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A nodule endophytic plant growth-promoting Pseudomonas and its effects on growth, nodulation and metal uptake in Medicago lupulina under copper stress

A nodule endophytic plant growth-promoting Pseudomonas and its effects on growth, nodulation and... Ann Microbiol (2017) 67:49–58 DOI 10.1007/s13213-016-1235-1 ORIGINAL ARTICLE A nodule endophytic plant growth-promoting Pseudomonas and its effects on growth, nodulation and metal uptake in Medicago lupulina under copper stress 1,2,3 4 3 1 1 Zhaoyu Kong & Zhenshan Deng & Bernard R. Glick & Gehong Wei & Minxia Chou Received: 27 April 2016 /Accepted: 9 September 2016 /Published online: 22 September 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract The aim of this study was to determine the plant translocation to shoots were observed in co-inoculated plants. growth-promoting potential of the nodule endophytic These results demonstrate that co-inoculation of M. lupulina Pseudomonas brassicacearum strain Zy-2-1 when used as a with S. meliloti and P. brassicacearum Zy-2-1 improves plant co-inoculant of Medicago lupulina with Sinorhizobium growth, nitrogen nutrition and metal extraction potential. This meliloti under copper (Cu) stress conditions. Strain Zy-2-1 can be of practical importance in the remediation of heavy was capable of producing ACC deaminase activity, IAA and metal-contaminated soils. 2+ siderophores, and was able to grow in the presence of Cu up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 en- Keywords Pseudomonas brassicacearum Sinorhizobium . . . hanced M. lupulina root fresh weight, total plant dry weight, meliloti Copper stress Co-inoculation Phytoremediation number of nodules, nodule fresh weight and nitrogen content 2+ in the presence of 100 or 300 mg/kg Cu . In the presence of 2+ 500 mg/kg Cu , co-inoculation with S. meliloti and strain Zy- Introduction 2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoc- Copper is an essential redox-active micronutrient for normal ulation alone. Furthermore, a higher amount of Cu accumula- growth and development of plants, as it is directly involved in tion in both shoots and roots and a higher level of Cu a variety of metabolic activities, including photosynthesis, respiration, protein synthesis, cell wall lignification and oxi- dative stress protection. Indeed, these properties make copper ions indispensable for the life of plants; however, they are also Electronic supplementary material The online version of this article (doi:10.1007/s13213-016-1235-1) contains supplementary material, the reason that the copper ion could be strongly toxic for which is available to authorized users. plants when it is present at even slightly higher than optimal levels. Over the centuries, as a result of industrial production, * Minxia Chou sewage irrigation and extensive use of feed additives, organic minxia95@126.com fertilizer, fungicides and urban sewage-sludge compost, cop- per pollution of both soil and water has become a major envi- State Key Laboratory of Soil Erosion and Dryland Farming on the ronmental problem, as it poses a significant direct toxicity Loess Plateau, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China threat to plants, which in turn impacts negatively on both human and environmental health (Figueira et al. 2002;Lu Key Laboratory of Poyang Lake Environment and Resource, Ministry of Education, College of Life Science, Nanchang et al. 2009;Manusadžianas et al. 2012; Srinivasa Gowd University, Nanchang 330022, China et al. 2010). For example, the East China Sea and Pearl Department of Biology, University of Waterloo, 200 University River estuary were subjected to heavy copper pollution as a Avenue West, Waterloo, ON, Canada result of the rapid development of information technology 4 2 College of Life Sciences, Yan’an University, Yan’an 716000, China (IT). In Jiangxi Province, up to 163 hm of farmland along 50 Ann Microbiol (2017) 67:49–58 the Le’an River has been contaminated by wastewater from the selection of the appropriate co-inoculation partners copper mining, resulting in severe crop losses (Tang 2006). and the traits that they encode. Phytoremediation, as a cost-effective and environmentally According to a field survey, we found that M. lupulina was friendly biotechnological approach for remediation of heavy a dominant plant growing in lead-zinc mine tailings in metal contamination of soil, has