Plasmids profiles , antibiotic and heavy metal resistance incidence of endophytic bacteria isolated from grapevine ( Vitis vinifera L . )

Little is known about the bacterial communities associated with the plant inhabiting desert ecosystem. In this study, the bacterial population associated with grapevine (Vitis vinifera L.) plant, growing desert soil was analyzed using the culture dependent approach. A total of 111 bacterial isolates were isolated from stems and leaves samples, collected from different locations and subjected to further analyses. Based on the identification methods, the bacterial isolates were grouped into 14 genera. The main genera are Acetobacter, Acinetobacter, Citrobacter, Enterobacter, Erwinia, Escherichia, Methylococcus, Xanthomonas, Vibrio, Bacillus, Micrococcus, Planococcus, Staphylococcus and Streptomyces. Significant differences in the endophytic communities were observed between plants collected from different sites and also between plant stems and leaves. All the isolates were examined for plasmid DNA content and resistance to antibiotics (Ampicillin, Kanamycin, Tetracyclin) and heavy metals. Minimum inhibitory concentrations (MICs) of Cu, Cd, Hg, Mn, Ni and Zn for isolates were also determined. Resistance was most frequent to Ampicillin (57%), followed by Kanamycin (53%) and Tetracycline (26%). The highest MICs observed were 10 μg/ml for mercury, 50 μg/ml for Cu and Cd and 200 μg/ml for other metals. On a percentage basis, 18.48% of total strains from leaves were found to harbour plasmids, whereas, 11.83% of the roots isolates contained plasmids.


INTRODUCTION
Bacterial endophytic species are common inhabitants of a wide range of plant species and reside either within cells (Jacobs et al., 1985), in the intercellular space (Patriquin and Dobereiner, 1978), or in vascular systems (Bell et al., 1995) of a plant.Microbial endophytes are typically defined as microorganisms that do not visibly harm the host plant but can be isolated from surfacedisinfested plant tissues or the inner parts of plant organ (Hallmann et al., 1997).Bacterial endophytes seem to be ubiquitous in plant tissues, having been isolated from flowers, fruits, leaves, stems, roots and seeds of various plant species (Kobayashi and Palumbo, 2000).Significant variations in the populations of both indigenous and introduced endophytes have been reported.These variations are attributed to plant source, plant age, tissue type and environment (Zinniel et al., 2002).
The potential to use plant-bacteria association or endophytes to remediate polluted soils has attracted considerable interest (Idris et al., 2004).Bacterial endophytes can stimulate contaminant disappearance by the accumulation and transformation of heavy metals and some xenobiotic compounds.Several authors have investigated the role of endophytes in phytoremediation and they have found that certain plant-bacterial associations can increase bioremediation processes (Burd et al., 2000).While there have been many studies on the microbial diversity associated with the plants of terrestrial environment (Kobayashi and Palumbo, 2000), virtually little information is available about the microbial diversity associated with plants in desert environments with special attention to the bacterial endophytes of grapevine.
My research goal was to determine the prevalence, properties, persistence and types of endophytic bacteria in grapevine.An additional goal was to identify and characterize the plasmid complement in several strains of the endophytic bacteria as a first step for determination of whether they have a role in the endophytic phenotype.

