The impact of using the Zingiber officinale extract in controlling the endogenous bacterial contamination of date palm during tissue cultures.

— Endogenous bacterial infections are the most critical problem hampers date palm (Phoenix dactylifera) micropropagation cycle. This work aimed to evaluate the influence of the antibacterial property of ginger(Zingiber officinale) extract on suppressing the endogenous bacterial contamination of date palm tissuecultures, to avoid the continuous using of the ordinary chemical antibiotic. The prepared aqueous extract ofZingiberofficinaleat different concentrations (0.5, 1, 2, 4, 6, 8, or 10 ml/l), in comparison toantibiotic, Chloramphenicol or Tetracycline at 100 mg/l, studied onmultiplication media of Gondelah cultivar.The residual effect of all studied treatments also followed up on the rooting media . The results showed that the concentrations of ginger extract at 8 or 10 ml/l (without significant result between) came in the second rank follow the Tetracycline in inhibition the growth of the endogenous bacterial contamination of shoot clusters during the multiplication stage, but subsequently, their residual effect during the rooting stage recorded the highest significant results over the use of antibiotics in preventing bacterial growth in plantlets’ cultures. Ginger extract at 8 or 10 ml/l gave the highest significant results of the vegetative growth of shoot clusters explants on multiplication media, as well as, the highest values of plantlets vegetative growth during the rooting stage. So, using the ginger extract as an organic antibacterial source for controlling the endogenous bacterial problem of the date palmensures that cultures are controlled protective, well vegetative growth, and avoiding the disadvantages of the continuously using of ordinary antibiotics.


INTRODUCTION
Date palm (Phoenix dactylifera L.) is a perennial tree, represents an essential fruit crop around the countries of the Middle East and North Africa. It possesses a great economic importance and has an obvious role in socioeconomic life in these countries. (El Hadrami and Al-Khayri, 2012). Because of all constraints associated with conventional date palm propagation using seeds or offshoots, in vitro culture has become attractive and alternative for mass propagation of commercial cultivars. This technique allows the rapid multiplication of healthy and true-to-type date palm planting materials (Al-Samir et al., 2015).Microbial contamination by fungi or bacteria, is one of the most obstacles hinder the date palm tissue culture process, which can destroy almost cultures in any stage during micro propagation protocols (Odutayo et al., 2007;Abass, 2013). Date palm Bacterial contamination may result from the plant explants materials, the surrounding environment of the laboratory, operators' performance, or inadequate sterilization processes, resulted in reduce growth rate and increase culture mortality .Date palm micro propagated cultures are occasionally highly contaminated with, endogenous bacteria, even well-sterilized cultured explants remain with them these contaminants during the in vitro cycle (Abahmane, 2017). In this respect,   (Thomas et al., 2017).Generally, these endogenous contaminants are not easily eliminated by the action of surface disinfectants, due to their location inside cell tissues. Endophytic bacterial contamination is remaining latent during in vitro growth and they expected to attack suddenly in any stage on plant cultures media (Cassells,2012  Effect of the residual effect of ginger extract on contamination during rooting: -At the end of multiplication stage, elongated plantlets (10 cm) were collected from all previous treatments of the three groups cultures, and transferred to rooting nutrient media which composed of the same components of multiplication nutrient medium but containing in addition of 1 g/L Activated charcoal and supplemented with 1 mg/L (NAA) and 1 mg/L Indole butyric acid (IBA) as plant growth regulators. The cultures were maintained by the regular subculture on fresh medium for tow subcultures (with 8 weeks intervals), in order to follow up the residual effect of the two types of ordinary antibiotics (Chloramphenicol and Tetracycline at 100mg/ L) compared to different concentrations of ginger extract on the appearance of endogenous bacteria and the growth development, during rooting stag. Each treatment presented in 30 jars and each jar contains 3 explants. All groups of cultured jars were incubated under the light condition, with 3000 lux, for 16 (hrs.) and 8(hrs.) dark at 27 ± 2 o C. Data were taken about the appearance degree of endogenous bacteria inthe cheeked cultures jars.The vegetative growth performance For all prepared media pH was adjusted to 5.7± 0.1 before adding agar at 6 g/l and medium dispensed at (40 ml/ jar) into small jars (150 ml) for multiplication media, or into (250 ml/ jars) for rooting media before autoclaving at 121 o C and 15 Ibs/in2 for 20 min. At the end of the experiments, all received healthy full rooted plantlets from all treatments transferred to the acclimatization stage.
Statistical analysis: -The analytical statics was performed in a complete randomized design with three replicates. All obtained results were determined by subjection to statistical analysis of variance, according to the method described by Snedecor and Cochran (1982)using the LSD test at 5%.

