Detection and Identification of Potato Soft Rot &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; Subspecies &lt;i&gt;carotovorum&lt;/i&gt; by PCR Analysis of 16S rDNA in Jordan

Abstract. Joko T, Soffan A, Muhammad Saifur Rohman MS. 2019. A novel subspecies-specific primer targeting the gyrase B gene for the detection of Pectobacterium carotovorum subsp. brasiliense. Biodiversitas 20: 3042-3048. Pectobacterium carotovorum subsp. brasiliense is one of the major causative bacterial pathogens of the soft rot disease in various crops. It has a high virulence and a wide range of hosts in the tropics and the subtropics. Most often, conventional methods are not able to accurately distinguish P. carotovorum subsp. brasiliense from other subspecies. Thus, this study aimed to design a specific gyrase B gene (gyrB) -based primers for the detection and identification of soft rot pathogen. The specific primers design was based on the alignment using gyrB gene sequence data from P. carotovorum subsp. brasiliense and other data from the GenBank. The primers comprised of F-gyr-Pcb (5’-CAC AGG CAC CGC TGG CTG TT-3’) and R-gyr-Pcb (5’-CGT CGT TCC ACT GCA ATG CCA-3’) with an amplicon of 336 base pairs. The specificity of the primers pair was verified both in silico and in polymerase chain reaction (PCR) assays, where the primers could only detect P. carotovorum subsp. brasiliense. Primers’ sensitivity was determined by qualitative PCR with a detection limit of less than 0.5 ng/µL of genomic DNA. Hence, the proposed detection tool can be beneficial to advance further studies on the ecology and epidemiology of soft rot diseases.

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First Report of Bacterial Soft Rot of Milk Thistle (Silybum marianum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China

Plant Disease ◽

10.1094/pdis-02-14-0137-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1152-1152 ◽

Cited By ~ 1

Author(s):

J. Gao ◽

N. Nan ◽

B. H. Lu ◽

Y. N. Liu ◽

X. Y. Wu ◽

...

Keyword(s):

16S Rdna ◽

Soft Rot ◽

Jilin Province ◽

Milk Thistle ◽

Pectobacterium Carotovorum ◽

Bacterial Soft Rot ◽

Silybum Marianum ◽

16S Rdna Sequence ◽

First Report ◽

Carotovorum Subsp

Milk thistle (Silybum marianum) is an annual or biannual plant of the Asteraceae family that produces the hepaprotectant silymarin. In 2012, almost all milk thistle grown in the medicinal herbal garden of Jilin Agricultural University (Changchun, Jilin Province, China) exhibited symptoms of a previously undetected soft rot disease. Initial symptoms on stems appeared as tan, semitransparent, and water-soaked, then became sunken. The rotted lesions expanded rapidly and inner stem tissues were rotten with a foul smell. Eventually, the whole plant became black, then collapsed and died. Economic losses were significant as the seed crop was almost completely lost. Nine bacterial strains were isolated from tissues on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies of the nine strains were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent, facultatively anaerobic, motile with two to four peritrichous flagella (observed by electron transmission microscope), positive for catalase and potato rot, but negative for oxidase and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose, and indole. Results were variable for the Voges-Proskauer test and production of H2S from cysteine. The strains utilized esculin, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or starch. Glycerol and maltose were only weakly utilized. Species identity was confirmed by molecular analysis of one of the strains, SMG-2. HPLC indicated a DNA GC content of 50.55%. The 16S rDNA sequence (KC207898) of SMG-2 showed 99% sequence identity to that of a Pectobacterium carotovorum subsp. carotovorum strain (DQ333384) and the sequence of the 16S-23S rDNA spacer region (KJ415377) was 95% similar to that of another known strain of P. carotovorum subsp. carotovorum (AF232684). Based on biochemical and physiological characteristics (2), as well as 16S rDNA gene analysis, the strains were identified as P. carotovorum subsp. carotovorum. Pathogenicity of the nine strains was evaluated by depositing a bacterial suspension (108 CFU/ml) on wounded stems (made with a disinfected razor blade) of 3-month-old milk thistle plants. Three plants were inoculated with each strain and three plants were treated with sterilized water as negative controls. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 48 h, the plants inoculated with bacteria showed similar symptoms as the naturally infected plants, while control plants remained symptomless. The symptoms observed on inoculated stems were rotten and sunken tissues. Bacteria were re-isolated from the inoculated plants and confirmed to be identical to the original strains based on 16S rDNA sequence analysis. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of milk thistle in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (2) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Bacterial Soft Rot of Horn Lian (Typhonium giganteum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China

Plant Disease ◽

10.1094/pdis-03-14-0329-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1268-1268 ◽

Cited By ~ 1

Author(s):

J. Gao ◽

N. Nan ◽

Y. N. Liu ◽

B. H. Lu ◽

W. Y. Xia ◽

...

