Khalil, Hadeel M.M. ; 2G. I.A. Mohamed; A. M.I. Eraky and ... · Khalil et al. 2014 30 Introduction: Bacterial soft rot disease, caused by Erwinia carotovora, is one of the most important

Assiut J. Agric. Sci., (45) No. (4) 2014 (29-44)

Genetic differentiation of Erwinia carotovora subsp. carotovora and subsp. atroseptica

Khalil, Hadeel M.M.1; G. I.A. Mohamed2; A. M.I. Eraky1 and M.A.A. Sallam1 1 Department of Plant Pathology, Faculty of Agriculture, Assiut University, Egypt.

2 Genetics Department, Faculty of Agriculture, Assiut University, Egypt.

Abstract: Erwinia carotovora subsp. atroseptica (Eca), the causes of blackleg of po-

tato, is closely resembles to E. carotovora subsp. carotovora (Ecc), the causes of

bacterial soft rot of potato, in many physiological and biochemical characteris-

tics. Nine isolates were obtained from potato tubers collected from different lo-

calities of El-Minia, Assiut and Sohag Governorates and subjected to this inves-

tigation. Three different molecular techniques were employed to differentiate the

two subsp. of Erwinia carotovora. The primers used in RAPD-PCR technique

generated an unique distinct bands which could be used as genetic markers to

distinguish the isolates in respect of their sub species (carotovora or atroseptica)

or their virulence (high or low virulence). Also, the 16s rRNA gene sequences

was obtained from the bacterial isolates of Ecc and Eca. The phylogenetic tree

couldn`t distinguish between the two subsp. of E. carotovora, where some iso-

lates of Ecc connected in the same cluster with Eca. Moreover, SDS-PAGE anal-

ysis technique was used. The application of protein (SDS-PAGE) analysis may

aid to detect the differences between the isolates of E. c. subsp. carotovora or

atroseptica in the base of the region of collection. Where, protein analysis

showed polypeptide fractions could be used as genetic markers to distinguish iso-

lates collected from same region.

Keywords: RAPD; SDS-PAGE; 16s rRNA gene; Erwinia carotovora. Received on: 9/11/2014 Accepted for publication on: 11/11/2014

Referees: Prof. Hamdy M. El-Arif Prof. Abdel-Razak A. Abdel-Razak

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Introduction: Bacterial soft rot disease, caused

by Erwinia carotovora, is one of the most important and widespread pa-thogenic bacterial disease either in the field (causing blackleg of potato stems) or during storage (causing soft rot of potato tubers) in both temperate and tropical zones showing wide host range to several economically impor-tant food crops including potatoes, tomatoes and Chinese cabbages (Toth et al., 1999 and El-Sheikh, 2011). Soft rot disease causing great losses in potato tubers (solanum tuberosum L.) which consider the important vegetable crops in the world. It is the 4th most important staple food and it's the second most important crop pro-duced energy. Soft rot of potato crop is primarily caused by two of the five subspecies (subsp. Ecc and subsp. Eca) (Helias et al., 1998). The bacte-ria occur as latent contamination on potato tubers and depend on envi-ronmental factors. Researchers seek-ing for methods that enable them to differentiate between the two sub species of Erwinia carotovora. Clas-sical methods based on biochemical and biological tests on selective me-dia have proved to be useful tools for identification of soft rot Erwinia spe-cies (Bdliya, 1995) but they are labo-rious and time consuming and rela-tively insensitive (Smid et al., 1995). Therefore, Immunological and sero-logical techniques have been devel-oped, and their efficiency is increased by culture on selective medium (Klopmeyer and Kelman, 1988), but the serological tests are complicated because of the high number of sero-groups within E. carotovora (De Boer et al., 1979). Serological and more recently molecular methods, such as polymerase chain reaction

