1 In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 9 (3), pp. 001-008, March, 2021. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Identity and genetic diversity of Pectobacterium spp., causal agents of potato soft rot in Zanjan, Iran E. Tavasoli1, A. R. Marefat2 and N. Hassanzadeh1* 1 Department of Plant Pathology, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2 Department of Plant Pathology, Faculty of Agriculture, Zanjan University, Zanjan, Iran. Accepted 09 October, 2020 Zanjan province is one of the areas of potato production in Iran with more than 6917 ha of cultivation. One of the most important factors to limit potato cultivation is soft rot bacteria belonging to the family Enterobacteriaceae. During the years 2007 to 2008, different potato fields and the most important potato storages in Zanjan province were surveyed. Phenotypic identification of the strains was performed using recommended biochemical and physiological tests. Genetic diversity was determined by BOX-PCR and ERIC-PCR. Also all of the strains were identified with specific primers (Y1, Y2 and ECA1f, ECA2r). The strains belonged to "carotovora" group and identified as Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. Although there were some atypical characteristics among the strains, but a very close relationship were found between DNA fingerprints and geographical origins of isolates. Key words: Pectobacterium, Soft rot, Black-leg, BOX-PCR, ERIC-PCR INTRODUCTION Potato is the fifth most important food crop worldwide. Likewise, Iran is one of the most important potato producing countries in Asia and Oceania. Zanjan province, located in North West of Iran, is one of the areas of potato production in Iran with more than 6917 ha of cultivation. The soft rot erwinias in the Enterobacteriaceae are economically important, because they cause serious damage worldwide on a wide variety of plants (Perombelon and Kelman, 1980). Pectobacterium carotovorum subsp. carotovorum and Dickeya chrysanthemi have a wide host range, infecting crops mainly in tropical and subtropical regions, whereas P. atrosepticum is restricted almost exclusively to potato in temperate regions, causing soft rot of tubers and blackleg of stems (Perombelon, 1992). Different studies have been done for recognition of these bacteria on potato in some parts of Iran, which were based on physiological and biochemical tests as well as the *Corresponding author. E-mail: hasanzadehr@yahoo.com. comparison of protein electrophoretic patterns that in most cases did not lead to a distinct group differentiation (Bahar and Danesh, 1986; Zohoor et al., 1998). One of the tools in studying the genetic diversity of bacteria including potato soft rot Pectobacteria is rep-PCR (Rademaker and de Bruijn, 1997; Xiu et al., 2006). To perform this, many potato soft rot bacteria were isolated from both farms and stores of Zanjan province and their genetic diversity were estimated. MATERIALS AND METHODS Sample handling and isolate collection During the years 2007 to 2008, different potato fields and the most important potato storages in Zanjan province (Abhar, Ijrood, Khodabandeh and Khoramdareh) were surveyed and sufficient samples were collected from plants suffering from soft rot disease. Nutrient agar, King’s B agar and Eosin methylene blue agar media were used to isolate the suspected bacteria (Schaad et al., 2001). The type strain of Erwinia carotovora 5702 was obtained from the International Collection of Micro-organisms from Plants (ICMP), Auckland, New Zealand. 