Keyword(s):

Cell Suspension ◽

16S Rdna ◽

Soft Rot ◽

Rdna Sequence ◽

Jilin Province ◽

Pectobacterium Carotovorum ◽

Bacterial Soft Rot ◽

16S Rdna Sequence ◽

First Report ◽

Carotovorum Subsp

Horn lian (Typhonium giganteum) is a perennial herb of the family Aracea and is commonly used for expelling phlegm and as an antispasmodic treatment. In August 2012, horn lian grown in Changchun, Jilin Province of China, exhibited soft rot disease with ~60% incidence and experienced great losses. Water-soaked and dark green lesions on leaves expanded along main veins. Semitransparent, water-soaked, and sunken lesions on stems expanded rapidly and caused the whole plant to collapse with a foul smell. Nine representative strains were isolated from infected leaves and stems on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent on King's B medium (KB), facultatively anaerobic, motile with three to six peritrichous flagella (observed by electron transmission microscope), positive for catalase and pectolytic activity test on potato slices, but negative for oxidase, urease, and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin and reduced nitrate, but did not reduce sucrose. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose and indole. Results were variable for the Voges-Proskauer test. The strains utilized sucrose, arabinose, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, maltose, raffinose, glycerol, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or dulcitol. Species identity was confirmed by molecular characterization of one of the nine strains, DJL1-2. DNA GC content indicated by high performance liquid chromatography (HPLC) was 51.7%. The 16S rDNA sequence (KC07897) of DJL1-2 showed 99% identity to that of a Pectobacterium carotovorum subsp. carotovorum (Pcc) strain (CP001657) and the sequence of the 16S-23S rDNA spacer region (KJ623257) was 93% similar to that of another known strain of Pcc (CP003776). As a result, the strains were identified as Pcc (2). Pathogenicity of the nine strains was evaluated by spraying 1 ml of bacterial cell suspension (108 CFU/ml) onto healthy leaves and injecting 0.1 ml of cell suspension into stems of 3-year-old horn lian plants with a sterile pipette tip. Three seedlings were used for each strain and sterilized water served as negative controls. Pcc SMG-2 reference strain (from milk thistle) was also inoculated into horn lian leaves and stems. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 72 h, water-soaked lesions similar to the naturally infected plants were observed on leaves and stems inoculated by the nine isolated strains and Pcc SMG-2, while negative control plants remained symptomless. Biochemical tests and 16S rDNA sequence analysis confirmed that the re-isolated bacteria were Pcc. To our knowledge, this is the first report of Pcc causing bacterial soft rot of horn lian in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press, 1998. (2) N. W. Schaad, et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

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Development of PCR-Based Detection System for Soft Rot Pectobacteriaceae Pathogens Using Molecular Signatures

Microorganisms ◽

10.3390/microorganisms8030358 ◽

2020 ◽

Vol 8 (3) ◽

pp. 358

Author(s):

Md Niamul Kabir ◽

Ali Taheri ◽

C. Korsi Dumenyo

Keyword(s):

Pathogen Detection ◽

Detection System ◽

Soft Rot ◽

Pcr Analysis ◽

Morphological Identification ◽

Specific Primers ◽

Detection And Identification ◽

Significant Yield ◽

Rot Disease ◽

Species Specific

Pectobacterium and Dickeya species, usually referred to as soft rot Enterobacteriaceae, are phytopathogenic genera of bacteria that cause soft rot and blackleg diseases and are responsible for significant yield losses in many crops across the globe. Diagnosis of soft rot disease is difficult through visual disease symptoms. Pathogen detection and identification methods based on cultural and morphological identification are time-consuming and not always reliable. A polymerase chain reaction (PCR)-based detection method with the species-specific primers is fast and reliable for detecting soft rot pathogens. We have developed a specific and sensitive detection system for some species of soft rot Pectobacteriaceae pathogens in the Pectobacterium and Dickeya genera based on the use of species-specific primers to amplify unique genomic segments. The specificities of primers were verified by PCR analysis of genomic DNA from 14 strains of Pectobacterium, 8 strains of Dickeya, and 6 strains of non-soft rot bacteria. This PCR assay provides a quick, simple, powerful, and reliable method for detection of soft rot bacteria.

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First Report of Artichoke Bacterial Stem Rot Caused by Pectobacterium carotovorum subsp. carotovorum in China

Plant Disease ◽

10.1094/pdis-04-11-0314 ◽

2011 ◽

Vol 95 (8) ◽

pp. 1026-1026 ◽

Cited By ~ 2

Author(s):

B.-D. Gao ◽

X.-L. Wang ◽

H. Xia

Keyword(s):

