INTRODUCTION

Over the last century,the rapid growth of the human population has created a global scarcity of water resources,increased salinization of soils,loss of biodiversity,and environmental degradation,and decreased nutrient availability(Shahbaz and Ashraf,2013).A-mong these problems,salt stress poses an increasing number of challenges to agriculture and food security,which has led to the increased exploitation of natural resources(Ashraf et al.,2012).Globally,6%of the Earth’s surface is affected by salinity,which corresponds to approximately 20%of irrigated regions,especially in the arid and semi-arid regions where rainfall is insufficient to leach salts from the upper layers of the soil.It has been estimated that more than 50%of arable land will be salinized by the year 2050(Jamil et al.,2011;Dikilitas and Karakas,2012;Stankovi´c et al.,2015).

Soil is considered saline if the electrical conductivity(EC)of the saturation extract in the area adjacent to the roots exceeds 4 dS m−1at 25 °C,and if it has 15%exchangeable sodium(Shrivastava and Kumar,2015).High concentrations of sodium and chloride,as well as other ions(potassium,calcium,carbonate,nitrate,and sulfate),decrease the acquisition of water by plants and negatively affect many physiological processes,such as photosynthesis,protein synthesis,and lipid metabolism(Kosov´a et al.,2011;Rojas-Tapias et al.,2012).

培养一批能够适应技术发展需要的专家型、复合型的管理人才、技术保障人才。在引进高级专业人才的同时，对现有人员进行持续教育培训，组织开展审计业务能力、计算机应用能力、大数据分析研究能力和内部管理能力经验交流活动与知识讲座，充分发挥审计干部教育学院与网络培训的作用，加强大数据与审计交叉学科的培训，提升工作人员数据采集、清洗、转换、模型构建能力。培养工作人员的团队合作意识，加强与不同审计小组的分工协作。

Salt stress also affects the microbial community in the soil,including the soil bacteria that are able to colonize the surface of the root system(and sometimes the root inner tissues)to stimulate the plant’s growth and health.This group of bacteria was named in 1981 by Kloepper and Schroth as plant growth-promoting rhizobacteria(PGPR)(Barea et al.,2005;Paul and Nair,2008;Dutta and Podile,2010;Bhattacharyya and Jha,2012;Vacheron et al.,2013).When they are exposed to solutions with increased osmolarity,the water activity in the bacterial cytoplasm decreases,consequently disturbing protein activity and inhibiting many biological processes,such as the synthesis of macromolecules or DNA replication(Nabti et al.,2010;Bhattacharyya and Jha,2012).

综上,宫颈息肉患者临床治疗中,治疗方式选择CO2激光治疗方法,可取得显著治疗效果,疼痛程度较低,且复发可能性小,应在临床实践中将该种方法推广应用。

Plant growth enhancement by PGPR occurs through several mechanisms such as nitrogen(N) fixation,siderophore production,the solubilization of minerals essential for plant nutrition,and the synthesis of enzymes implicated directly in the degradation of organic matter and soil fertility(such as proteases,ureases,chitinases,and cellulases)(do Vale Barreto Figueiredo et al.,2010;Reddy 2013).Recently,numerous studies reported the role of PGPR as an inducer of plant tolerance to abiotic stress(e.g.,drought and salinity)(Paul and Nair,2008;Yang et al.,2009;Lim and Kim,2013).The term “induced systemic tolerance(IST)”has been suggested to represent the tolerance conferred to plants by PGPR against these types of stress(Bhattacharyya and Jha,2012).

Ethylene is a plant hormone with an active role in various developmental processes,such as leaf senescence,leaf abscission,epinasty,and fruit ripening(Vogel et al.,1998;Gray and Smith,2005).However,high amounts of this hormone,produced under salt stress,lead to reductions in root and shoot growth(Shrivastava and Kumar,2015).The enzyme 1-aminocyclopropane-1-carboxylate(ACC)deaminase,synthesized by a wide range of rhizospheric bacteria,contributes to the reduction of ethylene levels in plant tissues.In the presence of ACC deaminase-producing bacteria,part of the plant’s ACC is sequestered and degraded to supply N and energy,contributing to the reduction of ethylene levels in the plant(Penrose and Glick,2003;Glick et al.,2007).

Under salt stress,PGPR and plants accumulate and/or synthesize compatible solutes or osmoprotectants;these metabolites are compatible with their cellular metabolism and protect their cells against plasmlysis(C´anovas et al.,1997;G¨oller et al.,1998;Wani et al.,2013).The compatible solutes comprise mainly sugars and derivatives(sucrose,trehalose,sulfotrehalose,and glucosylglycerol),some amino acids and derivatives(proline,glutamic acid,glutamine,γaminobutyric acid,and glycine betaine(GB)),ectoin and derivatives,and polyhydric alcohols(glycerol,arabitol,and mannitol)(Courtenay et al.,2000).

All the tested isolates were able to produce siderophores,except the strains BEB4,BOA5,and MEA4.The isolates BEB6 and SEA3 showed a weak ability to produce siderophores(Table III).

MATERIAL AND METHODS

Soil samples

Soil samples were aseptically collected from seven agricultural sites used for wheat cultivation in Algeria(Table I).Soil samples(500 g)from each site were collected at depths from 10 to 30 cm by using sterile metallic corers.The soil surface was disinfected with ethanol(70%,volume∶volume)before sampling.It is important to mention that the seven sites are situated at a range of different distances from the Mediterranean(approximately from 500 m to 200 km)and encompass a range of temperatures,to ensure the high diversity of the isolated bacteria.

Macroalgae and OFI cladodes

Marine macroalgae(Chlorophyta),UL and EI,and the cladodes of OFI,were harvested from Beja¨ıa Province,Algeria(Table I),and transported at 4°C to the laboratory for the preparation of the extracts.

Soil physicochemical properties

精矿铁品位提高0.23%，精矿产率提高6.62%，铁回收率提高12.61%；次级精矿品位降低了7.03%，产率降低15.84%。

TABLE I Date and location of soil,Ulva lactuca(UL),Enteromorpha intestinalis(EI),and Opuntia ficus-indica(OFI)sampling

Sample Location Latitude,longitude Date Soil BEA Beja¨ıa 36°42′11.60′′N,5°5′14.47′′E Jan.21,2014 BEB Beja¨ıa 36°41′30.19′′N,4°55′46.13′′E Jan.2,2014 BEC Beja¨ıa 36°40′44.54′′N,4°53′35.55′′E Jan.29,2014 BOA Bouira 36°7′41.29′′N,3°32′55.89′′E Jan.29,2014 MEA Medea 36°15′38.8′′N,3°23′29.25′′E Jan.31,2014 SEA Setif 36°12′3.81′′N,5°22′1.44′′E Feb.24,2014 SEB Setif 36°11′30.55′′N,5°22′16.79′′E Feb.24,2014 UL Beja¨ıa 36°48′54.5′′N,4°59′11.1′′E May 13,2014 EI Beja¨ıa 36°48′54.5′′N,4°59′11.1′′E May 13,2014 OFI Beja¨ıa 36°48′05.5′′N,5°00′15.7′′E May 15,2014

Isolation of diazotrophic bacteria from soil samples

Free-living N- fixing bacteria were isolated by using the N-free Jensen’s selective medium containing 20 g mannitol,1.0 g K2HPO4,2.0 g CaCO3,0.5 g MgSO4·7H2O,0.5 g NaCl,0.1 g FeSO4,0.005 g Na2MoO4,and 17 g agar in 1 L distilled water,with pH adjusted at 7.4±0.02(Thompson,1989).Ten grams of each sample,homogenized in a sterile manner,was thoroughly mixed with 90 mL of sterile distilled water to form the first dilution(10−1),from which successive dilutions from 10−2to 10−7were prepared.Each dilution was inoculated to three Petri dishes(100µL each)containing N-free Jensen’s medium.The Petri dishes were incubated for 7 d at 30°C.At the end of the incubation period,10 isolates with different appearances were purified from each soil sample,tested for their motility and oxidase and catalase activity,subjected to Gram staining,and stored for further studies.

