Impaginato 525 Adv. Hort. Sci., 2018 32(4): 525-534 DOI: 10.13128/ahs-22424 Diversity of plant growth promoting Rhizobacteria of Rhus tripartitus in arid soil of Algeria (Ahaggar) and their physio- logical properties under abiotic stresses A. Benaissa 1, 3 (*), R. Djebbar (1), A. Abderrahmani (2) 1 Laboratory of Plant Physiology, Department of Biology and Physiology of Organisms, Faculty of Biological Sciences, University of Sciences and Technologies of Houari Boumediene - El-Alia, BP 16011 Bab Ezzouar, Algiers, Algeria. 2 Laboratory of Microbiology, Department of Cellular and Molecular Biology, Faculty of Biological Sciences, University of Sciences and Technologies of Houari Boumediene - El-Alia, BP 16011 Bab Ezzouar, Algiers, Algeria. 3 Laboratory of Sciences and Environment Research, Universitary Center of Amine Elokkal ElHadj Moussa Eg. Akhamoukh, 11039 Sersouf, Tamanrasset, Algeria. Key words: abiotic stresses, Ahaggar, plant growth promoting Rhizobacteria, Rhus tripartitus. Abstract: Plant Growth Promoting Rhizobacteria (PGPR) associated with Ucria (Rhus tripartitus) represents a good alternative for including this crop in reveg- etation programs in arid area. In this study, 137 bacterial strains were isolated in Tryptic Soy Agar medium (TSA) from six samples of ucria’s rhizospheric soil (Ahaggar, Algeria), based on colony characteristics, Gram © reaction, oxidase and catalase tests. To evaluate their PGP activities and their physiological char- acteristics under stress environment, ten tests were made. Sixty strains of 16 genera were selected for their PGPR abilities, which represent 43.79% to the total of rhizobacteria isolated. The maximum bacterial population were Bacillus (35%). 71.66% of isolates were able to solubilize the phosphate, 31.66% were able to produce Indole Acid Acetic (IAA), 58.33% were siderophore producers, 28,33% were able to produce Cyanhydric Acid (HCN) and 70% were able to grow without any source of nitrogen. Indeed, PGPR strains have shown toler- ance and/or resistance to several experimental environmental conditions. As a conclusion, the PGPR strains of Ucria’s rhizosphere were shown a good poten- tial for biofertilization and biocontrol of crops, and their tolerance to abiotic stresses is an interesting step to support their utilities. 1. Introduction Rhus tripartitus (called Ucria, African sumac and Tahounek in tama- haq) is an important medicinal plant belonging to the Anacardiaceae fam- (*) Corresponding author: benaissa.asmaa@yahoo.fr Citation: BENAISSA A., DJEBBAR R., ABDERRHMANI A., 2018 - Diversity of plant growth promoting Rhizobacteria of Rhus tripartitus in arid soil of Algeria (Ahaggar) and their physiological proper- ties under abiotics stresses. - Adv. Hort. Sci., 32(4): 525-534 Copyright: © 2018 Benaissa A., Djebbar R., Abderrhmani A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 19 December 2018 Accepted for publication 27 June 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(4): 525-534 526 ily. This species often grows in areas of marginal agri- cultural capacity. It encountered in Algeria, in arid areas especially in the mountains of the Ahaggar where the indigenous people (Touaregs) use it to treat gastric disorders (Chermat and Gharzouli, 2015). This plant is also found in the North-Eastern part of Saudi Arabia and in Tunisia where it spreads in the center to the far southern part of the country. It is used in the Arabian traditional medicine for cen- turies to treat cardiovascular and gastrointestinal dis- orders and inflammatory conditions (Chetoui et al., 2013; Shahat et al., 2016). The study of rhizosphere bacteria from medicinal plants is very important, as they are well known to have impact on plant growth and also produce indus- trially important metabolites and improve quality of medicinal products (Bafana and Lohiya, 2013). Considerable numbers of studies were focused on the beneficial effects of bacterial species that colo- nize the rhizosphere of many plant species and proved their beneficial effects on plant growth, yield, and productivity as well as their role in the reduction of their susceptibility to diseases caused by phy- topathogenic bacteria, fungi, viruses and nematodes and even against abiotic stresses. These bacteria h a v e b e e n c a l l e d « P l a n t G r o w t h P r o m o t i n g Rhizobacteria» (Kloepper et al., 2004; Orhan et al., 2006; Miransari, 2014; Nadeem et al., 2014; Gupta et al., 2015). These PGPR’s can enhance the plant growth by direct mechanisms such as the fixation of atmospheric nitrogen, the solubilization of minerals l i k e p h o s p h o r u s a n d i r o n , t h e p r o d u c t i o n o f siderophores and enzymes, the synthesis of phyto- hormones like the auxin, indole-3-acetic acid (IAA), cytokinins and gibberellins, their role in lowering of ethylene levels and the induction of systemic resis- tance. Indirect mechanisms are used by PGPR to ben- efit the plant growth by the induction of the disease resistance by producing antibiotics or hydrogen cyanide, competition for nutrients, extracellular enzymes production and others (Glick, 1995; Vessey, 2003; Adesemoye et al., 2009; Saharan and Nehra, 2011; Saha et al., 2016). To the best of our knowledge, there are no stud- ies conducted on the rhizospheric bacteria associated with Rhus tripartitus, a medicinal plant that grows in Ahaggar (Algeria). We hypothesize, that this plant harbors a diverse group of rhizospheric bacteria that can help Ucria to cope with harsh environmental con- ditions. So, the main objectives of this study were to characterize the isolated rhizobacteria associated with the rhizosphere of Rhus tripartitus, their pro- preties as plant growth promoting bacteria and their capacities to tolerate abiotics stresses. 