Plant sample processing and isolation of endophytic bacteria
The host plant used in this study was grapevine (Vitis vinifera L.).This plant grows in the west Taif governate, Saudia Arabia.Grapevine plant was removed from soil with a trowel placed in plastic bags and immediately taken to the laboratory.Leaves and stems were washed in running tap water and graded by their size and surface appearance in order to exclude samples that showed symptoms of diseases or superficial damage.Endophytic bacteria were isolated from internal plant tissues using surface-triturationplating technique.This technique involves immersing tissue samples in a sequence of 70% ethanol for 1 min, sodium hypochlorite solution (2% available Cl-) for 4 min, 70% ethanol for 30 s and rinsed three times in sterile distilled water (Kuklinsky-Sobral et al., 2004).To ensure that the surfaces were sterile, samples were imprinted on tryptic soy agar plates (10% TSA) and water from the final washing step was spread on tryptic soy agar plates.After surface disinfection, the leaves or stems tissues were cut and triturated in 20 ml of sterile 20 mM potassium phosphate buffer, pH 7 and allowed to stand at room temperature for 20 min, after which 100 µl of 10-fold serial dilution (10 -1 , 10 -2 , 10 -3 ) of suspension in sterile buffer was plated on Plate Count Agar (PCA, Difco Laboratories, Detroit, MI) at full strength for colony forming units (CFU) enumeration.The duplicate dilution plates were incubated at 28°C, colony forming units (CFU) per gram were scored after 5 days because some slow growing bacteria took more than 3 days to appear.Representative bacterial colonies were selected from dilution plates based on colony size, shape, morphology and color and purified by restreaking onto fresh plates of the same medium used for primary inoculations.These colonies were also cultivated in 10% TSA, incubated at 28°C for 18 h and following each culture, were suspended in 20% glycerol solution and stored at -70°C.

Identification of bacteria
Colonies representing the most numerous members in each sample were subcultured on PCA by streaking on the same fresh medium and incubated at 30°C for 3 days.The identification of strains was carried out by using the classification given in the Bergey's Manual of systematic Bacteriology (Krieg and Holt, 1984).The strains were further identified with API 20 NE strips (biomerieux, Marcy I'Etoile, France).

Antibiotic resistance test
The test isolates were spot-inoculated on nutrient agar plates incur-porated with filter-sterilized ampicillin, kanamycin and tetracycline, at the rate of 100, 50 and 10 µg/ml respectively and incubated for 48 h at 30°C.The antibiotics resistance was recorded as positive if the test colony appeared on the plates, as compared to the control plate in which no antibiotic was added.

Motility test
Each isolate was spots-inoculated on the center of semi-solid nutrient agar plates (0.2% agar) and incubated at 30°C.The diffusion of colony was observed and recorded at 24 h (Elbeltagy et al., 2000).

Determination of minimum inhibitory concentrations (MIC) of heavy metals
The MIC of the metal for each isolate was determined by the plate dilution method as adopted by Summers and Silver (1972).The metals, Hg 2+ , Cd 2+ , Cu 2+ , Pb 2+ , Ni 2+ and zin 2+ were used as HgCl2, CdCl2, CuCl2.2H2O,PbCl2, NiCl2 and ZnCl2 in different concentrations ranging from 5 to 1000 ppm.Stock solutions of the metal salts were prepared in double distilled water and were added to sterilized nutrient agar plates.In each test, 5 µl of a liquid broth overnight broth culture (containing approximately 2 -4 x 10 5 CFU) was applied onto duplicate agar plates containing the appropriate heavy metal salts and incubation was at 30°C for 3 days.The lowest concentration of the metals, which inhibits the bacterial growth, was considered as MIC.Since there is no defining concentration of metal ions which can be used to distinguish metal resistant bacteria and metal sensitive bacteria, the concentration used in this study has been employed in similar studies reported on soil bacteria (Trevors et al., 1985;Malik and Ahmed, 2002;Malik and Jaiswal, 2000).

Plasmid isolation
Several plasmid isolation methods and modification of these methods were used including: alkaline lysis (Ausubel et al., 1987), the method of Kado and Liu (1981), Brenner et al. (1993) and Hansen and Olsen (1978).The isolated plasmids were characterized by agarose gel electrophoresis according to standard procedure (Maniatis et al., 1989).The size estimates of the isolated plasmids were obtained by comparing their relative mobilities on agarose gel with standard molecular weight DNA marker.

RESULTS
Incidence of endophytic bacteria isolated from stems and leaves of grapevine (Vitis vinifera L.) The endophytic bacterial communities of healthy looking leaves and stems of grapevine (V.vinifera L.) were assessed in surface disinfested plant parts upon cultivation in TSA medium.During this study, a total of 111 isolates of endophytic bacteria were recognized in both leaves and stems of grapevine.Preliminary characterization of these isolates indicated that studied parts of tested contained both gram-negative and gram-positive bacteria.