目前宏济堂中医药文化产业园区已建成多条不同主题的游览线路以适合不同群体的需求，旅游配套设施不完善是园区的短板，逐渐设置明确的路标、导览图、标示牌、景物介绍牌、安全提示牌等标识。园区内设有多辆观光游览车、摆渡车等，主题展区均配备充足的厕所，男女厕位比例合理。特色旅游商品主要有阿胶、阿胶枣、阿胶山楂、阿胶速溶粉、雪梨膏等企业自有产品。园区内虽然有高中低不同档次的餐厅，但与员工、游客期望的差距依然巨大，调查问卷对应得分是2.72和3.56，均为所有项目中的最低分。

Plant growth-promoting traits

Enzymatic activities. The seventy purified strains were screened for their potential hydrolytic enzyme activities using the agar disk method.Cellulolytic(Carder,1986),esterasic(Sierra,1957),lipolytic(Sierra,1957),chitinolytic(Kopeˇcn´y et al.,1996),proteolytic(Kavitha et al.,2013),amylolytic(Raj et al.,2009),and ureasic(Christensen,1946)activities were tested.Twenty-eight isolates with the best enzymatic scores(the sum of the positive results for each isolate)were chosen for further studies.To reveal any possible in fluence of the environmental parameters(pH,EC,and SOM)on the enzyme expression by the selected isolates,the correlations between the physicochemical properties of each soil sample and the total score of the enzymatic activities of their corresponding strains together(the sum of the positive results for all the strains purified from the same sample)were determined.

(4) 模块配置.根据具体需求结合配置规则库,检索出符合设计要求的模块,并对模块进行组合、替换,完成设备模块配置方案.配置规则库的建立需要对企业长期积累的设计经验进行归纳总计.

All soil samples were slightly alkaline.One sample from Beja¨ıa(BEA)was identified as a saline soil(EC ＞4 dS m−1);for the other samples,the EC was between 3.8 dS m−1for the samplefrom Bouira(BOA)and 2.1 dS m−1for for the samplefrom Setif(SEA).The samples collected from Medea(MEA)and Setif(SEA and SEB)were richer in SOM(11,13,and 15 g kg−1,respectively)than those collected from Beja¨ıa(BEA,BEB,and BEC)and Bouira(BOA).Other physicochemical properties were similar for the seven samples(Table II).

2.可操作性。指标体系指标数据易于获取，既体现完整性，又要进行必要的简化，突出关键指标，能够体现信息化对企业运营的支撑。

Indole 3-acetic acid(IAA)detection and quanti-fication. Indole 3-acetic acid was quantified on Luria-Bertani(LB)medium supplemented with 1 g L−1 tryptophan(Bric et al.,1991).After incubation,1.5 mL of the culture was transferred to microtubes and centrifuged under approximately 25°C(room temperature)at 9500 g for 2 min.One milliliter of the supernatant,together with 1 mL of Salkowski’s reagent(1 mL of 0.5 mol L−1FeCl3and 49 mL of 35%HClO4),was transferred to the tubes.After 30 min,the absorbance at 350 nm was measured.A standard curve of IAA(Sigma-Aldrich®)was prepared.The development of a pink color after the addition of Salkowski’s reagent indicated the production of auxin by the isolate;14 auxin-producing isolates were selected and tested for their ability to produce siderophores.

Siderophore production. The ability of the 14 selected strains to produce siderophores was tested by using chrome azurol S(CAS)agar medium prepared in accordance with the protocol originally proposed by Schwyn and Neilands(1986)and modified by Alexander and Zaberer(1991).The Fe-CAS indicator solution(Solution 1)was prepared by mixing 10 mL of 1 mmol L−1FeCl3·6H2O(in 10 mmol L−1HCl)with 50 mL of CAS solution(1.21 mg mL−1).The mixture was added slowly to 40 mL of an aqueous solution of hexadecyltrimethylammonium bromide(HDTMA)(1.82 mg mL−1).The buffer solution(Solution 2)was prepared by dissolving 30.24 g of 1,4-piperazinediethanesulfonic acid(PIPES)in 750 mL of a salt solution∶0.3 g KH2PO4,0.5 g NaCl,1.0 g NH4Cl,and 15 g agar.The pH was adjusted to 6.8 with 50%(weight∶weight)KOH,and water was added to bring the volume to 800 mL.The composition of Solution 3,per 70 mL water,was∶2 g glucose,2 g mannitol,493 mg MgSO4·7H2O,11 mg CaCl2,1.17 mg MnSO4·H2O,1.4 mg H3BO3,0.04 mg CuSO4·5H2O,1.2 mg ZnSO4·7H2O,and 1.0 mg Na2MoO4·2H2O.The three obtained solutions were autoclaved separately and cooled to 50°C.An aliquot of 30 mL of the filter-sterilized solution(Solution 4)containing 10%(weight∶volume)casamino acids was mixed with Solutions 3 and 2.The indicator solution was added last,with sufficient stirring to mix the ingredients without forming bubbles.Based on this test,eight siderophore-producing isolates were selected for further study.

Phosphate solubilization. The eight selected strains were tested for their ability to solubilize inorganic rock phosphate(Ca3(PO4)2).Pikovskaya’s medium(PKV)was used,which contained 10 g glucose,5 g Ca3(PO4)2,0.5 g(NH4)2SO4,0.2 g NaCl,0.1 g MgSO4·7H2O,0.2 g KCl,0.5 g yeast extract,0.002 g MnSO4·H2O,0.002 g FeSO4·7H2O,and 15 g agar in 1 L of distilled water.The capacity of an isolate to solubilize inorganic phosphate was indicated by a clear halo around the agar disk(Sagervanshi et al.,2012).Four strains considered to be phosphate solubilizers were molecularly identified and subsequently used in the second part of this work.

第一步分类。相比于HAPLR评价体系按图书馆服务人口数量分类，星级评价则是按各图书馆经费总数进行分类，划分为10 000～4.9999万、50 000万～9.9999万……3 000万以上9各级别。同样，只有在同一级别的公共图书馆才有资格进行评比。

Effective and titratable acidity(pH(water),pH(KCl)),EC,gravimetric moisture content(θg),and soil organic matter(SOM)were determined according to Rouiller et al.(1994),Hardie and Doyle(2012),Pepper and Gerba(2004),and Huang et al.(2009),respectively.

Use of ACC as a sole N source. The bacteria were cultivated on a rich medium containing 5 g tryptone,2.5 g yeast extract,and 2 g glucose in 1 L of distilled water and incubated with shaking(100 r min−1)for 48 h at 30 °C.The obtained biomass was washed three times with a sterile solution of 0.1 mol L−1MgSO4and finally resuspended in the same solution.In a 96-well plate,120µL of Dworkin and Foster(DF)salt medium,which contained(in 1 L)4.0 g KH2PO4,6.0 g Na2HPO4,0.2 g MgSO4·7H2O,2.0 g glucose,2.0 g gluconic acid,2.0 g citric acid,1 mg FeSO4·7H2O,10 mg H3BO3,11.19 mg MnSO4·H2O,124.6 mg ZnSO4·7H2O,78.22 mg CuSO4·5H2O,and 10 mg MoO3,at pH 7.2(Penrose and Glick,2003),was added to each well.Aliquots of 15 µL of 0.1 mol L−1 MgSO4or 15µL of 0.1 mol L−1(NH4)2SO4were added to the first and the second thirds of the microplates,respectively.The ACC solution(3 mmol L−1)was filtersterilized(0.2µm)and stored at−20°C until the assay.The ACC was thawed and 15µL was placed in the final third of the microplates.Each well was inoculated with 15µL of the washed bacterial culture.A control without bacteria was used(Shahzad et al.,2010).After incubation(48 h at 28°C),the bacterial growth was determined by the measurement of optical density at 600 nm(OD600).

Molecular identification

The selected strains were identified based on their coding DNA fragments for 16S rRNA sequences.The total DNA extraction was performed,and the 16S rRNA gene was amplified by using the universal primers 5′-S-D-Bact-0008-a-S-20-3′and 5′-S-D-Bact-1495-a-S-20-3′,with the sequences 5′-AGAGTTTGATCCTGGCTCAG-3′and 5′-AAGGAGGTGATCCAGCCGCA-3′,respectively(Daffonchio et al.,2000).

The amplification reaction mixture consisted of 2.5µL PCR reaction buffer,2.5µL MgCl2,0.2µL deoxynucleoside triphosphate,0.3µL of each primer,0.2µL Taq DNA polymerase,and 1µL of total DNA.The PCR program consisted of an initial step of 94°C for 3 min,35 cycles of denaturation at 94°C for 45 s,annealing at 55 °C for 1 min,and elongation at 72 °C for 2 min,followed by a final elongation step at 72°C for 8 min.

An agarose gel(1.5%,weight∶volume)was used for the separation of the PCR products in 0.5×Trisborate-EDTA buffer,stained for 30 min in 0.5 mg L−1 ethidium bromide solution,and developed by exposure to ultra-violet light.An enzymatic sequencing of the amplified DNA fragments was conducted in accordance with the method of Sanger(Sanger and Coulson,1975).The obtained sequences were deposited in GenBank and compared with sequences available from the National Center for Biotechnology Information(NCBI)database(http∶//www.ncbi.nlm.nih.gov)using the BLAST algorithm.A phylogenetic dendrogram was conducted by using the neighbor joining method,and tree topology was evaluated by performing bootstrap analysis of 1000 data sets using MEGA4.1(Kumar et al.,2004).

