INTRODUCTION

In the natural environment,plants are continuously subjected to a number of abiotic and biotic stresses.A-mong the abiotic stresses,drought is particularly detrimental to plant growth and yield in agricultural production,affecting the world’s food security(Gamalero and Glick,2012;Singh et al.,2015a;Vurukonda et al.,2016).If current trends continue,it is estimated that the area affected by drought will increase by 2-fold and water resources will decline by 30%by the year 2050(Kasim et al.,2013).In the last 100 years,the average temperature in the Indian subcontinent has risen by 0.57°C and is predicted to increase by up to 2.5°C by 2050 and 5.8 °C by 2100(Venkateswarlu et al.,2008).Plant growth is also often limited by insufficient levels of macro and micronutrients,plant genotype,physical and chemical properties of soil,and growth conditions,thereby contributing to a major decrease of harvest yield in majority of the crops worldwide.To some extent,the impact of biotic and nutritional factors can be overcome using pesticides and biofertilizers,respectively;however,the partial amelioration of the many difficulties that arise as a consequence of various biotic and abiotic factors by non-biological means is highly challenging(Singh et al.,2015a).

Biosynthesis of ethylene is generally increased during both biotic and abiotic stresses,including drought stress,resulting in a reduced rate of root and shoot growth.A number of strains of plant growth-promo-ting bacteria(PGPB)possess the enzyme 1-aminocyclopropane-1-carboxylic acid(ACC)deaminase(EC 4.1.99.4),a pyridoxal 5′-phosphate(PLP)-dependent enzyme that converts ACC,the immediate precursor of ethylene in all higher plants,to α-ketobutyrate(α-KB)and ammonia,thereby lowering the level of plant ethylene(Shaharoona et al.,2006;Glick et al.,2007;Nascimento et al.,2014;Singh et al.,2015b).Bacteriaproducing ACC deaminase exerts its beneficial effects its protecting the plant from the deleterious effects of environmental stresses(Glick,2004,2014),favors nodulation in legumes(Ma et al.,2003;Nascimento et al.,2012),delays flower senescence(Ali et al.,2012),and exhibits biocontrol activity against phytopathogens(Hao et al.,2011).Thus,bacteria with ACC deaminase activity are of great importance in reducing the deleterious effects of environmental stressors and recovering plant growth under various stressful conditions,such as drought.

Isolation of ACC deaminase-producing bacteria and their impact on plant growth under water stress conditions have been reported by a number of researchers(Mayak et al.,2004;Cheng et al.,2007;Shaharoona et al.,2007;Ahmad et al.,2011).Drought is one of the most important factors adversely affecting plant growth and productivity.Enhancement of growth and drought tolerance in PGPB-inoculated tomatoes(Mayak et al.,2004),peas(Arshad et al.,2008;Zahir et al.,2008),maize(Vardharajula et al.,2011),cucumber(Wang et al.,2012),Arabidopsis(Bresson et al.,2013),wheat(Bangash et al.,2013),mung bean(Sarma and Saikia,2014),soybean(Kang et al.,2014),chickpea(Kumar et al.,2016),and velvet bean(Saleem et al.,2015)has been reported.To our knowledge,this is the first report describing the effect of bacterial strains with ACC deaminase activity on finger millet(Eleusine coracana(L.)Gaertn.),one of the most economically important staple food crops in semi-arid areas of Eastern and Southern Africa and South Asia,belonging to the family Poaceae.The present work to aimed to isolate the efficient ACC deaminase-producing PGPB from the rain-fed agriculture soil with farming practices relying on rainfall for water of Central Himalaya of Kumaun region of Uttarakhand,India,and to characterize them for their plant growth-stimulating effects on finger millet under non-stressed and droughtstressed conditions.

地质体当中的不确定因素具有生命周期性，在潜伏期时，不会造成非常严重的安全事故。即在初期，不安全因素已经有所显现，但是其影响较小，容易被人忽略。但是在爆发阶段，会出现严重的安全事故，所有的安全事故因素也均浮出水面。最后这些风险进入结束阶段。从中可见，地质项目工程的风险往往具备一定的生命周期性。我们一定要认真了解地质工程的生命周期性，在隐患的初期将不安全因素解决，如果可以的话，在发展的潜伏期进行相关防范和规避。通过生命周期性的了解我们对于安全投资的认识也会进一步深刻，生命周期性的发展影响着安全投资的走向和发展，根据周期性不同阶段的特征，我们都要做出相对应得投资分析和措施。

MATERIALS AND METHODS

Isolation of ACC deaminase-producing PGPB

Three ACC deaminase-producing drought-tolerant PGPB strains used in this study were isolated from soil samples collected from the rain-fed area of Central Himalaya of the Kumaun region of Uttarakhand,India,using dilution plate technique on Dworkin and Fostor(DF)salt minimal medium containing 3 mmol L−1 ACC as a sole source of nitrogen(N).Plates containing DF salt minimal medium without ACC served as a negative control,while plates with(NH4)2SO4as a N source served as a positive control.The plates were incubated at 28°C for 3 d.Growth of isolates on ACC-supplemented plates was compared with the positive and negative control plates(Dworkin and Foster,1958;Penrose and Glick,2003).The isolates growing well on the ACC-supplemented plates were selected for further study.

Quantification of ACC deaminase activity in isolated PGPB

The acdS gene from the drought-tolerant bacterial strains DPB13,DPB15,and DPB16 was amplified using PCR,with the acdSf3 and acdSr3 primers.It is evident from Fig.3 that a∼680 bp amplicon specific to acdS gene,a functional gene encoding ACC deaminase,was obtained from the genomic DNA of Pseu-domonas sp.DPB13,Pseudomonas sp.DPB15,and Pseudomonas sp.DPB16 by using a specific pair of acdS primers.On performing gene alignment analysis using BLAST algorithm,the given amplified fragment showing 94%sequence similarity with Pseudomonas fluorescens F113(Accession No.CP003150.1)for all three strains DPB13,DPB15,and DPB16 was identified.The obtained gene sequences of acdS gene for strains DPB13,DPB15,and DPB16 were submitted to the NCBI GenBank database under Accession Nos.MF040747,MF040748,and MF040749,respectively.

