Available Online: http://www.ejpau.media.pl/volume19/issue4/art-09.html
FAST MOLECULAR DETECTION OF PECTOBACTERIUM ATROSEPTICUM, PECTOBACTERIUM CAROTOVORUM SUBSP. CAROTOVORUM, AND DICKEYA SPP. FROM POTATO (SOLANUM TUBEROSUM L.) STEM TISSUES
Małgorzata P. Oksińska1, Elżbieta G. Magnucka1, Stanisław J. Pietr2
1 Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
2 Laboratory of Agricultural Microbiology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
Detection of the pathogenic bacteria causing blackleg of potato
plants is important for seed producers, growers and potato industry. The conventional
PCR method using primers specific for Pectobacterium atrosepticum (Y45/Y46), Pectobacterium
carotovorum subsp. carotovorum (together with Pectobacterium wasabiae)
(INPCCF/INPCCR) and Dickeya spp. (Ech1/Ech1’) turned out to be useful
to determine the presence of these bacteria among the various microorganisms
inhabiting potato stems. In order to minimize the time and costs of a commercial
analysis, the pathogen detection method omitted bacterial DNA extraction step.
Moreover, universal organic medium was applied for enrichment of the microbial
cell number to detection level instead of commonly used isolation of pure bacterial
cultures on selective media. Results of our studies confirmed the rising problem
of the infection with Dickeya spp. and P. carotovorum subsp. carotovorum which
caused the potato plant wilting during hot summer.
Detection of the pathogenic bacteria causing blackleg of potato plants is important for seed producers, growers and potato industry. The conventional PCR method using primers specific for Pectobacterium atrosepticum (Y45/Y46), Pectobacterium carotovorum subsp. carotovorum (together with Pectobacterium wasabiae) (INPCCF/INPCCR) and Dickeya spp. (Ech1/Ech1’) turned out to be useful to determine the presence of these bacteria among the various microorganisms inhabiting potato stems. In order to minimize the time and costs of a commercial analysis, the pathogen detection method omitted bacterial DNA extraction step. Moreover, universal organic medium was applied for enrichment of the microbial cell number to detection level instead of commonly used isolation of pure bacterial cultures on selective media. Results of our studies confirmed the rising problem of the infection with Dickeya spp. and P. carotovorum subsp. carotovorum which caused the potato plant wilting during hot summer.
Key words: PCR detection, specific primers, potato plant wilting, pectinolytic Enterobacteriaceae.
Pectinolytic members of the Enterobacteriaceae family are widely distributed in the world. They are considered as phytopathogens of such economically important crops as: potato, carrot, tomato, maize, banana, and ornamental plants [19, 20]. These microorganisms cause the blackleg of potato (Solanum tuberosum L.) plants during vegetation period and the soft rot of potato tubers during storage. Tissue maceration caused by bacterial extracellular pectinase, cellulase, and protease is the main disease symptom . In plants showing the blackleg symptoms, the level of bacterial density exceed 106 cells g-1, whereas, for latent infections this value was not higher than 103 cells g-1. The pathogens can be found in tissues of stem, mainly 15–20 cm above ground level, roots, leaves, and tubers [4, 9]. They can also survive in soil and water . Therefore, besides seed tubers, infection sources may include irrigation waters, miscellaneous insects, and agricultural techniques [1, 8].
Pectolytic strains of Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora), Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica, P. carotovorum subsp. atrosepticum) and Pectobacterium wasabiae were recognized as important potato pathogens in Europe [7, 16, 17, 20]. Moreover, Dickeya spp. (previously Erwinia chrysanthemi), mainly D. dianthicola and D. solani, in the last decade became significant pathogens causing problems for potato production in several European countries [17, 19].
