EFFICACY OF MOLECULAR DIAGNOSIS TO DETERMINE GENETIC DIVERSITY AMONG INDIGENOUS ISOLATES of Erwinia carotovora UNDER EGYPTIAN CONDITION

Thirty-Four isolates of Erwinia carotovora subspecies carotovora (Ecc) causing soft rot of potato, were collected from tubers of potato plants grown in North and East Egypt. The frequency of the isolated species of soft rot-associated bacteria, being 10, 10, 8 and 6 isolates from Governorates of Sinai, Sharqia, Gharbia and Beheira, respectively. The isolates were identified as Ecc by biochemical method and molecular identity then was confirmed by polymerase chain reaction (PCR) using specific primers FEc1 and REc1 which amplified the Eca-specific band of 690-733 bp. Pathogenicity test divided these 9 isolates into 3 aggressiveness groups, High infection (2 isolates), moderate infection (5 isolates) and low infection (2 isolates), receptively. These isolates were also characterized using the technique of Inter Simple Sequence Repeats polymerase chain reaction (ISSRPCR) found that 61 amplified fragments, 56 were polymorphic (91.8%) and the percentage of polymorphism ranged from 100% (844B) to 84.62% (HB12); the fragment sizes were 224bp to 1.36 kb. The unweighted pair-group method with arithmetical average (UPGMA) cluster analysis based on pairwise genetic similarity coefficient revealed that the similarity between the selected 9 soft rot bacterial isolates ranged from 0.125 between Ec6 and Ec9 to 0.875 between Ec1, Ec3 and Ec4. The average of similarity among genotypes was 0.500 and divided the soft rot isolates into three welldefined clusters showing a great level of genetic diversity. However, these clusters were not specific to aggressiveness groups, origin or special potato variety. Isolates with different aggressiveness levels, originated from different potato varieties participate between the same clusters. This means that the isolates likely derived from the same source population and got scattered from one area to another through their hosts.


INTRODUCTION
In 2016, Egypt's potato (Solanum tuberosum) production -targeted within the River Delta within the North -has distended at a rate of over 5% a year. Between 1990 and 2016, annual output rose from 2.6 million tonnes to about 6.000.000 tonnes, creating Egypt Africa's Number one potato producer. Egypt conjointly ranks among the world's high potato exporters in 2016, exports destroyed over 380.000 tonnes of contemporary potatoes and 18.000 tonnes of frozen potato merchandise, destined principally for markets in Europe (FAOSTAT, 2016).
Soft rot area unit foremost diseases of potato that cause large losses to potato crop not solely within the field however additionally within the storage wherever the bacterium area unit transmitted from the pathologic tubers to the healthy ones. Multiple subsp. of Erwinia, together with (Erwinia carotovora subsp. http:/www.journals.zu.edu.eg/journalDisplay.aspx?Journalld=1&queryType=Master Biotechnology Research atroseptica and Erwinia carotovora subsp. carotovora and Erwinia carotovorum subsp. brasiliensis). Ecc and Eca area unit the first eubacterium in control of soft decay of potato in temperate climates (Pitman et al., 2010).
The genetic variability of Phyto-pathogenic bacteria are important to elucidate possible relationships between proven populations of the pathogen and the area from where they were originally isolated (Scortichini, 2005). Various techniques could be used to study genetic diversity present in a pathogen population. Inter Simple Sequence Repeats polymerase chain reaction (ISSR-PCR), using low strictness conditions and primers having short nucleotide sequences has been used efficiently to distinguish genetic diversity through some plant pathogenic bacteria (Mello et al., 2006).
The ISSR marker belongs to a class of multilocus, mostly dominant genetic markers that also include the amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) markers, and efficiency for studying diversity in pathogen populations (Hsiang and Wu, 2000;Jawhar et al., 2000).
Although the reproducibility of DNA fingerprints may be influenced by the reagents, thermocycler (Tyler et al., 1997) and intensity of amplicons used to score the fingerprints (Skroch and Nienhuis, 1995), under wellestablished parameters the results can be very reproducible within a laboratory (Toth et al., 1999;Mello et al., 2008) evaluated a number of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora and concluded that among the molecular identified and ISSR analysis was the most discriminatory.
Erwinia carotovora is an important pathogen of potato in Egypt, especially the North and East part of the country. However, no research work has been done so far on the population structure of this important pathogen. Therefore, our long term goal has been to develop an effective disease management strategy and understanding the variation in the pathogen population is a must to achieve this goal. In this paper we report the pathogenic and genetic diversity found, using ISSR techniques, among the different isolates of Erwinia carotovora collected from different geographical areas of the North and East Egypt.

Isolation and Purification of Bacteria Associated with Soft-rotted Tubers
Thirty-four samples of naturally infected potato tuber (Solanum tuberosum L.) were collected from four totally different farms situated in Sinai, Sharqia, Gharbia and Beheira Governorates. Infected tubers with soft rot bacteria Erwinia carotovora (Ec) were used (Yuan et al., 2004).

