Occurrence and Identification of Pectobacterium carotovorum subsp. brasiliensis and Dickeya dianthicola Causing Blackleg in some Potato Fields in Serbia

Plant Disease ◽  
2020 ◽  
Author(s):  
Sanja Marković ◽  
Slaviša Stanković ◽  
Aleksandra Jelušić ◽  
Renata Ilicic ◽  
Andrea Kosovac ◽  
...  
Keyword(s):  
Housekeeping Genes ◽  
Pathogenicity Test ◽  
Pectobacterium Carotovorum ◽  
New Genotype ◽  
Genetic Features ◽  
Carotovorum Subsp ◽  
Polymerase Chain ◽  
Causative Bacterium ◽  
Genetic Profiles ◽  
First Time

Blackleg outbreaks were noticed on three fields (total c. 100 ha) during two consecutive years (2018, 2019) in one of the main potato growing areas in Serbia (Bačka region, Vojvodina). The percentage of infected plants reached 40-70% with 10.5% to 44.7% yield reductions. From the three fields out of 90 samples Pectobacterium carotovorum subsp. brasiliensis was most frequently identified and diagnosed as causal agent of potato blackleg in Serbia for the first time (29 isolates). Dickeya dianthicola was a less frequently causative bacterium, which was also noticed for the first time (nine isolates). A total of 38 isolates were characterized based on their phenotypic and genetic features, including a pathogenicity test on potato. The repetitive element Polymerase Chain Reaction (rep-PCR) using BOX, REP and ERIC primer pairs differentiated five genetic profiles among 38 tested isolates. Multilocus sequence analysis (MLSA) of four housekeeping genes, acnA, gapA, icdA and mdh, revealed the presence of three so far unknown P. c. subsp. brasiliensis multilocus genotypes and confirmed clustering into two main genetic clades as determined in other studies. MLSA also revealed the presence of a new genotype of D. dianthicola in Serbia.

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

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1819-1819 ◽  
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.

scholarly journals First Report of Bacterial Stem Rot Caused by Pectobacterium carotovorum subsp. carotovorum and P. carotovorum subsp. atrosepticum on Grafted Eggplant in Italy

Plant Disease ◽  
2001 ◽  
Vol 85 (8) ◽  
pp. 921-921 ◽  
Author(s):  
V. Catara ◽  
P. Bella ◽  
G. Polizzi ◽  
A. Paratore
Keyword(s):  
Relative Humidity ◽  
Pathogenic Bacteria ◽  
Solanum Melongena ◽  
Fruit Rot ◽  
Pathogenicity Test ◽  
Pectobacterium Carotovorum ◽  
Carotovorum Subsp ◽  
Dark Green ◽  
Stem Lesions ◽  
Physiological Tests

In December 1999, widespread dieback of eggplant (Solanum melongena L., hybrid Mission Bell), grafted onto the interspecific hybrid Beaufort (Lycopersicon esculentum × Lycopersicon hirsutum) and on tomato hybrid Energy, was observed during four rootstock evaluation trials in greenhouses in Sicily, Italy. Dark brown to black, firm but sometimes fissured lesions, 1 to 20 cm long, were observed just above the grafting point. Water-soaked, soft, dark green lesions that turned brown with age were observed on the upper stem. Extensive discoloration of vascular tissues and, in some cases, breakdown of the pith and stem hollowness occurred. Eggplant seedlings, present in the same experimental trials, did not show any symptoms. From symptomatic tissues, numerous bacterial colonies were obtained on nutrient dextrose agar. Bacteria from purified colonies were gram-negative, oxidase-negative, facultatively anaerobic pectolitic on crystal violet pectate agar, which is nonfluorescent on King's B medium. On the basis of biochemical and physiological tests (1), seven of 10 isolates were identified as Pectobacterium carotovorum subsp. carotovorum (Jones 1901) Hauben et al. 1999, comb. nov. (2), and the remaining three were identified as P. carotovorum subsp. atrosepticum (van Hall 1902) Hauben et al. 1999, comb. nov. (2). Four days after prick inoculation of the stems of eggplant and tomato plants, all isolates caused extensive collapse of stems and internal brown discoloration and hollowness, respectively. In a second pathogenicity test, basil leaves of grafted eggplants and eggplant seedlings were either removed or left intact. These plants were then sprayed with bacterial suspensions (108 CFU/ml) of one isolate of each pathogen. Plants were held at 100% relative humidity or in a low-moisture incubator. After 6 days incubation, stem lesions and vascular discoloration, typical of symptoms observed in greenhouses, were observed on plants held at 100% relative humidity. No symptoms were observed in unwounded plants or plants maintained in low relative humidity. Similar symptoms on grafted eggplant have been attributed to physiological disorders (3). To our knowledge, only P. carotovorum subsp. carotovorum has been reported on eggplant as causing fruit rot. References: (1) N. W. Schaad, ed. 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 2nd ed. American Phytopathological Society, St. Paul, MN. (2) L. Hauben et al. Int. J. Syst. Bacteriol. 41:582, 1999. (3) G. Ginoux and H. Laterrot. Rev. Horticole (Paris) 321:49, 1991.