Screening of bacterial isolates for drought tolerance

Tryptic soy broth(TSB;Difco Laboratories,Detroit,USA)with different water potentials(−0.5,−0.75,and−1.0 MPa)was prepared by adding 20%,25%,and 30%of polyethylene glycol(PEG)8000(Michel,1983)and was inoculated with 1%of overnight bacterial cultures grown in TSB.Four replicates of each isolate were prepared at different water potentials.After incubation at 28°C under shaking conditions(120 r min−1)for 24 h,isolate growth was estimated by measuring the optical density at 600 nm(OD600)using a spectrophotometer(Ray Leigh UV-2601,BRAIC Co.Ltd.,Beijing,China).

Screening of bacterial isolates for plant growth-promoting traits

Phosphorus(P)-solubilizing activity was assessed according to Pikovskaya(1948).Indole acetic acid(IAA)production was measured using Salkowski’s reagent(Gordon and Weber,1951).Siderophore pro-duction was assayed following growth on Chrome Azurol S(CAS)agar plates at 28°C for 3 d(Schwyn and Neilands,1987),and ammonia production was measured as described by Cappuccino and Sherman(1992).

Characterization of bacterial isolates

Fatty acid methyl ester(FAME)analysis. Cellular fatty acids were extracted from the selected bacterial cells following the standard extraction procedure(Sasser,1990).Fatty acid methyl ester pro files were then obtained by running the samples on a gas chromatograph(GC)equipped with flame ionization detector(FID)and MIDI(R)Sherlock Microbial Identification System(MIDI Inc.,Newark,USA)software.Each bacterial strain was identified by comparing its FAME pro file with that in commercial databases(RTSBA6)using the MIS software package.

Isolation,PCR amplification,and sequencing of bacterial 16S rRNA gene. Bacterial strains were characterized by 16S rRNA gene sequence analysis.Bacterial genomic DNA was isolated from overnight grown cells using a genomic DNA extraction kit(HiPuraTMbacterial genomic DNA purification kit,Himedia,India).The sequence of the oligonucleotide primers used for amplification of 16S rRNA genes were forward primer(GM3f)5′-AGAGTTTGATCMTGG-3′ and reverse primer(GM4r)5′-TACCTTGTTACGACTT-3′(Muyzer et al.,1995).DNA samples were amplified on Veriti Thermal Cycler(Applied Biosystems,Foster City,USA).The following program was used for the amplification of 16S rRNA∶preheating at 95 °C for 5 min,denaturation at 95 °C for 1 min,annealing at 42 °C for 1 min,extension at 72 °C for 2 min,and final extension at 72°C for 10 min,followed by a chill at 4°C.The amplified PCR product was purified and sequenced.Obtained sequences were compared with GenBank database using BLAST program.Multiple sequence alignment for the homologous sequence was done by MEGA 5.10 software,and a phylogenetic tree was constructed using neighborjoining method(Tamura et al.,2013).

Guaiacol peroxidase(GPX,EC 1.11.1.7)activity was determined as described by Urbanek et al.(1991)in a reaction mixture(3.0 mL)containing 100 mmol L−1phosphate buffer(pH 7.0),0.1 mmol L−1EDTA,5.0 mmol L−1guaiacol,15 mmol L−1H2O2,and 100µL enzyme extract.The addition of enzyme extract started the reaction,and the increase in absorbance was recorded at 470 nm for 90 s.Enzyme activity was quantified by the amount of tetraguaiacol formed using its molar extinction coefficient(26.6 mmol L−1cm−1)and was expressed as nmol tetraguaiacol min−1mg−1 protein.

Plant materials and growth conditions

Seeds of finger millet(var.VL-149)were procured from the Vivekanand Parvartiya Krishi Anusandhan Sansthan(VPKAS),Almora,Uttarakhand,India.For the experiment, finger millet seeds were surface-disinfected by immersion, first in 3%sodium hypochlorite and then in 70%ethanol for 3 and 1 min,respectively.The seeds were then washed thoroughly three times with sterile distilled water before germination on sterilized Petri dishes containing one sheet of sterilized paper moistened with sterilized distilled water,and placed in an incubator at 30°C for 2 d.All steps were carried out aseptically.The plants were grown in a glass house under controlled conditions at a relative humidity of 60%,temperature of 28± 2°C,a photoperiod of 16 h/8 h day/night cycle,and a light intensity of 400 Em−2s−1between 400 and 700 nm.

Physicochemical characteristics of soil

The soil used in the present study was a 1∶3 mixture of soil and sand.Estimation of organic carbon(C)was done using the method described by Walkley and Black(1934).Available P was determined by the method of Olsen et al.(1954).Available N was determined by Kjeldahl digestion(Pelican Kelplus Kelvac VA equipment,Chennai,India),and potassium(K)content was estimated by flame photometry(Flame Photometer 128 Systronics,Ahmedabad,India).The mixture was characterized with pH of 6.98,1.9 g kg−1total N,10.97 mg kg−1available P,138.88 mg kg−1extractable K,and 7.4 g kg−1organic C.The potting mixture was steam-sterilized by autoclaving at 1.03 MPa at 121°C for 3 h in 3 consecutive days.The sterilized soil mixture was thoroughly mixed and then used to fill plastic pots(10 cm length and 10 cm internal diameter).