Determination of halotolerance

Compared with the control,in the absence of salt stress,OFI extract enhanced the growth of the isolates BEA4 and SEB9 on the N-Fb medium by 115.342%and 124.51%,respectively,and that of the isolates BEA4,BEC9,BOA4,and SEB9 by 109.231%,105.5%,103.115%,and 144.887%on the LB medium.The same extract increased the bacterial growth of the isolates BEA4,BEC9,BOA4,and SEB9 on LB medium with 1000 mmol L−1of NaCl by 141.085%,230.805%,215%,and 221%,respectively,whereas their growth was restored under 350 mmol L−1of NaCl on N-Fb medium to 187.877%,414.398%,183.321%,and 414.267%of the control(without extract).EI-extract ameliorated the growth of the isolates BEA4,BEC9,and BOA4 on LB medium with 1300 mmol L−1of Na-Cl by 135.529%,641.538%,and 949.895%,respectively.Glycine betaine stimulated the growth of the isolates BEA4,BEC9,BOA4,and SEB9 on N-Fb medium with 300 mmol L−1of NaCl by 127.286%,142.22%,113.269%,and 101.834%,respectively(Figs.4 and 5).

Marine macroalgae and OFI cladodes were cut into fine pieces to facilitate the improved release of intracellular substances.The finely cut pieces(2.5 g fresh weight)were immersed in vials containing 50 mL of N-Fb medium with the aforementioned NaCl concentrations.The vials were then autoclaved at 110°C for 30 min.After decantation,10 mL of the supernatant was distributed in sterile tubes to be loaded into microplates for the study of bacterial halotolerance(Ghoul et al.,1995).

开展人文社科类学科竞赛对于培养当代大学生所缺失的人文精神在目前看来是行之有效的举措。高校教育重视专业知识和技能的传授，思政课程与专业课程脱节，难以起到相互促进的效果。这在一定程度上导致了大学生在信仰缺失、价值危机、缺乏社会责任感等人文精神方面的缺憾。这就需要大学生通过学习践行社会主义核心价值观来加以疗救，从而不断领略社会主义核心价值观与我国传统文化精髓中蕴含共通的人文精神。而参与到人文社科类学科竞赛之中，在紧张的竞赛氛围内，在亲身体验的情境中，感受思想上的净化，浸染传统国学中文化的力量，有助于大学生对健康完善的人格的塑造。

Luria-Bertani broth(5 mL)was inoculated with a fresh culture(18 h)of the tested strains(BEA4,BEC9,BOA4,and SEB9).After incubation at 28°C for 24 h,the culture was centrifuged at 3000 r min−1for 10 min.The pellet was washed three times with 5 mL of normal saline solution(8.5 g L−1NaCl)and finally resuspended in 5 mL of the same solution.The obtained bacterial suspensions were inoculated to the culture medium already distributed in microplates.After incubation(28°C for 4 d),the bacterial growth was estimated through the measurement of the absorbance of the culture at 600 nm.

The above procedure was then repeated after the replacement of N-Fb with LB medium(0,300,500,800,1000,1300,1500,1800,and 2000 mmol L−1of NaCl)supplemented,or not,with GB(1 mmol L−1)or hydro-alcoholic extracts(10−2,volume∶volume)from OFI,UL,and EI.The hydro-alcoholic extracts were prepared through the immersion of 5 g(dry weight)of UL,EI,and OFI cladodes in vials containing 150 mL of 70%ethanol with shaking for 5 to 10 min.The obtained extract was filtered through a filter paper to eliminate solids,evaporated to dryness,and resuspended in 10 mL distilled water(Ghoul et al.,1995).

比较两组呼吸内科教学查房满意度；教师对学生病例处理的能力、基础操作的技能以及专业理论知识掌握度的考核评分（每一项满分均100分，分值和指标为正比）；学生对教学模式提高临床逻辑思维能力、提高资料查阅能力、提高主动学习能力、锻炼交流能力的认可度（每一项满分均100分，分值和指标为正比）。

Wheat germination assay

The bacterial cultures of the four strains finally selected were washed three times with sterile distilled water(OD600∶0.8)and used for the germination assays.The seeds of wheat(variety Boussalem)were surface sterilized(G¨otz et al.,2006)and imbibed in washed bacterial suspension for 30 min.The imbibed seeds were placed in Petri dishes layered with sterile filter paper and soaked(5 mL per Petri dish)in various NaCl solutions(100,200,and 300 mmol L−1).Hydro-alcoholic extracts from OFI and UL(1%,weight∶volume)and GB(1 mmol L−1)were added to the salt solutions(Ramadoss et al.,2013).All tests were repeated in triplicate(12 seeds per Petri dish).Controls without bacterial treatment,without GB,and without extracts were used.The experiment was conducted in darkness at 20°C.Readings were recorded every 2 d for 12 d,and the final germination percentage was determined at the end of the experiment.

Statistical analysis

Statistical analyses were computed by using GraphPad Prism software(version 6).The data from the halotolerance and germination assays were analyzed using two-way analysis of variance(ANOVA)subjected to a 95%con fidence limit.The correlations between the enzymatic scores and physicochemical properties of the soil samples were analyzed by the calculation of Pearson’s coefficient of correlation(r)and linear regression(P＜0.05).

RESULTS

Soil physicochemical properties

对于很多人来说，可能这是一句废话，难道你会开一个没有用的处方给养殖户吗，注意了，我这里所说的多开处方并不是“销售处方”，这个处方是对于池塘养殖过程中所出现的问题而开出的用药处方，是关乎养殖户最终效益的处方。简言之，开出来的处方要行之有效，在能够处理好鱼病、水质、营养、代谢等问题的同时，提供的是最简洁、最实惠，行之有效的技术服务指导方案，与此同时加强用药结果跟踪和个人技术总结，不断提升自身的技术水平，优化处方科学性和可操作性。

TABLE II Selected physicochemical propertiesa)of the collected soil samples

a)θg=gravimetric moisture content;EC=electrical conductivity;SOM=soil organic matter.

Sample pH(H2O)pH(KCl)θg EC SOM g kg−1 dS m−1 g kg−1 BEA 8.05 7.16 1.37 4.2 8 BEB 8.54 7.63 2.00 3.5 5 BEC 8.20 7.29 2.40 2.4 9 BOA 8.29 7.60 1.86 3.8 7 MEA 8.12 7.34 2.11 2.9 11 SEA 8.20 7.15 2.87 2.1 15 SEB 8.19 7.41 2.22 2.4 13

Enzymatic activities

Sixty-three strains(90%of the selected bacteria)were amylase positive.Ureasic activity was detected in 44.29%of the isolates,with less protease activity detected(37.14%).Lipolytic,esterasic,chitinolytic,and cellulolytic activities were detected in 45.71%,47.14%,70%,and 50%of the 70 tested strains,respectively(data not shown).The intensity of the enzymatic activities of the selected isolates from each soil sample and their respective pH(KCl)values were positively correlated;for a sample with a basic pH,the bacteria produced more hydrolytic enzymes(Fig.1a).The SOM of all samples,except BEA,and their θg,except BEA and SEB,were negatively correlated with the total enzymatic activities in each group of isolates,whereas the EC of all samples,except BEA,was positively correlated with the enzymatic activities of the isolates(Fig.1b,c).In all samples,both SOM and θg were negatively correlated with EC(Fig.1d).

Fig.1 Pearson’s coefficient of correlation(r)and linear regression between the total enzymatic activities(score)of isolates selected from each soil sample and soil pH(KCl)(a),electrical conductivity(EC)(b),and soil organic matter(SOM)(c)and EC and SOM and gravimetric moisture content(θg)of each soil sample(d).*,**Significant at P ＜ 0.05 and P ＜ 0.01,respectively.

IAA quantification

The phytohormone IAA was produced by all the tested strains,ranging from 76.31µg mL−1for the isolate BEA4 to 3.35 µg mL−1for the isolate BOA2.The isolates BEC9,BOA4,and SEB9 produced 25.7,47.4,and 32.2 µg IAA mL−1,respectively(Table III).