Identification of bacteria and frequency of occurrence
A more extensive phenotypic characterization was carried out with 111 different isolates of endophytic bacteria isolated from different organs (stems and leaves) of Grapevine plants collected from different locations.As whenever possible, the bacterial isolates were identified to the species level, but most isolates were only identified to the genus level.
The fourteen bacterial genera identified in a survey of 111 isolates; Acetobacter, Acinetobacter, Citrobacter, Enterobacter, Erwinia, Escherichia, Methylococcus, Xanthomonas, Vibiro, plus gram-positive bacteria; Bacillus, Micrococcus, Planococcus, Staphylococcus and Streptomyces were the most predominant (Table 1).There appears to be differences in the frequency of occurrence of specific genera of bacteria between the plant organs, age and the water sources from which the plants were collected.
The results in Table 1 revealed that four genera (1 gram-negative and 3 gram positive) were identified in young leaves compared to five genera (3 gram-negative and 2 gram-positive) recorded in old leaves collected from different locations.Among gram-negative bacteria, Acetobacter was recorded from both young and old leaves with different frequencies (2.32 and 4.41%, respectively).However, Enterobacteor and Erwinia were recorded only in old leaves.Of gram-positive bacteria, Bacillus and Planococcus were found in both young leaves (frequency, 11.76 and 1.47% respectively) and old leaves (frequency, 19.11 and 1.47% respectively).However, Altalhi 5875 Table 2. Endophytic bacterial population density (CFU g -1 FW) recovered from leaves and stems of grapevine (Vitis vinifera L.).
On the other hand, three genera of endophytic bacteria ( 2 gram-negative and one gram-positive ) were recorded in young stems compared to three genera (2 gram negative and one gram-positive) in old stems of grapevine plants collected from different locations.Two genera of gram-negative bacteria (Acintobacter and Enterobacter) were isolated from young stems and (Acetobacter and Methylococcus) were isolated from old stems.Species of Bacillus were the only Gram-positive isolates from young and old stems (Table 1).
The results presented in Table 1 show that three genera of endophytic bacteria were isolated from young leaves (2 gram-negative and 1 gram positive) compared to four genera were isolated from old leaves (3 gramnegative and 1 gram positive) of grapevine plants collected from irrigation canals.Bacillus and Enterobacter were isolated from young and old leaves with different frequencies.However, Acintobacter and Xanthomonas were found only in old leaves and Acetobacter in young leaves.
On the other hand, three genera were recorded in young stems (1 gram-negative and 2 gram-positive) compared to 4 genera (3 gram-negative and one grampositive) in old stems of grapevine (V.vinifera L.) plants collected from different locations.Among gram-negative bacteria, only Enterobacter was isolated from young and old stems.However, Citrobacter and Escherichia were isolated only from old stems (Table 1).Gram-positive bacteria were represented by Bacillus sp. in both young and old stems, whereas, Planococcus was only recovered from young stems.

Population density of endophytic bacteria
The number of colony-forming units per gram fresh weight (CFU) of culturable endophytic bacteria isolated from various plant tissues of grapevine (V.vinifera L.) collected from different locations (Table 2).The results showed that CFU value in old leaves (4.7 x 104 ) is much higher than in young leaves (2.4 x 10 3 ) of grapevine plants.In contrast, young stems (1.1 x 10 stems contained greater CFU values than young stems.

Activity of hydrolytic enzymes in endophytic bacterial population of Grapevine (Vitis vinifera L.)
A more extensive phenotypic characterization was carried out with 111 culturable bacterial isolates recovered from surface sterilized stems and leaves of grapevine plants collected from different locations.The in vitro activities of some hydrolytic enzymes of isolated strains were studied.The data presented in Table 3 revealed that all the tested strains were cellulase, pectinase, chitinase and protease negative.Amylase activity was only observed in Vibrio, Micrococcus and Staphylococcus strains.All the Gram positive strains except Streptomyces were lipase positive.In contrast, all Gramnegative strains recorded were lipase negative; however, Enterobacter and Erwinia were positive.