为实现快速测量，实验系统需要预先标定，标定的测量范围为100 mm(在正交光栅成像610 mm～710 mm之间)设710 mm处为参考平面高度为0，标定间隔为5 mm，标定调制度比值与高度对应关系已经在图2(b)给出.为了评估该方法的测量精度，标定后实测一个平面，检测平面放置在离参考面32.5 mm位置.图8(a)为CCD获取的检测平面上的正交光栅像，检测平面的恢复结果如图8(b) ，图8(c)为检测平面测量误差的放大图.平面恢复的平均高度为32.49 mm，平面恢复的标准差为0.03 mm,平面恢复的平均误差为-0.01 mm，最大误差为0.09 mm.

Inoculum preparation

Inoculum was prepared by growing PGPB strains in 250-mL flasks containing DF salt minimal medium containing 3 mmol L−1ACC as a sole source of N.The broth in each flask was inoculated with an isolated PGPB strain separately and incubated at 28°C for 24 h in a shaking incubator at 200 r min−1.Optical density at 600 nm was measured to achieve uniform population of bacteria(107–108colony-forming units(CFU)mL−1,OD600= ∼ 0.6)in the broth prior to inoculation.For pot trials,uniform sizes of germinated finger millet seeds were planted in the 1∶3 soil and sand mixture and then inoculated with 1 mL bacterial suspension.Sterile DF medium without inoculation was used as a control.After germination,thinning was carried out,and four uniform sized plants remained in each pot.

Experimental details

Pot trials were conducted in the glass house at the College of Basic Science and Humanities,Govind Ballabh Pant University of Agriculture and Technology,Pantnagar,Uttarakhand(29.05° N,79.52° E),India,to compare the effectiveness of the selected bacterial strains for promoting growth of finger millet under non-stressed and drought-stressed conditions.The experiment was performed in a completely randomized design with four replicates and four treatments(including a control).The moisture level of the soil and sand was maintained at 90%water-holding capacity(WHC)throughout the experiment by weighing the pots and adding water based on the weight loss.However,weight gain due to the growth of plants was ignored.In the present study,two sets of pot trials were set up∶one for non-stressed plants and the other for drought-stressed plants.After 5 weeks,one set of pot trials was subjected to drought stress for 5 d,while the other set of pots(non-stressed)was not drought stressed.In non-stressed pots,the moisture level maintained at 90%WHC till the end of the experiment.After 5 d of drought stress,the moisture level in pots was 35%WHC.The same growth parameters such as shoot and root fresh and dry biomass,plant height,and root elongation were recorded for plants subjected to both drought-stressed and non-stressed conditions on day 40 of plant growth.

Biochemical analysis of plants

Total pigment. The chlorophyll contents of leaves of finger millet plants were measured following the method of Arnon(1949).The absorbance at 470,645,and 663 nm was measured using a UV-VIS spectrophotometer(Ray Leigh UV-2601,BRAIC Co.Ltd.,Beijing,China).

Total proline content. The total proline content was estimated using the method of Bates et al.(1973).Leaf sample(0.2 g)was homogenized in 2.0 mL of 3%sulphosalicylic acid,and the residue was removed by centrifugation.Then,2.0 mL extract was reacted with 2 mL glacial acetic acid and 2 mL acid ninhydrin(prepared by warming 1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6 mol L−1phosphoric acid)for 1 h at 100°C.The reaction was terminated by immersion in an ice bath before the reaction mixture was extracted with 4 mL toluene.The chromophore-containing toluene was warmed to 28°C,its absorbance was measured at 520 nm,and the amount of proline was determined from a standard curve.

Lipid peroxidation. Lipid peroxidation was determined by estimating the amount of malondialdehyde(MDA)produced by the thiobarbituric acid reaction following the method of Heath and Packer(1968).The leaf samples were homogenized in a mortar and pestle with ice-cold extraction buffer and centrifuged at 14000×g for 30 min in a refrigerated centrifuge.The supernatant was used for the determination of MDA content.Four mL of 0.5%thiobarbituric acid solution containing 20%trichloroacetic acid was added to a 1 mL aliquot of the supernatant.The mixture was heated at 95°C for 30 min and then quickly cooled in an ice bath.The absorbance was measured at 532 and 600 nm.After subtracting the non-specific absorbance at 600 nm,the MDA content was determined by its molar extinction coefficient(155 mmol L−1cm−1),and the results were expressed asµg MDA g−1fresh weight.

Antioxidant enzymes of plants

Superoxide dismutase(SOD,EC 1.15.1.1)activity was assayed spectrophotometrically as the inhibition of photochemical reduction of nitro-blue tetrazolium(NBT)at 560 nm(Beauchamp and Fridovich,1971).The reaction mixture(3 mL)consisted of 50 and 13 mmol L−1Na-phosphate buffer(pH 7.8)and L-methionine,respectively,75,10,and 2.0 µmol L−1 NBT,ethylenediaminetetraacetic acid(EDTA),and ribo flavin,respectively,and 0.3 mL enzyme extract.Test tubes containing the reaction mixture were incubated for 10 min in 4000 lx at 35°C.One unit of SOD activity was defined as the amount of enzyme required to cause a 50%inhibition of the rate of NBT reduction measured at 560 nm.

Ascorbate peroxidase(APX)was extracted in 50 mmol L−1phosphate buffer(pH 7).The activity of APX(EC 1.11.1.11)was measured using the method of Nakano and Asada(1981).The reaction mixture consisted of 50 mmol L−1sodium phosphate buffer(pH 7)containing 0.2 mmol L−1EDTA,0.5 mmol L−1ascorbic,and crude enzyme extract.The reaction was initiated by the addition of H2O2to a final concentration of 0.1 mmol L−1.The oxidation of ascorbic acid as a decrease in the absorbance at 290 nm was followed 2 min after starting the reaction.The difference in absorbance was divided by the ascorbate molar extinction coefficient(2.8 mmol L−1cm−1),and the enzyme activity was expressed as nmol H2O2min−1mg−1protein,taking into consideration that 1.0 mol ascorbate was required for the reduction of 1.0 mol H2O2.