been highly touted (Ali et al. Northwest China (Wei and Ma 2010). The Cu-resistant strain 2013; Brígido and Glick 2015; Ma et al. 2016). However, Sinorhizobium meliloti CCNWSX0020 was isolated from the many of the plants used in phytoremediation are characterized root nodules of M. lupulina, and this symbiosis was found to by slow growth rates and/or low biomass production, thus display potential for use in Cu phytostabilization (Kong et al. reducing their remediation potential and restricting their prac- 2015a, b). The aim of the present study was the characteriza- tical use in this technology (Baker et al. 1994; Komárek et al. tion of a plant growth-promoting bacterium, Pseudomonas 2007). Plant growth-promoting bacteria (PGPB) can act as brassicacearum strain Zy-2-1, and its effects when co- adjuncts in heavy metal phytoremediation and signifi- inoculated with Sinorhizobium meliloti on the symbiotic per- cantly facilitate the growth of plants in the presence of formance and metal uptake of Medicago lupulina plants under otherwise inhibitory levels of metals (Glick 2010; copper stress. Gamalero and Glick 2011; Kong et al. 2015b). The as- sociation of PGPB with plants may confer a number of advantages upon host plants, including the production of Materials and methods the phytohormone indole-3-acetic acid (IAA), solubiliza- tion of phosphate, secretion of siderophores to mobilize Bacterial strains and cultures iron, and synthesis of the enzyme ACC deaminase to lower stress ethylene levels in plants. Pseudomonas brassicacearum strain Zy-2-1 was originally Legumes are well known for their ability to form nodules on isolated from the root nodules of the leguminous weed roots and stems with compatible rhizobial strains, within which Sphaerophysa salsula growing on the Loess Plateau in China atmospheric nitrogen is reduced to ammonia. The legume– (Deng et al. 2011). This strain was deposited in the Agricultural rhizobia symbiosis is of great environmental and agricultural Culture Collection of China and named ACCC19944. Strain importance, and has been studied extensively (Hao et al. 2014; P. brassicacearum Zy-2-1 inoculants were grown for 2 days at Naveed et al. 2015). However, environmental constraints such 30 °C with shaking at 150 rpm in tryptic soybean broth (TSB) as drought, freezing, high temperature, salinity and toxic metals medium (BD Difco, Detroit, MI, USA). can reduce or restrict the expected beneficial effects of rhizobial Sinorhizobium meliloti strain CCNWSX0020, which is re- 2+ symbiosis on plant growth, as both nodulation and nitrogen sistant to 1.4 mmol/L Cu , was isolated from Medicago fixation processes can be impaired (Tejera et al. 2005;Wani lupulina plants growing in lead-zinc mine tailings in China et al. 2008; Sánchez-Pardo et al. 2013). Enhancement of le- (Fan et al. 2010). This strain was deposited in the Agricultural gume nitrogen fixation by inoculation with both rhizobia and Culture Collection of China and named ACCC19736. The a PGPB is a way to overcome these environmental limitations S. meliloti CCNWSX0020 inoculant was grown for 2 days and improve plant growth (Yadegari et al. 2010; Fox et al. at 28 °C, with shaking at 150 rpm in tryptone yeast extract 2011; Hungria et al. 2013). In this regard, recent studies have (TY) liquid medium (5 g tryptone, 3 g yeast extract, and 0.7 g reported the use of legumes inoculated with rhizobia and metal- CaCl ·2H O per liter; pH 7.2). 2 2 resistant PGPBs for metal phytoremediation (Dary et al. 2010; Pseudomonas putida UW4 was originally isolated from the Fatnassi et al. 2013). Although most previous studies dealing rhizosphere of common reeds, based on its ability to utilize ACC with co-inoculation in legumes have reported plant growth pro- as a sole source of nitrogen (Glick et al. 1995;Duanet al. 2013). motion and enhancement of symbiotic parameters, contradic- Pseudomonas fluorescens 17400, obtained from the American tory results have also been observed, suggesting that co- Type Culture Collection, was previously reported to have inoculation might impair rhizobial colonization and interfere no plant growth-promoting activity (Shah et al. 1998). with the nodulation process (Lucas García et al. 2004a; Lucas The Pseudomonas spp. strains, growing in TSB medium García et al. 2004b; Berggren et al. 2005; Estévez et al. 2009). at 30 °C, were used as the positive and negative controls, For example, Estévez et al. (2009) reported