2. Materials and Methods Sample collection, isolation and characterization of rhizobacteria Six soil samples are taken from the rhizosphere of t h e w i l d u c r i a s h r u b s f r o m I l a m a n r e g i o n i n Tamanrasset, an arid area, which is located in the National Culturel Parc of Ahaggar in the south of Algeria (22° 49’ 59’’ north, 5° 19’ 59’’ east) during March, 2017. Each soil sample was collected at a depth of 15 cm, around the root and placed in a ster- ile container. The samples collection was transported to the laboratory in an ice box set at 4°C. Tenfold serial dilution of the samples was made by mixing the soil with sterile water, and plating on a Tryptic Soy Agar medium (TSA). The plates were inverted and incubated, at 30°C for five days. The maximum of bacterial colonies present on plates were purified and characterized. The Gram reaction, oxidase reaction and catalase test were performed as per standard procedure. In vitro screening and identification of Plant Growth Promoting Rhizobacteria The collected rhizobacterial isolates associated with Rhus tripartitus were tested for their capacities to produce plant growth promoting effects. The PGPR were identified by using API galeries E20, NE20, C H B , S t a p h a n d N H ( A P I , b i o M e r i e u x s a , L y o n , France). All the strains were preserved in nutrient broth added with 20% glycerol at -80°C. Nitrogen fixation. The fixation of molecular nitro- gen is tested on a free nitrogen medium. The bacteri- al isolates are inoculated on the plates and incubated at 25°C for 24-48 h. The growth on this medium after being transferred ten times in the same medium r e f l e c t s t h e a b i l i t y o f b a c t e r i a t o f i x n i t r o g e n (Haahtela et al., 1983). Production of HCN. The strains ability to produce Hydro Cyanic Acid (HCN) is carried out according to the method of Lorck (1948) on solid bennett agar amended with 4.4 g/l glycine is inoculated with a loop of the bacterial culture. 90 mm Whatman paper are dipped in sodium picrate solution (0.5% picric acid and 2% sodium carbonate) for one minute and then placed underneath the Petri plates lids. The plated were sealed with parafilm and incubated at 30°C for four days. The appearance of an orange to Benaissa et al. - PGPR of Rhus tripartitus 527 red color indicates the production of HCN. Solubilization of phosphates. Qualitative phos- phate solubilization activity was tested on NBRIP (National Botanical Research Institutes Phosphate) medium by applying a spot of 20 μl of bacterial sus- pension on the surface of the agar and incubated at 30°C for 15 days (Nautiyal, 1999). A clear halo zone around the colony is an indication of phosphate solu- bilization. The calculation of the solubilization index (S.I.) is carried out according to the formula devel- oped by Kumar and Narula (1999): S.I. = Diameter of the halo around the spot/Diameter of the spot Production of siderophores. The production of siderophores on solid medium is carried out qualita- tively on Chrome Azurol S (CAS) medium as described by Schwyn and Neilands (1987). The CAS plates were prepared and divided into Sectors and inoculated with bacterial culture spots (10 μl of 106 CFU/ml) and incubated at 25±2°C for 48-72 h. The development of an orange yellow halo around the colony was consid- ered positive for the siderophore production. The change in color is due to the transfer of ferric ions from the CAS to the siderophores. The calculation of the ratio (halo diameter/diameter of the bacterial colony) makes it possible to compare production dif- ferences between bacterial strains. Production of indole acetic acid (IAA). The produc- tion of IAA was determined according to the method of Holt et al. (1994). The principle is to inoculate the selected strains on the nutrient broth containing 0.1 g/l of L-tryptophan. The change of the solution color from yellow to pink or red when we add the reagent of Salkowski (50 ml, 35% perchloric acid; 1 ml 0.5 FeCl3) is an indication of positive result. Screening of PGPR isolates for stress tolerance Influence of salinity. To evaluate the strains ability of these strains to grow in salinity levels, different concentrations of NaCl (1%, 5% and 8%). The salt were added to TSA medium in the liquid stage (infu- sion at 40°C) and deposited on the magnetic stirrer to dilute it. Influence of pH and temperature. In order to test the ability of the bacteria to grow in alkaline and/or acidic environment, two media were prepared with the addition to 250 ml of TSA of 3 g of solid NaOH 6.4 M and 1 ml of KCl 12 N to obtain a pH of 8.8 and 6 respectively. For studying the