Catalase(CAT,EC 1.11.1.6)activity was assayed spectrophotometrically by monitoring the decrease in absorbance of H2O2at 240 nm according to the method of Beers and Sizer(1952).The enzyme was extracted in 50 mmol L−1phosphate buffer(pH 7).The assay solution contained 50 mmol L−1phosphate buffer and 10 mmol L−1H2O2.The reaction was started by the addition of an enzyme aliquot to the reaction mixture,and the change in absorbance was monitored 2 min after starting the reaction.The difference in absorbance was divided by the H2O2molar extinction coefficient(36 mol L−1cm−1).Enzyme activity was expressed asµmol H2O2min−1mg−1protein.

Amplification of acdS gene. The acdS gene encoding ACC deaminase was amplified by PCR using a pair of primers designed by Li et al.(2015).Sequences of primers were as follows∶forward primer(acdSf3)5′-ATCGGCGGCATCCAGWSNAAYCANAC-3′and reverse primer(acdSr3)5′-GTGCATCGACTTGCCCTCRTANACNGGRT-3′.The PCR reaction was carried out on Veriti Thermal Cycler.The reaction conditions of PCR included an initial denaturation step at 94°C for 4 min,35 amplification cycles of denaturation at 94°C for 45 s,annealing at 53 °C for 45 s,and primer extension at 72°C for 1 min,followed by a final extension at 72°C for 7 min.The PCR purified amplicon of acdS gene was purified,cloned,and sequenced.The identity of the acdS amplicon was con firmed using the National Center for Biotechnology Information(NCBI)BLAST algorithm.

Foliar nutrient content

Nitrogen,P,K+,Ca2+,and Na+contents in the oven-dried leaves of finger millet were assessed.Phosphorus content of the plants was estimated by the vanadomolybdate phosphoric acid method after wet digestion with HNO3and HClO4,and then estimated spectrophotometrically(Jackson 1973).The digested samples were also analyzed for K+,Ca2+,and Na+contents by flame photometry(Flame Photometer 128 Systronics,Systronics,Ahmedabad,India).Nitrogen content was estimated by Kjeldahl digestion(Pelican Kelplus Kelvac VA equipment,Chennai,India).

Statistical analysis

Statistical analysis was performed by analysis of variance(ANOVA)using SPSS software(IBM SPSS statistics 20).Comparisons between means were carried out using Duncan’s multiple range tests at a significance level of P＜0.05.

RESULTS

Characterization and identification of PGPB

Three newly isolated ACC deaminase-producing bacteria were used for the present study and tested for different functional properties,such as phosphate solubilization as well as IAA,siderophore,and ammonia production(Table I).These ACC deaminaseproducing PGPB were investigated for their growthpromoting activities by inoculation into the roots of finger millet in pots under non-stressed and droughtstressed conditions.The results indicated that inoculation with ACC deaminase-producing PGPB significantly improved the growth performance of finger millet under both non-stressed and drought-stressed conditions as compared to their respective non-inoculated controls.The ACC deaminase activity of these selected bacteria was quantified and is shown in Table I.

Inoculation of finger millet seeds with ACC deaminase-producing PGPB had a significant positive effect on root and shoot fresh and dry weights,shoot length,and root elongation compared with noninoculated plants under drought-stressed and nonstressed conditions(Fig.4).Under drought-stressed conditions,a maximum increase of 48.6%in shoot fresh weight was observed when the plants were inoculated with strain DPB15,followed by strains DPB16 and DPB13 showing 35.8%and 22.4%increase,respectively.On the other hand,when plants were not exposed to drought stress,inoculation of finger millet seeds with DPB15 yielded a 52.5%increase in shoot fresh weight as compared to non-inoculated plants,followed by strains DPB16 and DPB13 showing 48.2%and 39.0%increase in shoot fresh weight,respectively.

Growth of ACC deaminase-producing bacterial strains in TSB supplemented with PEG 8000

These ACC deaminase-producing bacterial strains(DPB16,DPB15,and DPB13)showed maximum growth in the absence of PEG,while their growth was significantly decreased with increasing concentrations of PEG.However,all strains were able to grow at ametric potential of−1.0 MPa(Fig.2).

TABLE I Functional propertiesa)of the selected bacterial strains

a)IAA=indole acetic acid;ACC=1-aminocyclopropane-1-carboxylic acid.b)α-KB= α-ketobutyrate. c)+and–=the presence and absence of activity,respectively. d)Means±standard errors(n=3).

Bacterial Phosphate Siderophore IAA ACC Ammonia ACC deaminase IAA strain solubilizer producer production deaminase production activity production nmol α-KBb)min−1mg−1protein µg mL−1 DPB16 +c) + + + + 147.31±5.88d) 41.24±1.73 DPB15 + + + + − 157.92±4.62 43.52±1.27 DPB13 + + − + + 51.60±4.41 Not detected

TABLE II Fatty acid methyl ester(FAME)and molecular identifications of the selected bacterial strains

Bacterial FAME Molecular identification strain identification Accession No. Closest NCBI match Similarity%DPB16 Pseudomonas putida biotype A KX267841 Pseudomonas palleroniana 99 DPB15 Pseudomonas putida biotype A KX267848 Pseudomonas fluorescens 99 DPB13 No match KX607475 Pseudomonas palleroniana 99

Fig.1 Identification of 16S rRNA gene sequence established by performing a similarity search against the GenBank database(http://www.ncbi.nih.gov/BLAST).The phylogenetic tree was constructed with existing 16S rRNA gene sequences from different bacteria by using Mega 5.10 software,and Tepidisphaera mucosa 2813(KM052380.1)was used as an out-group.The bacterial isolates described in this study are marked with closed circles.Bootstrap values(n=1000)are listed as percentages at the branching points.