Detection of siderophores

Marine macroalgae and Opuntia ficus-indica(OFI)are in the first line among the organisms known for their tolerance to stresses such as salinity and drought.Their adaptation to abiotic stress and chemical composition suggest that these plants might be used as efficient sources of a wide range of compatible solutes(Edwards et al.,1988;Ghoul et al.,1995;Kirst,1996;Pichereau et al.,1998;Nabti et al.,2007;S´aenz et al.,2013).Thus,the objectives of this study were∶1)to isolate,select,and identify diazotrophic bacteria with a good ability to produce“in vitro” metabolites of potential agricultural interest and 2)to evaluate the effect of the natural extracts from marine macroalgae,Ulva lactuca(UL)and Enteromorpha intestinalis(EI),and OFI on the growth of the selected bacteria and to examine the effect of these extracts and bacterial isolates on the germination of wheat(Triticum durum)seeds under salt stress conditions.

Phosphate solubilization

Only two isolates,BEA3 and SEA3,were unable to solubilize inorganic phosphate(Ca3(PO4)2).The solubilization index of the isolates BEA4,BEC9,BOA4,and SEB9 was 0.79,0.40,0.36,and 0.65,respectively(Table III).

Use of ACC as a sole N source

Four strains(BEA4,BEC9,BOA4,and SEB9)were able to grow with ACC as the sole N source.The growth of the two isolates BEA4 and BEC9 was much faster in the presence of ACC and(NH4)2SO4than in the absence of N.However,no significant difference was detected in the growth of the two strains BOA4 and BEC9 in the presence and absence of ACC as a N source(Fig.2).

Molecular identification

The selected strains BEA4,BEC9,BOA4,and SEB9 were molecularly similar to the species Flavobacterium johnsoniae,Pseudomonas putida,Achromobacter xylosoxidans,and Azotobacter chroococcum,respectively(Fig.3).

Fig.2 Utilization of 1-aminocyclopropane-1-carboxylate(ACC)and(NH4)2SO4as the sole N source by isolates BEA4,BEC9,BOA4,and SEB9.Readings were performed in four replicates.Error bars represent the standard deviations of means.A600=absorbance of the culture at 600 nm.*,**Significant at P＜0.05 and P＜0.01,respectively.

Halotolerance of bacteria

The LB medium conferred greater protection to bacteria against salt stress than the N-Fb medium in the absence of GB and extracts.At high salt concentrations,the OFI extract conferred better protection than the GB and UL and EI extracts.The isolate BEA4 seemed to be the most tolerant to salt stress.The isolate SEB9 was the most sensitive to salinity on the LB medium,whereas BEC9 expressed the weakest tolerance to NaCl on the N-Fb medium(Figs.4 and 5).

孔守真的棺木已从如玉豆腐坊移到了大成殿。棺首置了祭桌，祭桌上摆着碗筷杯盏，几荤几素，两旁点着高烛，燃了长香。棺木四周帷幔层层，非白即黑，透着一股悲凉，几幅挽联云：悲声难挽流云住，哭音相随野鹤飞。前世典范，后人楷模；名留后世，德及乡梓。

The halotolerance of the selected strains was studied using an N-free broth medium(N-Fb)containing 6 g K2HPO4,4 g KH2PO4,0.2 g MgSO4,0.1 g NaCl,0.02 g CaCl2,0.01 g FeCl3,0.002 g Na2MoO4,0.05 g yeast extract,and 5 g glucose(added after autoclaving)in 1 L of distilled water.This medium was supplemented with various concentrations of NaCl(0,250,300,350,400,450,500,550,600,and 650 mmol L−1)(Nabti et al.,2007).

大港油田提出了技术经济一体化，开放合作，有效建产的思路。油田创新开发模式，从开发方案设计入手，最大限度开展地质设计及过程优化来探讨未动用储量的效益开发；创新提出全生命周期模式，在沈家铺、舍女寺等20个区块动用地质储量1609万吨，钻新井109口，总进尺32.11万吨，建产能22.5万吨，形成日产520吨规模，累积产油24.8万吨，提高低渗油藏的开发效果；推进风险作业管理模式，6个风险合作区块最高日产达200吨以上，最高年产3万吨，累计产出原油10.5万吨，推动了低渗难采区块的有效动用。

TABLE III Hierarchical strain selection based on the production of indole 3-acetic acid(IAA)and siderophores and phosphate solubilization

a)−=negative;+=weakly positive;++=moderately positive;+++=highly positive.

Strain IAA Siderophore Phosphate solubilization code productiona)Colony zone diameter Halo zone diameter Solubilization indexµg mL−1cm BEA1 12.3±2.8 BEA3 38.0±2.7 +++ 1.1 1.1 1 BEA4 76.3±7.7 +++ 1.1 1.4 0.79 BEA6 12.3±4.2 BEB3 15.4±2.8 BEB4 23.1±4.1 BEB6 30.3±8.4 +BEB9 05.3±1.3 BEC1 10.2±4.2 BEC4 34.2±2.8 +++ 0.6 1.5 0.40 BEC5 34.4±5.9 BEC9 25.7±2.6 +++ 0.2 2 0.40 BOA2 03.3±2.8 BOA4 47.4±5.6 +++ 0.9 2.5 0.36 BOA5 40.2±5.5 −BOA8 20.3±2.7 MEA1 11.3±2.6 MEA2 05.3±2.5 ++MEA4 31.4±1.4 −MEA6 07.4±0.0 SEA3 26.9±1.4 + 1.2 1.2 1 SEA4 24.3±5.9 +++SEA6 03.4±0.8 SEA8 23.3±2.7 SEB1 12.7±0.7 SEB4 08.4±1.2 SEB7 32.6±5.0 +++ 1 1.2 0.83 SEB9 32.2±1.4 +++ 1.3 2 0.65

Fig.3 Neighbor-joining tree revealing the phylogenetic relationship of the analyzed isolates BEA4,BEC9,BOA4,and SEB9.The bar indicates 5%sequence divergence.

Wheat germination

Plant growth-promoting rhizobacteria may in fluence plant growth through several mechanisms,such as the increased availability of other nutrients and the production of phytohormones.In the first part of this work,we established a hierarchy of potential properties in order to isolate diazotrophic bacteria with high plant growth-promoting potential∶N fixation;catabolic activity(urease,protease,lipase,chitinase,esterase,amylase,and cellulase);IAA and siderophore production;and phosphate solubilization.Through this method,we selected four bacterial strains(Flavobacterium johnsoniae BEA4,Pseudomonas putida BEC9,Achromobacter xylosoxidans BOA4,and Azotobacter chroococcum SEB9)with a high in vitro potential to promote plant growth.

Fig.4 Maximum growth of isolates BEA4(a),BEC9(b),BOA4(c),and SEB9(d)achieved on N-free broth medium with different NaCl concentrations in the the absence(control)and presence of glycine betaine(GB)(1 mmol L−1)and hydro-alcoholic extracts from Ulva lactuca(UL)and Opuntia ficus-indica(OFI)(1%,weight:volume).The readings were performed in duplicate.A600=absorbance of the culture at 600 nm.

Fig.5 Maximum growth of isolates BEA4(a),BEC9(b),BOA4(c),and SEB9(d)achieved on Luria-Bertani medium with different NaCl concentrations in the absence(control)and presence of glycine betaine(GB)(1 mmol L−1)and aqueous extracts from Ulva lactuca(UL),Enteromorpha intestinalis(EI),and Opuntia ficus-indica(OFI).The readings were performed in duplicate.A600=absorbance of the culture at 600 nm.

DISCUSSION

Morrissey et al.(2014)indicated that salinity increased the microbial decomposition rates in lowsalinity wetlands,which was consistent with the positive correlation between the EC of soil samples and the enzymatic activities of their corresponding strains found in this work.The isolates from the saline soil BEA(EC＞4)showed a high enzymatic score when tested in non-saline medium.Despite this,BEA contained high levels of SOM,probably due to inhibition of the lytic enzymes under salt stress.Eivazi and Tabatabai(1988),Garc´ıa et al.(1994),and Batra and Manna(1997)showed that the activities of the soil enzymes were seriously reduced by salinity,which affected nutrient recycling and release for use by plants.pH(KCl)in soil assesses the exchangeable hydrogen and affects the N- fixing efficiency of diazotrophic bacteria(Nikli´nska et al.,2005;Lapinskas and Piaulokait˙e-Motuzien˙e,2006;Rousk et al.,2009).Generally,bacterial extracellular enzymes in soil are alkaline,in contrast to those released by fungi and plants(Caldwell,2005),which could explain the positive correlation observed between the pH of soil samples and the total detected enzymes.