Fast detection of these pathogenic bacteria is important for seed potato producers, growers, and potato industry. The most common identification methods rely on an isolation of pure cultures on selective media and then theirs morphological, biochemical, or immunological characterization. Currently, the molecular tools based on analysis of genomic DNA that offer benefits over traditional diagnostic tests become more and more useful . Detection of P. atrosepticum, P. carotovorum subsp. carotovorum (together with P. wasabiae), and Dickeya spp. by the polymerase chain reaction (PCR) based methods using specific primer pairs Y45/Y46, EXPCCF/EXPCCR, INPCCF/INPCCR, and Ech1/Ech1’ was described in literature [5, 6, 10, 14]. Until now, these methods required the extraction of bacterial DNA. However, the pure culture isolation on selective media as well as the bacterial DNA extraction is time-consuming and expensive processes, especially for commercial and mass analysis. Therefore, the aim of our study was the PCR detection without these steps.
MATERIAL AND METHODS
Few centimetres long stem samples of 55 potato plants growing during hot summer were collected in July 2012 from commercial potato fields located in the Province of Lower Silesia, in Strzelin, Ziębice, and Wrocław districts, as well as in the Province of Opole, in Brzeg district. Additionally, 10 stem samples were taken from potato plants cultivating under the same weather conditions in July 2015 in the Province of Pomerania, in Słupsk district. The plants that were collected in the first period showed wilting without the typical blackleg symptoms, whereas the plants harvested from the second stage did not show any disease symptoms. The potato plant stem fragments were cut using sterile disposable scalpels and next placed in sterile Falcon tubes. This material was transported to the laboratory in a cooler box at temperature not exceeding 10ºC and then it was immediately analysed.
In order to enrich the cell number of phytopathogenic bacteria, which can potentially colonized inner stem tissues, the samples were rinsed three times with sterile distilled water, 5–10 mm long fragments were placed in 1.5 mL of Luria Broth (LB) medium without glucose (Difco Laboratories, Detroit, USA), and incubated overnight at 28ºC . To provide oxygen-limiting conditions, 2.0 mL Eppendorf tubes were fully filed with the medium and the stem fragments . After the multiplication step, bacteria were detached from the plant tissues by sonication for 5 min at 16 kHz (Ultrasonic Disintegrator, Techpan, Poland), transferred (without any tissue scraps) to sterile Eppendorf tubes, centrifuged (5 min, 7,560 × g), washed three times in ultra-pure sterile water, and finally suspended in 1.0 mL of this water. The optical density (OD600) of cell suspensions was measured at 600 nm (BioPhotometr, Eppendorf AG, Hamburg, Germany). Each plant was tested in three replications.
Detection of P. atrosepticum, P. carotovorum subsp. carotovorum, and Dickeya spp. was done by the PCR-based method that used the specific primer pairs. The following referential strains were utilized as positive controls: P. atrosepticum DSM 18077 originating from the Culture Collection at Leibniz Institute DSMZ (Braunschweig, Germany), P. carotovorum subsp. carotovorum 1815 and 1822, as well as E. chrysanthemi 1452 (present scientific name Dickeya sp. Ech722) and E. chrysanthemi 1450 (actually Dickeya dadantii Ech3937) obtained from the Culture Collection of Plant Pathogens at Institute of Plant Protection (Poznań, Poland).