Fig.2 Growth of the bacterial strains(DPB16,DPB15,and DPB13)estimated by measuring the optical density at 600 nm(OD600)in tryptic soy broth with different water potentials(0.50,0.75,and 1.00 MPa)by adding polyethylene glycol 8000.Values are the means with standard errors shown by the vertical bars(n=3).Bars with different letters are significantly different at P ＜ 0.05 using Duncan’s test.CFU=colony-forming units.

Molecular characterization of ACC deaminase(acdS)gene from drought-tolerant bacterial strains

Activity of ACC deaminase in isolated PGPB was assayed according to the method of Penrose and Glick(2003)by measuring the amount of α-KB produced when the enzyme ACC deaminase cleaves ACC,which was determined by comparing the absorbance at 540 nm of a sample to a standard curve of α-KB ranging between 0.1 and 1.0µmol.To measure specific activity of the cultures,protein estimation was carried out according to Lowry et al.(1951).Protein content was estimated by reference to a standard curve of bovine serum albumin(0.1 to 1.0 mg mL−1).The activity of ACC deaminase was expressed in nmol α-KB min−1 mg−1protein.

Plant growth

On the basis of FAME analysis and 16S rRNA gene homology,three bacteria were identified,as listed in Table II.A phylogenetic tree was derived from the obtained sequences of the isolates DPB13,DPB15,and DPB16 with existing sequences in the NCBI databases(Fig.1).

Root fresh weight was significantly reduced in water deficit conditions;however,inoculation of seeds with the selected strains increased the root fresh weight.Under drought stress,the maximum increase in root fresh weight was found with strain DPB15,i.e.,about 49.8%higher than the control,followed by strains DPB16 and DPB13 showing 44.0%and 34.7%increases,respectively,as compared with inoculated plants(Fig.4).Similarly,under non-stressed conditions,strain DPB15 was the most efficient strain showing a 47%increase in root fresh weight compared to the control,followed by DPB16 and DPB13 with a 42.5%increase.Similarly,data comparing the dry weights of roots and shoots,under both drought-stressed and non-stressed conditions,showed a similar pattern in terms of the positive effect of PGPB compared to what was observed with root and shoot fresh weights.Importantly,following drought stress,the dry weights of the roots and shoots of plants inoculated with PGPB were significantly higher than the dry weights of nonstressed non-inoculated plants.

Inoculation of finger millet seeds with ACC deaminase-producing PGPB significantly increased the shoot length under water-deficit condition(Fig.4).Strain DPB16-inoculated plants showed a maximum increase in shoot length up to 24.1%compared to noninoculated plants under drought stress,followed by strains DPB15 and DPB13 showing 18.5%and 13.7%increases in shoot length,respectively.Under nonstressed condition,strain DPB16 exhibited a maximum increase in shoot length of 24.4%as compared to untreated control plants,followed by strains DPB15 and DPB13 showing 22.3%and 18.3%increases,respe-ctively.Under drought-stressed conditions,a maximum increase of 29.9%in root length was observed in plants with DPB15-inoculated,followed by DPB16(23.0%)and DPB13(14.4%),whereas under non-stressed condition,the DPB15 showed a maximum of 33.0%increase in root length,followed by DPB16(28.6%)and DPB13(24.3%).

环保清淤处理技术在施工过程中对于水体浊度的控制较好，近年来在中小型河道的清淤工作中取得较好的成绩，特别是在抑制污染物扩散、泄漏等方面。该技术的淤泥清除效果超过95% 以上，清淤效果较好。缺点是成本较高，实际运用较少。

Fig.3 Amplification(∼680 bp)of acdS gene from the droughttolerant bacterial strains(DPB16,DPB15,and DPB13).M=marker.

Fig.4 Growth of finger millet non-inoculated(control)and inoculated with bacterial strains(DPB16,DPB15,and DPB13)under non-stressed and drought-stressed conditions.Values are the means with standard errors shown by the vertical bars(n=3).Bars with different letters are significantly different at P ＜ 0.05 using Duncan’s test.

Plant leaf total pigment content

In order to examine the impact of the newly isolated PGPB strains on the photosynthetic efficiency of finger millet plants exposed to water-deficit conditions,we first determined the leaf chlorophyll content of finger millet plants.The total leaf chlorophyll content was significantly increased in finger millet plants inoculated with PGPB in comparison to non-inoculated plants,indicating that these bacteria maintained plant chlorophyll levels under drought-stressed conditions(Table III).This was consistent with the visual observation that finger millet plants inoculated with bacteria producing ACC deaminase had much darker green leaves than the control.A 59.2%increase in total pigment content was recorded in DPB16-inoculated plants under drought stress compared to non-inoculated plants,followed by a 47.2%increase with strain DPB15 and a 43.6%increase with strain DPB13.Under non-stressed conditions,strain DPB16 increased total chlorophyll content by 30.4%,followed by strains DPB13 and DPB15 showing 21.4%and 17.2%increases in total chlorophyll content,respectively,compared to noninoculated treatment.