effect of incubation temperature on growth of the isolates, the bacterial cultures were grown on TSA medium and incubated at a variables temperatures (10, 20, 30, 40 and 50°C). Tolerance of heavy metal. The sensitivity of the selected strains to Copper (Cu), Zinc (Zn), Bromine (Br), Cyanide (Cn), Fluorine (F) and Silicon (Si) were tested. For this test, six TSA media of 250 ml were prepared with respectively three grams of solid KBr, CaF2, SiO2 and one gram of K3[Fe(Cn)6], ZnSO4.7H2O and CuSO4.5H2O. Any growth on this media reflects the ability of strains to tolerate the heavy metal toxi- city. Antibiotics resistance. The selected strains were tested for their susceptibility to antibiotics on Muller- Hinton medium. It consists of bringing the germ into contact with disks of blotting paper impregnated with a given antibiotic at concentrations determined by the standardization of the antibiogram according to the Clinical and Laboratory Standards Institute (CLSI), to determine the sensibility of this germ to antibiotics which allows classifying it in the category: R (resistant), S (sensible) or I (intermediate). Ten dif- ferent antibiotics were used: Fosfomycin (FOS) 50 µg, Rifampin (RA) 5 µg, Nalidixicid (NA) 30 µg, Spiramycin (SP)100 µg, 30 µg, Novobiocin (NV) 30 µg, Teicoplycin (TEI) 30 µg, Kanamycin (K) 30 µg and Erythromycin (E) 15 µg (all from Sigma Chemical Co., St. Louis, Mo.). 3. Results In this study, we focused on the diversity of bacte- rial community of ucria’s rhizosphere and the evalua- tion of their plant growth promoting abilities, also under abiotic stresses. Isolation and characterization of rhizobacteria One hundred and thirty seven (137) culturable bacteria were isolated from the rhizosphere of six healthy ucria plants using TSA medium. The rhizobac- teria isolates showed a diversity of phenotypic and cultural characteristics of their colonies. Infact, 54.19% of the isolated rhizobacteria were Gram neg- ative. 100% of strains showed positive test to cata- lase and variability to oxidase reaction (67.88% nega- tive). Screening and identification of PGPR Biochimical characterization of PGPR isolates. The isolates were grouped into 16 genera of Bacillus, Adv. Hort. Sci., 2018 32(4): 525-534 528 Pseudomonas, Ewingella, Staphylococcus, Alcaligenes, Micrococcus, Kocuria, Chryseomonas, Chryseobacterium, Cedecea, Shigella, Yersinia, Providencia, Acinetobacter, Haemophilus and Aeromonas based on cultural, morphological and bio- chemical characteristics. The maximum and minimum populations were Bacillus (35%), and Haemophilus and Aeromonas with a percentage of 1.66% each (Table 1). Table 1 - Functional diversity of PGPR strains isolated from ucria’s rhizosphere Sample No Division/strains Functions 1 Gamma Proteobacteria Cedecea lapagei Solubilization of phosphate, production of HCN and siderophores Chryseomonas luteola Solubilization of phosphate, production of siderophores and nitrogen fixation Chryseomonas luteola Solubilization of phosphate, production siderophores and nitrogen fixation Firmicutes Bacillus megaterium Solubilization of phosphate, production of siderophores and nitrogen fixation Bacillus megaterium Solubilization of phosphate, production of AIA and siderophores and nitrogen fixation Bacillus circulans Solubilization of phosphate, production of AIA and siderophores, 2 Gamma Proteobacteria Ewingella americana Solubilization of phosphate, production of AIA and siderophores Chryseomonas luteola Solubilization of phosphate, production of AIA and siderophores and nitrogen fixation Cedecea lapagei Solubilization of phosphate, production of siderophores and nitrogen fixation Cedecea lapagei Solubilization of phosphate, production of AIA and siderophores and nitrogen fixation Firmicutes Bacillus licheniformis Solubilization of phosphate, production of AIA and siderophores Bacillus subtilis Solubilization of phosphate, production of siderophores and nitrogen fixation Bacillus licheniformis Solubilization of phosphate, production of AIA and siderophores and nitrogen fixation. Bacillus licheniformis Production of AIA and siderophores Actinobacteria Kocuria varians Production of AIA 3 Gamma Proteobacteria Chryseomonas luteola Solubilization of phosphate, production of AIA and siderophores and nitrogen fixation Providencia rattgeri Production of AIA Ewingella americana Production of siderophores Firmicutes Bacillus subtilis Production of HCN and fixation of azote Bacillus non reactiv Production of siderophores and fixation of azote Bacillus licheniformis Production of HCN and fixation of azote 4 Gamma Proteobacteria Yersinia pestis Solubilizationof phosphate, production of siderophores and nitrogen fixation Haemophilus aphrophilus Solubilization of phosphate, production of AIA, and nitrogen fixation Aeromonas salmonicida Solubilization of phosphate, production of HCN and siderophores Pseudomonas aeruginosa Solubilization of phosphate, production of HCN and nitrogen fixation Cedecea lapagei Solubilization of phosphate, production of siderophores and nitrogen fixation Acinetobacter colcoaticus Solubilization of phosphate and production of AIA, Acinetobacter baumanii Solubilization of phosphate, production of AIA and nitrogen