III. RESULTS
Obtained data for the endogenous bacterial appearance degree and the vegetative development of explant cultures during multiplication and rooting stages,under different concentrations of ginger extract at (0.5, 1, 2, 4, 6, 8, or 10 ml/L), or two ordinary antibiotics (Chloramphenicol or Tetracycline at 100 mg/ L), incorporated in cultures media of date palm shoot clusters explants, revealed as follows: -Obviously, from data, of Fig (2), untreated shoot cultures media group (control treatment) showed the highest significant value of endogenous bacteria appearance for three subcultures. Shoot cultures media group which treated with Tetracycline antibiotic for three subcultures recorded the lowest significant value for endophytic bacteria appearance. There was no significant difference between using ginger extract at 10 ml or 8 ml for controlling the endogenous bacteria appearance in shoot cultures media, which they ranked significantly after Tetracycline antibiotic treatment for suppressing endophytic bacterial contamination growth. Shoot cultures media which treated with ginger extract at 10 ml showed significantly less degree of endogenous bacteria appearance, to be more antimicrobial activity than using Chloramphenicol antibiotic. Data obtained revealed that by increasing ginger extract concentrations in shoot cultures media the inhibition of endophytic bacteria appearance increase significantly comparing to control treatment.  showed that maximum values of the average of shoot number shoot length, secondary embryo number and the growth vigor degree obtained with ginger extract treatments at 8 ml and 10 ml, without significant differences between them. Where the lowest significant results of the average of shoot number of date palm shoot cultures obtained on control media treatment, culture media supplemented with both of (Chloramphenicol or Tetracycline at 100 mg/ L) antibiotics treatments and culture media supplemented with ginger extract treatment at 0.5 ml treatment, without significant differences among them. Data indicated that the average of shoot length and growth vigor degree of shoot cultures increased significantly by increasing the ginger extract concentration, from 4 ml to 10 ml, comparing to control media treatment and, both of (Chloramphenicol or Tetracycline at 100 mg/ L).The addition of ginger extract at 8ml or 10 ml to shoot cultures media, during multiplication stage, gave a great positive effect on the vegetative growth parameters, compared to the addition of ordinary antibiotics (Chloramphenicol or Tetracycline at 100 mg/ L).   Fig (3) revealed that the highest degree of endogenous bacterial contamination appearance recorded with control treatment. The lowest significant results for endophytic bacteria appearance degree on plantlets cultures achieved when these plantlets cultures developed on multiplication media, whish supplemented with high concentrations of ginger extract at 8 and 10 ml, without significant differences in between. Where the residual effect of using a common antibiotic (Chloramphenicol or Tetracycline) on controlling endophytic bacterial contamination appearance degree during the rooting stage, were ranked significantly in the second order. The residual effect of using Tetracycline treatment was more effective significantly than the residual effect of using Chloramphenicol treatment in suppressing bacterial contamination growth. On the other hand explants, cultures received from multiplication cultures supplemented with a low concentration of ginger extract at 0.5 ml, hadn't inhibited endophytic bacterial contamination as a residual effect during the rooting stage, without significant difference with control treatment result. It seemed to be that, with the increasing of ginger extract concentrations in cultures media during the multiplication stage, endophytic bacterial contamination gradually showed decreasing in bacteria appearance as residual effects during the rooting stage. From data in Table (2) obtained results showed that the highest values of plantlets length and roots number/plantlets during the rooting stage recorded after using ginger extract at 8 ml or 10 ml as an antibacterial agent to control the endophytic appearance, during multiplication stage. Received plantlets from shoot cultures treated with ordinary antibiotic Chloramphenicol, Tetracycline at 100 mg/ L or control treatment gave the lowest results in increasing of plantlets length and root number/plantlets, during the rooting stage as a residual effect. It could be noted that all plantlets received from 8 ml and 10 ml as best treatments for controlling endogenous bacterial contamination of multiplied shoots cultures, could resume their development successfully for further acclimatization stage Fig (4).

