, a soft rot with a foul smell was noted in stored potato tubers of different cultivars in the Guangdong Province. Symptoms on tubers appeared as tan, water-soaked areas with watery ooze. The rotted tissues were white to cream colored. Stems of infected plants with typical inky black symptoms could also be found in the fields prior to harvest. Three different potato fields were surveyed, and 13% of the plants had the symptoms. Twenty-seven samples (three symptomatic tubers per sample) were collected. Bacteria were successfully isolated from all diseased tissues on nutrient agar media supplemented with 5% sucrose and incubated at 26 ± 1°C for 36 h. After purification on tripticase soy agar media, four typical strains (7-3-1, 7-3-2, 8-3-1, and 8-3-2) were identified using the following deterministic tests: gram-negative rods, oxidase negative, facultatively anaerobic, able to degrade pectate, sensitive to erythromycin, negative for phosphatase, unable to produce acid from α-methyl-glucoside, and produced acid from trehalose. Biolog analysis (Ver 4.20.05, Hayward, CA) identified the strains as P. carotovorum subsp. carotovorum (SIM 0.808, 0.774, 0.782, and 0.786, respectively). The identity of strains 7-3-1 (GenBank Accession No. JX258132), 7-3-2 (JX258133), and 8-3-1 (JX196705) was confirmed by 16S rRNA gene sequencing (4), since they had 99% sequence identity with other P. carotovorum subsp. carotovorum strains (GenBank Accession Nos. JF926744 and JF926758) using BLASTn. Further genetic analysis of strain 8-3-1 was performed targeting informative housekeeping genes, i.e., acnA (GenBank Accession No. JX196704), gabA (JX196706), icdA (JX196707), mdh (JX196708), mtlD (JX196709), pgi (JX196710), and proA (JX196711) (2). These sequences from strain 8-3-1 were 99 to 100%, homologous to sequences of multiple strains of P. carotovorum subsp. carotovorum. Therefore, strain 8-3-1 grouped with P. carotovorum subsp. carotovorum on the phylogenetic trees (neighbor-joining method, 1,000 bootstrap values) of seven concatenated housekeeping genes when compared with 60 other strains, including Pectobacterium spp. and Dickeya spp. (3). Pathogenicity of four strains (7-3-1, 7-3-2, 8-3-1, and 8-3-2) was evaluated by depositing a bacterial suspension (106 CFU/ml) on the potato slices of cultivar ‘Favorita’ and incubating at 30 ± 1°C. Slices inoculated with just water served as non-inoculated checks. The strains caused soft rot within 72 h and the checks had no rot. Bacteria were reisolated from the slices and were shown to be identical to the original strains based on morphological, cultural, and biochemical tests. Although this pathogen has already been reported in northern China (1), to our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of potato in Guangdong Province of China. References: (1) Y. X. Fei et al. J. Hexi Univ. 26:51, 2010.(2) B. Ma et al. Phytobacteriology 97:1150, 2007. (3) S. Nabhan et al. Plant Pathol. 61:498, 2012. (4) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

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First Report of Pectobacterium punjabense Causing Blackleg and Soft Rot on Potato in Hebei and Fujian Province, China

Plant Disease ◽

10.1094/pdis-12-21-2731-pdn ◽

2022 ◽

Author(s):

Utpal Handique ◽

Yaning Cao ◽

Dekang Wang ◽

Ruofang Zhang ◽

Wensi Li ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Housekeeping Genes ◽