Isolation and Pathogenicity Test
Potato tubers were surface disinfected by flaming, then cut exploitation with sterile knife into 1cm thick slices with virtually equal diameter. Every slice was placed on the surface of moistened sterilized paper into a Petri-dish. 0.5ml of bacterial inoculum of every isolate was used for artificial inoculation by pipetting at the middle of every slice. Three slices were used for every tested bacterial isolate likewise as management. All treatments were incubated at 30°±2°C for four days, then examined daily. Disease assessment of decayed tubers slices were expressed in keeping with 48 hours (Lelliott and Stead, 1987

Bacterial DNA Extraction
To extract DNA, bacteria were grown overnight at 27± 2 o C in 5ml LB broth in shaking incubator. Half ml of each isolate was poured in Eppendorf tubes, centrifuged at 7000 rpm at 4 o C for 10 minutes, supernatants were discarded, and the pellets were resuspended in 100 µl 0.5N NaOH solution (Wang et al., 1993). The suspension was centrifuged at 8000 rpm for 10 minutes, and supernatant was saved. For each isolate, 5µl of the supernatant were mixed with 45µl of 0.1 M TBE buffer and stored at 4 o C until used. When needed, 3 µl of this mixture was directly added to the PCR tube.

Identification of the Selected Isolates
Identity of isolates was confirmed using biochemical tests (Lelliott and Stead, 1987) and molecular identified by subspecies-specific PCR primers (Table 1), was obtained from DNA amplification was done according to the non-conventional method and the PCR reaction mix was in the final reaction volume of 25 µl contained; 5.0 µl 5X Taq buffer, 1.1 µl 25 mM MgCl 2 , 0.5 µl 10 mM dNTPs, 0.13 µl 5 U/µl Taq polymerase, 1.25 µl 10 µM of each primer and 2.0 µl DNA template finalized to 25 µl by adding 13.77 dH 2 O; PCR was performed in a thermal cycler T Professional (Biometra, Germany) using the following protocol and adjusted as needed. The reaction involved initial denaturation (94˚C, 5 min) followed by 35 cycles of denaturation (94˚C, 1 min), annealing (55˚C, 1 min), extension (72˚C, 1 min) with a final extension (72˚C, 7 min).
Amplification products (25µl) were mixed with 3µl loading buffer and separated on 1.3% agarose gel and stained with 0.5 µg/ml ethidium bromide and visualized under ultraviolet light and photographed. DNA fragment sizes were determined by comparisons with the 1kb plus DNA ladder marker.

ISSR-PCR Analysis
Similarity coefficients were calculated according to dice matrix (Nei and Li, 1979;Rohlf, 1993). Parents were grouped by cluster analysis with the similarity matrix and unweighted pair group method based on arithmetic mean (UPGMA).

Isolation and Biochemical Identification of the Pathogenic Isolates
Biochemical assays for the isolated bacteria revealed that the 34 isolates are distributed among three species belonging to three genera grouped as Erwinia sp., Bacillus sp., Pseudomonas sp. At a frequency of 12, 13, and 9 isolates, respectively. Different governorates did not show much difference in the frequency of the isolated species of soft rot-associated bacteria, being 10, 10, 8 and 6 isolates at Sinai, Sharqia, Gharbia, and Beheira, respectively (Table 2). (2007) found that the main bacteria causing potato tuber soft rot are Erwinia sp., P. carotovorum subsp. carotovorum. Pseudomonas syringae van Hall found only two species of Gram-negative bacteria isolated from rotting potatoes collected from clamps in England in 1945-7 (Jones and Dowson 1950). The isolates of Bacillus spp. are thought to be saprophytic rather than pathogenic, as Bacilli are not previously reported to be the primary causal agents of soft rots. Lund and Wyatt (1979) highlighted that potato tubers may harbor pectolytic saprophytic bacteria (Bacillus spp., Clostridium spp., Flavobacterium spp. and Pseudomonas spp.) which, if given the chance, can also cause rotting (Table 3).

Pathogenicity Test
In the previous section, we showed that bacterial isolation trial from rotted potato tubers resulted in 9 bacterial isolates ( Table 3). Results of pathogenicity assay showed that 9 of all the tested isolates were pathogenic on potato tubers. Out of them, only 2 isolates (Ec1 and Ec5) caused highly severe symptoms of soft rot on potato tubers, meanwhile, the remaining isolates (Ec2, Ec3, Ec7, Ec 8 and Ec9) showed moderate severity on potato tubers, while the Ec4 and Ec6 showed low infection.
Traditional identification of the 9 strains of the presumptive pathogen Erwinia carotovorum isolates resulted in the confirmation of all of them to be Erwinia carotovorum as they showed the following results: creamy, short rods, Gram negative, which did not have the ability to produce any pigment in Kings B medium. All the tested isolates were positive for motility, starch hydrolysis, growth at 37°C, growth in 5% NaCl, catalase activity, but negative for KOH 3%, pigment production. The strains also induced typical bacterial soft rot symptoms on tuber slices of cv. Spunta (Table 4).