fixation Chryseomonas luteola Solubilization of phosphate, production of HCN and siderophores. Acinetobacter baumanii Solubilization of phosphate and nitrogen fixation Firmicutes Staphylococcus lentus Solubilization of phosphate and production of AIA. Staphylococcus lentus Solubilization of phosphate, production of siderophores and nitrogen fixation Actinobacteria Micrococcus ssp Solubilization of phosphate, production of siderophores and nitrogen fixation Béta Proteobacteria Alcaligenes faecalis Production of AIA and nitrogen fixation 5 Gamma Proteobacteria Chryseomonas luteola Production of siderophores and nitrogen fixation Chryseomonas luteola Solubilization of phosphate, production of AIA and siderophores and nitrogen fixation Chryseomonas luteola Solubilization of phosphate, production of HCN and AIA. To be continued Benaissa et al. - PGPR of Rhus tripartitus 529 Plant growth promoting traits.The isolated rhi- zobacteria were screened for various PGP features responsible for plant growth promotion. The PGPR isolates represent 43.79% of the total of the rhizos- p h e r i c b a c t e r i a . T h e y b e l o n g t o f o u r divisions (Gamma-Proteobacteria, Firmicutes, Actino-bacteria and Béta-Proteobacteria). The strains mostly belonging to the Gamma-Proteobacteria, most of them affiliated to the Enterobacteriaceae. According to the figure 1, species of Gamma Proteobacteria division were pre- dominant with 45% among the isolats of PGPR of Ucria behind firmicutes with 38.33%. Infact, the species Cedecea lapagei, Chryseomonas luteola and Ewingella americana were the most representative PGPR of Gammaproteobacteria division. The species Chryseomonas luteola found in five samples of six rhi- zospheric soils (Table 1). Different combinations of PGP effects have been found, 18.33% strains have been able to produce up to 4 PGP effects against 40% were able to produce 3 PGP effects, 25% showed 2 traits of PGP and 16.66% were able to produce only one PGP effect. In fact, the genera Bacillus, Ewingella, Alcaligenes, The Rhizobacteria were identified through six samples and five Plant Growth Promoting treatments: solubilization of phosphate, produc- tion of Cyanhydric Acid, production of Indol-Acid-Acetic, fixation of nitrogen and production of siderophores. Fig. 1 - Classification of rhizospheric bacteria of Ucria on the PGP traits. The main bacteria divisions of plant growth pro- moting rhizobacteria from the rhizosphere of ucria. Sample No Division/strains Functions 5 Firmicutes Bacillus circulans Solubilization of phosphate, production of HCN and siderophores and nitrogen fixation Bacillus circulans Solubilization of phosphate, production of siderophores and nitrogen fixation Bacillus licheniformis Solubilization of phosphate, production of siderophores and nitrogen fixation Bacillus subtilis Solubilization of phosphate, production of siderophores and nitrogen fixation Bacillus licheniformis Solubilization of phosphate, production of HCN and siderophores and nitrogen fixation Bacillus licheniformis Solubilization of phosphate and nitrogen fixation Bacillus circulans Production of HCN Bacteroidetes Chryseobacterium meningosepticum Nitrogen fixation Actinobacteria Kocuria varians Nitrogen fixation Micrococcus ssp Solubilization of phosphate, production of HCN and siderophores and nitrogen fixation 6 Gamma Proteobacteria Ewingella americana Production of AIA and nitrogen fixation Shigella spp Solubilization of phosphate and nitrogen fixation Pseudomonas aeruginosa Solubilization of phosphate, production of siderophores and nitrogen fixation Ewingella americana Solubilization of phosphate, production of siderophores and HCN and nitrogen fixation Ewingella americana Solubilization of phosphate and nitrogen fixation Providencia rattgeri Production of AIA Firmicutes Bacillus subtilis Solubilization of phosphate and nitrogen fixation Bacillus licheniformis Solubilizationof phosphate, production of HCN and siderophores and nitrogen fixation Bacillus subtilis Solubilization of phosphate, production of HCN and nitrogen fixation Bacillus non reactiv Nitrogen fixation Actinobacteria Micrococcus ssp Production of HCN and siderophores and nitrogen fixation Béta Proteobacteria Alcaligenes faecalis Solubilization of phosphatase, production of HCN and siderophores Alcaligenes faecalis Production of HCN Table 1 - Functional diversity of PGPR strains isolated from ucria’s rhizosphere continued Chryseomonas and Cedecea have showed positive screening for all the PGP traits, according to the Table 2. Screening and assessment of phosphate solubliz- ers and IAA producers. Among the total isolates screened for phosphate solublization, 71.66% were able to solubilize inorganic phosphate (Fig. 2) and were identified as potential phosphate solubilizing 530 Adv. Hort. Sci., 2018 32(4): 525-534 bacteria that showed a clear halo zone around the colonies on NBRIP’s agar plates amended with bro- mophenol blue. 20 isolates out of total rhizospheric flora had the capacity to produce were IAA in the presence of L-Tryptophane. Molecular nitrogen fixation. The ability of the iso- lates to grow on N-free medium indicated positive results for nitrogen fixation. In fact 70% of