16S Rdna ◽

Chinese Cabbage ◽

Vascular Tissue ◽

Soft Rot ◽

Hunan Province ◽

Pectobacterium Carotovorum ◽

Globe Artichoke ◽

Bacterial Cells ◽

First Report ◽

Carotovorum Subsp

A new disease on globe artichoke (Cynara scolymus L.) was observed in the springs of 2008 and 2009 and during the spring and fall seasons of 2010 in commercial fields (nearly 1,000 ha) in Changde, Hunan Province, China. Characteristic symptoms were wilting and necrosis of the outermost leaves and dark brown discoloration of the vascular tissue and pith of the stem base. Eventually, the plants wilted and died. Nearly 5, 35, and 4% (2008, 2009, and 2010, respectively) of the artichoke fields were destroyed because of the disease. Manual weeding and cuttings often led to the development of typical soft rot during propagation. To investigate the causal agent of the disease, isolations were made from rotted stems of field artichoke plants on nutrient agar (NA). Bacteria consistently isolated from the diseased tissues formed gray-white, glossy, convex, translucent, and round colonies on NA. The bacterial cells were gram-negative rods with two to eight peritrichous flagella. Ten isolates were negative for oxidase, arginine dehydrolase, H2S, gelatin liquefaction, and tryptophan ammonialyase. Isolates were positive for catalase, reduced NO3 to NO2, indole, glucuroide, galactosidase, Voges-Proskauer test, and β-galactosidase, along with being facultatively anaerobic and insensitive to erythromycin (40 μg/ml). Negative results were obtained for utilization of maltose, gluconate, and phenylacetic acid, and positive results were obtained from arabinose, glucose, mannose, N-acetyl-glucosamine, mannitol, and sodium citrate for all isolates. Acid was produced from glucose, inositol, rhamnose, melibiose, arabinose, mannitol, sucrose, and amarogentin. All test results were similar to reference strain PCC1000 (GenBank Accession No. JF721959) of Pectobacterium carotovorum subsp. carotovorum. These isolates could also cause soft rot of Chinese cabbage stem, carrot slice, pepper, lettuce and artichoke stems, and tomato and potato slices within 48 h at 28°C in an artificial inoculation test (3). PCR amplification was carried out by utilizing universal 16S rDNA primer pair 16SF/16SR and pel gene primers Y1/Y2 (1). The 16S rDNA and pel gene sequences of isolate HNXDT002 (GenBank Accession Nos. JF721958 and JF721960, respectively) had 99 and 93% nucleotide identity with strains of P. carotovorum subsp. carotovorum (GenBank Accession Nos. U80197 and CP001657, respectively). Pathogenicity was confirmed by needle-stab inoculation (1 × 108 CFU/ml) at the stem on three healthy artichoke plants held at 28°C for 48 h. Sterile distilled water was used as a negative control. Within 72 h after inoculation, water-soaking and soft-rot symptoms were observed on all inoculated artichoke plants, while controls remained healthy. The bacterium was recovered only from rotted stems of inoculated plants. In recent years, P. carotovorum was reported on such plants as Pinellia ternata (4) and Chinese cabbage (2) in China. To our knowledge, this is the first report of bacterial rot disease caused by P. carotovorum subsp. carotovorum on artichoke in China. References: (1) D. J. Brenner et al. Bergey's Manual of Systematic Bacteriology. Vol. 2. Springer, NY, 2005. (2)Y. Fang et al. Acta Microbiol. Sinica 44:136, 2004. (3) H. Yi-Bo et al. Acta Phytopathol. Sinica 37:338, 2007. (4) F. X. Ying et al. Plant Dis. 91:1359, 2007.

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Isolation and efficacy of two bacterial strains antagonists of Erwinia amylovora and Pectobacterium carotovorum

Egyptian Journal of Biological Pest Control ◽

10.1186/s41938-021-00443-0 ◽

2021 ◽

Vol 31 (1) ◽

Author(s):

M’hamed BENADA ◽

Boualem BOUMAAZA ◽

Sofiane BOUDALIA ◽

Omar KHALADI

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Bacterial Species ◽

Crop Protection ◽

Soft Rot ◽

Causal Agent ◽

Plant Diseases ◽

Ecological Niches ◽

Pectobacterium Carotovorum ◽

Bacterial Strains

Abstract Background The development of ecofriendly tools against plant diseases is an important issue in crop protection. Screening and selection process of bacterial strains antagonists of 2 pathogenic bacterial species that limit very important crops, Erwinia amylovora, the causal agent of the fire blight disease, and Pectobacterium carotovorum, the causal agent of bacterial potato soft rot, were reported. Bacterial colonies were isolated from different ecological niches, where both pathogens were found: rhizosphere of potato tubers and fruits and leaves of pear trees from the northwest region of Algeria. Direct and indirect confrontation tests against strains of E. amylovora and P. carotovorum were performed. Results Results showed a significant antagonistic activity against both phytopathogenic species, using direct confrontation method and supernatants of cultures (p<0.005). In vitro assays showed growth inhibitions of both phytopathogenic species. Furthermore, results revealed that the strains of S. plymuthica had a better inhibitory effect than the strains of P. fluorescens against both pathogens. In vivo results on immature pear fruits showed a significant decrease in the progression of the fire blight symptoms, with a variation in the infection index from one antagonistic strain to another between 31.3 and 50%, and slice of potato showed total inhibition of the pathogen (P. carotovorum) by the antagonistic strains of Serratia plymuthica (p<0.005). Conclusion This study highlighted that the effective bacteria did not show any infection signs towards plant tissue, and considered as a potential strategy to limit the fire blight and soft rot diseases.

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