RS技术最早发展于上个世纪60年代，起初该技术主要是以航空摄影技术为基础的一门技术，其最大特点在于大范围同步观测。将RS技术应用在建筑工程测量中，对测量数据的有效性、科学性都起着重要作用，而且掌握RS技术，并了解其各项优势，对于提高建筑工程测量水平，在工程项目中进行全面、大范围观测起着重要的实践意义。

The presence of GB and UL and OFI extracts markedly promoted seed germination under salt stress.The bacterial treatment,especially with BOA4 and SEB9,improved seed germination success.The germination percentage was restored from 2.77%to 22.23%,38.89%,and 30.57%under 300 mmol L−1of NaCl and from 22.2%to 47.23%,44.45%,and 47.23%under 200 mmol L−1NaCl in the presence of GB and OFI and UL extracts,respectively.Bacterial inoculation with the strains BEA4,BEC9,BOA4,and SEB9 enhanced the germination success from 22.2%to 44.43%,41.66%,61.1%,and 63.86%under 200 mmol L−1NaCl and from 2.77%to 25%,22.23%,50%,and 44.43%under 300 mmol L−1NaCl,respectively(Fig.6).

One of the mechanisms by which rhizobacteria ameliorate plant growth is N fixation(James et al.,1994;Glick et al.,1997;Vessey 2003;Gray and Smith 2005).Several strains belonging to the species F.johnsoniae,P.putida,A.xylosoxidans,and A.chroococcum,are known to be plant growth promoters;they are diazotrophic bacteria in the soil(Altman and Waisel,1997;Malik et al.,1997;Kader et al.,2002;Jha and Kumar,2009;Ma et al.,2009;Sgroy et al.,2009;Laskar et al.,2013;Vacheron et al.,2013;Pathani et al.,2014).

Fig.6 Seed germination success(percentage)of wheat in the presence and absence of glycine betaine(GB)(1 mmol L−1)and hydro-alcoholic extracts from Opuntia ficus-indica(OFI)and Ulva lactuca(UL)(1%,weight:volume).Seeds other than controls were treated with isolate BEA4,BEC9,BOA4,or SEB9.Controls without bacterial,without GB,and without extracts were used.Error bars are the standard deviations of means.Different letters above the bars indicate significant differences at P ＜ 0.05 within each NaCl concentration.

The degradation of organic matter through catabolic activity is one of the most important means through which bacteria increase plant growth.Mi-crobial enzymes,such as ureases,esterases,lipases,proteases,chitinases,amylases,and cellulases,play key roles in biological transformation processes(Glick,1995;Mart´ınez-Viveros et al.,2010;Rana et al.,2012;Xun et al.,2015).Enzymes such as chitinase and cellulose play important roles as biocontrol agents through the degradation of fungal cell walls(Mitchell and Alexander,1963;Sindhu and Dadarwal 2001).Kathiresan et al.(2011)reported that a bacterium of the genus Azotobacter produces important quantities of amylases,cellulases,lipases,chitinases,and proteases.Ahmad et al.(2013)revealed that several strains of the genus Pseudomonas produce proteases,cellulases,chitinases,and lipases,that are implicated in SOM degradation and the biocontrol of plant pathogens.

Indole 3-acetic acid is the most commonly produced phytohormone whose effect on plant growth depends on the sensitivity of the plant and the amount of IAA produced(Pant and Agrawal,2014).Our results with respect to IAA production are in agreement with previous works(Torres-Rubio et al.,2000;Tsavkelova et al.,2006;Jha and Kumar,2009;Soltani et al.,2010;Kumar et al.,2014),which reported that bacteria of the genera Flavobacterium,Pseudomonas,Achromobacter,and Azotobacter can produce large quantities of IAA and promote plant growth.

Siderophores are iron-chelating compounds whose biosynthesis is regulated by the availability of iron in the surrounding medium(Sim˜oes et al.,2007).In soil,they play a crucial role in plant growth improvement,enhancing iron uptake by roots.Recent work has demonstrated that iron-chelating siderophores suppress soil-borne fungal pathogens(Beneduzi et al.,2012).Others have shown that the bacterial species F.johnsoniae,P.putida,A.chroococcum,and A.xylosoxidans produce siderophores(Suneja et al.,1996;Hynes et al.,2008;Moretti et al.,2008;Sgroy et al.,2009;Tian et al.,2009;Ahemad and Kibret,2014).

After N,phosphorus is the most limiting element for plant nutrition.Phosphate-solubilizing microorganisms(PSM)in soil are considered the most ecofriendly option to provide inexpensive phosphorus to plants(Kumar et al.,2000;Chen et al.,2006;Hussain et al.,2013;Sharma et al.,2013;Khan et al.,2014;Nosrati et al.,2014).Sashidhar and Podile(2010)highlighted the phosphate solubilization capacity of the bacteria A.xylosoxidans,A.chroococcum,P.putida,and F.johnsoniae.

All the discussed traits(N fixation,hydrolytic enzymes,IAA synthesis,solubilization of inorganic insoluble phosphorus,ACC degradation,and siderophore production)are known to be involved in the enhancement of plant tolerance to salt stress(Egamberdieva,2009;Walpola and Arunakumara,2010;Kizildag et al.,2012;Shrivastava and Kumar,2015).

The use of PGPR is becoming a widespread practice.However,they may be even more beneficial under stress conditions.It is therefore important to ensure that the tested inoculant is able to survive in the particular conditions under which its use is proposed.Microbial tolerance to salt stress may be improved using compatible solutes synthesized by the microorganism and/or taken up from the medium(Madkour et al.,1990);the latter mechanism is preferred when the appropriate substances are present in the environment(Lamosa et al.,1998;Paul and Nair 2008).

In the second part of this work,we demonstrated that OFI extract was,in most cases,better at promoting bacterial growth(BEA4,BEC9,BOA4,and SEB9)and wheat seed germination under high salinity than GB.The LB medium most likely conferred greater protection to bacterial cultures than N-Fb medium in the absence of other sources of compatible solutes owing to the presence of proline,betaines,and other molecules known for their osmoprotectant role,especially in the yeast extract(Nagata et al.,1996;Wood,2007).It is also important to report the positive results obtained by the addition of the extracts of UL and EI.Both marine macroalgae have been reported to synthesize and accumulate a wide variety of osmoprotectants under salt stress(Dickson et al.,1982;Edwards et al.,1987;Plettner et al.,2005).Marine macroalgal extracts have already been used to improve the halotolerance of bacteria such as Sinorhizobium meliloti,Azospirillum brasilense,and Escherichia coli(Ghoul et al.,1995;Pichereau et al.,1998;Nabti et al.,2007).Dimethylsulfoniopropionate and other tertiary sulfoniums,sugars,polyols,and N-organic compounds are the most abundant compatible solutes accumulated under salt stress conditions by marine macroalgae,including UL and EI(Edwards et al.,1988;Kirst,1996).

It is pertinent to note that this is the first recorded use of OFI as a source of natural osmoprotectants.No prior reports have detailed the chemical nature of these compounds,nor the conditions of their synthesis by this plant,which is known for its resistance to environmental stress.However,the total content of free amino acids(257.24 mg per 100 g)was higher than the average for other fruits.Opuntia ficus-indica has relatively high levels of serine,g-amino butyric acid,glutamine,proline,arginine,and histidine,and contains methionine(S´aenz et al.,2013),suggesting that the amino acids may constitute the majority of the natural osmoprotectants provided by the extract of this plant.In addition,OFI cladodes are rich in minerals such as potassium,calcium,phosphorus,and iron(Hadj Sadok et al.,2008),which are involved in the enrichment of the culture media,and thus promote bacterial growth.Tryptophan,the direct precursor of IAA,is an abundant compound in OFI cladodes(1.04 g per 100 g)(El-Mostafa et al.,2014).The production of such elements plays a key role in the promotion of plant growth and the amelioration of salt stress(Egamberdieva 2009;Kaya et al.,2009;Liu et al.,2013).

Nabti et al.(2007)reported the use of PGPR and natural compatible solutes from UL to promote wheat seed germination and growth under salt stress.However,our report on the use of extracts of OFI and EI to promote plant growth under high salinity is new.Basavaraju et al.(2002),Kaymak et al.(2009),Tam and Diep(2014),and Rathi et al.(2014)reported the role of A.xylosoxidans,Flavobacterium sp.,P.putida,and A.chroococcum in the amelioration of seed germination and plant growth under salt stress conditions.The extract from OFI cladodes appears to be an economically viable,natural alleviator of salt stress in agriculture.These plants are distributed worldwide in many countries with arid and semi-arid zones,the most extensive dry lands on the planet,where there is a need for plant species that can adapt to provide food and materials under abiotic stress conditions(S´aenz et al.,2013).