the iso- lates were able to grow even being transferred ten times in this medium which indicated their capacity to fix molecular nitrogen (Fig. 2). Siderophore and HCN production. Siderophore production was registered at 58.33% of the strains based on the appearance of a halo zone of yellow orange color around the colony inoculated on CAS- agar plates. Seventeen isolates were positive for HCN production (Fig. 2). Physiological properties of PGPR isolates under abiotic stresses. The isolated bacteria were tested for their ability to tolerate abnormal growth conditions after incubation in a wide range of salt, pH and tem- perature stress condition, heavy metal toxicity and antibiotics. Influence of salinity. According to the figure 3, the most percentages of strains which can grow in the 1% and 5% salinity are respectively 96.66% and 81.66%. In addition, only 16.66% of strains can toler- ate % of salinity. Influence of pH and temperature. The rhizobacter- ial isolates seems to tolerate the alkalinity better than the acidity with 66.66% and 46.66% respective- ly. However, these isolates could grow up to higher temperature of 50°C with 85%, which means that the temperature hadn’t a remarkable effect on bacterial growth (Fig. 3). Tolerance to heavy metal. Depending on the metal tested and the species, the tolerance to heavy metals is different. In fact, we recorded a very good tolerance to silicon and bromine with respectively 80% and 78.33%. 55% of our strains have well toler- ated fluoride followed by 30% for cyanide. The low- est percentages recorded were for copper and zinc with 13.33% and 3.33%, respectively. These results showed that heavy metals affected the bacterial growth (Fig. 4). Table 2 - Plant growth promotion activities of rhizobacteria gen- era isolated from ucria’s rhizosphere Genera N2 Fe 2 HCN AIA PO2 Bacillus + + + + + Pseudomonas + + + - + Ewingella + + + + + Staphylococcus + + - + + Alcaligenes + + + + + Micrococcus + + + - + Kocuria + - - + - Chryseomonas + + + + + Chryseobacterium + - - - - Cedecea + + + + + Shigella + - - - + Yersinia + + - - + Providencia - - - + - Acinetobacter + - - + + Haemophilus + - - + + Aeromonas - + + - + N2 = nitrogen fixation, Fe2= siderophore production, HCN produc- tion, AIA and PO= phosphate solubilisation. Fig. 2 - Siderophores production, phosphate solubilization, fixa- tion of nitrogen, HCN and AIA productions of the Rhizobacteria isolates of ucria (Ahaggar). Fig. 3 - Estimation of tolerance of PGPR strains to alkalinity, aci- dity, salinity and temperature. Fig. 4 - Estimation of tolerance of PGPR strains to heavy metals toxicity. Br= Bromine, Zn= Zinc, Si= Silicon, F= Fluorine, Cn= Cyanide, Cu= Copper. 531 Benaissa et al. - PGPR of Rhus tripartitus Antibiotics resistance. The collection of PGPR iso- lates was tested for susceptibility to 10 antimicro- bials. High frequency of resistance was observed for Métronidazole (MT) followed by Amoxyclav (AMC) with respectively 93.33% and 91.66% of strains. The minimum resistance was recorded for Fosfomycin (FOS) with 16.66% (Fig. 5). 4. Discussion and Conclusions The diversity of native bacterial species in the southern Algerian soils remains largely unknown, especially for the rhizosphere of Rhus tripartitus. Most of the rhizobacteria isolated from ucria’s rhizos- pheric soils have been dominated by Gram-negative (54.19%) and all were catalase positif. These results are in-line with studies realized on rhizosphere of Brassica campestris (Poonguzhali et al., 2006) and Eragrostis tef Zucc. Trotter (Woyessa and Assefa, 2011). Plant growth promoting rhizobacteria (PGPR) rep- resent a diverse range of soil bacteria that stimulate the growth of their host when grown in association. Such rhizosphere microbes benefit by utilization of metabolites secreted by plant roots as a nutrient for their growth and promote plant growth through more than one mechanism, including production of phytohormones and biocontrol of plant pathogens (Rana et al., 2011). This present study showed a high diversity among the isolates of these species under different combi- nation of PGP effects. The most often genera identi- fied was Bacillus (35%) with a high diversity of species, such as Bacillus megaterium, B. licheni- formis, B. subtilis, B. circulans and B. non-reactiv. PGPR of the genera Bacillus have been reported in many studies (Trivedi and Pandey 2008; Zou et al., 2010; Liang et al., 2011; Woyessa and Assefa, 2011; Nadeem et al., 2012; Mishra et al., 2014; Susilowati et al., 2015). The second PGPR group found through- o u t t h i s s t u d y w a s b e l o n g i n g t o s p e c i e s Chryseomonas luteola (11.6%) and Ewingella ameri- cana (8.3%). The latter was able to increase the growth of pipper and spinach (Hou and Oluranti, 2 0 1 3 ) . T h e t h i r d a n d l a s t P G P R g r o u p ( 4 5 . 