(PCR), 16S rRNA gene sequences analysis and SDS-PAEG, have been developed to detect and differentiate the main Erwinia pathogens infecting potato including Ecc and Eca. New molecular tools like hybridization probes have been demonstrated to be very efficient in plant disease diagno-sis (Miller and Martin, 1988), and some probes have been developed for pectolytic erwinias (Ward and De Boer, 1990). However, the use of such probes for routine tests is lim-ited. Moreover, not all of these meth-ods can be used routinely for analysis or specific detection of each Erwinia sub-species (Helias et al., 1998). In the PCR-based a target DNA frag-ments is amplified enzymatically. Thus, PCR combines a high degree of specificity with a high degree of sen-sitivity (Saiki et al., 1988; Steffan and Atlas, 1991 and Wink and Mo-hamed, 2003). Also, it has been suc-cessfully developed as a tool for spe-cific and sensitive detection of micro-organisms (Hartskeer et al., 1989; Bel et al., 1991 and Atlas et al., 1992). The random amplified polymorphic DNA (RAPD) technique was devel-oped for the genetic analysis of DNA (Williams et al., 1990). The tech-nique is based on the enzymatic am-plification of non selected DNA fragments, initiated by arbitrarily chosen DNA primers. With this tech-nique, individual genomes can be grouped and classified by screening primers and defining those that gen-erate adequate levels of DNA poly-morphism. The RAPD technique is simple and fast, and could be an al-ternative to biochemical identification of soft rot bacteria (El-Sheikh, 2011). The 16S rRNA gene sequences are conserved with stable copies. In gen-eral, the entire 16S rRNA is amplified


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and sequenced with universal primers to identify species, subspecies and strains. The rRNA locus (rrn) is high-ly conserved, and may therefore be used to analyze evolutionary relation-ships. In prokaryotes, rrn loci contain the genes for all three rRNA subunits, namely 16S, 23S and 5S genes and they are separated by a large number of various sequences and intergenic spacer regions (Cedergren et al., 1988 and Zhu et al., 2010).

Based on assay of protein pro-files of isolates by sodium-dodecyl sulphate polyacrylamide gel electro-phoresis (SDS-PAGE) technique, the molecular weights of the isolates from different areas differed in pro-tein profiles according their environ-mental conditions (Poerwadikata, 1998). For intraspecific grouping of Ralstonia solanacearum, 23 strains isolated from Brazil had different protein bands and showed various po-lypeptide patterns associating with both original hosts and biovar (Melo and Furuya, 1998). In addition, El-Ariqi (2001) and El-Sheikh (2011) found that the visible differences in protein profiles of R. solanacearum were correlated with differences in virulence, not in colony morphology.

The aims of this study were compare and differentiate the two subspecies of Erwinia carotovora by using three different molecular meth-ods. Materials and Methods: Isolation and pathogenicity tests:

Isolation was carried out from naturally infected potato tubers, let-tuce, carrot and cabbage showing soft rot symptoms. Nine bacterial isolates were obtained from potato tubers col-lected from different localities of El-Minia, Assiut and Sohag Gover-norates (Table 1). Diseased potato

tubers were washed and surface ster-ilized by soaking in 1% sodium hy-pochlorite solution for two minutes, then, a small portion of the diseased tissues were macerated in 5 ml of ste-rilized 0.05 M potassium phosphate buffer, after 10 minutes, a loopful of the resulting suspension was streaked onto nutrient sucrose agar medium (NSA) (Dowson, 1957). Plates were incubated at 28°C for 48 h. The sin-gle colony technique was used to ob-tain pure culture. Ability of isolated bacteria to cause soft rot to potato slices as well as blackleg disease to potato plants was examined. In order to determine the ability of the organ-isms to infect potato plants and cause blackleg disease, 7 weeks old potato plants grown in pots were used. Plants were inoculated by injuring the stem above the ground level and in-serting 48 hours old bacterial growth into stem injury by means of a sterile dissecting needle. At least 10 plants were inoculated with each organism. Injured stems under aseptic condition were used as control (Sellam, 1974, De Boer and Kelman, 1978, and MuGuire and Kelman, 1984). Sever-ity of disease was recorded as disease index using the method of Saleh et al. (1996). Identification of isolates was carried out according to Bergey's Manual of systematic bacteriology (Krieg and Holt, 1984 and Garrity et al., 2005). Molecular examination:

Three different molecular tech-niques were employed to differentiate nine different isolates of Erwinia ca-rotovora, which collected from com-pletely separated areas. DNA Extraction:

Total DNA was extracted from the nine Erwinia carotovora isolates via Bioflux DNA/RNA extraction pu-

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rification Kit (Sigma scientific ser-vices company). DNA concentration was measured using spectrophotome-ter and gel electrophoresis methods (Sambrook et al., 1989). Random Amplified Polymorphic DNA (RAPD) technique:

The molecular differences among Erwinia carotovora isolates were detected by RAPD technique. Ten decamere arbitrary primers were employed under modified PCR con-ditions. Only three primers showed satisfied results. Preparation of the amplification reaction was done un-der the biosafety cabinet in a separate room rather than that in which the amplification and the extraction were done. In Eppendorf tube, the compo-nents of the PCR were prepared as a master mix containing the reagents needed to amplify the required num-ber of samples as well as positive and negative control, then 23 µl of the master mix were dispensed in each PCR tube. The components of the PCR tube will be as: (17.8 µl) sterile nuclease free water; (2.5 µl) 10 X Taq buffer; (2.5 µl) 4 mM PCR nu-cleotide mix (dNTPs); (0.2 µl) Taq DNA polymerase (5 u/µl), then 1 µl (25 ng) of the DNA and 1 µl proper primer were added in the PCR tubes to reach 25 µl as a final volume of the reaction. The thermocycler was pro-grammed as the following: Reaction consists of 40 cycles; each cycle con-sisted of denaturation at 94°C for 1 min followed by annealing at 55°C for 30 sec and extension at 72°C for 30 sec. There was an initial delay for 15 min at 95°C at the beginning of the first cycle and 10 min delay at

72°C at the end of the last cycle as a post extension step, then the PCR product was stored at 4°C or -20°C. The RAPD-PCR products were sepa-rated on 1 % Agarose gel and photo-graphed after visualized under UV transilluminator. The banding profiles were analyzed using Gene Profiler 3.1 software and SPSS statistical software system to estimate similarity and genetic distances. PCR amplification of 16S rRNA gene:

Full length (1550 bp) of 16s rRNA gene was amplified from six isolates of Erwinia spp. by using two specific primers (Table 2). The PCR reaction was carried out in a total vo-lume 50 µl as following (Sambrook et al., 1989): 4 µl 25 mM Mg CL2; 5 µl 10 X Taq buffer; 4 µl 2.5 mM dNTPs; 2 µl of each forward and re-verse primers (10 Pmol/µl); 0.4 µl Taq DNA polymerase (5 u/µl) and 2 µl DNA extracted sample (50 ng/µl). The thermocycler was programmed for one cycle at 95°C for 5 min fol-lowed by 34 cycles each cycle con-sisted of 45 sec at 95°C for denatura-tion, 1 min at 50°C for annealing and 2 min at 72°C for elongation. Reac-tion mixture was then incubated at 72°C for 10 min for final extension; the products were stored at -20°C or 4°C (Wink and Mohamed, 2003). Then, the PCR products of 16s rRNA gene were examined on 1.5 % aga-rose gel. QIA quick PCR purification kit (Qiagen Germany) was used to purify the amplification products of 16s rRNA gene. Then, 30-90 ng of PCR product was sequenced by Big Dye terminator cycle sequencing kit.


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Table (2): Primers used successfully for PCR amplification and sequence of 16s rRNA gene in Erwinia ssp. isolates.

Primer code Nucleate sequence GC content % TM

value P0 (F) 5'-GAAGAGTTTGATCCTGGCTCAG-3' 50 50°C P6 (R) 5'-CTACGGCTACCTTGTTACGA-3' 50 50°C Sequence 5'-AGGTTTGATCCTGGCTCAG-3' 52.6 56

The reaction mixture was as previously described for amplifica-tion with additional use of ddNTPs and primer of sequence (Table 2). The reaction was made in four sepa-rated tubes; each reaction tubes con-cerned one of the four nucleotide (A, T, C and G). The programmed of the sequence reaction (25 cycles) was as the following: Denaturation at 96°C for 10 seconds followed by annealing at 56°C for 5 seconds and extension at 72°C for 2 minutes. There was an initial delay for 5 min at 96°C at the beginning of the first cycle and 10 min delay at 72°C at the end of the last cycle. Then, sequence products were purified using Centri-Sep Spin columns and resolved on an ABI-PRISM model 310 automated DNA sequencer at the sigma scientific ser-vices company. Protein (SDS-PAGE) analysis:

Nine isolates of Erwinia caroto-vora were subjected to protein analy-sis. SDS polyacrylamide gel electro-phoresis (SDS-PAGE) was performed on total water soluble protein fraction extracted for Erwinia isolates accord-ing to the method of Laemmli (1970). 100 µl of bacterial culture were cen-trifuged at 5000 rpm for 15 min. Then, supernatant was discarded and 100 µl of loading sample buffer (50 mM Tris-HCl, pH 8, 5 mM dithio-thereitol and 1 mM EDTA) was added to the pellet, then vortexes for 2 minutes and incubated in water bath at 95°C for 10 min. Finally, centri-

fuged at 5000 rpm for 5 min and the supernatant was ready for loading on SDS-PAGE gel wells. Protein electrophoresis: The stock solutions used for electro-phoresis were prepared as follows:

Acrylamide and bis-acrylamide solution (30:0.8), freshly prepared ammonium per sulphate (1.5% w/v), SDS (10% w/v); TEMED and 2-mercaptoethanol were used as undi-luted solution. Resolving gel buffer (3.0 M Tris-HCl, pH 8.8), Staking gel buffer (0.5 M Tris-HCl, pH 6.8) and resolving buffer [10X, pH 8.6 as: 30.3 g Tris (0.25 M); 144 g glycine (1.92 M) and 10 g SDS (1%) in one litter of distilled water].

Preparation of 15% Slab gel: 10 ml acrylamide-bisacrylamide; 3.7 ml resolving buffer stock; 0.3 ml 10% SDS; 1.5 ml freshly prepared 1.5% ammonium per sulphate; 14.45 ml distilled water and 0.015 ml TEMED.

4% stacking gel: 2.5 ml acry-lamide-bisacrylamide; 5 ml stacking buffer; 0.2 ml 1% SDS; 1 ml freshly prepared 1.5% ammonium per sul-phate; 11.3 ml distilled water and 0.015 ml TEMED. Loading of samples and electro-phoresis:

After gel polymerization, for each sample 30 µl protein solutions were loaded and electrophoresis was running at 75 volt through stacking gel followed by 125 V during ap-proximately 2 h. Gel was stained us-ing avidin-alkaline phosphatase (De

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Prado et al., 2000) to probe the pro-tein fractions. Then, gel was photo-graphed and protein molecular mass was determined using Gel-Pro Ana-lyzer package. Results:

Nine bacterial isolates of Er-winia carotovora were isolated from naturally diseased potato tubers, let-tuce, carrot and cabbage. Diseased

samples were collected from different localities of El-Minia, Assiut and So-hag Governorates. Only nine of fifty isolates was used for this study. The tested isolates (No. 4, 48, 11 and 37) proved to be belong to Erwinia caro-tovora subsp. carotovora and isolates (No. 25, 35, 40, 42 and 26) proved to be belong to Erwinia carotovora subsp. atroseptica (Table 1).

Table (1): Source and locality of bacterial isolates of Erwinia carotovora

subsp. carotovora (Ecc) and E. carotovora subsp. atroseptica (Eca). Number of

isolates Source Locality Disease index Identification of isolates

1 Potato Assiut 20.6 Ecc 4 2 Lettuce Assiut 15.5 Ecc 48 3 Potato El-Minia 7.8 Ecc 11 4 Potato Sohag 21.5 Eca 25 5 Cabbage Sohag 8.3 Eca 35 6 Carrot Sohag 3.6 Eca 40 7 Cabbage Sohag 10.9 Ecc 37 8 Lettuce Assiut 25.7 Eca 42 9 Potato Sohag 11.6 Eca 26

* Random amplified polymorphic DNA (RAPD) technique:

Ten arbitrary oligonucleotide primers were used to generate RAPD profiles from the tested six isolates of Erwinia carotovora (Ecc 4, Ecc 48, Ecc 11, Eca 25, Eca 35 and Eca 40). Only Three primers (CUGEA-4, CUGEA-5, and CUGEA-6) showed reliable results. All three primers were amplified successfully on the genomic DNA from taken samples yielding distinct RAPD patterns (Fig-ures 1 to 3). The number of the am-plified fragments per primer varied between 29- 39, with an average 22.34 bands per primer. These frag-ments have a size ranged from 1156 to 218 bp. A total number of 98 bands were amplified with no band being monomorphic (shared by all exam-ined samples) and all bands were po-