For material obtained from the plant samples, the PCR was performed without genomic DNA extraction. Heat-killed (98ºC, 15 min) bacterial cells were used as a template. In the case of the referential strains, the heat-killed cells as well as total bacterial DNA extracted with the GeneEluteTM Bacterial Genomic DNA Kit (Sigma-Aldrich Inc. St. Louis, USA) were tested. Amounts of the referential DNA were calculated on the basis of the absorbance measurement at 260 nm (A260) (BioPhotometr, Eppendorf, Germany). From 1.7 to 22.0 ng DNA µL-1 were obtained after the extraction from pure cultures with OD600 ranged from 0.19 to 2.02 units. The Pearson correlation coefficient calculated for the OD600 values and the DNA amounts was equal to 0.9657 (p = 0.002). The amplification was conducted in the final volume of 20 µL, using 4 mL of 5 × hot FIREPol® Blend Master Mix (Soils BioDyne, Tartu, Estonia), 0.5 µM of each primer (Blirt S.A., Gdańsk, Poland), and 5 µL of cell suspension (OD600 from 0.98 to 3.00) or referential DNA (the final concentration from 4.4 to 7.4 ng µL-1). The following primer pairs were utilized: Y45/Y46 specific to P. atrosepticum, INPCCF/INPCCR, and EXPCCF/EXPCCR unique to P. carotovorum subsp. carotovorum (together with P. wasabiae), and Ech1/Ech1’ typical of Dickeya spp. [5, 6, 10]. Characteristics of the primers were presented in Table 1. Forty PCR cycles were carried out to amplify specific target regions from genomic DNA of P. atrosepticum and Dickeya spp., according to the following procedure: initial denaturation of DNA and activation of polymerase for 15 min at 95 oC, denaturation for 30 sec at 94ºC, annealing for 45 sec at 65ºC, elongation for 45 sec at 72ºC, and final extension for 5 min at 72ºC (Mastercycler® gradient, Eppendorf AG, Germany) . Additionally, the primer pairs Y45/Y46 and Ech1/Ech1’ were used for the simultaneous detection both of the pathogens by multiplex PCR under the above mentioned conditions . The detection of P. carotovorum subsp. carotovorum (together with P. wasabiae) by conventional PCR with the primer sets INPCC and EXPCC as well as by nested PCR (the amplification with the primers INPCC within a product obtained previously with the set EXPCC) was performed over the following 30 cycles: initial denaturation for 15 min at 95ºC, denaturation for 1 min at 94ºC, annealing for 1 min at 60ºC, elongation for 2 min at 72ºC, and final extension for 7 min at 72ºC . In order to verify the specificity of tested primer pairs, combination of the DNA that was extracted from two or three pure cultures of the referential strains or suspensions of theirs heat-killed cells were used as the template.
Table 1. Specification of the PCR primers used in the study
The presence of PCR products was confirmed by electrophoresis in a 1.5% (w/v) agarose gel supplemented with ethidium bromide (0.5 µg mL-1) and the DirectLoad Step Ladder 50 bp was used as molecular weight marker (Sigma-Aldrich Inc., USA).
The visualization and documentation of PCR products was done using the biostep® UV light transilluminator (UXDT-47SM-15PC, biostep® GmbH, Jahnsdorf, Germany), the biostep® Dark Hood (DH-40/50, biostep® GmbH, Germany), and the Canon PowerShot digital camera.
RESULTS AND DISCUSSION
The applicability of the PCR-based method that utilizes primer sets specific for P. carotovorum subsp. carotovorum (together with P. wasabiae), P. atrosepticum, and Dickeya spp. for detection of these phytopathogenic bacteria in planta was positively verified in our studies. In order to minimize the time and costs of the analysis, the PCR amplification was performed without the prior genomic DNA extraction. This method has been employed for potato plants, which did not show the typical blackleg disease symptoms. It can therefore be assumed that the cell number of pathogens that colonized potato steam tissues was rather low [4, 9]. According to literature data, the detection limit of PCR method varied from 1 to 15 cells mL-1 for bacterial cultures suspended in water to 104 – 105 cells for plant extracts [5, 15]. In this work, in order to enrich the amounts of bacteria existing in potato tissues, microorganisms were pre-cultured in universal LB medium consisting of yeast extract and tryptone as nutrient sources and then suspended in ultra-pure sterile water. This procedure allows avoiding false negative results of the PCR method caused by some inhibitors that can be presented in plant tissues. The fact that LB medium is easy to prepare and allows the growth of various bacteria, including the Enterobactericeae family, was the decisive factor in the choice of this medium instead of routinely used crystal violet pectate (CVP) or pectate enrichment medium (PEM). These two media are mainly utilized to isolate pure cultures of Pectobacterium spp. or Dickeya spp. from plant and soil samples as well as to enrich their low cell number to detection levels . The use of universal organic medium instead of these selective or differentiating media can result in the growth inhibition of target bacteria through microorganisms coexisting in plant tissues. On the other hand, CVP is only selective for pectolytic bacteria, therefore, besides the genus Pectobacterium and Dickeya it can also support the growth of some microorganisms from the genera Pseudomonas and Flavobacterium [2, 11] as well as isolates of Xanthomonas campestris, Cytophaga johnsonae, and Bacillus spp. . It was also found that majority of stressed bacteria that originated from natural environments may not be able to grow on CVP unlike non-selective organic media . Meneley and Stanghellini  have found that low oxygen conditions inhibited the multiplication of aerobic microorganisms and have not affected the growth of facultative anaerobic bacteria from the Enterobacteriaceae family. Therefore, in our work, tightly closed and fully filled with the medium Eppendorf tubes were used for the multiplication of tested bacteria.