Antibiotic resistance
All the 111 endophytic isolates were screened for growth on LB agar amended with ampicillin 100 µg/ml, tetracycline 50µg/ml, or kanamycin 10 µg/ml.Analyses of resistance to various antibiotics are shown in Table 3.For gram-negative bacteria, resistant to ampicillin was most common among the strains isolated from plants (57%) followed by resistance to kanamycin (53%) and resistance to tetracycline (26%).Gram-positive strains demonstrated a similar pattern of antibiotics resistance.However, the isolated strains were more likely to be resistant to kanamycin than to ampicillin (Table 3)

Heavy metal resistance
All the isolates were examined for presence of plasmids and were also checked for tolerance to all metals studied.The tolerance of the plasmid-containing strains isolated from different organs of Grapevine (V.vinifera L.) collected from different locations expressed as MICs (The lowest concentration at which no growth was observed) is shown in Table 4.

Hg 2+ resistance
Mercury was the most toxic since only two isolates, Erwinia13 isolated from old leaves and Enterobacter isolated from old stems, were able to grow in the presence of 10 ppm.

Pb 2+ resistance
All the tested strains isolated from leaves and stems of Grapevine plants collected from different locations were resistant to high levels of Pb 2+ with MIC values ranged between 800 and 1200 ppm.

Cu 2+ resistance
Most of the tested bacterial isolates isolated from both organs are less tolerant to Cu 2+ ions.However, Entero-bacter26, Escherichia19, Citrobacter 54, Vibrio7 and Bacillus 61 grew in the presence of 50 ppm Cu 2+ ions.

Cd 2+ resistance
All of the tested strains isolated from different organs of plants collected from target sites showed the same sensitivity to Cd 2+ ions and MIC was (25 ppm).However, Enterobacter18 and Micrococcus11 exhibited MIC (50 ppm).

Ni 2+ resistance
Most of the tested strains isolated from both organs exhibited MIC ranging between 200 and 350 ppm.However, Citrobacter54 and Vibrio7 were less resistant to Ni 2+ ions with MIC values of 100 and 70 ppm, respectively.On the other hand, bacterial strains isolated from different organs of grapevine plant were tested for nickel resistance, Bacillus strains showed the same response to Ni 2+ ions with MIC 200 ppm, whereas, the MIC for Micrococcus11 was 300 ppm.However, Enterobacter26 and Escherichia19 were sensitive to Ni 2+ ions.

Mn 2+ resistance
Most of the tested strains isolated from leaves and stems had similar MICs to Mn 2+ ions (200 ppm).However, Citrobacter54 was more resistant to Mn 2+ and exhibited MIC of 400 ppm, whereas, Vibrio was sensitive to Mn 2+ (50 ppm).On the other hand, gram-positive bacteria isolated from different plant organs responded differently to the presence of Mn 2+ ions.Bacillus3 and Bacillus9 strains had the same MIC (250 ppm), while the MICs for Bacillus14 and Bacillus42 were 100 ppm and 200 ppm respectively.The MIC for Micrococcus11 was 100 ppm (Table 4).

Zn 2+ resistance
All the tested strains recovered from different organs of plant, collected from different target sites exhibited MIC which ranged between 200 to 300 ppm.However, Citro-bacter, Enterobacter and Escherichia were sensitive to Zn ions and the MICs were 75, 50 and 60 ppm, respectively.

Plasmids incidence
Although the aforementioned parameters strongly suggest the presence of plasmid DNA in the bacterial isolates, yet remains confirmation of this suggestion.In present investigation all the metals and antibiotic resistant strains were screened for the presence of plasmid DNA.Figure 1 shows a typical electrophoretic seperation of the plasmids in some of the isolates having more than one plasmid.Approximate size and in some cases, size ranges, for the plasmids were determined by comparing their mobilities with those of the known multiple plasmid in Shigella sp.
The results in Table 5 show the number and type of plasmid possessed by endophytic bacterial strains isolated from leaves and stems of plants collected from different locations.Among Gram-negative bacteria, Enterobacter 47 and Erwinia 13 harboured two large plasmids, whereas, Enterobacter23 and Enterobacter18 possessed one large plasmid.In the case of Citrobacter 54, an additional small plasmid was observed.However, Vibrio7, recorded from young stems possessed only two small plasmids.In addition, Gram-positive bacteria represented by Bacillus species harboured only large plasmids.
On the other hand, plasmid screening of bacterial strains isolated from different plant organs and collected from different locations revealed that all plasmidcontaining strains harboured large plasmids (Table 5).Among gram-negative bacteria, Escherichia 19 harboured two large plasmids whereas; Enterobacter 26 possessed two large and two small plasmids.Among gram-positive strains, Bacillus sp and Micrococcus 11 possessed only one large plasmid.However, Bacillus 14 contained one large and one small plasmid.

