Impaginato 19 Adv. Hort. Sci., 2018 32(1): 19-26 DOI: 10.13128/ahs-21174 The impact of Sinorhizobium meliloti and Pseudomonas fluorescens on growth, seed yield and biochemical product of fenugreek under water deficit stress S. Bolandnazar 1 (*), A. Sharghi 2, H. Naghdi Badhi 3, A. Mehrafarin 3, M.R. Sarikhani 4 1 Department of Horticulture, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. 2 Department of Horticulture Science, Islamic Azad University, Science and Research Branch, Teheran, Iran. 3 Medicinal Plants Research Centre, Institute of Medicinal Plants, ACECR, Karaj, Iran. 4 Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. Key words: nicotinic acid, PGPR, seed, trigonelline, water use efficiency. Abstract: Bacteria that colonize plant roots and promote plant growth are referred to as plant growth-promoting rhizobacteria (PGPR). For a long-serving period, the PGPRs have been applied as biofertilizers in crops culture. Recent studies indicated the importance of PGPR for controlling the water deficit. The present study investigates the effects of two different PGPRs on some morpho- physiological characteristics in fenugreek under water deficit stress. The first factor was application of four PGPR levels including (1. Sinorhizobium meliloti, 2. Pseudomonas fluorescens, 3. combination of S. meliloti and P. fluorescens and 4. control without bacterial inoculation) and four levels of soil water con- tent including 40%, 60%, 80% and 100% of field capacity (FC) was considered as second factor. The results showed that, leaf area, shoot fresh and dry weight, nitrogen, phosphorus and potassium content, and water use efficacy (WUE) were significantly improved by PGPR inoculation and individual use of PGPR was more effective. Decreasing of soil water content up to 0.40 FC and inocula- tion of two bacteria led to increase of secondary metabolites such as nicotinic acid and trigonelline. However seed yield was decreased in PGPR treated plants. 1. Introduction Insufficient water induces a stress in plants called water deficit stress (Dodd and Ryan, 2016). Water deficit stress has major effects on plant growth and development, limiting crop production in the worldwide. (*) Corresponding author: bolandnazar@tabrizu.ac.ir Citation: BOLANDNAZAR S., SHARGHI A., NAGHDI BADHI H., meHRAfARIN A., SARIkHANI m.R., 2018 - The impact of Sinorhizobium meliloti and Pseudomonas fluorescens on growth, seed yield and biochemical product of fenugreek under water deficit stress. - Adv. Hort. Sci., 32(1): 19-26 Copyright: © 2018 Bolandnazar S., Sharghi A., Naghdi Badhi H., mehrafarin A., Sarikhani m.R. This is an open access, peer reviewed article published by firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 12 September 2017 Accepted for publication 8 November 2017 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(1): 19-26 20 Water deficit negatively affects the plant growth and reproduction and disrupts the whole-plant functions (Bray, 2004; Hummel et al., 2010). It causes cellular changes such as solutes concentration, cell volume alteration, disruption of water potential gradients, changes in membrane shape and disrupting its integrity, loss in turgor pressure, and protein denatu- ration (Bray, 1997; Bartels and Sunkar, 2005). Water deficit is a major threat to agricultural production and tolerance to drought conditions is one of the main targets for crop improvement (Salekdeh et al., 2009). Plant growth-promoting rhizobacteria (PGPR) are rhizosphere bacteria which constitute symbiotic rela- tionships in large varieties of plants and are used as a biofertilizer (Shaukat et al., 2006). PGPR have been reported to confer positive effects and induce plant resistances to environmental stresses and diseases caused by pathogens (kloepper et al., 2004; mayak et al., 2004 a, b; Compant et al., 2005; He and Yang, 2007; Dimkpa et