lymorphic with a polymorphism ratio of 100%. Primer 2 (CUGEA-5) gen-erated the largest number of frag-ments (24 bands). Primer 3 (CUGEA-6) produced 23 fragments, while primer 1 (CUGEA-4) generated the lowest number of fragments (20 bands). The three primers produced a total number of 98 bands in the six samples of Erwinia carotovora iso-lates. Primer 1 (CUGEA-4) produced 29 bands ranged in size from 1108 to 376 bp in the six samples (Figure 1). The number of fragments generated by this primer varied among samples where the lowest number was three observed in isolate number 6, while the highest number was eight in iso-late number four. Primer 2 (CUGEA-5) produced thirty bands having sizes of 1156 and 247 bp in the six samples (Figure 2). The number of fragments


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generated by this primer varied among samples from two in isolate number 5, to eight in isolate number one. Primer 3 (CUGEA-6) produced 39 bands which ranged in size from 1068 to 218 bp in the six samples (Figure 3). The number of fragments generated by this primer varied among samples where the lowest number was five observed in isolate number 3, while the highest number was eight in isolate number two.

A dendrogram, generated from the RAPD data (Figure 4), indicating genetic relationships between the six isolates of Erwinia carotovora showed that all samples divided into two main clusters. The first cluster contains isolate No 1 which repre-sents the main root of dendrogram. The second cluster composed also two clusters at 20% of genetic simi-larity, isolate No 2 represent one of them and the other cluster divided into two sub-clusters at 25% of ge-netic similarity, the first sub-cluster composed isolate No 4 and the sec-ond sub-cluster divided into two groups at 40% genetic similarity. The first group contains isolate No 3 and the second group contains two iso-lates (No 5 and 6) which represent the most recent isolates in the dendro-gram with 70% genetic similarity. 16s rRNA gene sequence of Erwinia carotovora isolates:

The amplification results of the 16s rRNA gene of the six Erwinia carotovora isolates (Figure 5) showed the same molecular weight for all bands in the six isolates. Sequence analysis of the 16s rRNA gene:

The nucleotide sequences of all six Erwinia carotovora isolates were performed and the results of sequenc-ing employed to constructed a phy-

logenetic tree, figure (6). Two main clusters could be recognized. The first cluster represent the root of the phylogeny tree compressing two iso-lates (No 3 and 6). The second cluster compresses the other isolates (No 1, 2, 4 and 5). Moreover, the results of sequencing used to constructed a phy-logenetic tree by Neighbor-Joining method (500 replicates for bootstrap), in addition with twenty sequences of 16s rRNA gene of Erwinia caroto-vora available in the GenBank, (fig-ure 7). Three groups could be recog-nized, the first group compressed the isolate number 6 (E. c. subsp. atroseptica 40) which represents one cluster with E. chrysanthemi. The second group contains isolates No. 3 and 4 (E. c. subsp. carotovora 48 and E.c. subsp. atroseptica 35, respec-tively). Isolate No. 3 represent the ancestor clad of the isolates No. 4 and E. c. subsp. carotovora E112 from GenBank. The third group contains isolates No. 1, 2 and 5 (E. c. subsp. carotovora 4, E. c. subsp. atroseptica 25 and E. c. subsp. carotovora 11, respectively). Total soluble protein analysis by polyacrylamide gel electrophoresis (SDS-PAGE):

All nine isolates of Erwinia ca-rotovora were used. Protein of purifi-cation fractions separated by SDS-PAGE (Figure 8) showed that the number of protein fractions per sam-ple varied between 11 to 18 with an average of 14.44 fractions per sam-ple. These polypeptide fractions have a size ranged about 185 to 56 KDa. Some samples to be distinguished by some polypeptide fractions as frac-tions with 176.7 and 170 KDa were found to the present only in isolates 7 and 9, respectively, and absent in the other isolates.

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1- DNA 1 2 3 4 5 6 2- DNA 1 2 3 4 5 6 Ladder Ladder

3- DNA 1 2 3 4 5 6 Ladder

Figure (1-3): Agarose-gel electrophoresis Figure (4): UPGMA dendrogram of the six of RAPD products generated by primer isolates of Erwinia carotovora based on (1): CUGEA-4, primer (2): CUGEA-5 values of genetic distances calculated from and primer (3): CUGEA-6 in the isolated data of all three primers in RAPD analysis. samples of Erwinia carotovora subsp.