1 % ) b e l o n g e d t o t h e g e n e r a C e d e c e a , P r o v i d e n c i a , Yersinia, Heomophilus, Aeromonas, Acinetobacter, Micrococcus, Alcaligenes, chryseobacterium, Shigella, Staphylococcus and Pseudomonas. Earliers studies showed that Pseudomonas PGPR is a producer of HCN (Castric, 1975), improving the availability of nec- essary nutrient (Islam et al., 2014) and an agent of biocontrol (Weller and Thomashow, 1993). The gen- era Acinetobacter promotes production of wheat, pea, chickpea, maize and barley through nitrogen fix- ation, siderophore production and mineral solubiliza- tion (Gulati et al., 2009; Sachdev et al., 2010). In con- strat, Cedecea have never shown in the litterature as PGPR potential and molecular analysis is necessary to confirm the identification. The industrialization of chemical fertilizers such phosphate and nitrogen has increased in the agricul- tural sector. These minerals are considered impor- tant limiting factors for many crops (Ahmad et al., 2008). However, the aim of this study is also to evalu- ate the ability of rhizobacteria to promote the biodisponibility of N and P, in order to reduce of industrial fertilizers. In fact, most of ucria’s rhizobac- teria could fix atmospheric nitrogen (70%) and 71.66% were able to solubilize inorganic phosphate in NBRIP’s medium. Similary, many studies shown that phosphorus-solubilizing microorganisms are ubiquitous in soils (Chandra et al., 2007; Tsavkelova et al., 2007; Banerjee et al., 2010). PGPR can produce auxin-like compounds that increase the development of root system thus improving nutrient uptake by plants (Voisard et al., 1989); However, out of total isolated rhizobacteria, 31.66% exhibited as producers of IAA in medium supplemented with l-tryptophan. The number of PGPR producers of HCN represent 28.33% of total isolates strains. These PGP traits have the capacity to enhance indirectly plant growth and protect them from phytopathogens (Lugtenberg and Dekker, 1999; Shahat et al., 2016). Rhizobacteria pro- ducing siderophores are of great importance for the p l a n t b e c a u s e t h e y m a k e b i o a v a i b l e i r o n . T h e siderophores decreases the metal bond and forma- tion of free radicals in the roots zone, which prevent- ed the degradation of IAA (Yang et al., 2009). Among the isolates of PGPR obtained in this study, 58.33% Fig. 5 - Estimation of resistance of PGPR strains to antibiotics. Adv. Hort. Sci., 2018 32(4): 525-534 532 are producers of siderophores. The production of siderophores in the rhizosphere increases the bacter- ial competition as well the root colonization (Abrol et al., 1988). The rhizosphere is characterized by large environ- mental fluctuations, which may promote high diversi- ty in the rhizosphere microbial community by main- taining high niche diversity. Thus, microbial commu- nity diversity may be important especially in extreme condition, like high temperature, salinity or pH changes. Under stress conditions, bacterial rhizos- phere may promote the plant growth (Cheikh and Jones, 1994). In the present research, these PGPR isolates were traited under several environmental conditions. They have showed a good potential of tolerance for the previuosly conditions, which is a value, added to their beneficial effect. Therefore, the aim of this work is not only to deal with the diversity of PGPR but also the selection of resistant strains at the most extreme conditions. The ucria’s rhizobacteria, were isolated from an arid area of Ahaggar (Algeria) and salinity is a natural feature of ecosystems in arid and semi-arid regions (Curl and Truelove, 1986). The 81.66% of PGPR stud- ied exhibited as tolerant to the presence of 5% NaCl and 85% could growth until 50°C. The variation on temperature is an important factor that can affect the hormonal balance of the plant (Lovley, 1995). Then, certain beneficial microorganisms can influ- ence plants response to abiotic stresses like drought a n d h i g h t e m p e r a t u r e ( G r o v e r e t a l . , 2 0 1 1 ) . Therefore, the necessity of discovering species able to grow under salt stress conditions and in a high temperature, are important to include them in reveg- etation system in arid area. The pH is one the obvi- ous influencing factors of microbial activity and pop- ulations in soil (Woyessa and Assefa, 2011). Most of these isolates (66.66%) can growth over alkaline pH and more than 46% of growth recorded in acidity condition, it suggests that there is a good potential to inoculate them over a range of wide pH. The growth o f r h i z o b a c t e r i a a t a c i d i c p H v a l u e s c o u l d b e explained by their adaptation at arid soil. Effectively, low rainfall can probably cause an increase in acidity of soil. On the other hand, an acidic environment of roots due to CO2 and organic acid can be included in soil acidity (Gururani et al., 2012). Moreover, the PGPR isolates of this work, demon- strated a good tolerance in-vitro for heavy metals toxicity, with an average of 43.33% for chemicals forms of six metals. The accumulation in soil of heavy metals can perturbate the growth and the diversity of bacterial communities. Many studies are shown the potential of application of PGPR in resistance and uptake of heavy metal by certain plants (Lovley, 1995; Yang et al., 2009; Gururani et al., 2012). The ability to colonize roots and antibiotic resistance are other parameters needed to detect effective PGPR strains (Siddiqui, 2005). Effectively, the study of the sensitivity of rhizobacteria to antibiotics adds PGP potential. Indeed, our