2 Biochemical and physiological tests All strains were tested for gram reaction using the KOH test, potato soft rot, oxidase reaction, catalase reaction, gelatin hydrolysis, production of gas from glucose, production of reducing substances from sucrose, growth in 5 and 6% NaCl, ability to grow at 37ºC and phosphatase (Schaad et al., 2001), fermentative metabolism (Hugh and Leifson, 1953), lecithinase (Fahy and Hayward, 1983), production of indole (Dye, 1968), and sensitivity to erythromycin (Psallidas, 1993). Additional biochemical tests were starch hydrolysis, esculin hydrolysis, nitrate reduction, arginine dihydro- lase, action on litmus milk, urease production, H2S production from cysteine (Schaad et al., 2001), Tween-80 hydrolysis (Misaghi and Grogan, 1969), casein hydrolysis (Fahy and Hayward, 1983), 3- ketolactose production (Cowan, 1974), methyl red reaction and acetoin production (Dye, 1968) and acid production from carbohydrates utilized as a source of carbon (Schaad et al., 2001). Preparation of DNA and Rep-PCR genomic fingerprinting Genomic DNA was extracted using the whole cell alkaline lysis method (Rademaker and de Bruijn, 1997). The DNA was stored at - 20°C until it was required. All strains were subjected to BOX-PCR using the BOX A1R primer (5´-CTA CGG CAA GGC GAC GCT GAC G-3´) (Martin et al., 1992). ERIC-PCR was performed for 10 selected strains from different groups of BOX-PCR by using the ERIC 1R primer (5´-TGT AAG CTC CTG GGG ATT CAC-3´) and ERIC 2 primer (5´-AAG TAA GTG ACT GGG GTG AGC G-3´) (Versalovic et al., 1991). The method developed by Rademaker and de Bruijn (1997) was performed with slight modification in cycling conditions. The PCR master mix (25 µl) containing: 1 µl DNA, 0.3 µl Taq DNA polymerase (0.1 U), 2.5 µl 10 × PCR buffer, 0.5 µl dNTPs (25 mM), 1 µl MgCl2 (25 mM), 1.1 µl of each primer (10 pmol). DNA amplification was performed on a thermocycler (Corbett, Germany) under the following conditions: 4 min at 94ºC for initial denaturation, 34 cycles of 40 s at 94ºC, 40 s at 50ºC and 1 min at 72ºC, followed by a final elongation step of 10 min at 72ºC. PCR products (6 µl) were separated by gel electrophoresis in 1.5% agarose gels in TBE buffer. Following staining with ethidium bromide, the gels were viewed and photographed under UV Transilluminator. A 1-kb marker (Fermentas, Germany) was included on every gel. Captured photographs were subjected to the Gel-Pro® Analyzer (Media Cybernetics, MD, USA) computer program. Bands were scored in binary form, 1 and 0, indicating the presence and absence of a band, respectively. A similarity matrix was obtained using the Jaccard coefficient and the software package NTSYS-pc (version 2.02K, Applied Biostatistics, Inc., NY, USA). To determine the relationship among the strains, cluster analysis was performed with UPGMA (unweighted pair-group method, using arithmetic averages) in the SAHN program of the NTSYS-pc software. Dendrograms were generated for the BOX and ERIC binary matrixes individually. Identification and detection with specific primers Primers Y1 (5´- TTA CCG GAC GCC GAG CTG TGG CGT-3') and Y2 (5'-CAG GAA GAT GTC GTT ATC GCG AGT-3') selected from the pectate lyase-encoding pel gene sequences of E. carotovora (Darrasse et al., 1994), were used. A specific PCR assay for P. atrosepticum was performed using primers ECA1f (5'-CGG CAT CAT AAA AAC ACG-3') and ECA2r (5'-GCA CAC TTC ATC CAG CGA-3') following the protocol of De Boer and Ward (1995), with slight modifications. PCR master mix (25 µl) with Y1 and Y2 primers containing: 2.5 µl DNA, 0.1 U Taq DNA polymerase, 2.5 µl 10 × PCR buffer, 2 µl dNTPs (1 Mmol), 1.5 µl MgCl2 (25 mmol), 0.1 µmol of each primer. DNA amplification was performed on a icycler (BIO-RAD) under the following conditions: 5 min at 94ºC for initial denaturation, 34 cycles of 30 s at 94ºC, 45 s at 55ºC and 45 s at 72ºC, followed by a final elongation step of 7 min