al., 2009; Yang et al., 2009). A wide variety of mechanisms that can improve the plant growth, have been suggested to be impressed by PGPR. These involved mechanisms are as follows: nitrogen fixation (Van Loon, 2007), production of 1- Aminocyclopropane-1-carboxylate deaminase (ACC) (Govindasamy et al., 2008), production of volatile organic compounds (Ryu et al., 2003), induction of systemic resistance (Chandler et al., 2008), phytohor- mone production (Vessey, 2003), siderophore pro- duction (el-Tarabily and Sivasithamparam, 2006), phosphate solubilization (Ryu et al., 2003) and potas- sium releasing (Sarikhani et al., 2016). fenugreek (Trigonella foenum-graecum L.) is a member of the fabaceae family, cultivated world- wide as a semiarid crop, and traditionally used as a medicinal plant. fenugreek is grown as a spice and a vegetable crop and also have been used as a tradi- tional therapy for the remedy of diabetes (miraldi et al., 2001; Smith, 2003; fernández-Aparicio et al., 2008). Its effect as an antidiabetic and antiathero- sclerotic have been documented (Ajabnoor and Tilmisany, 1988; Sharma and Raghuram, 1990). fenugreek’s leaves are a rich source of iron, calcium, β-carotene and other vitamins and its seeds contain tannic acid, diosgenin, trigocoumarin, alkaloids trigonelline, trigomethyl coumarin, gitogenin and vit- amin A (Warke et al., 2011). Recently, published liter- atures indicated that PGPR ameliorate the plants tol- erance to abiotic stresses through a variety of mech- anisms (Srivastava et al., 2008; Sandhya et al., 2010). Also beneficial effects of PGPRs on medicinal plants have been reported (Jaleel et al., 2007). Shafighi et al. (2014) reported that inoculation of fenugreek with PGPR increases plant height, vegeta- tive growth and seed yield in both well watered and water limited condition. Rubin et al. (2017) empha- sized that application of PGPR decreases abiotic stress especially drought stress in various plants. They summarized that PGPR inoculation not only improves root and shoot biomass in non-stress condi- tion but also it can enhance aerial biomass and reproductive yield under drought stress condition, however root mass was not increased under stress. In enormous studies, the synergistic effect of nitro- gen fixing bacteria especially Rhizobia and phosphate solubilizing bacteria such as Pseudomons has been reported but application of these PGPR in stressed condition needs more attention. Therefore, in this study we focused to the inoculation effect of two native and endogenous PGPR (Sinorhizobium meliloti Tabriz and Pseudomonas fluorescens Tabriz) in fenu- greek under drought stress condition. 2. Materials and Methods The seeds of fenugreek with good germination quality was provided from “Jahad Daneshgahi-Iranian Institute of medicinal Plants”, karaj, Iran. The present investigation, carried out in research greenhouse of faculty of Agriculture at the University of Tabriz dur- ing 2015-2016. Two bacteria, including Pseudomonas fluorescens Tabriz and Sinorhizobium meliloti Tabriz were obtained from the Laboratory of Soil Biology, University of Tabriz (Tabriz, Iran). Nutrient Broth (NB) and Yeast mannitol Broth (YmB) were used to pre- pare a primary culture of Pseudomonas and Sinorhizobium respectively to inoculate seeds of plant. The experiments were conducted in a factorial design based on completely randomized block design with three replications. The first factor was applica- tion of PGPR in 4 levels including 1. S. meliloti, as nitrogen fixing bacterium 2. P. fluorescens, as phos- phorous solubilizing bacterium 3. combination of S. meliloti and P. fluorescens 4. negative control with- out any bacteria and fertilizer treatment. The second factor was soil water content treatment based on field capacity (fC) in 4 levels (100, 80, 60 and 40% of fC). Seed of fenugreek was sown in a plastic