2

3

5

6

4

1


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1 2 3 4 5 6 7 8 9 M

Figure(8):Total protein purification fractions patterns separated by SDS-PAGE electrophoresis for nine Erwinia carotovora isolates.

Figure (9): Phylogeny tree for nine Figure (7): Dendrogram of the six Erwinia Erwinia carotovora isolates of protein carotovora isolates (with Arabic numerals 1-6) purification fractions separated and twenty related sequences from GenBank by SDS-PAGE electrophoresis. (with GenBank accession numbers) from data of 16s rRNA gene.

Also, fraction with about 150 KDa present only in isolates 1 and 4. The polypeptide fractions with about 134 and 137 KDa were found to be present only in isolates 9 and 5, re-spectively, and absent in the other isolates. Polypeptide fractions with 115.4 and 112.2 KDa present only in isolates 5 and 9, respectively.

In addition, the polypeptide fractions with about 78 and 79 KDa were found to be present only in iso-lates 5, 9 and 8, respectively. More-over, polypeptide fraction with 56.8 is unique marker for the isolate 8. On the other hand, some isolate samples are distinguished by absence of poly-peptide fraction which present in all

E. c. subsp. wasabiae 88 E. c. subsp. carotovora 2766 2

E. c. subsp. carotovora E4 1

E. c. subsp. atroseptica 32 E. c. subsp. carotovora 22 E. c. subsp. carotovora E2809 E. c. subsp. carotovora 441 5

E. c. subsp.wasabiae 92

E. c. subsp. carotovora E2807 3

E. c. subsp. carotovora E112

4

E. c. subsp. atroseptica 9224

E.c. subsp. atroseptica z445092 E. c. subsp. carotovora S334 E. c. subsp. carotovora E162 E. c. subsp. carotovora E161 E.c.subsp. atroseptica z9602

E. c. subsp. carotovora E145

E. chrysanthemi 344

6 E. c. subsp. atroseptica 8665

E.c.subsp. atroseptica z9602

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others isolates. For instance, polypep-tide fraction with about 165 KDa is absent in isolates 4 and 9 but present in the others. Also, polypeptide frac-tions with about 155, 145, 130, 107, 88, 70 and 58 KDa were absent in samples 3, 6, 3, 1, 2, 7, 1 and 1, 6, respectively.

Cluster analysis of protein frac-tions (Figure 9) showed that the iso-lates were classified into two groups.

The first group containing isolates number 1, which represents the main root of this cluster. Also, the first group compresses isolates number 6, 7, 2 and 3. The second group contain-ing isolates number 5, 8, 4 and 9.

This group divided into two clusters, one of them containing iso-lates number 5 and 8 and the other containing isolates number 4 and 9.

Figure (5): Agarose-gel electrophoresis Figure (6): Dendrogram of the six Erwinia of PCR products of the 16s rRNA gene carotovora isolates from data of in six Erwinia carotovora isolates. 16s rRNA gene.

Discussion: Potato soft rot disease causes

huge economic losses estimated to be between 40 to 80% depending on climatic conditions (Chigumira wa Ngwerume, 2002). Erwinia caroto-vora subsp. carotovora (Ecc) is the causal agent of the soft rot disease of potato tubers and Erwinia carotovora subsp. atroseptica (Eca) is the causal agent of the blackleg disease of po-tato plants (Pérombelon and Kelman, 1980; Zhijian et al., 2000; Chigumira wa Ngwerume, 2002 and Manzira 2010). In this study; pathogenicity tests of fifty isolated soft rot and blackleg bacterial isolates, collected from different host plants (potato, let-tuce, cabbage and carrot) and differ-ent locations (Assiut, Sohag and EL-Minia), were carried out. The isolates of Ecc and Eca differed in their viru-lence on potato, cabbage, carrots and

lettuce. These results are in agree-ment with those reported by El-Kazazz, 1984; Smith and Bartz, 1990; Saleh and Huang, 1997 and Abd El-Sayed et al., 2003. The morphologi-cal, physiological and cultural charac-teristics as well as pathological be-haviors of the isolated bacteria were conformity with those known for all soft rot and blackleg diseases. On the basis of the obtained data and those reported by Holt et al., (1994) and Staley et al., (2005) it could identi-fied these isolates as Erwinia caroto-vora subsp. carotovora and Erwinia carotovora subsp. atroseptica. More-over, it could be classified the isolates according to their virulence to three classes: high, intermediate and weak virulence. To differentiate between the two subsp. of Erwinia, six iso-lates, three isolates of E. c. subsp. ca-rotovora and three isolates of E. c.