Soft Rot ◽

Rrna Gene ◽

Pectobacterium Carotovorum ◽

Fujian Province ◽

First Report ◽

Potato Plants ◽

Carotovorum Subsp

Pectobacterium spp. and Dickeya spp. cause blackleg and soft rot on potato worldwide (Charkowski, 2018). Potato plants (cv. Favorita or Jizhang 8#) with blackleg symptoms (vascular browning of crown stems, Fig. S1) were observed in the field in Zhangjiakou, Hebei province in 2018, and in Ningde, Fujian Province in 2019, in China. The disease incidence was around 50% and 10% in Zhangjiakou (5 ha) and Ningde (4 ha), respectively. Diseased plants (3 from each site) were collected to isolate the pathogen. Blackleg symptomatic stems were soaked in 75% ethanol for 2 min, rinsed and ground in sterile distilled water. Serial tenfold dilutions of the above solution were plated onto the crystal violet pectate agar (CVP) plate (Ge et al., 2018). Two to 3 days after incubation at 28°C, 4 bacterial colonies in total which digested pectin from the media and developed pit on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al., 2002). Two colony sequences that showed more than 99% sequence identity to Pectobacterium punjabense type strain SS95 (MH249622) were submitted to the GenBank ( accession numbers: OK510280, MT242589). Additionally, six housekeeping genes proA (OK546205, OK546199), gyrA (OK546206, OK546200), icdA (OK546207, OK546201), mdh (OK546208, OK546202), gapA (OK546209, OK546203), and rpoS (OK546210, OK546204) of these two isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with MH249622T . Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 2 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch’s postulate was performed on potato seedlings and potato tubers (cv. Favorita) by injecting 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems or the tubers and kept at 100% humidity and 21°C for 1 day. Four days after inoculation, the infected area of the inoculated seedlings rotten and turned black, while the controls were symptomless (Fig. S4). Two days after inoculation, the infected tubers rotten and turned black, while the controls were symptomless (Fig. S4). Bacterial colonies were reisolated from these symptomatic tissues and identified using the same methods described above. Blackleg on potato plants or soft rot on potato has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium parmentieri, Pectobacterium polaris in China (Zhao et al., 2018; Cao et al., 2021; Wang et al., 2021). To our knowledge, this is the first report of blackleg/soft rot of potato caused by Pectobacterium punjabense in China. We believe that this report will draw attention to the management of this pathogen in China.

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First Report of Pectobacterium wasabiae Causing Soft Rot of Cabbage in Malaysia

Plant Disease ◽

10.1094/pdis-01-13-0112-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1110-1110 ◽

Cited By ~ 2

Author(s):

E. Golkhandan ◽

K. Sijam ◽

S. Meon ◽

Z. A. M. Ahmad ◽

A. Nasehi ◽

...

Keyword(s):

16S Rrna ◽

Dna Sequences ◽

Acid Production ◽

Soft Rot ◽

Economic Losses ◽

Economic Damage ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

First Report ◽

Carotovorum Subsp

Soft rot of cabbage (Brassica rapa) occurs sporadically in Malaysia, causing economic damage under the hot and wet Malaysian weather conditions that are suitable for disease development. In June 2011, 27 soft rotting bacteria were isolated from cabbage plants growing in the Cameron Highlands and Johor State in Malaysia where the economic losses exceeded 50% in severely infected fields and greenhouses. Five independent strains were initially identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and elicit hypersensitive reaction (HR) on Nicotiana tabaccum and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG-positive and positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-D-glucoside, and D-arabitol. All the strains exhibited pectolytic activity on potato slices. PCR assays were conducted to distinguish P. wasabiae from P. carotovorum subsp. brasiliensis, P. atrosepticum, and other Pectobacterium species using primers Br1f/L1r (2), Eca1f/Eca2r (1), and EXPCCF/EXPCCR, respectively. DNA from strains did not yield the expected amplicon with the Br1f/L1r and Eca1f/Eca2r, whereas a 550-bp amplicon typical of DNA from P. wasabiae was produced with primers EXPCCF/EXPCCR. ITS-RFLP using the restriction enzyme, Rsa I, produced similar patterns for the Malaysian strains and the P. wasabiae type strain (SCRI488), but differentiated it from P. carotovora subsp. carotovora, P. atrosepticum, P. carotovorum subsp. brasiliensis, and Dickeya chrysanthemi type strains. BLAST analysis of the 16S rRNA DNA sequence (GenBank Accession No. KC445633) showed 99% identity to the 16S rRNA of Pw WPP163. Phylogenetic reconstruction using concatenated DNA sequences of mdh and gapA from P. wasabiae Cc6 (KC484657) and other related taxa (4) clustered Malaysian P. wasabiae strains with P. wasabiae SCRI488, readily distinguishing it from other closely related species of Pectobacterium. Pathogenicity assays were conducted on leaves and stems of four mature cabbage plants for each strain (var. oleifera) by injecting 10 μl of a bacterial suspension (108 CFU/ml) into either stems or leaves, and incubating them in a moist chamber at 80 to 90% relative humidity at 30°C. Water-soaked lesions similar to those observed in the fields and greenhouses were observed 72 h after injection and bacteria with similar characteristics were consistently reisolated. Symptoms were not observed on water-inoculated controls. The pathogenicity test was repeated with similar results. P. wasabiae was previously reported to cause soft rot of horseradish in Japan (3). However, to our knowledge, this is the first report of P. wasabiae infecting cabbage in Malaysia. References: (1) S. H. De Boer and L. J. Ward. Phytopathology 85:854, 1995. (2) V. Duarte et al. J. Appl. Microbiol. 96:535, 2004. (3) M. Goto and K. Matsumoto. Int. J. Syst. Bacteriol. 37:130, 1987. (4) B. Ma et al. Phytopathology 97:1150, 2007.