pot which had 5 kg soil and after establishment 5 plants remained in each pot. Soil water content was main- tained as aforementioned values by daily weighting of pots by digital scale and water loss by evapotran- Bolandnazar et al. - The impact of S. meliloti and P. fluorescences on fenugreek 21 spiration was added to each pot. Plants kept in a greenhouse under a 16 h photoperiod, 24±4/18±3°C day/night temperatures, and 40-60% relative humidi- ty. At the end of the experiment leaf area was mea- sured, by the leaf area meter (LI 3100C area meter, LI-COR, USA). Dry weight of each part was deter- mined after drying at 72˚C until constant weight. The fresh and dry weight plants were determined using a digital weighing scale. The composition of potassium and phosphorus was determined by nitric perchloric and nitric acid diges- tion methods (Zasoski and Burau, 1977; Havlin and Soltanpour, 1980). Phosphorous was measured by a vanadate-molybdate method using a spectropho- tometer (motic, CL-45240-00, China) and k was determined using a flame photometer (model 405G, Iran). Nitrogen was measured according to the kjeldahl method that involves changing the form of organic nitrogen to ammonium (NH4) by concentrat- ed sulfuric acid and then measuring the amount of ammonium production (Baker and Thompson, 1997). Also the seed yield was recorded at maturity. Water use efficiency (WUe) was calculated by the following formula: WUe = DW/UW In this formula, DW and UW represent dry mass production and the amount of consumed water, respectively (karimi and Roosta, 2014). Analysis and quantization of trigonelline Trigonelline in the seed sample was measured according to modified method of Zheng and Ashihara (2004). The samples were ground with 80% methanol and magnesium oxide (mgO) in a mortar and pestle. After incubation at 60°C for 30 min, the homo- genates were centrifuged and the supernatant was collected. After complete evaporation of methanol, the methanol-soluble extracts were dissolved in dis- tilled water. The samples were filtered using a dis- posable syringe filter unit and the aliquots were used for determination of trigonelline (TG) by HPLC. The analyses of the samples were carried out using a knauer k2600A liquid chromatography (Germany), equipped with a Nucleosil C18 (150 mm × 4.6 mm I.D, 5 μm) column. A mixture of methanol: water (50:50 v/v) served as the mobile phase and pH of solution adjusted to 5.0 with 50 mm sodium acetate. The elution has been made in an isocratic mode at a flow rate of 1 mL min-1 and the detection made at 268 nm by UV detector from the above mentioned company (koshiro et al., 2006). One analysis requires 20 min. The retention time of this alkaloid was 4.4 min. Before carrying out HPLC analysis, we made cali- bration curve by using different concentrations (0.1, 0.2, 0.5, 0.7 and 1.0 mg mL-1) of trigonelline in phase media. Then calibration curve made with trigonelline and the correlations were excellent for trigonelline. This process was performed according to United States Pharmacopoeia (U.S. Pharmacopeia, USA) by cold extraction method as directed for alcohol solu- ble material, except where water was used in place of alcohol. Measuring nicotinic acid for the measurement of nicotinic acid, it was car- ried out according to modified martin et al. (1997). The 0.5 g of fenugreek seed powder was mixed with 0.5 g of magnesium oxide (mgO) and 30 ml of dis- tilled water was added to it. The resulting mixture for 30 minutes at 100°C was placed in bath water bath. After cooling, the resulting mixture was filtered using filter paper (1) and was brought to a volume of 50 ml with distilled water. finally absorption at a wave- length of 263 nm of the samples was measured by a spectrophotometer. Nicotinic acid concentrations were determined using the standard curve. Statistical analysis All collected data were subjected to two-way analy- sis of variance (ANOVA) through PROC GLm proce- dure, using a SAS statistical package (SAS Institute, software Version 9.4, Cary, NC, USA). If interactions were significant, means were compared by Duncan’s multiple range test to determine whether means of the dependent variable were significantly different at P<0.05. 