at 72ºC. PCR reaction mix in final reaction volumes of 25 µl was prepared containing 2.5 µl DNA, 0.5 U Taq DNA polymerase, 2.5 µl 10 × PCR buffer, 100 µM each of dNTPs, 2 mM MgCl2, 0.5 µM of each primer. DNA amplification was performed on a icycler (BIO-RAD) under the following conditions: 5 min at 95ºC for initial denaturation, 40 cycles of 30 s at 94ºC, 45 s at 62ºC and 45 s at 72ºC, followed by a final elongation step of 8 min at 72ºC. PCR products (6 µl) were separated by gel electrophoresis in 1.5% agarose gels in TBE buffer. Following staining with ethidium bromide, the gels were viewed and photographed under UV transilluminator. RESULTS Biochemical and physiological tests On the basis of biochemical profiles, 67 and 2 strains were identified as P. carotovorum and P. atrosepticum, respectively. All of them were gram negative, facultatively anaerobic, oxidase negative, catalase positive, non- fluorescent on King´s B (KB) medium and showed soft rot symptoms on slices of potato. All strains grew at 37ºC and in 5% NaCl. Gelatin hydrolysis and lecithinase were negative. Some of them produced gas from glucose (22.3%), indole (59.2%) and phosphatase (41.8%). 10.4% of strains showed sensitivity to erythromycin. 3% were able reduce substances from sucrose and 94% were able to tolerate 6% NaCl. Thus these strains were identified as the "carotovora" group belonging to the family Enterobacteriaceae (Tables 1 and 2). Results of complementary tests on 22 strains (selected by results of BOX-PCR), showed that no strains produced urease, 3- ketolactose and acetoin, also no strains were able to hydrolyse Tween-80. They hydrolyzed starch (22.7%), casein (27.3%) and esculin (77.3%). Litmus milk reaction was acid (86.4%) and arginine dihydrolase was positive (72.7%). All strains could reduce nitrate and produced H2S from cysteine. 50% of strains were positive in methyl red reaction. All of them produced acid from arabitol, arabinose, trehalose, rhamnose, ribose, cellobiose, glucose, fructose, sucrose or lactose, but none produce acid from insulin. In addition they could produce acid from α-methyl-D-glucoside and raffinose (81.8%), maltose (86.4%), sorbitol and myo-inositol (77.3%), melibiose and palatinose (68.2%). All of them utilized citrate and 86.4% utilized malonate. No strains utilized D-tartrate. Results showed a high variability among strains. Results also revealed that P. carotovorum was the main pathogen and D. chrysanthemi was absent from the strains tested. Rep-PCR genomic fingerprinting PCR using the BOX primer gave genomic fingerprints with 22 bands, ranging from about 250 to 3000 bp. Results revealed two main clusters among the pathogen strains and their similarity value, based on UPGMA, was 3 Table 1. Bacterial strains used in this study. Strain Location of origon Year isolated Z1, Z2, Z3, Z4 Kheirabad 2007 Z5, Z6, Z7, Z8, Z9 Khoramdareh 2007 Z10, Z11, Z12, Z13 Abhar 2007 Z14, Z15, Z16, Z17, Z18 Gonbad soltanieh 2007 Z19, Z20, Z21, Z22, Z23, Z24 Khodabandeh 2007 I1, I2, I3, I4, I5, I6, I7, I8, I9, I10, I11, I12, I13, I14, I15, I16, I17, I18, I19, I20, I21 Ijrood 2007 E2, E15, E26, E29, E24, E25, E32, E34, E35, E41, E40, E37 Ijrood 2008 B5, D8, D9, D10, D20, D22, D25, D34, D17, D24 Zanjan 2008 Table 2. Phenotypic characteristics of the strains of Pectobacterium isolated from potato in comparison with standard isolate. Results Test Number of strains positive or Positive P. carotovorum negative/number of strains tested strains (%) 5702 Fermentative growth +(67/67) 100 + Gram reaction -(67/67) 0 - Potato soft rot +(67/67) 100 + Oxidase -(67/67) 0 - Catalase +(67/67) 100 + Lecithinase -(67/67) 0 - Gelatin hydrolysis -(67/67) 0 + Gas from glucose +(52/67) 77.6 - Reducing substances