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First Report of Broccoli Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Serbia

Plant Disease ◽

10.1094/pdis-03-13-0258-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1504-1504 ◽

Cited By ~ 1

Author(s):

K. Gašic ◽

V. Gavrilović ◽

Ž. Ivanović ◽

A. Obradović

Keyword(s):

Pathogenic Bacteria ◽

Soft Rot ◽

Hypodermic Needle ◽

Bacterial Suspension ◽

Rrna Gene ◽

Pectobacterium Carotovorum ◽

Vegetable Crops ◽

Bacterial Strains ◽

Carotovorum Subsp ◽

Its Pcr

In September 2012, soft rot symptoms on broccoli (Brassica oleracea L. var. italica Plenck) were observed in several commercial fields in the western part of Serbia. Following the first harvest, water-soaked areas developed on broccoli stem tissue and progressed into soft rot decay of entire plants. The incidence of disease was approximately 30%. In Serbia, broccoli is grown on smaller fields compared to other vegetables, but its production and consumption increased significantly in recent years. From the diseased tissue, shiny, grayish white, round colonies were isolated on nutrient agar. Six non-fluorescent, gram-negative, facultative anaerobic, oxidase-negative, and catalase-positive bacterial strains were chosen for further identification. All strains caused soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew at 37°C and in yeast salts broth medium containing 5% NaCl (2), did not produce acid from α-methyl glucoside, but utilized lactose and trehalose, and did not produce indole or lecitinase. Investigated strains formed light red, 1.5-mm-diameter colonies on Logan's medium (2), and did not produce blue pigmented indigoidine on glucose yeast calcium carbonate agar (2) nor “fried egg” colonies on potato dextrose agar. Based on biochemical and physiological characteristics (1) and ITS-PCR and ITS-RFLP analysis (4), the strains were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rRNA gene sequence from two strains (GenBank KC527051 and KC527052) showed 100% identity with sequences of P. carotovorum subsp. carotovorum previously deposited in GenBank (3). Pathogenicity of the strains was confirmed by inoculation of broccoli head tissue fragments. Three florets per strain were inoculated by pricking the petals with a syringe and hypodermic needle and depositing a droplet of bacterial suspension (approx. 1 × 108 CFU/ml) at the point of inoculation. Sterile distilled water was used as a negative control. Inoculated florets were placed in a sealed plastic container and incubated in high humidity conditions at 28°C. Tissue discoloration and soft rot developed around the inoculation point within 48 to 72 h. No symptoms developed on control florets. Identity of bacterial strains reisolated from inoculated plant tissues was confirmed by ITS-PCR using G1/L1 primers followed by digestion of PCR products with Rsa I restriction enzyme (4). In Serbia, P. carotovorum subsp. carotovorum has been isolated from potato, some vegetable crops, and ornamentals, but not from broccoli until now. References: (1) S. H. De Boer and A. Kelman. Page 56 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. (2) P. C. Fahy and A. C. Hayward. Page 337 in: Plant Bacterial Diseases: A Diagnostic Guide. P. C. Fahy and G. J. Persley eds. Academic Press, New York, 1983. (3) S. Nabhan et al. J. Appl. Microbiol. 113: 904, 2012. (4) I. K. Toth et. al. Appl. Environ. Microbiol. 67:4070, 2001.

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First Report of Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum on Cucumber in Malaysia

Plant Disease ◽

10.1094/pdis-10-10-0754 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1474-1474 ◽

Cited By ~ 3

Author(s):

E. Nazerian ◽

K. Sijam ◽

M. A. Zainal Abidin ◽

G. Vadamalai

Keyword(s):