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

Plant Disease ◽  
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.

scholarly journals Characterization of Pectobacterium carotovorum subsp. carotovorum isolates from a recent outbreak on cabbage in Bosnia and Herzegovina

2019 ◽  
Vol 34 (3-4) ◽  
pp. 211-222
Author(s):  
Tatjana Popovic ◽  
Aleksandra Jelusic ◽  
Sanja Markovic ◽  
Renata Ilicic
Keyword(s):  
Bosnia And Herzegovina ◽  
Disease Incidence ◽  
Housekeeping Genes ◽  
Soft Rot ◽  
Pectobacterium Carotovorum ◽  
Preliminary Identification ◽  
Band Size ◽  
Carotovorum Subsp ◽  
Primer Sets ◽  
Rot Disease

The causal agent of soft rot disease associated with a cabbage outbreak in Semberija region, Bosnia and Herzegovina, in 2018 was identified and characterized. Symptoms appeared in the form of water-soaked lesions on leaves and specific odour. Disease incidence ranged from 20% to 30%. The causal pathogen was isolated on nutrient agar (NA), King?s B and crystal violet pectate (CVP) media. Eight creamy-white, round and convex bacterial isolates, which produced characteristic pits on CVP medium were taken as representative. They were gram negative, facultative anaerobe, oxidase negative, catalase positive, nonfluorescent on King?s B medium, levan and arginine dehydrolase negative. The isolates were able to cause soft rot on cabbage and potato tuber slices 24 h after inoculation under conditions of high relative humidity. Polymerase chain reaction (PCR) was performed for preliminary identification by using three specific primer sets: F0145/E2477 (specific for Pectobacterium carotovorum subsp. carotovorum), Br1f/L1r (specific for P. carotovorum subsp. brasiliensis) and ECA1f/ECA2r (specific for P. atrosepticum). All isolates produced the band size of 666 bp with F0145/E2477 primer pair, indicating that they belong to the species P. carotovorum subsp. carotovorum. Further genetic characterization was based on sequence analysis of the gapA and mdh housekeeping genes. BLAST analysis confirmed 99.39% (Q. cover 100%, E. value 0.0) and 100% (Q. cover 100%, E. value 0.0) identity of the isolates with P. carotovorum subsp. carotovorum strains deposited in the NCBI database as M34 (KY047594) for gapA and Pcc t0437 (KC337296) for mdh genes, respectively. Phylogenetic analysis showed genetic homogeneity among the cabbage isolates.