Molecular Identification of the Pathogenic Isolates
Amplifications with the FEc1 and REc1 primers were positive in all strains (Ec1 to Ec9) and negative control B (-): Bacillus spp., P (-): Pseudomonas spp., respectively. Also, the Fec1and Rec1 primers detected Erwinia carotovorum subsp. carotovorum DNA in the ten tested strains (Fig. 1). It found that PCR product was 690 -733 bp long, which was the same size as the region on the Erwinia carotovorum subsp. carotovorum between the two primers. Results illustrated in (Figure 1) showed the sequence of this 690-733 bp PCR amplicon. These results were confirmed with those obtained by (De Boer and Ward, 1995) who proved that for definitive identification of Erwinia carotovorum strains.

Genetic Diversity of Erwinia carotovorum Strains Inter simple sequence repeats (ISSR) polymorphism of Erwinia carotovorum isolates varied in their virulence on potato tubers
Amplification of soft rot (Erwinia carotovora) DNA from 9 bacterial strains were done with  five ISSR primers of 61 amplified fragments, 56 were polymorphic (91.8%) and the percentage of polymorphism ranged from 100% (844 B ) to 84.62% (HB 12 ); the fragment sizes were 224 bp to 1.36 kb (Table 5 and Fig. 2).

Acid prod. Gas glucose
The number of bands which obtained by each primer was 9 with 814 A primer, 16 with the 844 B primer, 12 with 17898A primer, 11 with both HB 9 primer and 13 with HB 12 primer. The 844 B primer had the higher polymorphism (100%) but HB 12 primer gave the lowest polymorphism (84.62%). Finally, 61 bands loci were amplified by ISSR primers which 56 loci of them were polymorphic and 5 loci were monomorphic.

Jaccard's Similarity Coefficient:
The UPGMA cluster analysis based on pairwise genetic similarity coefficient revealed that the similarity between the 9-soft rot bacterial isolates ranged from 0.125 between Ec6 and Ec9 to 0.875 between Ec1, Ec3 and Ec4; the average of similarity among genotypes was 0.500 (Table 6 and Fig. 3).
The dendrogram constructed from ISSR analysis of Erwinia carotovora isolates collected from geographically diverse zones of the North and East Egypt separated the 9 isolates into three major ISSR groups (Fig. 3).   The first group was formed by Ec 5, 8 and 7; Only Ec 7 and 8 belongs to "Moderate infection". The second group consisted of five Erwinia carotovora isolates, two isolates (Ec4 and Ec6) of which belongs to "low positive infection" and Ec2 and Ec3 strains to "Moderate infection"; Only one isolate (Ec1) of this group belong to "High infection". Finally, the isolate Ec9 formed the third group and only Ec1 strain of this group belong to "Moderate infection". Found the different within groups, the various bacterial strains diverged slight from each other. Never close relationship was observed among the ISSR and infection groups or between ISSR groups and geographic origin and conformable results were reported by Uddin et al. (2013).
The actuality that various Erwinia carotovora isolates can cause different amounts of soft rot under similar experimental conditions propose that the population of the pathogen is not homogeneous and there is pathogenic variation between the isolates examined.
The less virulent isolates might have evolved from extremely virulent ones just by losing virulence gene clusters as is that the case of P. wasabiae isolates (Kim et al., 2009;Pitman et al., 2010). instead, the weakly virulent isolates might be due to the existence of lower virulent phenotypes in natural environments along with the aggressive strains, as a part of population diversity (Yap et al., 2004). It can't be dominated out that these weakly virulent strains might possess a role in the survival and institution of the population underneath specific environmental conditions (Yishay et al., 2008). Yahiaoui-Zaidi et al. (2010) found that Erwinia carotovora isolates were a lot of aggressive on their origin plant of than on alternative potato varieties. Our information showed that there was variation within the pathogenicity of Erwinia carotovora.
Similar results were reported by other researchers (Arabi and Jawhar, 2007;Jawhar and Arabi, 2009) studying different plant pathogens. This can be due to genotype-isolate interactions where different virulence genes are operating in the pathosystem (Van der plank,  1984).
ISSR analysis potentially provides information across the entire genome as it uses non-specific primers which bind randomly to regions over the whole genome. ISSR analysis would even detect smaller changes caused by point mutations, thus offering a higher degree of sensitivity as compared to that obtained by other methods. As compared to other related Erwinia sp. is relatively homogeneous (Parent et al., 1996). Such relatively low levels of genetic diversity may be due to a subspecies having more recent origins, limited geographical distribution and limited host range. Avrova et al., (2002) however, found quite a high level of genetic diversity among 9 strains of Erwinia carotovora using amplified fragment length polymorphism. Other researchers studying genetic differences among isolates of enterobacteria on potatoes also demonstrated a greater diversity of pectolytic soft rot infecting potatoes than previously thought (Oliveira et al., 2003