that soybean respectively, for the measurement of plant growth- plants co-inoculated with Chryseobacterium balustinum promoting characteristics. and rhizobia did not always show better symbiotic per- formance under moderate saline conditions. These con- Presence and activity of ACC deaminase trary results indicate that in order to optimize the phytoremediation potential of the system under particular The presence of the ACC deaminase structural gene (acdS) in environmental conditions, close attention must be paid to P. brassicacearum Zy-2-1 was tested by polymerase chain Ann Microbiol (2017) 67:49–58 51 reaction (PCR). Genomic DNA from P. brassicacearum Zy-2- spotted onto solid seawater yeast extract (SWYE) medium 1 was extracted according to the method described by (Nieto et al. 1987). In this way, 20 cultures could be tested Terefework et al. (2001). The acdS DNA fragment was am- per plate. The medium was supplemented with a filter- plified from the genomic DNA of strain Zy-2-1 by PCR using sterilized CuSO solution at concentrations of 0.005, 0.01, the oligonucleotides 5’-GGCAAGGTCGACATCTATGC-3’ 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mmol/L to determine and 5’-GGCTTGCCATTCAGCTATG-3’ as primers. The the minimum inhibitory concentration (MIC) of each PCR product was electrophoresed at 100 V for 40 min in bacterial strain. The MIC was defined as the lowest con- 1 % w/v agarose gel, and the band corresponding to the ex- centration of metal that prevented bacterial growth. 2+ pected size (approximately 1 kb) was excised from the gel, Duplicate plates were prepared for each Cu concentra- purified and sequenced directly. The sequence that was ob- tion and incubated at 28 °C for 10 days. The agar plates tained was deposited in the GenBank database (http://blast. without CuSO were used as controls. For the purpose of ncbi.nlm.nih.gov/Blast.cgi) and was aligned with related defining copper resistance, a strain that could grow in the 2+ sequences. presence of 1 mmol/L Cu was considered to be resis- ACC deaminase activity was determined by spectrophoto- tant (Nieto et al. 1987). metrically measuring the production of α-ketobutyrate as de- scribed by Penrose and Glick (2003), with a standard curve of α-ketobutyrate from 0.05 to 0.5 μ moles. The protein concen- Plant growth and treatments tration of the disrupted cell suspension was determined ac- cording Bradford (1976) using the Bio-Rad protein reagent Medicago lupulina seeds (provided by Gansu Agricultural (Bio-Rad Laboratories, Hercules, CA, USA), according to University, China) were surface-sterilized by treatment with the manufacturer’s instructions. 75 % v/v ethanol for 2 min, followed by 10 min in 20 % v/v NaClO (containing 8 % available chlorine). After the seeds Indoleacetic acid (IAA) production were thoroughly rinsed with several changes of sterile dis- tilled water, they were germinated on moist sterile filter paper IAA production was measured as described by Patten and in the dark at 25 °C for 3 days. The 3-day-old sterilized Glick (2002), with minor modifications. Aliquots of 20 μlof seedlings were planted in plastic pots (10 cm diameter) filled overnight bacterial cultures were used to inoculate 5 mL TSB with 100 g of a sterilized perlite–vermiculite (1:1) mixture. medium without and with tryptophan (100, 250 and 500 μg/ The seedling medium was supplemented with copper in the mL; Sigma-Aldrich, St. Louis, MO, USA) and incubated at form of CuSO . Copper stock solution (0.1 mol/L, pH 3.97) 30 °C for 24 h. When the cell cultures reached stationary was made in distilled water, sterilized by filtration through a phase, they were centrifuged (5,500×g, 10 min), and 1 mL 0.22-μm-pore membrane filter. The seedling medium was of supernatant was mixed with 4 mL Salkowski’s reagent thoroughly watered by properly diluting this stock solution (150 mL of concentrated H SO , 250 mL distilled H O, with distilled water to produce Cu (II) concentrations of 2 4 2 7.5 mL of 0.5 mol/L FeCl ·6H O) and incubated for 100 mg/kg (lightly polluted), 300 mg/kg (moderately pollut- 3 2 20 min at room temperature, after which the absorbance was ed) and 500 mg/kg (heavily polluted). The range of copper measured at 535 nm. The concentration of IAA in each culture concentrations was determined in preliminary experiments was determined by comparison with a standard curve of pure (data not shown). After it was thoroughly mixed with the IAA (Sigma-Aldrich) from 0.1 to 40.0 μg/mL. copper solution, the soil medium was packed into the plastic pots and allowed to equilibrate for 1 week. The seedlings Siderophore production were then maintained in a plant growth incubator at 25 °C at 200 μmol/(m /s) light for 16 h, and 21 °C in the dark for Siderophore levels in the bacterial culture were assayed ac- 8 h. Fåhraeus nitrogen-free mineral nutrient solution cordingto the universalchemicalassayofSchwynand (Fåhraeus 1957) was used to water the plants when necessary Neilands (1987). A 5-μL of aliquot of overnight bacterial (approximately 150 mL every 5–6 days). Six seedlings were culture in King’s B medium was spotted onto a chrome azurol planted in each pot, and four replicates were conducted for S (CAS) agar plate (Alexander and Zuberer 1991)in triplicate, each treatment. After 5 days, seedlings were inoculated with and incubated at 30 °C for 48 hours. cell suspensions of S. meliloti CCNWSX0020 or a co- inoculant cell suspension of S. meliloti CCNWSX0020 with Tolerance of bacteria to copper P. brassicacearum Zy-2-1, respectively. The bacterial cultures were standardized to an optical density of 0.8 at 600 nm, and Pseudomonas strains were grown overnight at 30 °C in TSB 1 mL of the bacterial cell suspension was inoculated onto medium at pH 7.3 ± 0.2, and 10 μL of each culture was then each seedling. 52 Ann Microbiol (2017) 67:49–58 Plant growth, nodulation, N content and Cu content Results Plants were harvested after 50 days, separated into above- Isolation and characterization of ACC deaminase gene ground plant tissues and roots, carefully rinsed with distilled water and dried at 65 °C for 48 hours before determining the The expected amplification product of approximately 1 kb dry weight. The fresh weight, dry weight, plant height, nodule representing the ACC deaminase structural gene (acdS) was fresh weight and the number of effective nodules (pink-red observed following PCR amplification of the genomic DNA colour) were recorded. The pink-red colour, because of the of P. brassicacearum Zy-2-1 (Supplemental, Fig. S1). The presence of leghemoglobin, was considered as an index of DNA sequence of this gene was deposited in the GenBank potential N fixation (Ott et al. 2005; Reichman 2007). database under accession number JN624298. Based on se- The N content of plant tissue samples was determined quence alignments, the acdSgeneof P. brassicacearum Zy- based on the Kjeldahl method using an automatic 2-1 had a high degree of similarity, 94.18 % and 94.65 %, to Kjeltec™ 8400 analyzer unit (FOSS A/S, Hilleroed, the acdS genes from P. brassicacearum strain Am3 Denmark). Nitrogenase activity in nodules was measured (AY604528) and P. fluorescens strain FY32 (FJ465155), by a acetylene reduction assay as described by Weaver respectively. and Danso (1994). Acetylene and ethylene were quanti- fiedthrough anHP-AL/M column(30m, I.D.0.53nm, Copper tolerance and plant growth-promoting 15 μm; J&W Scientific/Agilent Technologies, Folsom, characteristics of P. brassicacearum Zy-2-1 CA, USA) using a Shimadzu GC-17A gas chromato- graph (Shimadzu Corporation, Kyoto, Japan) and a flame 2+ The MIC of Cu for P. brassicacearum Zy-2-1 was ionization detector. Helium was used as the carrier gas, 2.0 mmol/L, which was higher than either P. putida UW4 or with a flow rate set at 6 mL/min and 36 kPa total pres- P. fluorescens 17400 (1.0 and 0.005 mmol/L, respectively) sure. The injector, column and detector temperatures (Table 1). Therefore, P. brassicacearum Zy-2-1 was consid- were 120 °C, 100 °C and 150 °C, respectively. ered to be resistant to CuSO . Ethylene elutes after 1.9 min, and acetylene elutes after P. brassicacearum Zy-2-1 was capable of producing ACC 3.0 min. The amount of ethylene produced by each nod- deaminase, IAA and siderophores, all to a greater extent than ule sample (0.20 g) was calculated using a standard either P. putida UW4 or P. fluorescens 17400 (Table 1). curve of ethylene. The above-ground plant tissues and roots were separated and rinsed three times with sterilized deionized distilled water Measurement of plant growth, nodulation and N content 2+ (ddH O) to remove any loosely bound Cu , and then dried at 65 °C for 48 hours. Aliquots of precisely 0.2 g powdered plant The total fresh and dry weights of plants decreased significant- tissue samples were digested with an acid mixture ly with an increased amount of Cu in the medium. No signif- (HNO :HClO = 3:1), and the copper content was determined icant differences were observed in plant biomass between the 3 4 by atomic absorption spectrophotometry (Z-5000; Hitachi, Sinorhizobium inoculation alone or in combination with Zy-2- Tokyo, Japan). To evaluate the transport behavior of Cu from 1 under control conditions (Fig. 1). However, in the presence 2+ plant roots to shoots under excess Cu conditions, the translo- of 100 or 300 mg/kg Cu , co-inoculation of plants with Zy-2- cation factor (Singh and Agrawal 2007) was calculated using 1 and Sinorhizobium produced a significantly greater plant the following formula: biomass than the single inoculation with Sinorhizobium.For the aerial parts, although the differences were not statistically Translocation factor ¼ Cu =Cu s r significant for the fresh weight (Fig. 1a), the dry weight of co- inoculated plants increased by 53.04 % and 78.3 % in the where Cu and Cu are Cu content in shoots and roots, s r 2+ presence of 100 and 300 mg/kg Cu , respectively, compared respectively. with the plants inoculated with Sinorhizobium alone (Fig. 1b). Similarly, the fresh weight of roots of co-inoculated plants Statistical analyses increased by 50.42 % and 65.99 % in the presence of 100 2+ and 300 mg/kg Cu , respectively, and the dry weight of roots All statistical analyses were performed with SPSS for was 39.97 % and 73.02 % greater in the co-inoculated plants Windows, version 16.0 (SPSS Inc., Chicago, IL, USA), sta- than in the plants with Sinorhizobium inoculation alone. The tistical software. Data were analyzed by one-way analysis of plant height was significantly increased, by 30.79 % and variance (ANOVA), followed by Duncan’stest (p <0.05). All 46 %, in co-inoculated plants compared to the plants inoculat- 2+ data were analyzed using OriginPro v8.0 (OriginLab ed with Sinorhizobium alone in the 300 and 500 mg/kg Cu Corporation, Northampton, MA, USA) to create figures. treatments, respectively (Fig. 2). Ann Microbiol (2017) 67:49–58 53 Table 1 Plant growth-promoting characteristics of P. brassicacearum Zy-2-1, P. putida UW4 and P. fluorescens 17400. Values indicate the mean ± SE of three replicates Strains ACCD activity IAA production (μg/mL) Siderophore MIC (mmol/L) 2+ [μmol α-keto/ production of Cu (mg Tryptophan Tryptophan Tryptophan Tryptophan protein · hr)] conc. 0 μg/mL conc. conc. conc. 100 μg/mL 250 μg/mL 500 μg/mL P. brassicacearum Zy-2-1 5.29 ± 0.50 5.18 ± 0.41 5.97 ± 0.05 7.51 ± 0.34 10.97 ± 0.79 ++ 2.0 P. putida UW4 3.38 ± 0.19 4.62 ± 0.14 6.49 ± 0.31 6.56 ± 0.20 8.90 ± 0.24 + 1.0 P. fluorescens 17400 — 0.52 ± 0.32 1.46 ± 0.51 4.58 ± 0.19 5.87 ± 0.33 + 0.005 2+ The pink-red colour of nodules could be observed, 500 mg/kg Cu (Fig. 3). Sinorhizobium inoculation alone or indicting the establishment of effective symbioses. The num- in combination with Zy-2-1 had little effect on the number of ber of effective nodules, nodule fresh weight and nitrogenase effective nodules, nodule fresh weight or nitrogenase activity activity were significantly reduced by treatment with 300 or under control conditions. However, dual inoculation of Sinorhizobium with Zy-2-1 produced 50 %, 100 % and 100 % more effective nodules per plant in the presence of 2+ 100, 300 and 500 mg/kg Cu , respectively, compared with Sinorhizobium inoculation alone (Fig. 3a). Similarly, nodule fresh weight of co-inoculated plants also showed significant 2+ increases in the presence of 100, 300 and 500 mg/kg Cu ,in comparison to the single inoculation with Sinorhizobium (Fig. 3b). No significant alterations were observed in the ni- trogenase activity of co-inoculated plants in either the absence or presence of Cu compared with the plants inoculated with Sinorhizobium alone under the same conditions (Fig. 3c). However, significant positive effects on N content were ob- served in both shoots and roots of co-inoculated plants under Cu stress conditions (Fig. 4). Cu content The Cu content in both shoots and roots of plants inoculated with either Sinorhizobium or Sinorhizobium + Zy-2-1 was sig- nificantly elevated with the increased level of Cu in the medi- um, an effect that was more pronounced in roots than in shoots (Fig. 5a). Furthermore, co-inoculation with Zy-2-1 dramati- cally increased the Cu content in both shoots and roots in the presence of excess Cu. The Cu content increased by 145.99 %, 209.56 % and 289.08 % in shoots, and by 108.29 %, 102.35 % and 89.40 % in roots of