3. Results Analysis of data variances indicated that the effect of PGPR and soil water content and their interaction on leaf area, shoot fresh and dry weight were significant (P≤0.01). means comparison showed that PGPR inoculation increased fenugreek leaf area, shoot dry and fresh weight especially S. meliloti (Table 1). By increasing of water deficit stress, leaf area, shoot fresh and dry weight was decreased (Table 2). In aspect of interaction between PGPR inoculation and water stress, the highest and lowest leaf area, shoot fresh and dry weight was observed in well watered (100% fC) and combination of S. meliloti and P. fluorescens treated plants and severe water stressed control plants respectively (fig. 1, 2 and 3). It seems that in normal condition S. meliloti Adv. Hort. Sci., 2018 32(1): 19-26 22 improved aerial growth of fenugreek better than P. fluorescens, whereas combination use of two PGPR bacteria was successful in enhancement of shoot growth better than individual using (fig. 1). Un-inoculated control plants produced significantly higher seed yield per pot than PGPR treated fenu- greek (Table 1). Water limitation led to decrease in seed yield (Table 2). The maximum seed weight was observed in control plants (fig. 4). The nitrogen, phosphorus and potassium concen- tration was significantly affected by PGPRs and water deficit. As shown in the table of mean comparison (Table 1) for the effects of PGPR treatments, the highest N, P and k were observed in plants treated with S. meliloti follows by P. fluorescens and treat- Table 1 - Results of mean comparison of different PGPR treatments SxP= treatment containing both S. meliloti and P. fluorescens. Dissimilar letters indicating significant differences (Duncan’s multiple range test P≤0.01). N, P and k were measured in dry shoot of plant. Bacteria Leaf area (cm2) Shoot fresh weight (g) Shoot dry weight (g) N (mg g-1) P (mg g-1) k (mg g-1) WUe (g kg-1) Seed yield (g pot-1) Trigonelline (mg g-1) Nicotinic acid (mg g-1) Control 756 c 17.71 c 4.18 c 7.36 c 0.54 d 25.84 c 0.139 c 26.53 a 6.84 b 12.34 ab S. meliloti (S) 1212 a 32.14 a 6.00 a 14.27 a 0.71 a 31.73 a 0.226 a 14.23 b 6.97 b 12.69 b P. fluorescens (P) 959 ab 29.74 a 4.37 b 12.93 b 0.67 b 30.40 b 0.213 a 13.25 c 7.65 a 14.11 a SxP 937 b 21.14 b 5.05 ab 12.17 b 0.63 c 29.43 c 0.181 b 14.19 b 7.74 a 14.05 a Table 2 - Results of mean comparison of different irrigation treatments Dissimilar letters indicating significant differences (Duncan’s multiple range test P≤0.01). N, P and k were measured in dry shoot of plant. Soil water content (fC) Leaf area (cm2) Shoot fresh weight (g) Shoot dry weight (g) N (mg g-1) P (mg g-1) k (mg g-1) WUe (g kg-1) Seed yield (g pot-1) Trigonelline (mg g-1) Nicotinic acid (mg g-1) 100% 1189 a 33.78 a 6.48 a 13.60 a 0.63 c 29.35 b 0.138 c 24.18 a 6.26 c 11.10 d 80% 1025 b 24.76 b 5.79 b 12.20 b 0.59 d 29.05 b 0.165 b 23.52 b 6.63 c 12.07 c 60% 913 c 18.17 c 3.97 c 12.19 b 0.65 b 30.25 a 0.169 b 12.83 c 7.55 b 13.84 b 40% 737 d 13.93 d 3.36 d 9.73 c 0.69 a 30.37 a 0.287 a 10.20 d 8.76 a 16.18 a fig. 1 - Interaction of PGPR and soil water content on leaf area of fenugreek. fig. 2 - Interaction of PGPR and soil water content on the shoot fresh weight fenugreek. fig. 3 - Interaction of PGPR and soil water content on the shoot dry weight of fenugreek. Bolandnazar et al. - The impact of S. meliloti and P. fluorescences on fenugreek 23 ments containing both S. meliloti and P. fluorescens. By decreasing of soil water content, N concentration was