CONCLUSIONS

The bacterial strains F.johnsoniae BEA4,P.putida BEC9,A.xylosoxidans BOA4,and A.chroococcum SEB9 were able to fix atmospheric N,produce a wide range of hydrolytic enzymes,solubilize inorganic insoluble phosphorus,produce siderophores,synthesize useful amounts of IAA,and use ACC as their sole N source.Aqueous and hydro-alcoholic extracts from OFI,UL,and EI improved the halotolerance of the aforementioned strains.The germination of wheat seeds was markedly enhanced by bacterial inoculation and the application of the OFI and UL extracts.In vivo studies are required to determine the effect of the extracts and the strains on extended stages of the plant growth,and to determine the nature of the compounds implicated in the enhancement of bacterial and plant halotolerance.

ACKNOWLEDGEMENTS

The authors are grateful to Mr.Steve Houghton,a civil and sanitary engineer based in Portugal,graduated from the University of Leeds,Lisbon Area,Portugal,for his critical review of the manuscript,and to Prof.Ghoul Mostefa from the University of Setif-1(Algeria)for his advices.This work is dedicated to him on the occasion of his retirement.

REFERENCES

Ahemad M,Kibret M.2014.Mechanisms and applications of plant growth promoting rhizobacteria:Current perspective.J King Saud Univ Sci.26:1–20.

Ahmad N,Shinwari Z K,Bashir S,Yasir M.2013.Function and pylogenetic characterization of rhizospheric bacteria associated with GM and non GM maize.Pak J Bot.45:1781–1788.

Alexander D B,Zuberer D A.1991.Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria.Biol Fertil Soils.12:39–45.

Altman A Y,Waisel Y.1997.Biology of Root Formation and Development.Plenum Publishing Co.,New York.

Ashraf M,Ahmad M S A,¨Ozt¨urk M,Aksoy A.2012.Crop improvement through different means:Challenges and prospects.In Ashraf M,¨Ozt¨urk M,Ahmad M S A,Aksoy A(eds.)Crop Production for Agricultural Improvement.Springer,Dordrecht.pp.1–15.

Barea J M,Pozo M J,Azc´on R,Azc´on-Aguilar C.2005.Microbial co-operation in the rhizosphere.J Exp Bot.56:1761–1778.

Basavaraju O,Rao A R M,Shankarappa T H.2002.Effect of Azotobacter inoculation and nitrogen levels on growth and yield of radish(Raphanus sativus L.).In Rajak R C(ed.)Biotechnology of Microbes and Sustainable Utilization.Scientific Publishers,Jabalpur.pp.155–160.

Batra L,Manna M C.1997.Dehydrogenase activity and microbial biomass carbon in salt-affected soils of semiarid and arid regions.Arid Soil Res Rehabil.11:293–303.

Beneduzi A,Ambrosini A,Passaglia L M P.2012.Plant growthpromoting rhizobacteria(PGPR):Their potential as antagonists and biocontrol agents.Genet Mol Biol.35:1044–1051.

Bhattacharyya P N,Jha D K.2012.Plant growth-promoting rhizobacteria(PGPR):Emergence in agriculture.World J Microbiol Biotechnol.28:1327–1350.

Bric J M,Bostock R M,Silverstone S E.1991.Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane.Appl Environ Microbiol.57:535–538.

Caldwell B A.2005.Enzyme activities as a component of soil biodiversity:A review.Pedobiologia.49:637–644.

C´anovas D,Vargas C,Iglesias-Guerra F,Csonka L N,Rhodes D,Ventosa A,Nieto J J.1997.Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes.J Biol Chem.272:25794–25801.

Carder J H.1986.Detection and quantitation of cellulase by Congo red staining of substrates in a cup-plate diffusion assay.Anal Biochem.153:75–79.

Chen Y P,Rekha P D,Arun A B,Shen F T,Lai W A,Young C C.2006.Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities.Appl Soil Ecol.34:33–41.

Christensen W B.1946.Urea decomposition as a means of di-fferentiating Proteus and Paracolon cultures from each other and from salmonella and Shigella types.J Bacteriol.52:461–466.

Courtenay E S,Capp M W,Anderson C F,Record Jr M T.2000.Vapor pressure osmometry studies of osmolyte-protein interactions:Implications for the action of osmoprotectants in vivo and for the interpretation of“osmotic stress” experiments in vitro.Biochemistry.39:4455–4471.

Daffonchio D,Cherif A,Borin S.2000.Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the “Bacillus cereus Group”.Appl Environ Microbiol.66:5460–5468.

Dickson D M J,Jones R G W,Davenport J.1982.Osmotic adaptation in Ulva lactuca under fluctuating salinity regimes.Planta.155:409–415.

Dikilitas M,Karakas S.2012.Behaviour of plant pathogens for crops under stress during the determination of physiological,biochemical,and molecular approaches for salt stress tolerance.In Ashraf M,¨Ozt¨urk M,Ahmad M S A,Aksoy A(eds.) Crop Production for Agricultural Improvement.Springer,Dordrecht.pp.417–441.

do Vale Barreto Figueiredo M,Seldin L,de Araujo F F,de Lima Ramos Mariano R.2010.Plant growth promoting rhizobacteria:Fundamentals and applications.In Maheshwari D K(ed.)Plant Growth and Health Promoting Bacteria.Springer-Verlag,Berlin.pp.21–43.

Dutta S,Podile A R.2010.Plant growth promoting rhizobacteria(PGPR):The bugs to debug the root zone.Crit Rev Microbiol.36:232–244.

Edwards D M,Reed R H,Chudek J A,Foster R,Stewart W D P.1987.Organic solute accumulation in osmotically-stressed Enteromorpha intestinalis.Mar Biol.95:583–592.

Edwards D M,Reed R H,Stewart W D P.1988.Osmoacclimation in Enteromorpha intestinalis:Long-term effects of osmotic stress on organic solute accumulation.Mar Biol.98:467–476.

Egamberdieva D.2009.Alleviation of salt stress by plant growth regulators and IAA producing bacteria in wheat.Acta Physiol Plant.31:861–864.

Eivazi F,Tabatabai M A.1988.Glucosidases and galactosidases in soils.Soil Biol Biochem.20:601–606.

El-Mostafa K,El Kharrassi Y,Badreddine A,Andreoletti P,Vamecq J,El Kebbaj M S,Latruffe N,Lizard G,Nasser B,Cherkaoui-Malki M.2014.Nopal cactus(Opuntia ficusindica)as a source of bioactive compounds for nutrition,health and disease.Molecules.19:14879–14901.

Garc´ıa C,Hern´andez T,Costa F,Ceccanti B.1994.Biochemical parameters in soils regenerated by the addition of organic wastes.Waste Manage Res.12:457–466.

Ghoul M,Minet J,Bernard T,Dupray E,Cormier M.1995.Marine macroalgae as a source for osmoprotection for Escherichia coli.Microb Ecol.30:171–181.

Glick B R,Cheng Z Y,Czarny J,Duan J.2007.Promotion of plant growth by ACC deaminase-producing soil bacteria.Eur J Plant Pathol.119:329–339.

Glick B R,Ghosh S,Liu C P,Dumbroff E B.1997.Effects of a plant growth-promoting rhizobacterium(Pseudomonas putida GR12-2)on the early growth of canola seedlings.In Altman A,Waisel Y(eds.)Biology of Root Formation and Development.Springer,Boston.pp.253–258.

Glick B R.1995.The enhancement of plant growth by free-living bacteria.Can J Microbiol.41:109–117.

G¨oller K,Ofer A,Galinski E A.1998.Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis.FEMS Microbiol Lett.161:293–300.

G¨otz M,Nirenberg H,Krause S,Wolters H,Draeger S,Buchner A,Lottmann J,Berg G,Smalla K.2006.Fungal endophytes in potato roots studied by traditional isolation and cultivation-independent DNA-based methods.FEMS Microbiol Ecol.58:404–413.

Gray E J,Smith D L.2005.Intracellular and extracellular PGPR:Commonalities and distinctions in the plant-bacterium signaling processes.Soil Biol Biochem.37:395–412.

Hadj Sadok T,Aid F,Bellal M,Abdul Hussain M S.2008.Composition chimique des jeunes cladodes D’opuntia Ficus indica et possibilites de valorisation alimentaire.Agric,Sci Pract Sci J(in French).65:65–66.

Hardie M,Doyle R.2012.Measuring soil salinity.In Shabala S,Cuin C T(eds.)Plant Salt Tolerance:Methods and Protocols.Humana Press,Totowa.pp.415–425.

Huang P T,Patel M,Santagata M C,Bobet A.2009.Classification of Organic Soils.FHWA/IN/JTRP-2008/02.Joint Transportation Research Program.Indiana Department of Transportation and Purdue University,West Lafayette.