from sucrose -(65/67) 3 - Growth in 5% NaCl +(67/67) 100 + Growth in 6% NaCl +(63/67) 94 + Growth at 37ºC +(67/67) 100 + Production of Indole +(39/67) 58.2 - Sensivity to erythromycin -(60/67) 10.4 + Phosphatase -(39/67) 41.8 + Starch hydrolysis -(17/22) 22.8 - Tween-80 hydrolysis -(22/22) 0 - Casein hydrolysis -(16/22) 27.3 + Urease production -(22/22) 0 - Methyl red reaction +(11/22) 50 + Acetoin production -(22/22) 0 - Esculin hydrolysis +(17/22) 77.3 + 3-ketolactose production -(22/22) 0 - Nitrate reduction +(22/22) 100 + Arginine dihydrolase +(16/22) 72.8 - H2S from cysteine +(22/22) 100 + Action on Litmus milk: Acid reaction (19/22) 86.4 + Alkaline reaction (3/22) 13.6 - Acid production from: Glucose +(22/22) 100 + Fructose +(22/22) 100 + Sucrose +(22/22) 100 + 4 Table 2. Contd. α-methyl-D-glucoside +(18/22) 81.8 + Sorbitol +(17/22) 77.3 + Melibiose +(15/22) 68.2 + Raffinose +(18/22) 81.8 + Arabitol +(22/22) 100 + Lactose +(22/22) 100 + Trehalose +(22/22) 100 + Rhamnose +(22/22) 100 + Maltose +(19/22) 86.4 + Palatinose +(15/22) 68.2 - Ribose +(22/22) 100 + Inulin -(22/22) 0 - Cellobiose +(22/22) 100 + Myo-inositol +(17/22) 77.3 + Arabinose +(22/22) 100 + Utilization of: Citrate +(22/22) 100 + D-tartrate -(22/22) 0 - Malonate +(19/22) 86.4 + +, positive reaction; -, negative reaction. M B5 E2 E15 E24 E25 E26 E29 E32 E34 E35 E37 E40 E41 TT C M 3000 bp 2000 1500 1000 750 500 250 Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Z11 Z12 Z13 Z14 Z15 Z16 M Figure 1. Genomic DNA fingerprinting patterns from strains of Pectobacterium isolated from potato, generated by BOX-PCR. M: Marker (1-kb); C: Control (without DNA); TT: P. carotovorum 5702. about 51.5%. The first group was divided into two subgroups at 65.5% similarity. The second group was also divided into two subgroups at 75.5% similarity. This protocol revealed 18 genotypes within the pathogen (Figures 1 and 2). Based on BOX-PCR results, 10 strains selected from these groups, were further analyzed with 5 Z17 TT Z18 Z21 Z22 Z4 Z7 Z8 Z10 D8 D9 D10 D20 I8 Z19 D17 Z20 I3 Z23 I9 Z1 Z2 Z3 Z5 Z9 Z11 Z12 D34 D22 D25 Z13 Z16 Z14 Z6 Z24 I13 Z15 D24 I7 I1 E34 E25 E2 I16 I14 I11 I6 I4 B5 E32 I12 I10 I5 E35 I2 E24 I15 I17 E29 E15 I20 I18 E26 I21 I19 E40 E37 E41 0.50 0.60 0.70 0.80 0.90 1.00 Coefficient Figure 2. Similarity dendrogram of Pectobacterium strains based on BOX-PCR. ERIC-PCR using the ERIC 1R primer and ERIC 2 primer. Genomic fingerprints in ERIC-PCR showed 28 bands, ranging from about 250 to 3500 bp including 9 genotypes. Based on UPGMA, the similarity value bet-ween the two main clusters was about 46.5%. The first group divided into two subgroups at 50% similarity and the second group showed one genotype (Figures 3 and 4). Identification and detection with specific primers All strains from the Zanjan province yielded a 434 bp DNA fragment in PCR with the Y1 and Y2 primers (Figure 5) and only two strains (E2 and D24) yielded a 690 bp DNA fragment in PCR with the ECA1f and ECA2r primers (Figure 6). Therefore all of the studied strains were identified as Pectobacterium carotovorum and only two strains as P. atrosepticum. DISCUSSION In this research, the characteristics of pectolytic Erwinias of potato in the Zanjan province and their genetic diversity were determined both biochemically and by the 6 M D8 D20 D22 D34 B5 E2 E15 E29 E24 E35 TT C M 3000 bp 2000 1500 1000 750 500 250 Figure 3. Genomic DNA fingerprinting patterns from representative strains of Pectobacterium isolated from potato, generated by ERIC-PCR. M: Marker (1-kb); C: Control (without DNA), and TT: P. carotovorum 5702 D8 D20 B5 E2 E15 E29 E24 E35 TT D22 D34 0.40 0.55 0.70 0.85 1.00 Coefficient Figure 4. Similarity dendrogram of Pectobacterium strains