the co- inoculated plants in the presence of 100, 300 and 500 mg/kg 2+ Cu , respectively, compared with the plants inoculated with Sinorhizobium alone. Interestingly, co-inoculation with Zy-2- 1 also increased the Cu content in both shoots and roots under 2+ control (with no Cu in the medium) conditions. Plant dry weight and Cu content were calculated to obtain the total Fig. 1 Fresh weight (a) and dry weight (b)of shoots (white bar)and roots (grey bar)of Medicago lupulina plants with increasing amount of Cu uptake in each plant. The total Cu uptake in concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti both shoots and roots of co-inoculated plants were significant- CCNWSX0020; SM + Zy-2-1 = Co-inoculated with S. meliloti ly increased in comparison to the single-inoculated plants in CCNWSX0020 and P. brassicacearum Zy-2-1. The values indicate the 2+ the presence of 100 or 300 mg/kg Cu , while a significant mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test increase occurred only in the shoots of co-inoculated plants in 54 Ann Microbiol (2017) 67:49–58 P. putida UW4, also displayed a higher level of IAA produc- tion and positive siderophore activity. This can be understood in terms of the observation that stressful growth conditions Fig. 2 Plant height of Medicago lupulina plants with increasing concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = Co-inoculated with S. meliloti CCNWSX0020 and P. brassicacearum Zy-2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test 2+ the presence of 500 mg/kg Cu (Fig. 5b). With respect to the transport behavior of Cu from the roots to shoots of plants, the translocation factor of co-inoculated plants increased in the 2+ presence of 300 and 500 mg/kg Cu , although the difference was not statistically significant for the co-inoculated plants 2+ treated with 100 mg/kg Cu (Fig. 5c). A significant increase was similarly observed in the translocation factor of co- inoculated plants under control conditions. Discussion Most of the bacterial endophytes that have been isolated from various plants are also capable of living outside plant tissues as rhizospheric bacteria, and thus the beneficial effects on their host plants appear to occur through mechanisms similar to those described for other PGPB (Zablotowicz et al. 1991; Höflich et al. 1994;DiFiore andDel Gallo 1995). P. brassicacearum Zy-2-1 was isolated as an endophytic bac- terium from root nodules of the wild legume Sphaerophysa salsula growing on the Loess Plateau in China, which has a dry monsoonal climate with sandy loam and saline/alkaline soil. The results presented here show that P. brassicacearum Zy-2-1, which has a greater level of ACC deaminase activity than the well-studied plant growth-promoting bacterium Fig. 3 Number of effective nodules (a), nodule fresh weight (b)and nitrogenase activity (c)of Medicago lupulina plants with increasing concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = Co-inoculated with S. meliloti CCNWSX0020 and P. brassicacearum Zy-2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test Ann Microbiol (2017) 67:49–58 55 Fig. 4 N content of shoots (white bar) and roots (grey bar)of Medicago lupulina plants with increasing concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = Co- inoculated with S. meliloti CCNWSX0020 and P. brassicacearum Zy- 2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test may select for plant-associated bacteria that possess an in- creased level of ACC deaminase, thereby protecting plants and facilitating both bacterial and plant survival (Belimov et al. 2001; Timmusk et al. 2011). In this regard, both siderophores and IAA produced by bacteria in the rhizosphere enhance plant growth and mineral uptake, resulting in in- creased plant nutrition (Wani et al. 2007;Nimnoi etal. 2014). Moreover, the IAA released by PGPB may be involved at different levels in plant–bacteria interactions. In particular, root development and nodulation are both significantly influ- encedbyIAA (Wanietal. 