Hussain M I,Asghar H N,Akhtar M J,Arshad M.2013.Impact of phosphate solubilizing bacteria on growth and yield of maize.Soil Environ.32:71–78.

Hynes R K,Leung G C Y,Hirkala D L M,Nelson L M.2008.Isolation,selection,and characterization of beneficial rhizobacteria from pea,lentil,and chickpea grown in western Canada.Can J Microbiol.54:248–258.

James E K,Reis V M,Olivares F L,Baldani J I,D¨obereiner J.1994.Infection of sugar cane by the nitrogen- fixing bacterium Acetobacter diazotrophicus.J Exp Bot.45:757–766.

Jamil A,Riaz S,Ashraf M,Foolad M R.2011.Gene expression pro filing of plants under salt stress.Crit Rev Plant Sci.30:435–458.

Jha P,Kumar A.2009.Characterization of novel plant growth promoting endophytic bacterium Achromobacter xylosoxidans from wheat plant.Microb Ecol.58:179–188.

Kader M A,Mian M H,Hoque M S.2002.Effects of Azotobacter inoculant on the yield and nitrogen uptake by wheat.J Biol Sci.2:259–261.

Kathiresan K,Saravanakumar K,Anburaj R,Gomathi V,Abirami G,Sahu S K,Anandhan S.2011.Microbial enzyme activity in decomposing leaves of mangroves.Intl J Adv Biotechnol Res.2:382–389.

Kavitha T,Nelson R,Jesi S J.2013.Screening of rhizobacteria for plant growth promoting traits and antifungal activity against charcoal rot pathogen Macrophomina phaseolina.Int J Pharm Bio Sci.4:177–186.

Kaya C,Tuna A L,Yoka¸s I.2009.The role of plant hormones in plants under salinity stress.In Ashraf M,Ozturk M,Athar H R(eds.)Salinity and Water Stress.Springer,Dordrecht.pp.45–50.

Kaymak H C¸,G¨uven¸c I,Yarali F,D¨onmez M F.2009.The effects of bio-priming with PGPR on germination of radish(Raphanus sativus L.)seeds under saline conditions.Turk J Agric For.33:173–179.

Khan M S,Zaidi A,Ahmad E.2014.Mechanism of phosphate solubilization and physiological functions of phosphate-solubilizing microorganisms.In Khan M S,Zaidi A,Musarrat J(eds.)Phosphate Solubilizing Microorganisms.Springer,Cham.pp.31–62.

Kirst G O.1996.Osmotic adjustment in phytoplankton and macroalgae:The use of dimethylsulfoniopropionate(DMSP).In Kiene R P,Visscher P T,Keller M D,Kirst G O(eds.)Biological and Environmental Chemistry of DMSP and Related Sulfonium Compounds.Springer,Boston.pp.121–129.

Kizildag N,Sagliker H A,Kutlay A,Cenkseven S¸,Darici C.2012.Some soil properties and microbial biomass of Pinus maritima,Pinus pinea and Eucalyptus camaldulensis from the Eastern Mediterranean coasts.Eurasia J Biosci.6:121–126.

Kopeˇcn´y J,Hodrov´a B,Stewart C S.1996.The isolation and characterization of a rumen chitinolytic bacterium.Lett Appl Microbiol.23:195–198.

Kosov´a K,V´ıt´amv´as P,Pr´aˇsil I T,Renaut J.2011.Plant proteome changes under abiotic stress-contribution of proteomics studies to understanding plant stress response.J Proteomics.74:1301–1322.

Kumar A,Kumar K,Kumar P,Maurya R,Prasad S,Singh S K.2014.Production of indole acetic acid by Azotobacter strains associated with mungbean.Plant Archives.14:41–42.

Kumar S,Tamura K,Nei M.2004.MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment.Brief Bioinform.5:150–163.

Kumar V,Aggarwal N K,Singh B P.2000.Performance and persistence of phosphate solubilizing Azotobacter chroococcum in wheat rhizosphere.Folia Microbiol.45:343–347.

Lamosa P,Martins L O,Da Costa M S,Santos H.1998.Effects of temperature,salinity,and medium composition on compatible solute accumulation by Thermococcus spp.Appl Environ Microbiol.64:3591–3598.

Lapinskas E,Piaulokait˙e-Motuzien˙e L.2006.The in fluence of soil acidity on symbiotic and non-symbiotic nitrogen fixation.ˇZemdirbyste.93:210–220.

Laskar F,Sharma G D,Deb B.2013.Characterization of plant growth promoting traits of diazotrophic bacteria and their inoculating effects on growth and yield of rice crops.Glob Res Anal.2:3–5.

Lim J H,Kim S D.2013.Induction of drought stress resistance by multi-functional PGPR Bacillus licheniformis K11 in pepper.Plant Pathol J.29:201–208.

Liu Y,Shi Z Q,Yao L X,Yue H T,Li H,Li C.2013.Effect of IAA produced by Klebsiella oxytoca Rs-5 on cotton growth under salt stress.J Gen Appl Microbiol.59:59–65.

Ma Y,Rajkumar M,Freitas H.2009.Inoculation of plant growth promoting bacterium Achromobacter xylosoxidans strain Ax10 for the improvement of copper phytoextraction by Brassica juncea.J Environ Manage.90:831–837.

Madkour M A,Smith L T,Smith G M.1990.Preferential osmolyte accumulation:A mechanism of osmotic stress adaptation in diazotrophic bacteria.Appl Environ Microbiol.56:2876–2881.

Malik K A,Bilal R,Mehnaz S,Rasul G,Mirza M S,Ali S.1997.Association of nitrogen- fixing,plant-growth-promoting rhizobacteria(PGPR)with kallar grass and rice.Plant Soil.194:37–44.

Mart´ınez-Viveros O M,Jorquera M A,Crowley D E,Gajardo G,Mora M L.2010.Mechanisms and practical considerations involved in plant growth promotion by rhizobacteria.J Soil Sci Plant Nutr.10:293–319.

Mitchell R,Alexander M.1963.Lysis of soil fungi by bacteria.Can J Microbiol.9:169–177.

Moretti M,Gilardi G,Gullino M L,Garibaldi A.2008.Biological control potential of Achromobacter xylosoxydans for suppressing Fusarium wilt of Tomato.Int J Bot.4:369–375.

Morrissey E M,Gillespie J L,Morina J C,Franklin R B.2014.Salinity affects microbial activity and soil organic matter content in tidal wetlands.Glob Change Biol.20:1351–1362.

Nabti E,Sahnoune M,Adjrad S,van Dommelen A,Ghoul M,Schmid M,Hartmann A.2007.A halophilic and osmotolerant Azospirillum brasilense strain from algerian soil restores wheat growth under saline conditions.Eng Life Sci.7:354–360.

Nabti E,Sahnoune M,Ghoul M,Fischer D,Hofmann A,Rothballer M,Schmid M,Hartmann A.2010.Restoration of growth of durum wheat(Triticum durum var.Waha)under saline conditions due to inoculation with the rhizosphere bacterium Azospirillum brasilense NH and extracts of the marine alga Ulva lactuca.J Plant Growth Regul.29:6–22.Nagata S,Adachi K,Sano H.1996.NMR analyses of compatible solutes in a halotolerant Brevibacterium sp.Microbiology.142:3355–3362.

Nikli´nska M,Chodak M,Laskowski R.2005.Characterization of the forest humus microbial community in a heavy metal polluted area.Soil Biol Biochem.37:2185–2194.

Nosrati R,Owlia P,Saderi H,Rasooli I,Malboobi M A.2014.Phosphate solubilization characteristics of efficient nitrogen fixing soil Azotobacter strains.Iran J Microbiol.6:285–295.

Pant G,Agrawal P K.2014.Isolation and characterization of indole acetic acid producing plant growth promoting rhizobacteria from rhizospheric soil of Withania somnifera.J Boil Sci Opin.2:377–383.

Pathani N,Gosal S K,Saroa G S,Vikal Y.2014.Molecular characterization of diazotrophic bacteria isolated from rhizosphere of wheat cropping system from central plain region of Punjab.Afr J Microbiol Res.8:862–871.

Paul D,Nair S.2008.Stress adaptations in a Plant Growth Promoting Rhizobacterium(PGPR)with increasing salinity in the coastal agricultural soils.J Basic Microbiol.48:378–384.

Penrose D M,Glick B R.2003.Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria.Physiol Plant.118:10–15.

Pepper I L,Gerba C P.2004.Environmental Microbiology:A Laboratory Manual.2nd Edn.Elsevier Academic Press,San Diego.