strains have showed some resistance to the majority of antibiotics tested which can be involve high microbial competition in the rhi- zosphere. This resistance increased the chances of survival and colonization of the rhizospheric soil. The present study reflects the preliminary work done on the rhizosphere of Rhus tripartitus in arid soils of Algeria. Indeed, the selection of PGPR strains, which can effectively grow under abiotics stresses conditions, can be used as promising biofertilizers and biocontroling of plants and useful in revegeta- tion system of arid area. Before that, others investi- gations should be done like a bioassay in vitro and in vivo of these strains on crops according to the inocu- lation treatments. The study of mechanisms of toler- ation to toxic substances or hard environment is an interesting step to support their utilities. References ABROL I.P., YADAV J.S.P., MASSOUD F.L., 1988 - Salt affect- ed soils and their management. - Food and Agriculture Organization (FAO), UN, Soils Bulletin, Rome, Italy, pp. 39. ADESEMOYE A.O., TORBERT H.A., KLOEPPER J.W., 2009 - Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. - Microbiol. Ecol., 58(4): 921-929. AHMAD F., AHMAD I., KHAN M.S., 2008 - Screening of free- living rhizospheric bacteria for their multiple plant growth promoting activities. - Microbiol. Res., 163(2): 173-181. BAFANA A., LOHIYA R., 2013 - Diversity and metabolic potential of culturable root-associated bacteria from Origanum vulgare in sub-Himalayan region. - World. J. Microbiol. Biotechn., 29: 63-74. BANERJEE S., PALIT R., SENGUPTA C., STANDING D., 2010 - S t r e s s i n d u c e d p h o s p h a t e s o l u b i l i z a t i o n b y Arthrobacter sp. and Bacillus sp. isolated from tomato rhizosphere. - Austral. J. Crop. Sci., 4(6): 378. CASTRIC P.A., 1975 - Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa. - Can. J. Microbiol., 21(5): 613-618. CHANDRA S., CHOURE K., DUBEY R.C., MAHESHWARI D.K., 2007 - Rhizosphere competent Mesorhizobium loti Benaissa et al. - PGPR of Rhus tripartitus 533 MP6 induces root hair curling inhibits Sclerotinia sclero- t i o r u m a n d e n h a n c e s g r o w t h o f I n d i a n m u s t a r d (Brassica campestris). - Braz. J. Microbiol., 38(1): 124- 130. CHEIKH N., JONES R.J., 1994 - Disruption of maize kernel g r o w t h a n d d e v e l o p m e n t b y h e a t s t r e s s ( r o l e o f cytokinin/abscisic acid balance). - Plant Physiol., 106: 45-51. CHERMAT S., GHARZOULI R., 2015 - Ethnobotanical study of medicinal flora in the North East of Algeria - An empirical knowledge in Djebel Zdimm (Setif). - J. Mat. Sci. Eng., 5 (1-2): 50-59. CHETOUI I., MESSAOUD C., BOUSSAID M., ZAOUALI Y., 2013 - Antioxidant activity, total phenolic and flavonoid content variation among Tunisian natural populations of Rhus tripartita (Ucria) Grande and Rhus pentaphylla Desf. - Ind. Crop. Prod., 51: 171-177. CURL E.A., TRUELOVE B., 1986 - Microbial interactions, pp. 140-166. - In: CURL E.A., and B. TRUELOVE (eds.) The Rhizosphere. Springer-Verlag, Berlin, Germany, pp. 288. GLICK B.R., 1995 - The enhancement of plant growth by free-living bacteria. - Can. J. Microbiol., 41(2): 109-117. G R O V E R M . , A L I S . Z . , S A N D H Y A V . , R A S U L A . , VENKATESWARLU B., 2011 - Role of microorganisms in adaptation of agriculture crops to abiotic stresses. - World J. Microbiol. Biotech., 27(5): 1231-1240. GULATI A.P., VYAS P.R., KASANA R.C., 2009 - Plant growth promoting and rhizosphere-competent Acinetobacter rhizosphaerae strain BIHB 723 from the cold deserts of the Himalayas. - Curr. Microbiol., 58: 371-377. GUPTA G., PARIHAR S.S., AHIRWAR N.K., SNEHI S.K., SINGH V., 2015 - Plant growth promoting rhizobacteria (PGPR): current and future prospects for development of sustainable agriculture. - J. Microbiol. Biochem. Technol., 7(2): 96-102. G U R U R A N I M . A . , U P A D H Y A Y A C . P . , B A S K A R V . , VENKATESH J., NOOKARAJU A., PARK S.W., 2012 - Plant growth-promoting rhizobacteria enhance abiotic stress tolerance in Solanum tuberosum through inducing changes in the expression of ROS-scavenging enzymes and improved photosynthetic performance. - J. Pl. Grow. Regul., 32(2): 245-258. HAAHTELA K., HELANDER I., NURMIAHA-LASSILA E.L., SUNDMAN V., 1983 - Morphological and physiological characteristics and lipopolysaccharide composition of N2-fixing (C2H2-reducing) root-associated Pseudomonas sp. - Can. J. Microbiol., 29(8): 874-880. H O L T J . G . , K R I E G N . R , S N E A T H P . H . A . , S T A L E Y J . T . , WILLIAMS S.T., 1994 - Bergey’s manual of determina- tive bacteriology. - 9th Edition, Williams and Wilkins, Baltimore, USA, pp. 518-537. HOU M.P., OLURANTI B.O., 2013 - Evaluation of plant growth promoting potential of four rhizobacterial species for indigenous system. - J. Cent. South. Univer., 20: 164-171. ISLAM F., TAHIRA Y., QASIM A., SHAFAQAT A., MUHAM- MAD S.A., SABIR H., HINA R., 2014 - Influence of Pseudomonas aeruginosa as PGPR on oxidative stress tolerance in wheat under Zn stress. - Ecotoxicol. Environ. Safety, 104: 285-293. KLOEPPER J.W., RYU C.M., ZHANG S., 2004 - Induced sys- temic resistance and promotion of plant growth by Bacillus species. - Phytopathology, 94: 