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subsp. atroseptica, representing the three virulence classes were subjected to the molecular analysis, RAPD-PCR, 16s-rRNA gene sequence and SDS-PAGE protein analysis.

Concerning the RAPD-PCR technique, the primers used showed clear differences among the six iso-lates, that differed in their virulence. The high and intermediate virulent isolates had more bands than the low virulent ones.

Our results are in harmony with the findings of Coplin et al., (2002). The primers used generated an unique distinct band which could be used to distinguish the isolates in respect of their sub species (carotovora or atro-septica) or their virulence (high or low virulence). Primer No. 1 could be used as a genetic marker to distin-guish E. c. subsp. carotovora, where it generated bands with size about 412, 431 and 542 bp, that size of bands only generated with sub spe-cies carotovora. Also, primer No. 1 could be used to distinguish isolates of E. c. subsp. carotovora according to their virulence, where it generated a unique band with 542 bp in low vi-rulent isolates, and generated two unique bands with 412 and 431 bp in high and intermediate virulent iso-lates, respectively. In addition, prim-ers No. 3 and 2 could be used as ge-netic markers to distinguish the iso-lates of E. c. subsp. atroseptica ac-cording to their high or low virulence, respectively. Where primer No. 3 generated band with size about 964 bp which could be sign to the high virulent isolates and it doesn`t gener-ated any band with the low virulent isolates. While, primer No. 2 gener-ated band with the same size (about 962 bp) which could be sign to the low virulent isolates, and it doesn`t

generated band has that size with high or intermediate virulent isolates. Our results are in agreement with the findings of Parent et al., (1996) and Valkama and Rkarjalainen, (1994) who reported that RAPD markers are a convenient method for identifica-tion of E. carotovora at species lev-els. The dendogram is shown that highly virulence isolates (No. 1 and 2) represent the ancestor of the other isolates. Also, the isolates No. 5 and 6 represent the most recent isolates in the dendogram with 70% genetic si-milarity, both isolates are low viru-lence. These results confirm that high virulent isolates are representing the ancestor of the low virulent one, which means the evolution direction was toward the low virulence.

Concerning the 16s rRNA gene, the sequences have been successfully obtained from six isolates of the Er-winia carotovora representing the two sub sp. carotovora and atrosep-tica. The PCR amplification results of the 16s rRNA gene of the six isolates produced bands with similar molecu-lar weight, that indicate there isn`t insertion or deletion mutations. The dendrogram constructed from the da-ta of the six sequences of isolates showed that isolates No 3 and 6 rep-resent the ancestor for the other iso-lates, these two isolates are low viru-lence. The other isolates form one cluster and the isolates No 1 and 2 represent the more recent isolates in the tree, they are Ecc and isolated from Assiut area. Moreover, we in-cluded twenty sequences of the 16s rRNA gene available in the GenBank for constructing the phylogeny tree to compare our sequences. In the com-plete data set (26 sequences) inser-tions, deletions or stop codons were not encountered. The phylogenetic

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tree Fig. (9) showed that E. c. isolates distributed in different clads within the phylogeny tree. Distribution of the isolates in the constructed phy-logeny tree reflecting that our isolates showed similar sequences of Erwinia carotovora. The phylogenetic analy-sis indicated that the isolate No 6, that clustered with E. chrysanthemi-344 and close to E. c. subsp. atrosep-tica- 8665 and Eca-z9602, was dis-tinct from other isolates. However, the morphological and physiological characteristics suggested that this iso-late could be identified as Eca. This result suggest that the isolate No 6 is probably from a new evolutionary taxonomy of Erwinia. Meanwhile, the phylogenetic tree couldn`t distin-guish between the two subsp. of E. carotovora (subsp. carotovora and atroseptica), where some isolates of Ecc connected in the same cluster with Eca. However, it has been de-bated that phylogenetic analysis based on a single molecular marker gene could lead to misleading phy-logenetic results, since these phylo-grams represent gene trees and don`t necessarily reflect the phylogeny of the corresponding species (Doyle, 1992). The employment of a different molecular marker could help to assess and overcome this problem.