Restriction Enzyme ◽

Soft Rot ◽

Bacterial Suspension ◽

Pectobacterium Carotovorum ◽

Agarose Gel ◽

First Report ◽

Appl Environ ◽

Pcr Products ◽

Carotovorum Subsp ◽

Its Pcr

Cucumber (Cucumis sativus L.) is one of the most important vegetable fruits in Malaysia. Cucumber is principally grown in the states of Johor, Kelantan, and Perak. The broad host range Enterobacteriaceae pathogen, Pectobacterium carotovorum, can cause soft rot on stems or cucumber fruit. In Malaysia, cucumber is produced in a warm, humid climate, thus the plant is susceptible to attack by P. carotovorum at any time during production. In 2010, cucumber samples with wilted and chlorotic leaves, water-soaked lesions, and collapsed fruits were found in multiple fields. Small pieces of infected stems and fruit were immersed in 5 ml of saline solution (0.85% NaCl) for 20 min and then 50 μl of this suspension was spread onto nutrient agar (NA) and incubated at 27°C for 24 h. White-to-pale gray colonies with irregular margins were selected for analysis. For pathogenicity tests, cucumber fruits were surface sterilized by ethyl alcohol 70%, washed with sterilized distilled water, cut into small pieces, and inoculated with 20 μl of 108 CFU/ml suspensions of five representative strains. Cucumber plants were grown for 3 weeks in sterilized soil and their stems were inoculated with 20 μl of 108 CFU/ml of bacterial suspension. Inoculated samples and control (noninoculated) plants were placed in a growth chamber with 80 to 90% relative humidity at 27°C. Symptoms occurred on fruit slices and stems after 1 to 3 days and appeared the same as naturally infected samples, but the control samples remained healthy. Koch's postulates were fulfilled with the reisolation of cultures with the same characteristics as described earlier. Hypersensitivity reaction (HR) assays were done by infiltrating 108 CFU/ml of bacterial suspension into tobacco leaf epidermis and HR developed. All strains were subjected to biochemical and morphological assays, as well as molecular assessment. The strains were gram negative, facultative anaerobes, rod shaped, able to macerate potato slices and growth at 37°C; catalase positive; oxidase and phosphatase negative; able to degrade pectate; sensitive to erythromycin; negative for utilization of α-methyl glycoside, indole production, and reduction of sugars from sucrose; acid production from arabitol, sorbitol, and utilization of citrate were negative, but positive for raffinose and melibiose utilization. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment on agarose gel 1% (1). Amplification of intergenic transcribed spacer region by G1 and L1 primers gave two main bands at approximately 535 and 580 bp on agarose gel 1.5%. The ITS-PCR products were digested with RsaI restriction enzyme (3). On the basis of biochemical and morphological characteristics, PCR-based pel gene and characterization of the ITS region, and digestion of the ITS-PCR products with RsaI restriction enzyme, all isolates were identified as P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of soft rot caused by P. carotovorum subsp. carotovorum on cucumber from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.

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First Report of Pectobacterium carotovorum subsp. carotovorum on Spathiphyllum wallisii in Argentina

Plant Disease ◽

10.1094/pdis-93-8-0842c ◽

2009 ◽

Vol 93 (8) ◽

pp. 842-842 ◽

Cited By ~ 4

Author(s):

A. M. Alippi ◽

A. C. López

Keyword(s):

16S Rrna ◽

Natural Infection ◽

Soft Rot ◽

Unknown Etiology ◽

Rrna Gene ◽

Pectobacterium Carotovorum ◽

Distilled Water ◽

Strain Atcc ◽

First Report ◽

Carotovorum Subsp

Peace lily (Spathiphyllum wallisii Regel) is a popular ornamental potted plant in Argentina. During May of 2008 (austral autumn), necrotic lesions of unknown etiology were observed on S. wallisii in a nursery in Pontevedra (34°45′6″S, 58°42′42″W). Plants first showed water-soaked areas starting from the leaf tips. Infected tissue became irregular, brown, dark-to-black lesions on leaves ~12 to 14 mm in diameter surrounded by yellowish haloes. Disease incidence approached 30%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria isolated from lesions formed white-to-cream, glistening, convex colonies on yeast dextrose calcium carbonate agar. Three bacterial strains isolated from different symptomatic plants were selected for comparative analysis with Pectobacterium carotovorum subsp. carotovorum type strain ATCC 15713. All were facultatively, anaerobic, gram-negative rods, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All strains were oxidase and arginine dihydrolase negative, fermented glucose, did not hydrolyze starch, did not produce lecithinase, indole or the blue pigment indigoidine, reduced nitrates, hydrolyzed gelatin and esculin, able to rot onion slices, caused soft rot of potato tubers, resistant to erythromycin, and grew at 37°C. Acid was produced from cellobiose, d-glucose, d-melibiose, d-mannitol, d-mannose, l-rhamnose, d-sucrose, and l-arabinose but not from inositol and d-sorbitol. Bacteria utilized N-acetyl-glucosamine and citrate but not tartrate, benzoate, or propionate. Their identity was confirmed by 16S rRNA gene sequencing of strain F402Pcc (GenBank Accession No. FJ717337) showing a 99% homology with that of strain ATCC 3326 (FJ 5958691). Pathogenicity was verified on S. wallisii, Dieffenbachia picta, Aglaonema commutatum, and Anthurium andraeanum within the Araceae family by spraying two plants per strain tested with bacterial suspensions (108 CFU/ml) in sterile distilled water with and without wounding the leaves with sterile needles. Controls were sprayed with sterile distilled water. After 48 h in a humidity chamber, inoculated plants and controls were maintained at 25 ± 3°C in a greenhouse. Water-soaked areas developed from 24 to 48 h after inoculation and became necrotic within 4 to 5 days. Lesions expanded to resemble natural infection in S. wallisii within 20 days, while in the rest of the hosts tested, lesions were smaller and remained brown surrounded by yellowish haloes. All strains were reisolated from each host tested. The original and all reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (4) confirming that DNA fingerprints of the reisolated strains were identical to those of the original strains. No lesions were observed on controls. The pathogen was identified as P. carotovorum subsp. carotovorum based on biochemical, physiological, pathogenicity tests, and 16S rRNA sequencing (1–3).To our knowledge, this is the first report of this pathogen on S. wallisii in Argentina although it has been reported as causing tomato pith necrosis (1) and soft rot of vegetables after harvest (3). References: (1) A. M. Alippi et al. Plant Dis. 81:230, 1997. (2) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:381, 2003. (3) L. Halperin and L. S. Spaini. Rev. Argent. Agron. 6:261, 1939. (4) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994.