Distribution of plasmid patterns among endophytic bacterial strains isolated from grapevine (Vitis vinifera L.)
On the percentage basis 9 (13.23%)out of 111 isolates recovered from old stems and leaves collected from different sites were found to harbour plasmids (Table 6).Only 3 (10.34%)strains out of 29 isolates recorded from stem tissues contained plasmids, whereas only 6 (15.38%) isolates out of 39 isolates recorded from leaf tissues possessed plasmids.On the other hand, 7 (16.27%) out of 43 endophytic bacterial isolates obtained from young organs collected from target sites; 4 (20%) out of 20 isolates from leaf tissues possessed plasmids compared to 3 (13 %) out of 23 isolates from stem tissues.Moreover, 8 isolates (88.8%) out of 9 isolates harboured large plasmids whereas one isolate (11%) among these isolates contained small plasmid from old organs of plants collected from different sites.On the other hand, 7 isolates (100%) of total strains possessed plasmids harboured large plasmids whereas, only 28.57% of these isolates possessed small plasmids from plants collected young organs (Table 7).

DISCUSSION
Endophytic bacterial strains were defined as isolates that were obtained from surface-sterilized plants, displayed differentiable colony morphologies and were recovered from initial survey of grapevine (V.vinifera L.) plant.
It is well established that plant bacterial endophytes are to be found in most healthy plant tissues (Frommel et al., 1993;McInory and Kloepper, 1995;Sturz, 1995).In this study, we isolated more than hundred bacterial strains from stems and leaves of Grapevine (V.vinifera L.) plant collected from different locations at Taif governate.Similarly, other workers have reported isolation of indigenous endophytic bacteria from grapevine (Bell et al., 1995).To my knowledge, this study is the first to describe indigenous bacterial endophytes isolated from grapevine (V.vinifera L.) in Saudia Arabia to evaluate populations of potentially endophytic bacteria, and a total of 111 isolates were collected over 1-year period.
There is a significant variation in the types of indigenous bacteria isolated from diverse host plant species (Zinniel et al., 2002).However, there is similarity in the types of indigenous bacteria isolated from different parts of the same host plant (Mocali et al., 2003).Preliminary characterization of endophytic bacteria in this study showed that a slight variation in the type of indigenous bacteria isolated from stems and leaves.Several factors may explain these differences, including plant age and tissue type (Kobayashi and Palumbo, 2000).
Morphological and biochemical characterization of these bacteria indicated that Gram-negative and grampositive bacteria were recovered from stems and leaves of Grapevine (V.vinifera L.) plant (Kobayashi and Palumbo, 2000).Furthermore, gram-negative bacteria were more diverse than gram-positive bacteria either those isolated from tissues of stems or leaves.In this context, similar results were obtained with other plant species for the diversity of Gram-negative bacteria isolated from different parts of the plant (Mocali et al., 2003).Interestingly, Gram-positive bacteria (61.73%) were isolated more frequently than gram-negative bacteria from grapevine plant tissues.Earlier workers have reported the predominance of gram positive bacteria in the tissues of various plants (Lalande et al., 1989;Leifert et al., 1989).In contrast, other investigators have reported the predominance of gram negative bacteria (Elbeltagy et al., 2000).However, Zinniel et al. (2000) reported an equal presence of gram-negative and Gram-positive bacteria.
The population densities of endophytic bacteria obtained in this study using TSA medium ranged from 1.15 x 10 3 to 5.2 x 10 4 colony forming units/g (CFU/g) of fresh tissues.Similar-sized populations were isolated from other host plants (Kobayashi and Palumbo, 2000;Zinniel et al., 2002).Furthermore, the host plant has an optimum carrying capacity of endophytic populations, which fluctuates depending on the plant age and different environmental factors (Hallmann et al., 1997).In present investigation, endophytic population was highest in the old tissues (Kuklinsky-Sobral et al., 2004).