IV. DISCUSSION
Endogenous or endophytic bacterial contamination is a serious problem in date palm tissue cultures due to competition for nutrients, the release of toxins and overgrowth of plant tissue which could destroy the whole propagation process. This source of contamination in date palm tissue culture is not extensively studied spot. It could be suggested that these endophytic bacteria can remain undetected due to some conditions which are not optimal for their growth, such as the composition of the culture medium of the nutrient elements concentrations, the carbon source concentration, the pH of the growth culture medium, or the surrounded temperature ( are considered beneficial as a substitution to antibiotics. (Savoia, 2012, Compean, and Ynalvez, 2014).Hydrophobicity is a significant feature, for plant extracts and their ingredients, which qualify them to split the lipids of the bacterial cell membrane and mitochondria, troubling the whole-cell construction, and causing more cell permeability (Taghizadeh et al.,2016). In this regard, antimicrobial property and phytochemical contents of ginger were reviewed by many types of research (Ali et al.,  2008; Sendanayake, 2017).
The obtained results of this study revealed that ginger extract at 10 ml/l showed significantly less value of endophytic bacteria appearance in shoot cultures of date palm cv. Gondelah, than those treated with Chloramphenicol antibiotic. This is on the other side of Abass et al.,(2016) who found thatboth Gentamicin and Chloramphenicol showed, high defense performance for date palm callus culture. However, Sabra et al., (2014)mentioned that antimicrobial activity of ginger rhizomes extract, versus both bacterial isolates of Grampositive and Gram-negative, refers that chemical components of extract may responsible for a disturbance of the membrane and/or genetic structure of the isolates. The antimicrobial activity of ginger may be owing to the considerable quantity of phenols as gingerol paradol, shogaols and zingerone (Rahmani et al., 2014).Beside also the existent of flavonoids in ginger, which found to be affective as in vitro antimicrobial substances, against an ample range of microorganisms (Fullerton et al.,2011).At this point, Dina and Hussein (2017) reported that, highest amount of total phenolic and flavonoids were detected in extract of both ginger and cinnamon. This was confirmed by Miklasińska-Majdanik(2018) who reported that,the increase in the number of hydroxyl groups presented on the phenolic ring, leading tohydroxylation property of plant extracts, that resulted in the increasing in the antimicrobial activity. These findings may explain the antimicrobial activity of ginger extract.
From our results, it could be noted that shoot cultures group treated with Tetracycline antibiotic at 100 mg/L for three subcultures recorded the lowest significant value for endophytic bacteria appearance during multiplication stage, but less and weak vegetative growth were observed. This is confirmed by Khafagi et al., (2001)who reported that antibiotics are currently used but are not always effective, can alter plant growth, and are costly, and resistant strains can result in extensive use. However, it has to be taken into consideration the phytotoxicity of some antibiotics(Oliveira and Scherwinski-Pereira, 2016).
Thus antibiotics should be restricted to very limited culture stages. Prolonged exposure to Tetracycline or Chloramphenicol at 100 mg/ L adversely affected shoot formation. The same result was obtained with shoots of Pelargonium cultures grown in the presence of cefotaxime antibiotic for long periodshowed a low rate of shoot formation, shorter and pale, shoots, finally caused the end of cultures by death (Wojtania et al., 2005).Growth value and plant shape followed up to check whether using antibiotics or ginger extracts, led to phytotoxicity on the later plantlet's growth. From resultsit could be observed that the residual effect of using Tetracycline or Chloramphenicol antibiotics treatment on controlling endophytic bacterial contamination appearance degree during rooting stage, came at the second rank significantly after using ginger treatment at 8 ml or 10 ml, which showed superiority in suppressing the endophytic bacterial growth during rooting stage as residual effect for being used as antibacterial agent during multiplication stage. This was contrary to Cassells (2012),who reported that bacterial growth is only repressed by antimicrobial curing and when chemical compounds are stopped to using, the bacteria colonies recapture their growth. In general ginger extract addition at 8 ml or 10 ml to date palm cultures during multiplication stage and also their residual effect during the rooting stage showed well enhancement for vegetative growth and plantlets development, this may be due to the nutrition value of Ginger rhizome contents of fats, carbohydrates, vitamins, minerals, proteins, vitamins and minerals as sodium and calcium as reported by (

V. CONCLUSION
Under the light of the presented study, the antibacterial potential of ginger aqueous extract was investigated as a primary study. Satisfying results obtained from studied concentrations at 8ml/L and 10 ml/L to eliminate endophytic bacterial contamination attack during the multiplication stage and their residual effects were resumed during the rooting stage. Moreover, well development and good vegetative growth of received plantlets were obtained. These results can be a diver to limit the repeated use of antibiotics. Therefore, more studies with ginger extract antibacterial potential can be carried out. Also, studies of using other plant extracts and phytochemicals sources of defined antimicrobial properties would have a great significance in date palm tissue culture, to eliminate or control microbial contamination and to restrict using antibiotics. Characterize and identify of date palmassociated bacteria which lead to more successful antibacterial therapies, intensive studies in this concern are needed. The lack of descriptive information and antibiotic susceptibilities of a large number of date palm endophytic