Based on the analysis of referential strains of P. atrosepticum, P. carotovorum subsp. carotovorum, Dickeya sp. and D. dadantii, the relationship between the concentration of total bacterial DNA that was extracted from their cells (y) and the values of optical density units (OD600) was expressed by the following equation: y = 11.58 × OD600 – 2.51. The OD600 values that were obtained for bacteria originated from plant material ranged from 0.98 to 3.00 units. Thus, the approximate DNA amounts should be in the range from 8.85 to 32.22 ng µL-1. On account of the presence of some competitor DNA in PCR mix, the primer sets Y45/Y46 and Ech1/Ech1’ allow the detection with the limit of 0.1–1.0 ng DNA µL-1 [5, 14]. In the case of conventional PCR with the primer set EXPCC, the detection limit was calculated as 1.2 × 103 colony-forming units (cfu) of pure bacterial cultures per reaction volume  or for 0.1 ng DNA µL-1 . For nested PCR, the detection level was 2–4 cfu per reaction . Moreover, the final DNA concentration in PCR mix, recommended by the producer of 5 × hot FIREPol® Blend Master Mix, ranges from 5 to 50 ng µL-1. Therefore, in this work 5 µL of the cell suspension was used for the PCR.
Detection of the pathogenic bacteria was narrowed to classical PCR with commonly used primers. The set Y45/Y46 allowed the amplification of the periplasmic pectate lyase gene (pelY) of P. atrosepticum (Fig. 1). Additionally, this set also amplified the gene of Pectobacterium batavasculorum strains which are commonly associated with sugar beet and were seldom found in potato plant tissues . The primers Ech1 and Ech1’ enable for amplification of DNA fragment that is located in the pectate lyase gene (pelI) of D. dadantii (Fig. 2). However PCR products with the same molecular weight were also obtained for strains of D. dianthicola and D. chrysanthemi and distinction among them was not possible . Based on literature data, the primer pairs Y45/Y46 and Ech1/Ech1’ can be also used simultaneously in multiplex PCR . However, in this work, apart from the right PCR products, unspecific DNA bands with molecular weights from 200 to 500 bp were also observed both when the heat-killed cells of pure bacterial cultures and their extracted DNA were used as the template. So, these two primer pairs have been finally used separately.
The primers INPCCF and INPCCR align to the genome of P. carotovorum subsp. carotovorum and P. wasabiae . They were mainly utilized together with the EXPCC primer pair for nested PCR, under the same reaction conditions . The set of INPCC anneal within the product, which was previously obtained in the reaction with the EXPCC primers for P. carotovorum subsp. carotovorum and P. wasabiae. Palacio-Bielsa et al.  found that in the analysis by the nested PCR the risk of false positive reactions by the possibility of cross-contamination increased. In this work, the nested PCR and the classical PCR with the set INPCC resulted in the intensive DNA bands of referential P. carotovorum subsp. carotovorum, which were not always visible after the reaction with the primers EXPCC. Moreover, 3 to 4 of some unspecific bands ranged from 300 to 600 bp were found for the plant bacterial mixtures when they were tested by the nested PCR. These unspecific bands were also observed when the DNA extracted from the pure cultures of referential strains was used as the template. In the case of two samples only, the appropriate PCR products were found as the result of the nested PCR. Based on these data, the conventional PCR method with the set INPCC was chosen (Fig. 3). In relation to the finally selected primer sets, the amplification specificities were confirmed used mixed cultures of the heat-killed cells of two or three referential strains. For each of the analyses, only the specific PCR product was obtained.