decreased significantly, but inverse, P and k con- centration was increased significantly with an excep- tion at 80% of fC treatment (Table 2). The highest and the lowest N concentration was observed in plants treated with S. meliloti and dual application of PGPR bacteria at well watered (100% fC) and control plant at severe water stress treatment (40% fC) respectively (fig. 5) and the highest and the lowest P concentration was related to plants treated with S. meliloti and P. fluorescens in single form at well watered (100% fC) and control plant at severe water stress treatment (40% fC) respectively (fig. 6). According to the interaction effects between PGPRs and drought stress the highest and lowest k concen- tration was observed in dual application of PGPR bac- teria at well watered (100% fC) and control plant under sever water stress (40% fC) respectively (fig. 7). Water use efficiency (WUe) was significantly affect- ed by PGPR and soil water content. mean compari- son indicated that PGPR inoculate plants produced more shoot biomass per water unit than control ones (Table 1). By increasing water deficit stress WUe was increased significantly (Table 2). In aspect of interac- tion between PGPR and water stress it was shown that both dual application of P. fluorescens and S. meliloti and separate application of bacteria under severe water deficit stress (40% fC) led to highest WUe and well watered control plants produced the lowest WUe (fig. 8). As it was shown in the table of mean comparison (Table 1), individual and dual PGPR treatments improved trigonelline and nicotinic acid. By increas- ing water deficit stress trigonelline and nicotinic acid was significantly increased (Table 2). The highest and lowest trigonelline and nicotinic acid was observed in P. fluorescens inoculated at 40% fC soil water con- tent treatment and control in 100% fC soil water content treatment respectively (figs. 9 and 10). fig. 4 - Interaction of PGPR and soil water content on the seed yield of fenugreek. fig. 5 - Interaction of PGPR and soil water content on N concen- tration of fenugreek shoot. fig. 6 - Interaction of PGPR and soil water content on P concen- tration of fenugreek shoot. fig. 7 - Interaction of PGPR and soil water content on k concen- tration of fenugreek shoot. Adv. Hort. Sci., 2018 32(1): 19-26 24 4. Discussion and Conclusions Water deficit limits many crop production world- wide and negatively affects the plant growth and reproduction; recently published literatures indicated that PGPR ameliorate the plants tolerance to abiotic stresses through a variety of mechanisms (Srivastava et al., 2008; Sandhya et al., 2010). In keeping with our results, mishra et al. (2010) indicated that PGPRs could ameliorate the negative effects of salinity stress conditions by positive effects on parameters such as increasing the germination in plants, and also increasing the yield, drought tolerance, and growth. It has also been reported that even in the presence of optimum levels of nitrogenous fertilizers, inoculat- ing with PGPR containing ACC-deaminase activity can improve the yield and growth of inoculated plants (Shaharoona et al., 2006). According to the results of this investigation inoculation with PGPR containing ACC-deaminase considerably decreased the damages caused by drought stress on the growth and yield. They reported that un-inoculated plants exposed to drought stress at vegetative growth stage had signifi- cantly decreased shoot growth by 41%, while in the inoculated plants the decreased shoot growth was only by 18 per cent. In present study control plants without any inocula- tion produced higher seed yield per pot than PGPR treated fenugreek (Table 1). It should be noted that the experiment duration was 5 month and fenugreek has indeterminate flowering habit, so while plants flowering were continued it was harvested. It