从上述定义可以看出，这里的样本值与随机试验的样本点有关但又有所不同，从随机试验的角度看，所谓总体实际上是随机试验所有可能的结果，也就是样本空间，由于随机变量是样本空间到实数域的映射，所以也把随机变量称为总体．这里的随机样本指的是n个随机变量的笛卡尔积，所以也可以说随机样本是一个随机向量(X1, X2, …, Xn)，且每一个随机分量都有相同的分布函数．这样说可能会让人难以理解，通俗地说，所谓随机样本就是从总体中随机抽取n个样本点构成的集合．

Plant leaf total proline content

利用道德矛盾情境进行判定的方式检测青少年道德判断能力以及道德判断能力与价值观之间的关系。结果表明：青少年道德判断能力与价值观之间没有明显的关联。具体而言，道德矛盾情境判断高水平及低水平之间的学生在价值观上无较大差异。其中，高分组对于社会公正观的态度明显低于低分组，而在人权观上高于低分组。因此，从一定角度而言，道德判断能力建立在学生对周围事物固有的认知上，而价值观则更多属于学生日积月累所形成的对生活及道德的态度。

奉献精神是鞭策我们牢记立党为公、执政为民本质要求和全心全意为人民服务根本宗旨的强大道德力量。“水可载舟，亦可覆舟”。奉献精神昭示我们，只有坚持以人民为中心，始终保持同人民群众的血肉联系，我们党才能保持其根本性质、恪守根本宗旨、牢记根本使命、巩固执政地位。

图6反映了地震波在地下倾斜状矿体模型中传播状态。t=80 ms时，由于倾斜体自身特征原因，当S波到达倾斜矿体，以一定的倾角入射到介质体分界面，P波与S波在遇到分界面处发生反射、透射以及散射，各种波之间相互叠加。

TABLE III Total pigment,malondialdehyde(MDA),and proline contents of finger millet plants non-inoculated(control)and inoculated with bacterial strains(DPB16,DPB15,and DPB13)under non-stressed(NS)and drought-stressed(DS)conditions

a)FW=fresh weight. b)Means±standard errors(n=3). c)Means in the same column followed by different letters are significantly different at P ＜ 0.05 using Duncan’s test.

Treat- Total pigment MDA Proline ment NS DS NS DS NS DS mg g−1FWa)µg g−1FW Control 1.03±0.050b)ac) 0.466±0.009a 15.48±0.90b 18.49±0.07c 6.08±0.138a 9.10±0.318a DPB16 1.48±0.038b 1.143±0.048c 13.33±0.66ab 13.48±0.62ab 8.41±0.238b 12.68±0.177bc DPB15 1.24±0.042ab 0.883±0.067b 11.86±0.62a 12.04±0.99a 9.14±0.225b 14.22±0.341c DPB13 1.31±0.084b 0.826±0.115b 14.19±0.25b 15.20±0.14b 7.65±0.212ab 11.71±0.273b

Plant leaf lipid peroxidation

Since the imposition of drought stress on plants leads to enhanced membrane peroxidation in leaf tissues,increasing the leaf MDA content,we determined the leaf MDA content of finger millet plants following various treatments.Under drought stress,the leaf MDA content of finger millet plants inoculated with ACC deaminase-producing PGPB strains was decreased by 53.5%in DPB15-inoculated plants,followed by 37.2%in DPB16-inoculated plants and 21.7%in DPB13-inoculated plants,as compared with non-inoculated plants(Table III).Under non-stressed condition,MDA contents of finger millet plants inoculated with strains DPB16 and DPB13 did not show any significant difference compared with non-inoculated plants;however,we observed a small reduction in MDA content of plants inoculated with strain DPB15.

Increasing leaf proline content is crucial for main-taining the osmotic potential of leaf tissues,which plays an important role in protecting leaves from severe dehydration under drought stress.Inoculation of ACC deaminase-producing PGPB resulted in a remarkable 1.56-fold increase in the leaf proline content of finger millet plants inoculated with strain DPB15,followed by strains DPB16(1.39-fold increase)and DPB13(1.29-fold increase)under drought-stressed conditions,as compared with non-inoculated control(Table III).Under non-stressed conditions,the bacterial treatments increased the leaf proline content by a small extent as compared with non-inoculated control.

Antioxidant enzymes(SOD,APX,CAT,and GPX)of plants

There was no significant difference observed between inoculated and non-inoculated treatments under non-stressed conditions with respect to the activities of antioxidant enzymes(SOD,APX,CAT,and GPX),except in a few cases(Table IV).The maximum increase in SOD activity under drought stress was observed in the plants inoculated with strain DPB15(33.8%),followed by strains DPB16 and DPB13 showing 29.9%and 28.1%increases,respectively.Also,increases in the activities of CAT(25.5%–36.2%),APX(24.9%–33.5%),and GPX(36.4%–47.5%)in inoculated finger millet plants were observed compared to noninoculated control under drought stress.A correlation study between non-enzymatic and enzymatic antioxidants under drought-stressed condition demonstrated significant positive correlations between CAT and chlorophyll(correlation coefficient(r)=0.978,P ＜ 0.05),SOD and proline(r=0.963,P＜0.05),GPX and SOD(r=0.988,P＜0.05),APX and SOD(r=0.986,P＜0.05),APX and GPX(r=0.980,P＜0.05),and proline and APX(r=0.977,P＜0.05)(Table V).Significant negative correlations between SOD and MDA(r=−0.962,P＜0.05),GPX and MDA(r=−0.990,P＜0.01),and APX and MDA(r=−0.974,P＜0.05),and also a significant negative perfect correlation between proline and MDA(r=−1.000,P＜0.05)were observed.

Plant foliar nutrient content

Contents of N,P,K+,Ca2+,and Na+in the shoots of finger millet plants was negatively affected by drought stress(Table VI).Plant nutrient analysis showed that the N(41.8%),P(28.0%),K+(18.9%),Ca2+(19.6%),and Na+(46.3%)contents were significantly lower in drought-stressed plants compared to non-stressed plants.However,inoculation of seeds with the three selected bacterial strains increased the N(16.9%–35.7%),P(32.7%–45.2%),K+(10.5%–14.6%),Ca2+(12.2%–21.9%),and Na+(16.9%–30.9%)contents of finger millet plants under drought stress.Similarly,under non-stressed conditions,the inoculation of finger millet seeds with bacterial strains significantly increased the N(26.3%–42.1%),P(26.9%–37.7%),K+(8.2%–17.6%),Ca2+(9.0%–36.5%),and Na+(15.4%–27.5%)contents as compared to non-inoculated control.