based on ERIC-PCR. 7 C TT Z18 D17 Z14 D24 E34 E2 E32 I18 I21 E41 M 434 bp Figure 5. Amplification of a specific 434 bp band in 10 strains generated using Y1 and Y2 primers. C: Water control; M: Marker (100 bp); TT: P. carotovorum 5702. C E15 E40 Z2 Z15 D22 D24 E2 M 690 bp Figure 6. Amplification of a specific 690 bp band in 7 strains, generated using ECA1f and ECA2r primers. C: Water control; M: Marker (100 bp). rep-PCR technique. The specific primers were used for the recognition of these pathogens. There was a considerable variation in phenotypic traits and gene electrophoretic clusters among the strains. In this case, 97% of strains were similar to P. carotovorum and only 3% were similar to P. atrosepticum. Properties of a few strains did not correspond with defined characteristics of authentic species and subspecies of Pectobacterium. These findings agree with Louw's justification on Rep-PCR and its high efficiency towards discrimination of pathogen population diversity (Louws et al., 1994). In this study, some correlations between biochemical characteristic and BOX-PCR based genetic fingerprints among some of the strains were observed. Thus strains related to a defined genetic group with the same genotype, showed the same phenotype characteristics and vice versa. An exceptional example is the D24 strain, which was phenotypically different from other strains showed 85% similarity in genotype to those of the same group. Comparing the results of genetic fingerprint BOX-PCR and ERIC-PCR, some similarities and differences were observed in their classification. In both cases, strains were placed in two main fingerprinting groups. In none of the current classifications has complete similarity between the standard strain and the examined one been reported. Thus there was no complete correspondence between the BOX-PCR results and ERIC-PCR and even those strains which showed the same genotype in BOX- PCR showed different genotypes in ERIC-PCR. Geographically, the group created by BOX-PCR was closely related to their sample collection areas. Because of this, most of strains of a single area were placed in one of the main groups. In ERIC-PCR, although only a limited number of strains were compared, these correlations were defined. Linkage between rep-PCR results and the geographic origin of bacterial strains has been recognized in various studies (Scortichini et al., 2001; Mkandawire et al., 2004). Louws et al. (1994) believe that one of the important reasons for this phenomenon is that the selection for one geographically suitable area can have influence on the genetic map of bacterium and also dispersion of these repetitive units in the genome of bacterium. This work supports this idea and in some cases artifacts, these relations were observed. No doubt rep-PCR is a reliable tool for epidemiological studies of diseases and one can use the information as a device for detection of pathogens. Despite this, more recent work with ISSR-PCR of bacteria such as Clavibacter michiganensis subsp. michiganesisis proved the greater sensitivity, specificity and reliability of this technique as another helpful informative tool in epidemiological studies (Baysal et al., 2011). Examining strains in Zanjan province, with special primers of Y1/Y2, specialized for P. carotovorum 8 (Darrasse et al., 1994) and ECA1f, ECA2r, specialized for P. atrosepticum (De Boer and Ward, 1995), the main pathogen of potato soft rot in the Zanjan province is P. carotovorum and in some cases P. atrosepticum. Failing to isolate D. chrysanthemi is not strange in the province, since this bacterium was reported in delimit potato cultivation areas of the Hamedan province. 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