1259-1266. KUMAR V., NARULA N., 1999 - Solubilization of inorganic phosphates and growth emergence of wheat as affect- ed by Azotobacter chroococcum mutants. - Bio. Fertil. Soil, 28(3): 301-305. LIANG J.G., TAO R.X., HAO Z.N., WANG L.P., ZHANG X., 2011 - Induction of resistance in cucumber against seedling damping-off by plant growth-promoting rhi- zobacteria (PGPR) Bacillus megaterium strain L8. - Afr. J. Biotech., 10(36): 6920-6927. LORCK H., 1948 - Production of hydrocyanic acid by bacte- ria. - Physiologia Plantarum, 1(2): 142-146. LOVLEY D.R., 1995 - Bioremediation of organic and metal contaminants with dissimilatory metal reduction. - J. Ind. Microbiol. Biotech., 14(2): 85-93. LUGTENBERG B.J.J., DEKKERS L.C., 1999 - What makes Pseudomonas bacteria rhizosphere competent. - Env. Microbiol., 1: 9-13. MIRANSARI M., 2014 - Plant growth promoting rhizobacte- ria. - J. Pl. Nut., 37(14): 2227-2235. MISHRA P.K., JOSHI P., SUYAL P., BISHT J.K., BHATT J.C., 2014 - Potential of phosphate solubilising microorgan- isms in crop production. - Bior. Sus. Plant Nut. Man., 8: 201-212. NADEEM S.M., AHMAD M., ZAHIR Z.A., JAVAID A., ASHRAF M., 2014 - The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop pro- ductivity under stressful environments. - Biotech. Adv., 32(2): 429-448. NADEEM S.M., SHAHAROONA B., ARSHAD M., CROWLEY D.E., 2012 - Population density and functional diversity of plant growth promoting rhizobacteria associated with avocado trees in saline soils. - App. Soil Ecol., 62: 147-154. NAUTIYAL C.S., 1999 - An efficient microbiological growth medium for screening phosphate solubilizing microor- ganisms. - FEMS Microbiol. Let., 170(1): 265-270. ORHAN E., ESITKEN A., ERCISLI S., TURAN M., SAHIN F., 2006 - Effects of plant growth promoting rhizobacteria (PGPR) on yield, growth and nutrient contents in organ- ically growing raspberry. - Sci. Hort., 111(1): 38-43. P O O N G U Z H A L I S . , M A D H A I Y A N M . , S A M . , 2 0 0 6 - Cultivation-dependent characterization of rhizobacteri- al communities from field grown Chinese cabbage Brassica campestris ssp. pekinensis and screening of traits for potential plant growth promotion. - Pl. Soil, 286: 167-180. RANA A., SAHARAN B., JOSHI M., PRASANNA R., KUMAR K., NAIN L., 2011 - Identification of multi-trait PGPR iso- lates and evaluating their potential as inoculants for wheat. - Ann. Microbiol., 61(4): 893-900. SACHDEV D.P., NEMA P., DHAKEPHALKAR S., ZINJARD E., Adv. Hort. Sci., 2018 32(4): 525-534 534 CHOPADE B., 2010 - Assessment of 16S rRNA gene- based phylogenetic diversity and promising plant growth-promoting traits of Acinetobacter community from the rhizosphere of wheat. - Microbiol. Res., 165: 627-638. SAHA M., SARKAR S., SARKAR B., SHARMA B.K., BHAT- TACHARJEE S., TRIBEDI P., 2016 - Microbial sidero- phores and their potential applications: a review. - Env. Sci. Pol. Res., 23(5): 3984-3999. SAHARAN B.S., NEHRA V., 2011 - Plant growth promoting rhizobacteria: a critical review. - Life Sci. Med. Res., 21(1): 30. SCHWYN B., NEILANDS J.B., 1987 - Universal chemical a s s a y f o r t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f siderophores. - Analytical Biochemistry, 160(1): 47-56. SHAHAT A.A., IBRAHIM A.Y., AL-GHAMDI A.A., ALSAID M.S., 2016 - Phytochemical investigation of Rhus tripar- tita and its activity against cyclooxygenases and acetyl- cholinesterase. - Tro. J. Pha. Res., 15(8): 1697-1706. SIDDIQUI Z.A., 2005 - PGPR: prospective biocontrol agents of plant pathogens, pp. 111-142. - In : SIDDIQUI Z.A. (ed.) PGPR biocontrol and biofertilization, Springer, Dordrecht, The Netherlands. SUSILOWATI D.N., SUDIANA I.M., MUBARIK N.R., SUWAN- TO A., 2015 - Species and functional diversity of rhi- zobacteria of rice plant in the coastal soils of Indonesia. - Ind. J. Agr. Sci., 16(1): 39-50. TRIVEDI P., PANDEY A., 2008 - Plant growth promotion abilities and formulation of Bacillus megaterium strain B 3 8 8 ( M T C C 6 5 2 1 ) i s o l a t e d f r o m a t e m p e r a t e Himalayan location. - Ind. J. Mic., 48(3): 342-347. TSAVKELOVA E.A., CHERDYNTSEVA T.A., BOTINA S.G., NETRUSOV A.I., 2007 - Bacteria associated with orchid roots and microbial production of auxin. - Mic. Res., 162(1): 69-76. VESSEY J.K., 2003 - Plant growth promoting rhizobacteria as biofertilizers. - Plant and Soil, 255(2): 571-586. VOISARD C., KEEL C., HAAS D., DE´FAGO G., 1989 - Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under genotobiotic conditions. - The EMBO J., 8: 351-358. WELLER D.M., THOMASHOW L.S., 1994 - Current challenges in introducing beneficial microorganisms into the Rhizosphere, pp. 1-18. - In: O’GARA F., D.N. DOWLING, and B. BOESTEN (eds.) Molecular ecology of Rhizosphere microorganisms. VCH, Basel, Switzerland, pp. 177. WOYESSA D., ASSEFA F., 2011 - Effects of plant growth promoting rhizobaceria on growth and yield of Tef (Eragrostis tef Zucc. Trotter) under greenhouse condi- tion. - Res. J. Microbiol., 6(4): 343. YANG J., KLOEPPER J.W., RYU C.M., 2009 - Rhizosphere bacteria help plants tolerate abiotic stress. - Tr. Pl. Sci., 14(1): 1-4. ZOU C., ZHIFANG L., DIQIU Y., 2010 - Bacillus megaterium strain XTBG34 promotes plant growth by producing 2- Pentylfuran. - The J. Mic., 48(4): 460-466.