TABLE IV Antioxidant enzymea)activities of finger millet plants non-inoculated(control)and inoculated with bacterial strains(DPB16,DPB15,and DPB13)under non-stressed(NS)and drought-stressed(DS)conditions

a)SOD=superoxide dismutase;CAT=catalase;APX=ascorbate peroxidase;GPX=guaiacol peroxidase. b)FW=fresh weight. c)Means±standard errors(n=3). d)Means in the same column followed by different letters are significantly different at P ＜ 0.05 using Duncan’s test.

Treat- SOD CAT ment NS DS NS DS units mg−1FWb)µmol H2O2mg−1protein Control 6.68±0.133c)ad) 8.70±0.087a 127.62±4.25a 146.18±6.10a DPB16 7.03±0.136ab 12.42±0.092c 125.76±9.35ab 229.09±5.06c DPB15 7.10±0.204ab 13.14±0.034d 128.50±6.81ab 196.26±4.30d DPB13 7.20±0.126b 12.10±0.074b 123.96±3.79b 201.37±3.77b Treat- APX GPX ment NS DS NS DS nmol H2O2min−1mg−1protein nmol tetraguaiacol min−1mg−1protein Control 860.85±30.81a 1334.38±44.80a 273.61±15.04a 507.13±12.91a DPB16 930.37±52.71a 1795.99±45.90b 300.08±19.99ab 899.64±15.63c DPB15 911.46±52.57a 2005.87±72.61c 318.48±17.84ab 965.74±12.30d DPB13 905.86±45.01a 1776.43±18.61b 336.57±14.05b 797.70±19.75b

TABLE V Correlation coefficient among studied parameters of non-enzymatic and enzymatic antioxidantsa)of finger millet plants under droughtstressed conditions

*and**Significant at P＜0.05 and P＜0.01,respectively. a)Chl=chlorophyll;MDA=malondialdehyde;SOD=superoxide dismutase;CAT=catalase;GPX=guaiacol peroxidase;APX=ascorbate peroxidase. b)ns=not significant.

Variable Chl MDA Proline SOD CAT GPX APX Chl 1.000 MDA −0.838nsb) 1.000 Proline 0.834ns −1.000** 1.000 SOD 0.926ns −0.962* 0.963* 1.000 CAT 0.978* −0.786ns 0.779ns 0.854ns 1.000 GPX 0.903ns −0.990** 0.990* 0.988* 0.851ns 1.000 APX 0.851ns −0.974* 0.977* 0.986* 0.759ns 0.980* 1.000

DISCUSSION

The bacterial strains isolated from the rain-fed soil of Central Himalaya of Uttarakhand,India exhibited different growth rates on DF salt minimal medium containing ACC,indicating that bacterial strains possess different enzymatic potentials to hydrolyze ACC(Table I),and thus could have differential PGPB activity on inoculated finger millet plants.Biochemical assay of ACC deaminase revealed the secretion of this enzyme by all three drought-tolerant bacterial strains,which was con firmed by PCR amplification of the acdS gene using the same primers as reported earlier by Li et al.(2015).Amplification of acdS gene provided proof of the presence of the gene in the form of a∼680 bp gel band.The ACC deaminase gene encoding the ACC deaminase enzyme has been isolated from different soil bacteria under both non-stress and abiotic stress conditions,such as drought,water logging,salinity,and heavy metals.Therefore,the acdS gene coding for enzyme ACC deaminase can be a very useful candidate gene for the development of bio-inoculants for abiotic stress tolerance in plants.

Our findings are supported by the study of Magnucka and Pietr(2015),who revealed that inoculation with PGPR having ACC deaminase activity significantly increased root elongation,coleoptile length,and shoot biomass of wheat.Several studies have also demonstrated that PGPB strains possessing ACC dea-minase can improve the growth of plants under drought-stressed condition(Lim and Kim,2013;Naveed et al.,2014).In addition to ACC deaminase activity,strains DPB16 and DPB15 also had the ability to synthesize IAA in the presence of L-trp in the growth medium.The IAA secreted by a bacterium may promote root growth directly by stimulating plant cell elongation or indirectly by in fluencing bacterial ACC deaminase activity(Patten and Glick 2002;Barnawal et al.,2013).In fact,it has been suggested that IAA and ACC deaminase likely work in a concerted manner to stimulate plant growth,especially root elongation(Glick et al.,1998;Noreen et al.,2012;Glick,2014).The IAA acts as a transcription factor for ACC synthase activity,which catalyzes the production of ACC in plants,and ACC stimulates ACC deaminase activity in bacteria(Glick et al.,2007).Therefore,ACC deaminase activity and IAA production traits of rhizobacteria are considered as one of the main plant growth-promoting attributes(Glick et al.,2007;Belimov et al.,2009).

The results of the present study are consistent with the results of Arshad et al.(2008),who showed that inoculation of pea seedling with ACC deaminaseproducing P. fluorescens and P.putida caused an increase in root elongation and root biomass over the respective non-inoculated control under drought stress.Additionally,these results were consistent with the observations of Mayak et al.(2004),who found that an ACC deaminase-producing strain,Achromobacter piechaudii,protected tomato plants from drought stress by lowering the levels of stress ethylene.In all of these instances,the data were consistent with PGPB-mediated amelioration of higher level of stress ethylene,so the root system became more prominent in plants under drought stress when treated with ACC deaminase-producing PGPB.Under non-stressed condition,significant increases in shoot length and weight were also recorded in response to inoculation with ACC deaminase-producing PGPB,as compared with respective non-inoculated controls.