Fig. 1–3. The PCR detection of Pectobacterium atrosepticum (Fig. 1), Dickeya spp. (Fig. 2), and P. carotovorum subsp. carotovorum (Fig. 3) with the specific primer pairs: Y45/Y46, Ech1/Ech1’, and INPCCF/INPCCR, respectively.
Lanes: (1 and 20) molecular weight marker (the Direct Load Step Ladder 50 bp), (2) water; (3) referential strains of P. carotovorum subsp. carotovorum 1815, (4) P. atrosepticum DSM 18077, (5) Dickeya sp. Ech722, (6–19) heat-killed cultures of microorganisms inhabiting potato stem tissues. The material from the same referential strains and potato stem samples was included in lanes with the same numbers.
Bacteria belonging to the genus Dickeya and P. carotovorum subsp. carotovorum are widely distributed in the world, unlike P. atrosepticum strains that are mainly found in cool or temperate regions [4, 5, 10]. On the basis of genetic analysis, that was preceded by isolation of pure cultures on CVP agar medium and several morphological and biochemical tests, Waleron et al.  have found that potato plants, which were collected in different regions of Poland during the season of 1996/1997 and showed the blackleg and soft rot symptoms, were infected with P. atrosepticum, P. carotovorum subsp. carotovorum, and P. wasabiae. Dickeya spp. strains were not found in this material, despite the fact that the presence of these bacteria has been denoted in Europe since 1972 . In Poland, in turn, the first report of the appearance of Dickeya spp. was published by Sławiak et al. in 2009 . In the opinion of these authors, this bacterium was probably introduced from the Netherlands via seed potato tubers. Methods used by them included bacterial isolation of pure cultures on CVP agar medium, purification on Trypticase soy agar (TSA), identification based on gram staining, the activity of oxidases, pectate lyase, or facultative anaerobic type of growth and finally genetic analysis with specific PCR primers. The method presented in this work was minimized to the microbial cultivation in low amounts of universal organic liquid medium and then the conventional PCR reaction. Application of this method lets us successful detected that 49.09% of plants showed wilting without the typical blackleg symptoms were infected simultaneously with Dickeya spp. and P. carotovorum subsp. carotovorum (and/or P. wasabiae). Additionally, 23.64, 20.00 and 7.27% of the remaining plants were infected with P. carotovorum subsp. carotovorum (and/or P. wasabiae), Dickeya spp., and P. atrosepticum, respectively. The absence of pathogens was confirmed in the plant material without wilting and disease symptoms. However, the introduction of the DNA extracted from pure cultures of referential strains to these samples allowed to appear the appropriate bands. So, this result confirms the effectiveness of our method as well as indicates the lack of non-specific amplification of other bacteria existing in plant tissues.
In order to minimize the time and costs of commercial analysis of potato plants, which showed wilting without the typical blackleg symptoms, the conventional PCR method omitting the step of genomic DNA extraction was used. The heat-killed cells of microorganisms existing in plant tissues were utilized instead of pure bacterial cultures obtained on selective media. Enrichment of bacterial amounts to the detection level was performed by pre-culturing of microorganisms existing in tissues of potato steam in universal organic medium under the oxygen limitation conditions. On the basis of the method, that was simpler and less expensive than routinely applied procedures, we have found that the majority of potato plants were infected with P. carotovorum subsp. carotovorum (and/or P. wasabiae) and with some of Dickeya species (D. dadantii, D. solani, D. dianthicola, or D. chrysanthemi). In our opinion this method may be successfully used for commercial detection of the presence of these pathogens in plant material.
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Accepted for print: 15.12.2016
Małgorzata P. Oksińska
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
phone/fax: +48 71 320 5621
Elżbieta G. Magnucka
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
Stanisław J. Pietr
Laboratory of Agricultural Microbiology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
phone/fax: +48 71 320 6521
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