Pichereau V,Pocard J A,Hamelin J,Blanco C,Bernard T.1998.Differential effects of dimethylsulfoniopropionate,dimethylsulfonioacetate,and other S-methylated compounds on the growth of Sinorhizobium meliloti at low and high osmolarities.Appl Environ Microbiol.64:1420–1429.

Plettner I,Steinke M,Malin G.2005.Ethene(ethylene)production in the marine macroalga Ulva(Enteromorpha)intestinalis L.(Chlorophyta,Ulvophyceae):Effect of light-stress and co-production with dimethyl sulphide.Plant Cell Environ.28:1136–1145.

Raj S V,Raja A K,Vimalanathan A B,Tyagi M G,Shah N H,Justin N A J A,Santhose B I,Sathiyaseelan K.2009.Study of starch degrading bacteria from kitchen waste soil in the production of amylase by using paddy straw.Recent Res Sci Technol.1:8–13.

Ramadoss D,Lakkineni V K,Bose P,Ali S,Annapurna K.2013.Mitigation of salt stress in wheat seedlings by halotolerant bacteria isolated from saline habitats.SpringerPlus.2:6.

Rana A,Saharan B,Nain L,Prasanna R,Shivay Y S.2012.Enhancing micronutrient uptake and yield of wheat through bacterial PGPR consortia.Soil Sci Plant Nutr.58:573–582.

Rathi M S,Paul S,Thakur J K.2014.Response of wheat to inoculation with mycorrhizae alone and combined with selected rhizobacteria including Flavobacterium sp.as a potential bioinoculant.J Plant Nutr.37:76–86.

Reddy P P.2013.Recent Advances in Crop Protection.Springer,India.

Rojas-Tapias D,Moreno-Galv´an A,Pardo-D´ıaz S,Obando M,Rivera D,Bonilla R.2012.Effect of inoculation with plant growth-promoting bacteria(PGPB)on amelioration of saline stress in maize(Zea mays).Appl Soil Ecol.61:264–272.

Rouiller J,Souchier B,Bruckert S,Feller C,Toutain F,Verdy J C.1994.M´ethodes D’analyses des sols.In Bonneau M,Souchier B(eds.)P´edologie:2.Constituants et Propri´et´es Du Sol(in French).Masson,Paris.pp.619–654.

Rousk J,Brookes P C,B˚a˚ath E.2009.Contrasting soil pH effects on fungal and bacterial growth suggest functional redundancy in carbon mineralization.Appl Environ Microbiol.75:1589–1596.

S´aenz C,Berger H,Rodr´ıguez-F´elix A,Galletti L,Garc´ıa J C,Sep´ulveda E,Varnero M T,Cort´azar V G,Garc´ıa R C,Arias E,Mondrag´on C,Higuera I,Rosell C.2013.Agro-Industrial Utilization of Cactus Pear.Food and Agriculture Organization,Rome.

Sagervanshi A,Kumari P,Nagee A,Kumar A.2012.Isolation and characterization of phosphate solubilizing bacteria from and agriculture soil.Int J Life Sci Pharma Res.2:256–266.

Sanger F,Coulson A R.1975.A rapid method for determing sequences in DNA by primed synthesis with DNA polymerase.J Mol Biol.94.441–446.

Sashidhar B,Podile A R.2010.Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenase.J Appl Microbiol.109:1–12.

Schwyn B,Neilands J B.1987.Universal chemical assay for the detection and determination of siderophores.Anal Biochem.160:47–56.

Sgroy V,Cass´an F,Masciarelli O,Del Papa M F,Lagares A,Luna V.2009.Isolation and characterization of endophytic plant growth-promoting(PGPB)or stress homeostasis-regulating(PSHB)bacteria associated to the halophyte Prosopis strombulifera.Appl Microbiol Biotechnol.85:371–381.

Shahbaz M,Ashraf M.2013.Improving salinity tolerance in cereals.Crit Rev Plant Sci.32:237–249.

Shahzad S M,Khalid A,Arshad M,Kalil-ur-Rehman.2010.Screening rhizobacteria containing ACC-deaminase for growth promotion of chickpea seedlings under axenic conditions.Soil Environ.29:38–46.

Sharma S B,Sayyed R Z,Trivedi M H,Gobi T A.2013.Phosphate solubilizing microbes:Sustainable approach for managing phosphorus deficiency in agricultural soils.Springer-Plus.2:587.

Shrivastava P,Kumar R.2015.Soil salinity:A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation.Saudi J Biol Sci.22:123–131.

Sierra G.1957.A simple method for the detection of lipolytic activity of micro-organisms and some observations on the influence of the contact between cells and fatty substrates.Antonie Van Leeuwenhoek.23:15–22.

Sim˜oes M,Cleto S,Sim˜oes L C,Pereira M O,Vieira M J.2007.Microbial interactions in bio films:Role of siderophores and iron-dependent mechanisms as biocontrol strategies.In Gilbert P,Allison D,Brading M,Pratten J,Spratt D,Upton M(eds.)Bio films:Coming of Age.Bio film Club,Manchester.pp.157–165.

Sindhu S S,Dadarwal K R.2001.Chitinolytic and cellulolytic Pseudomonas sp.antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp.Cicer in chickpea.Microbiol Res.156:353–358.

Soltani A A,Khavazi K,Asadi-Rahmani H,Omidvari M,Dahaji P A,Mirhoseyni H.2010.Plant growth promoting characteristics in some Flavobacterium spp.isolated from soils of Iran.J Argric Sci.2:106–115.

Stankovi´c M S,Petrovi´c M,Godjevac D,Stevanovi´c Z D.2015.Screening inland halophytes from the central Balkan for their antioxidant activity in relation to total phenolic compounds and flavonoids:Are there any prospective Medicinal Plants?J Arid Environ.120:26–32.

Suneja S,Narula N,Anand R C,Lakshminarayana K.1996.Relationship of Azotobacter chrooccum siderophores with nitrogen fixation.Folia Microbiol.41:154–158.

Tam H M,Diep C N.2014.Isolation,characterization and identification of endophytic bacteria in sugarcane(Saccharum spp.L.)cultivated on soils of the Dong Nai Province,Southeast of Vietnam.Am J Life Sci.2:361–368.

Thompson J P.1989.Counting viable Azotobacter chroococcum in vertisols:II.Comparison of media.Plant Soil.117:17–29.

Tian F,Ding Y Q,Zhu H,Yao L T,Du B H.2009.Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere.Braz J Microbiol.40:276–284.

Torres-Rubio M G,Valencia-Plata S A,Bernal-Castillo J,Mart´ınez-Nieto P.2000.Isolation of enterobacteria,Azotobacter sp.and Pseudomonas sp.,producers of indole-3-acetic acid and siderophores,from colombian rice rhizosphere.Rev Latinoam Microbiol.42:171–176.

Tsavkelova E A,Klimova S Y,Cherdyntseva T A,Netrusov A I.2006.Microbial producers of plant growth stimulators and their practical use:A review.Appl Biochem Microbiol.42:117–126.

Vacheron J,Desbrosses G,Bouffaud M L,Touraine B,Mo¨enne-Loccoz Y,Muller D,Legendre L,Wisniewski-Dy´e F,Prigent-Combaret C.2013.Plant growth-promoting rhizobacteria and root system functioning.Front Plant Sci.4:356.

Vessey J K.2003.Plant growth promoting rhizobacteria as biofertilizers.Plant Soil.255:571–586.

Vogel J P,Woeste K E,Theologis A,Kieber J J.1998.Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction,respectively.Proc Natl Acad Sci USA.95:4766–4771.

Walpola B C,Arunakumara K K I U.2010.Effect of salt stress on decomposition of organic matter and nitrogen mineralization in animal manure amended soils.J Agric Sci.5:9–18.

Wani S H,Singh N B,Haribhushan A,Mir J I.2013.Compatible solute Engineering in plants for abiotic stress tolerance-role of glycine betaine.Curr Genomics.14:157–165.

Wood J M.2007.Bacterial osmosensing transporters.In H¨aussinger D,Sies H(eds.)Methods in Enzymology,Volume 428:Osmosensing and Osmosignaling.Elsevier,Amsterdam.pp.77–109.

Xun F F,Xie B M,Liu S S,Guo C H.2015.Effect of plant growth-promoting bacteria(PGPR)and arbuscular mycorrhizal fungi(AMF)inoculation on oats in saline-alkali soil contaminated by petroleum to enhance phytoremediation.Environ Sci Pollut Res.22:598–608.

Yang J,Kloepper J W,Ryu C M.2009.Rhizosphere bacteria help plants tolerate abiotic stress.Trends Plant Sci.14:1–4.