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First Report of Cabbage Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Malaysia

Plant Disease ◽

10.1094/pdis-09-10-0683 ◽

2011 ◽

Vol 95 (4) ◽

pp. 491-491 ◽

Cited By ~ 7

Author(s):

E. Nazerian ◽

K. Sijam ◽

Z. A. Mior Ahmad ◽

G. Vadamalai

Keyword(s):

Pcr Amplification ◽

Soft Rot ◽

23S Rrna ◽

Bacterial Suspension ◽

Pectobacterium Carotovorum ◽

Disease Symptoms ◽

First Report ◽

Appl Environ ◽

Carotovorum Subsp ◽

Its Pcr

Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetables cultivated in Pahang and Kelantan, Malaysia. Pectobacterium carotovorum can cause soft rot on a wide range of crops worldwide, especially in countries with warm and humid climates such as Malaysia. Cabbage with symptoms of soft rot from commercial fields were sampled and brought to the laboratory during the winter of 2010. Disease symptoms were a gray to pale brown discoloration and expanding water-soaked lesions on leaves. Several cabbage fields producing white cultivars were investigated and 27 samples were collected. Small pieces of leaf samples were immersed in 5 ml of saline solution (0.80% NaCl) for 20 min to disperse the bacterial cells. Fifty microliters of the resulting suspension was spread on nutrient agar (NA) and King's B medium and incubated at 30°C for 48 h. Purification of cultures was repeated twice on these media. Biochemical and phenotypical tests gave these results: gram negative, rod shaped, ability to grow under liquid paraffin (facultative anaerobe); oxidase negative; phosphatase negative; positive degradation of pectate; sensitive to erythromycin; negative to Keto-methyl glucoside utilization, indole production and reduction sugars from sucrose were negative; acid production from sorbitol and arabitol was negative and from melibiose, citrate, and raffinose was positive. Hypersensitivity reaction on tobacco leaf with the injection of 106 CFU/ml of bacterial suspension for all strains was positive. Four representative strains were able to cause soft rot using cabbage slices (three replications) inoculated with a bacterial suspension at 106 CFU/ml. Inoculated cabbage slices were incubated in a moist chamber at 80% relative humidity and disease symptoms occurred after 24 h. Cabbage slices inoculated with water as a control remained healthy. The bacteria reisolated from rotted cabbage slices on NA had P. carotovorum cultural characteristics and could cause soft rot in subsequent tests. PCR amplification with Y1 and Y2 primers (1), which are specific for P. carotovorum, produced a 434-bp band with 15 strains. PCR amplification of the 16S-23S rRNA intergenic transcribed spacer region (ITS) using G1 and L1 primers gave two main bands approximately 535 and 580 bp and one faint band approximately 740 bp when electrophoresed through a 1.5% agarose gel. The ITS-PCR products were digested with RsaI restriction enzyme. According to biochemical and physiological characterictics (2), PCR-based pel gene (1), and analysis by ITS-PCR and ITS-restriction fragment length polymorphism (3), all isolates were identified as P. carotovorum subsp. carotovorum. This pathogen has been reported from Thailand, Indonesia, and Singapore with whom Malaysia shares its boundaries. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum in cabbage from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.

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