Detection and characterization of bacteria from the potato rhizosphere degradingN-acyl-homoserine lactone

2006 ◽  
Vol 52 (10) ◽  
pp. 1006-1015 ◽  
Author(s):  
S Jafra ◽  
J Przysowa ◽  
R Czajkowski ◽  
A Michta ◽  
P Garbeva ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Acid Methyl Ester ◽  
Soft Rot ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Pectobacterium Carotovorum ◽  
In Planta ◽  
Lower Efficiency ◽  
Carotovorum Subsp ◽  
First Time

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA–DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.Key words: AHL degradation, Ochrobactrum sp., Pectobacterium carotovorum.

scholarly journals First Report of Pectobacterium polaris Causing Blackleg on Potato in Inner Mongolia and Sichuan Province, China

Plant Disease ◽  
2021 ◽  
Author(s):  
UTPAL HANDIQUE ◽  
Yaning Cao ◽  
Zhiwen Feng ◽  
Qinghua Sun ◽  
Ruofang Zhang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Inner Mongolia ◽  
Housekeeping Genes ◽  
Sichuan Province ◽  
Rrna Gene ◽  
Pectobacterium Carotovorum ◽  
First Report ◽  
Potato Plants ◽  
Carotovorum Subsp

Pectobacterium spp. and Dickeya spp. cause Blackleg on potato worldwide (Charkowski, 2018). Potato plants (cv. Innovator V4 or Favorita) with blackleg symptoms (vascular browning of crown stems or curled leaves, Fig. S1) were observed in the field in Xilingol League, Inner Mongolia in 2018, and in Chengdu, Sichuan Province in 2020, in China. The disease incidence were around 10% and 20% in Xilingol League (20 ha) and Chengdu (40 ha), respectively. Diseased plants (5 from Xilingol League, and 2 from Chengdu) 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, the bacterial colonies 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). Three colony sequences that showed more than 99% sequence identity to Pectobacterium polaris type strain NIBIO1392 (NR_159086.1) were submitted to the GenBank ( accession numbers: MT242579, MT242580, and MZ489432). Additionally, six housekeeping genes proA (MZ39581–MZ395583), gyrA (MZ395569–MZ395571), icdA (MZ395572–MZ39574), mdh (MZ395575–MZ395577), gapA (MZ395578– MZ395580), and rpoS (MZ39584–MZ395586) of these three isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with Pectobacterium polaris strain NIBIO1392. Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 3 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch’s postulate was performed on potato seedlings (cv. Favorita) by injecting 100 μl bacterial suspension (107 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems and kept at 80% humidity and 21°C for 2 days. Seven days after inoculation, the infected area of the inoculated seedlings rotten and turned black or even lodged, while the controls were symptomless (Fig. S4). It was observed that isolate MZ489432 from Chengdu, Sichuan Province was more virulent than the isolates from Xilingol League (Fig. S4). Bacterial colonies were reisolated from these symptomatic seedlings and identified using the same methods described above. Blackleg on potato plants has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, and Pectobacterium parmentieri in China (Zhao et al., 2018; Cao et al., 2021). To our knowledge, this is the first report of blackleg of potato caused by Pectobacterium polaris in China. We believe that this report will draw attention to the management of this pathogen in China.

scholarly journals First Report of Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot on Squash and Watermelon in Serbia

Plant Disease ◽  
2019 ◽  
Vol 103 (10) ◽  
pp. 2667-2667 ◽  
Author(s):  
N. Zlatković ◽  
A. Prokić ◽  
K. Gašić ◽  
N. Kuzmanović ◽  
M. Ivanović ◽  
...  
Keyword(s):  
Soft Rot ◽  
Pectobacterium Carotovorum ◽  
First Report ◽  
Carotovorum Subsp

scholarly journals Pectobacterium spp. Associated with Bacterial Stem Rot Syndrome of Potato in Canada

2012 ◽  
Vol 102 (10) ◽  
pp. 937-947 ◽  
Author(s):  
S. H. De Boer ◽  
X. Li ◽  
L. J. Ward
Keyword(s):  
Gene Sequence ◽  
Blackleg Disease ◽  
Chain Reaction ◽  
Biochemical Tests ◽  
Pectobacterium Atrosepticum ◽  
Physiological Characterization ◽  
Specific Polymerase Chain Reaction ◽  
Sequence Identification ◽  
Carotovorum Subsp ◽  
Polymerase Chain

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.

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