The plant-associated habitat is a dynamic environment in which many factors may affect the structure and species composition of the microbial communities that colonize, stems branches and leaves (Kuklinsky-Sobral et al., 2004).Some of these factors are plant tissue type (Mocali et al., 2003), the habitats type and other environmental factors (Dalmastri et al., 1999).In the present study, endophytic bacteria were isolated from leaves and stems of grapevine; some genera such as Erwinia, Planococcus and Staphylococcus were recorded from leaf tissues and missed from the stem tissues collected from the same location.However, Acinetobacter and Xanthomonas were recorded from both tissues.Similarly Acetobacter, Acinetobacter, Vibrio and Streptomyces were recorded from stem tissues and never from plant leaves, whereas, Escherichia and Micrococcus were recovered from stems and were absent in plant stems.This is in agreement with previous results indicating the population fluctuation in plants grown in different sites (Picard et al., 2000).
It has been previously shown that fluctuation of endophytic communities depends also on the type of plant organ (Mocali et al., 2003).In the current study, certain endophytic genera were limited to stem tissues such as Vibrio, Escherichia, Micrococcus and Streptomyces, whereas other genera such as Xanthomonas and Staphylococcus were specific to leaf tissues.This could be related to the different environments involved; the leaves which are a source of nutrients or roots which are a nutrient sink (Mocali et al., 2003).Gardner et al. (1982) observed that fluctuation of endophytic bacteria not only depends on the type of organ but also on the age of the plant organ.Siciliano et al. (1998) also suggested that young tissues had morphological structure or chemical composition that affected the ability of certain bacteria to colonize these tissues.In the present study, Staphylococcus, Vibrio and Streptomyces were detected in young tissues whereas certain genera such as Erwinia, Xanthomonas, Escherichia, Methylococcus and Citrobacter were isolated from old tissues.
To evaluate the function and persistence of endophytic bacteria in plants, hydrolytic enzymes and motility were assayed.Hydrolytic enzymes, pectinase and cellulase may play role in the mechanisms by which endophytic bacteria penetrate into and persist in the host plant (Hallmann et al., 1997).However, release of plant cell wall constitutive degrading enzymes by endophytic bacteria is undesirable since this would confer pathogencity (Collmer et al., 1988).In this study, in vitro activity of extra-cellular hydrolytic enzymes was investigated.All isolates recovered during this study were pectinase, cellulase and chitinase negative.More than (70%) of the isolated strains were motile.Thus finding is in accordance with of Elbeltagy et al. (2000).The motility of these endophytes may confer advantages for intercellular entrance and spreading into host tissues (Hallmann et al., 1997).
The potential to use plants to remediate polluted sites has attracted considerable interest over the last few years (Zaurov et al., 2001;Salt et al., 1995;Siciliano et al., 1998).Several authors have investigated the role of microorganisms in phytoremediation and found that certain plant-bacterial associations (endophytic bacteria) can stimulate disappearance of contaminants by metal accumulation (Barac et al., 2004;So et al., 2003;Zaurov et al., 2001) and by extracellular transformation (Siciliano et al., 1998;Garcia, 1987).From the standpoint of environmental pollution, heavy metals and metalloids are extremely toxic because of their relative accessibility to biological systems (Taylor, et al., 1989).The ability of bacteria to adapt through mutation and selection to the presence of toxic metal can be used to indicate whether certain metals are present or have been present in a particular environment (Taylor et al., 1989).Therefore, we examined the natural metal tolerance levels of bacterial community isolated from Grapevine (V.vinifera L.).In the current study, some isolates susceptible to various concentrations of heavy metals were obtained.The MICs of endophytic bacterial strains isolated from leaves and stems of Grapevine (V.vinifera L.) plants.Mercury was the most toxic since 99% of the isolates were inhibited by only 10 µg/ml.Similar observations have been reported earlier for mercury toxicity to bacteria (Trevors et al., 1985).