have been reported that PGPR could delay the flowering time (Jaleel et al., 2007). Water stress led to decrease in fenugreek seed yield. The reason may be due to this fact that in the absence of stress conditions, more photosynthesis material have been stored in the organs such as stems and leaves which by trans- ferring to the seeds increased the grain weight. In contrary, under stress conditions, the water and min- eral absorption by the plant is disrupted which decreases the plant growth and reduces the trans- mission of photosynthesis material in leaf and other organs to the grain (Jaleel et al., 2007). Similar to our findings, root bacterial inoculations significantly affected the plant nutrient element con- tents in apple compared to controls and increased the phosphorus content of treated plants (karlidag et al., 2007). Ordookhani et al. (2010) reported that P. fluorescens improved potassium in tomato plant. Water use efficiency was increased by PGPR in fenugreek in present investigation. Similar to the pre- sent findings Jaleel et al. (2007) demonstrated that the minimum WUe was related to un-inoculated cases which was improved by inoculation with Rhizobium and PGPR. In the present investigation leaf area, shoot weight, dry weight, nitrogen, phos- phorus and potassium content, and WUe increased significantly by treatment with both PGPRs. PGPR fig. 8 - Interaction of PGPR and soil water content on the WUe of fenugreek. fig. 9 - Interaction of PGPR and soil water content on the Trigo- nelline of fenugreek seed. fig. 10 - Interaction of PGPR and soil water content on nicotinic acid of fenugreek seed. Bolandnazar et al. - The impact of S. meliloti and P. fluorescences on fenugreek 25 colonizes the plant’s root system and modulates its growth through increasing the availability of nutri- ents it also protects the plants from phytopathogens (Lee et al., 2013). It has been reported that in induced drought stress condition, fixed nitrogen dur- ing photosynthesis, spend the production of sec- ondary metabolites (Aliabadi farahani et al., 2009). Also beneficial effects of PGPRs on medicinal plants have been reported (Jaleel et al., 2007). It has been reported that the synthesis of secondary metabolites in medicinal plants is induced specific pathway by the impact of microorganisms (Bouchereau et al., 1996). Integrative use of PGPRs and water deficit stress could be an enhance the eco-friendly strategy of PGPRs and plants and could increasing the alkaloid yields in medicinal plants (Lee et al., 2013). Since the fenugreek is used as a medicinal plant this strategy could be applied for increasing its useful secondary metabolites. In conclusion, the results of the present investiga- tion indicate that both S. meliloti and P. fluorescens could effectively increase vegetative growth, nitro- gen, phosphorus and potassium content, secondary metabolites and WUe in fenugreek regardless of water stress. Also under water stress condition PGPR increased plant growth. However in present study seed yield because of delaying bolting time was decrease by application of PGPR. References AJABNOOR m.A., TILmISANY A.k., 1988 - Effect of Trigonella foenum graceum on blood glucose levels in normal and alloxan-diabetic mice. - J. ethnopharma- col., 22: 45-49. ALIABADI-fARAHANI H., VALADABADI S.A., DANeSHIAN J., kHALVATI m.A., 2009 - Evaluation changing of essential oil of balm (melissa officinalis L.) under water deficit stress conditions. - J. med. Plants Res., 3: 329-333. BAkeR W.H., THOmPSON T.L., 1997 - Determination of total nitrogen in plant samples by Kjeldahl. - In: PLANk C.O. (ed.) Plant analysis reference procedures for the southern region of the United States. University of Georgia, Athens, USA, pp. 13-16. BARTeLS D., SUNkAR R., 2005 - Drought and salt tolerance in plants. - Crit. Rev. Plant Sci., 24: 23-58. BRAY e.A., 1997 - Plant responses to water deficit. - Trends Plant Sci., 2: 48-54. BRAY e.A., 2004 - Genes commonly regulated by water- deficit stress in Arabidopsis thaliana. - J. exper. Bot., 55: 2331-2341. BOUCHeReAU A., CLOSSAIS B.N., BeNSAOUD A., BePORT L. ReNAR m., 1996 - Water stress effects on rapeseed quality. - europ. J. Agron., 5: 19-30. CHANDLeR D., DAVIDSON G., GRANT W., GReAVeS J., TATCHeLL G., 2008 - Microbial biopesticides for inte- grated crop management: an assessment of environ- mental and regulatory sustainability. - Trends food Sci. Technol., 19: 275-283. COmPANT S., DUffY B., NOWAk J., CLÉmeNT C., BARkA e.A., 2005 - Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects . - Appl . environ. microbiol., 71: 4951-4959. DImkPA C., WeINAND T., ASCH f., 2009 - Plant-rhizobacte- ria interactions alleviate abiotic stress conditions. - Plant Cell environ., 32: 1682-1694. DODD I.C., RYAN A.C., 2016 - Whole plant physiological responses to water deficit stress. - Wiley & Sons Ltd, Chichester, Uk., pp. 1-9. eL-TARABILY k.A., SIVASITHAmPARAm k., 2006 - Non- streptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. - Soil Biol. Biochem., 38: 1505-1520. feRNÁNDeZ-APARICIO m., emeRAN A.A., RUBIALeS D., 2008 - Control of Orobanche crenata in legumes inter- cropped with fenufreek (Trigonella foenum-graceum). - Crop Protec., 27: 653-659. GOVINDASAmY V., SeNTHILkUmAR m., GAIkWAD k., ANNAPURNA k., 2008 - Isolation and characterization of ACC deaminase gene from two plant growth-promot- ing rhizobacteria. - Curr. microbiol., 57: 312-317. HAVLIN J.L., SOLTANPOUR P., 1980 - A nitric acid plant tis- sue digest method for use with inductively coupled plas- ma spectrometry 1. - Commun. Soil Sci. Plant Anal., 11: 969-980. He Z.L., YANG X.e., 2007 - Role of soil rhizobacteria in phy- toremediation of heavy metal contaminated soils. - J. Zhejiang Univ. Sci. B, 8: 192-207. HUmmeL I., PANTIN f., SULPICe R., PIQUeS m., ROLLAND G., DAUZAT m., CHRISTOPHe A., PeRVeNT m., BOUTeILLÉ m., STITT m., 2010 - Arabidopsis plants acclimate to water deficit at low cost through changes of carbon usage: an integrated perspective using growth, metabolite, enzyme and gene expression analysis. - Plant Physiol., 154: 357-372. JALeeL C.A., mANIVANNAN P., SANkAR B., kISHORekUmAR A., GOPI R., SOmASUNDARAm R., PANNeeRSeLVAm R., 2007 - Pseudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress. - Colloids Surf. B: Biointer- faces, 60: 7-11. kARImI H.R., ROOSTA H., 2014 - Evaluation of inter-specific hybrid of P. atlantica and P. vera L. cv. ‘Badami riz-e- Zarand’as pistachio rootstock to salinity stress accord- ing to some growth indices and eco-physiology and bichemichal parameters. - J. Stress Physiol. Biochem., 10(3): 5-17. kARLIDAG H., eSITkeN A., TURAN m., SAHIN f., 2007 - Effects of root inoculation of plant growth promoting rhi- Adv. Hort. Sci., 2018 32(1): 19-26 26 zobacteria (PGPR) on yield, growth and nutrient element contents of leaves of apple. - Sci. Hortic., 114: 16-20. kLOePPeR J., ReDDY m., RODRÍGUeZ-kABANA R., keNNeY D., kOkALIS-BUReLLe N., mARTINeZ-OCHOA N., VAVRI- NA C., 2004 - Application for rhizobacteria in transplant production and yield enhancement . - Acta Horticulturae, 631: 179-188. kOSHIRO Y., ZHeNG X.Q., WANG m., NAGAI C., ASHIHARA H., 2006 - Changes in content and biosynthetic activity of caffeine and trigonelline during growth and ripening of Coffea arabica and Coffea canephora. - Plant Sci., 171(2): 242-250. Lee k.J., OH B.T., SeRALATHAN k.k., 2013 - Advances in plant growth promoting Rhizobacteria for biological control of plant diseases, pp. 1-13. - In: mAHeSHWARI D.k. (ed.) Bacteria in agrobiology: Disease manage- ment. Springer-Verlag, Berlin, Heidelberg, Germany, pp. 495. mARTIN m.J., PABLeS f., BeLLe m.A., GONZALeS A.G., 1997 - Determination of trigonelline in green and roasted coffee from single column ionic