On the basis of the results ob-tained from protein (SDS-PAGE) analysis, it could be detected that there are differences in protein pro-files and molecular weights (M.W.) among the nine isolates of Erwinia carotovora. Many polypeptide frac-tions could be used to distinguish col-lected from special area, as fraction with M.W. of 172.7, 176.7 and 170 kDa, that assign isolates collected from Sohag. Also, fractions with M.W. about 137 and 115.4 kDa only

present in isolate collected from El-Minia. On the other hand, isolates of E. c. subsp. carotovora could be dis-tinguished by absence of band which present in all other isolates, as bands with M.W. about 155, 130, 110 and 75 kDa. Also, isolates of E. c. subsp. atroseptica could be distinguished by absence of band with M.W. about 145 kda. The cluster analysis of pro-tein fractions supported the differen-tiation of isolates according to their isolate location as isolates No. 6 and 7, also intermediate virulent isolates No. 4 and 9, which were collected from Sohag. The application of pro-tein (SDS-PAGE) analysis may aid to detect the differences between the isolates of E. c. subsp. carotovora or atroseptica in the base of area of col-lection, which we didn`t found with RAPD-PCR technique. These results may be due to the protein contents of isolates more sensitive and depending on the environment of the isolates, but the results of RAPD depending on the DNA structure, which is more stable. References: Abd El-Sayed, Wafaa, M., Bayoumi,

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البكتریا إیروینیا كاروتوفورا تحت نوع كاروتوفورا و إیروینیا فىوراثیة ال االختالفات

كاروتوفورا تحت نوع أتروسبتیكا

، محمد عاطف احمد 1، آمال محمد إبراهیم عراقي2، جمال إبراهیم احمد محمد1هدیل مجدي محمد خلیل 1سالم

قسم أمراض النبات كلیة الزراعة جامعة اسیوط1 جامعة أسیوط- كلیة الزراعة-قسم الوراثة2

:الملخص

ا ال سببة ل Erwinia carotovora subsp. atrosepticaبكتری ي الم سوداء ف ساق ال مرض ال

شبه ا الالبطاطس ت ن Erwinia carotovora subsp. carotovoraبكتری سبب مرض العف ى ت الت

ى .ة والبیوكیمیائیةبكتیرى في البطاطس في كثیر من الصفات الفسیولوجی الطري ال م الحصول عل ت

اطق ممرضه من بكتیریة تعزال 9 ن من ا م م تجمیعه ات مصابه ت درنات البطاطس وذلك من عین

ات ن محافظ ة م امختلف یوط و المنی وهاجوأس تخدام .س م اس ه مختلف 3ت ات جزیئی ة ة تقنی لدراس

ین ات ب تاالختالف وعى تح نس ن ي .Erwinia carotovoraج ستخدمه ف وادئ الم د ان الب ووج

ه زأ RAPDتقنی ورق متمی دةظهرت ف ات ة فری ستخدم كعالم ن ان ت ى یمك ا والت ن نوعه ة م وراثی

نس وعي ج ت ن ین تح ز ب یه Erwiniaللتمیی شده المرض ذلك ال عیفة (وك ة او ض م أ). قوی ضا ت ی

سي 16s rRNAاستخدام تقنیه تحدید تتابع القواعد لجین ة لجن م Ecc, Ecaمن العزالت البكتیری ل

اروتوفورا عزالت لجنس ا عدم تمییز شجرة النسب العرقیة دراسة ظهرت ا ك ث یروینی ن بعض أ حی

ات دراسة فصل ال تم علي ذلكةوعالو .Eca في نفس المجموعه مع كانت تقع Eccعزالت بروتین

ه تخدام طریق ة باس ین (SDS-PAGE) الذائب ات ب ن االختالف شف ع ي الك ساعد ف ه ت ذه التقنی وه

ز ة عالمات وراثی إظهارفي حیث یمكن استخدام هذه التقنیه E.C عزالت ین للتمیی ي العزالت ب الت

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Khalil, Hadeel M.M. ; 2G. I.A. Mohamed; A. M.I. Eraky and ... · Khalil et al. 2014 30 Introduction: Bacterial soft rot disease, caused by Erwinia carotovora, is one of the most important