Large numbers of strains isolated from plants collected from different locations were resistant to lead (100%).The high resistance to lead could be attributed to the water lead pollution or lead accumulation by the plant tissue.Nearly, all tested strains isolated from different plant organs collected from different locations exhibited resistance to nickel, zinc and manganese.Increased industrialization has resulted in environmental contamination by these metals in many aquatic systems (Gazso, 2001;Sabry et al., 1997;Taylor et al., 1989).The tested strains isolated from plants collected from different locations showed similar sensitivity to Cd +2 and Cu +2 ions.Finally, there was no difference between the minimum inhibitory concentration exhibited by isolates from different tissues.Therefore, the similarity of heavy metals susceptibility among the tested strains might be attributed to the capacity of tolerance of metal level accumulated by the plant itself (Idris et al., 2004).This hypothesis needs further study.
The lack of a well-established genetic system is a major obstacle in the elucidation of the mechanism of endophytes or plant-microbe interactions.There is a paucity of genetic markers for endophytic bacteria.Some of the most useful genetic markers include resistance to antibiotics, antimetabolites and resistance to heavy metals (Van Elsas et al., 2003).Plasmid incidence and characterization among endophytic bacteria are useful in genetic studies and are also useful in the development of cloning vectors.Examination of the plasmid content and establishing the plasmid profiles is the first step of genetic investi-gation for diversity among endophytic bacterial popula-tion.In the present study, several kinds of plasmids and a diversity of plasmid profiles were found among endophytic bacteria isolated from different organs of grapevine plants collected from different locations.The plasmid frequency in bacteria isolated from leaves and stems of plants collected from different locations were variable, 9 (13.23%)out of 68 isolates harboured plasmids.Only 3 (10.34%)out of the 29 isolates recovered from stem tissues contained plasmids, compared to 6 (15.38%) out of 39 isolates recovered from leaf tissues possessed plasmids.Furthermore, the majority of plasmids recovered from plants collected from both sites were large enough (> 50 kb) to carry genes for conjugal transfer (Piotrowska-seget et al., 2005) suggesting the possibility of such transfer in this environment.
Altalhi 5881 Modification of plants, to acquire organisms with improved genetic capabilities and tolerance to different environmental conditions is generally carried out by plant breeding and by integrating foreign DNA into plant genomes to produce transgenic plants (Barac et al., 2004).Although successful for certain plants, these methods are costly and are dependent on the plant variety being studied and takes several years to reach market.As an alternative approach, beneficial endophytes have been used to express and secrete useful products without requiring integration of foreign DNA into the plant genome (Barac et al., 2004).Moreover, endophytic bacteria have a multitude of applications to enhance agricultural production; e.g.wheat growth was found to be enhanced through production of phytohormones increased resistance of cotton to diseases nitrogen fixation in rice and wheat and increasing potato tuber formation under heat stress condition (Hallmann et al., 1997).
Finally, this study demonstrated the occurrence and diversity of culturable endophytes in grapevine (V.vinifera L.).The roles of these endophytes in this habitat remain to be elucidated.Future work will address the effect of selected bacteria on plant growth and the uptake of heavy metals by the plant as well as the mechanisms involved.

Table 1 .
Frequency (%) and genus diversity of endophytic bacteria recorded from young and old organs of Grapevine (Vitis vinifera L.).

Table 3 .
Number of isolates reacted in screening.

Table 4 .
Plasmid-containing endophytic bacteria and their MIC a values against the metals isolated from different organs of Grapevine (Vitis vinifera L.).

Table 5 .
Plasmid profiles of endophytic bacterial strains recorded from leaves and stems of grapevine (Vitis vinifera L.).

Table 6 .
Distribution of plasmid patterns among endophytic bacterial strains isolated from grapevine (Vitis vinifera L.).

Table 7 .
Distribution of plasmids (large / small or both) in endophytic-plasmid containing isolates.