监督范围不够明确。就农商行而言，既有纪委监督，又有监事会的监督，那么二者之间的职责定位是什么，如何规避重复监督，制度上没有明确。而站在提升监督效率、避免出现重复监督或监督“真空”的角度，显然要予以明确或建立协调机制；职责范围内，哪些应实行全程监督，哪些可采取事后监督方式，单纯从效率和竞争的需要来讲，也应进行制度安排；只有这样，纪检监督才更加科学、合理、有效。

In the present study,the shoot and root fresh and dry weights of all plants inoculated with ACC deaminase-producing PGPB were significantly higher under both drought-stressed and non-stress conditions,as compared with non-inoculated controls.In addition,ACC deaminase-producing PGPB have a positive in fluence on the total pigment content of plants,thereby improving their photosynthetic efficiency under drought-stressed conditions(Gamalero et al.,2010;Barnawal et al.,2013).The newly isolated PGPB strains(such as DPB16)considerably increased the total pigment content in plants exposed to both droughtand non-stressed conditions,as compared to their respective non-inoculated controls.A significant reduction in MDA content,a product of lipid peroxidation under stress conditions,was also observed in finger millet plants inoculated with strains DPB16 and DPB15 as compared to non-inoculated controls under both non-stressed and drought-stressed conditions.The decrease in MDA content of drought-stressed plants treated with ACC deaminase-producing PGPB correlates management of lipid peroxidation to better stress tolerance mechanisms(Arbona et al.,2008;Evelin et al.,2012).

设计原则包括：（1）该区排水体制采用雨、污分流制。雨水分散排放，污水集中收集处理达标后排放。（2）采用重力流模式，雨水与排洪系统统一考虑，就近分散排放。合理布置管道标高，预留适量的支管接口，便于周边雨、污水的接入，并且可以避免与其他管线发生冲突。（3）海绵城市设计依据《武汉市海绵城市规划设计导则》（以下简称“导则”）相关内容执行。

在核心素养背景下，想要提升小学数学生活化探究教学质量，教师首先应当结合教材内容引入有效的生活案例，加强学生的理解。还要积极利用多媒体教学设备对数学问题进行生活化的展现，帮助学生联系生活实际进行教学思考。同时在教学课堂中，教师应当积极为学生提供多种生活化教学工具，让他们在实践中获得对知识的加深理解，然后通过积极地创建生活化教学情境，让学生专注于教学课堂，结合生活经验进行对问题的探究。最后还要将课堂内容进行课外的拓展，让学生通过探究结合生活经验对数学问题进行思考与探讨。通过这一系列的数学生活化教学措施，可以有效地提升学生的数学探究与学习能力，促进学生核心素质能力的全面提升。

TABLE VI Contents of N,P,K+,Ca2+,and Na+in the shoots of finger millet plants non-inoculated(control)and inoculated with bacterial strains(DPB16,DPB15,and DPB13)under non-stressed(NS)and drought-stressed(DS)conditions

a)Means±standard errors(n=3). b)Means in the same column followed by different letters are significantly different at P ＜ 0.05 using Duncan’s test.

Treat- N P K+ment NS DS NS DS NS DS g kg−1 Control 16.9±0.117a)ab) 9.8±0.056a 11.0±0.131a 7.0±0.043a 20.1±0.056a 16.3±0.067a DPB16 29.2±0.187c 15.2±0.070c 17.7±0.066b 12.8±0.152c 24.4±0.054c 18.2±0.048b DPB15 22.9±0.093b 12.3±0.093b 19.3±0.123b 11.2±0.175bc 21.9±0.048b 19.1±0.051b DPB13 23.8±0.166b 11.8±0.017ab 15.0±0.109b 10.4±0.164b 22.3±0.047b 18.3±0.039b Treat- Ca2+Na+ment NS DS NS DS g kg−1 Control 21.7±0.046a 17.5±0.064a 10.7±0.032a 5.7±0.028a DPB16 34.2±0.053d 20.3±0.068bc 13.5±0.029b 7.4±0.037bc DPB15 29.0±0.047c 22.4±0.071c 14.8±0.033c 8.3±0.0394c DPB13 23.8±0.061b 19.9±0.066b 12.6±0.039b 6.9±0.027b

Plants respond to water-deficit conditions by increasing their antioxidant potential and osmolyte production,which in turn increases the synthesis of defense-related enzymes and osmotic potential within the cell(Farooq et al.,2009).The present study also correlated the water stress-induced increased proline content,enzymatic antioxidant activities,and chlorophyll content,and decreased MDA content following inoculation of ACC deaminase-producing bacteria.Activities of SOD,CAT,APX,and GPX as well as proline and chlorophyll contents had negative correlations with MDA content,indicating that the selected strain might ameliorate the effects of drought-induced reactive oxygen species(ROS)accumulation on photosynthesis up to a certain level.The increased amount of photosynthetic pigments in inoculated plants probably could be a cause of improved plant growth under waterdeficit conditions.Therefore,inoculation of plants with selected PGPB with ACC deaminase activity induces the over-production of these ROS-scavenging enzymes,which in turn reduces levels of over-produced ROS,thereby conferring drought tolerance(Arbona et al.,2008).Improvement in the foliar nutrient uptake of N,P,K+,Ca2+,and Na+,and consequently increased root growth and biomass production in response to selected strains demonstrated their PGPB activity in inoculated finger millet plants.The outcome obtained from this study suggest that ACC deaminaseproducing bacterial strains can be used to build up a consortium for enhancing crop production and cut down dependence on synthetic chemical fertilizers.

同时，随着以物质需求为主的基础需求相对满足，对自我价值或虚荣的需求会越来越大，所以，技术进步使竞争加剧且成功率极低的社会很容易使人失落，缺乏幸福感。这种现象在前人工智能的当代已经表露。上述分析可以局部解释当代发达国家的某些社会现象，例如，美国抑郁症患者比例极高，日本陷入低欲望社会。未来，随着技术的日益进步，尤其是人工智能的进步，强竞争社会带给人类的不幸福感、失落感和失败感会越来越严重，这不限于那些失败者和不成功者，也包括部分成功者。

ACKNOWLEDGEMENT

The authors are thankful to the Indo-Australian project for providing financial support for this study.

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