chromatography. - fresenius J. Anal. Chem., 357: 357-358. mAYAk S., TIROSH T., GLICk B.R., 2004 a - Plant growth- promoting bacteria confer resistance in tomato plants to salt stress. - Plant Physiol. Biochem., 42: 565-572. mAYAk S., TIROSH T., GLICk B.R., 2004 b - Plant growth- promoting bacteria that confer resistance to water stress in tomatoes and peppers. - Plant Sci., 166: 525- 530. mIRALDI e., feRRI S., mOSTAGHImI V., 2001 - Botanical drugs and preparations in the traditional medicine of West Azerbaijan (Iran). - J. ethnopharmacol., 75: 77-87. mISHRA m., kUmAR U., mISHRA P.k., PRAkASH V., 2010 - Efficiency of plant growth promoting rhizobacteria for the enhancement of Cicer arietinum L. growth and ger- mination under salinity. - Adv. Biol. Res., 4: 92-96. ORDOOkHANI k., kHAVAZI k., mOeZZI A., ReJALI f., 2010 - Influence of PGPR and AMF on antioxidant activity, lycopene and potassium contents in tomato. - Afric. J. Agric. Res., 5: 1108-1116. RYU C.m., fARAG m.A., HU C.H., ReDD m.S., WeI H.X., PARÉ P.W., kLOePPeR J.W., 2003 - Bacterial volatiles promote growth in Arabidopsis. - Proceed. National. Acad. Sci., 100: 4927-4932. RUBIN R.L., VAN GRONIGeN k.J., HUNGATe B.A. 2017 - Plant growth promoting rhizobacteria are more effec- tive under drought: a metha-analysis. - Plant Soil, 416(1-2): 309-323. SALekDeH G.H., ReYNOLDS m., BeNNeTT J., BOYeR J., 2009 - Conceptual framework for drought phenotyping dur- ing molecular breeding. - Trends Plant Sci., 14: 488- 496. SANDHYA V., ALI S.Z., GROVeR m., ReDDY G., VeNkATeSWARLU B., 2010 - Effect of plant growth pro- moting Pseudomonas spp. on compatible solutes, antioxidant status and plant growth of maize under drought stress. - Plant Growth Regul., 62: 21-30. SARIkHANI m.R., kHOSHRU B., OUSTAN S., 2016 - Efficiency of some bacterial strains on potassium release from micas and phosphate solubilization under in vitro conditions. - Geomicrobiol. J., 33(9): 832-838. SHAfIGHI A., PAZOkI A., ASLI D.e., 2014 - Alleviation of water stress in fenugreek (Trigonella foenum-graceum L.) using different PGPR application methodes. - Adv. environ. Biol., 8(24): 275-280. SHAHAROONA B.m., ARSHAD Z., ZAHIR A., kHALID A., 2006 - Performance of Pseudomonas spp. containing ACC- deaminase for improving growth and yield of maize (Zea mays L.) in the presence of nitrogenous fertilizer. - Soil Biol. Biochem., 38: 2971-2975. SHARmA R., RAGHURAm T., 1990 - Hypoglycaemic effect of fenugreek seeds in non-insulin dependent diabetic subjects. - Nutri. Res., 10: 731-739. SHAUkAT k., AffRASAYAB S., HASNAIN S., 2006 - Growth responses of Triticum aestivum to plant growth pro- moting rhizobacteria used as a biofertilizer. - Res. J. microbiol., 1: 330-338. SmITH m., 2003 - Therapeutic applications of fenugreek. - Altern. med. Rev., 8: 20-27. SRIVASTAVA S., YADAV A., Seem k., mISHRA S., CHAUD- HARY V., NAUTIYAL C., 2008 - Effect of high tempera- ture on Pseudomonas putida NBRI0987 biofilm forma- tion and expression of stress sigma factor RpoS. - Curr. microbiol., 56: 453-457. VAN LOON L., 2007 - Plant responses to plant growth-pro- moting rhizobacteria. - europ. J. Plant Patholol., 119: 243-254. VeSSeY J.k., 2003 - Plant growth promoting rhizobacteria as biofertilizers. - Plant Soil, 255: 571-586. WARke V.B., DeSHmUkH T.A., PATIL V.R., 2011 - Development and validation of RP-HPLC method for estimation of diosgenin in pharmaceutical dosage form. - Asian J. Pharm. Res., 4: 126-128. YANG J., kLOePPeR J.W., RYU C.m., 2009 - Rhizosphere bacteria help plants tolerate abiotic stress. - Trends Plant Sci., 14: 1-4. ZASOSkI R., BURAU R., 1977 - A rapid nitric-perchloric acid digestion method for multi-element tissue analysis. - Commun. Soil Sci. Plant Anal., 8: 425-436. ZHeNG X.Q., ASHIHARA H., 2004 - Distribution, biosynthe- sis and function of purine and pyridine alkaloids in Coffea arabica seedlings. - Plant Sci., 166: 807-813.