2007). The present study also revealed that P. brassicacearum strain Zy-2-1 has a high level of copper resistance, thereby facilitating the use of this bacte- 2+ rium for co-inoculation with Cu -resistant rhizobia in con- taminated environments. The present study shows that plant biomass and nodulation parameters under light or moderate Cu stress conditions were enhanced in co-inoculated plants compared to plants inoculat- ed with Sinorhizobium alone. Even though in some cases there were no significant differences between co-inoculated and single-inoculated plants under severe Cu stress conditions, co-inoculation of Sinorhizobium and P. brassicacearum Zy- 2-1 significantly increased plant height, number of effective Fig. 5 Cu content (a), total Cu uptake (= dry weight × Cu content) (b) and translocation factor (c)ofshoots(white bar)and roots(grey bar)of nodules and nodule fresh weight compared to single inocula- Medicago lupulina plants with increasing concentrations of Cu. SM = tion. Similarly, many previous studies working on co- Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = inoculation with PGPBs in legumes have reported increased Co-inoculated with S. meliloti CCNWSX0020 and P. brassicacearum plant growth and enhanced symbiotic performance (Hungria Zy-2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test et al. 2013;Nimnoi et al. 2014; Sánchez et al. 2014). On the other hand, plant growth and nodulation parameters in the 2+ absence of Cu showed no significant differences whether plants were inoculated with Sinorhizobium or with the co- growth-promoting characteristics of S. meliloti strain inoculation of Sinorhizobium and P. brassicacearum Zy-2-1. CCNWSX0020, including positive siderophore activity, a This may be due to the presence of the endogenous plant high level of IAA production and a moderate level of ACC 56 Ann Microbiol (2017) 67:49–58 deaminase activity (Kong et al. 2015a). Although an increase consistent with those of previous studies in which inoculation was found in the effective nodule number and nodule fresh with PGPB increased metal uptake by plant organs compared weight, the co-inoculation pattern did not promote nitrogenase with non-inoculated plants (Mastretta et al. 2009; Prapagdee activity of Medicago plants under either control or Cu stress et al. 2013). In addition to plant growth-promoting activities, conditions. However, significant increases were found in the certain metal-resistant soil microorganisms have been shown N content of both shoots and roots of co-inoculated plants to possess other traits that can alter heavy metal mobility and under Cu stress conditions. This means that the copper- availability to the plants. For instance, the inoculation of ca- resistant strains P. brassicacearum Zy-2-1 and S. meliloti nola (Brassica napus) with Pb-resistant endophytic bacteria CCNWSX0020 are able to survive under the Cu concentra- enhanced the availability of Pb to B. napus by bacterial tions used in this study, and thus the co-inoculation pattern siderophores or by solubilization of Pb (Sheng et al. 2008). promotes an increased level of plant nitrogen. Moreover, the Although an increased heavy metal concentration in plant tis- co-inoculated plants had a healthy green colour, indicting the sues has toxic effects on plants, increased levels are valuable establishment of effective symbioses, as supported by the ob- for effective phytoremediation of soils (Fan et al. 2010). These servation of pink-red colour of nodules. The observed benefits results demonstrate that the application of selected metal- to Medicago plants from the combined inoculation of resistant PGPBs can improve the metal extraction potential Sinorhizobium and P. brassicacearum Zy-2-1 may be due to of plants, which can be useful for the remediation of heavy improved N nutrition in addition to growth-promoting sub- metal-contaminated soils. Similarly, a higher translocation stances. Similar positive effects of bacterial co-inoculation factor was also observed for the co-inoculated plants under on the N content of associated legumes have been reported control conditions. Since Cu is also an essential micronutrient in previous studies (Tilak et al. 2006; Fox et al. 2011;Nimnoi for plants when it is present at optimal level, it is assumed that et al. 2014). These findings indicate that multiple plant plants grown under the growth-limiting conditions used in the growth-promoting properties, such as nitrogen fixation, experiments might eventually take advantage (i.e. a more ef- siderophore activity and IAA biosynthesis, together with ficient micronutrition uptake) of the root apparatus in the pres- ACC deaminase activity, are responsible for the observed ence of bacterial plant growth-promoting activities. plant growth promotion and yield increases (Sarathambal In summary, our results suggest positive effects (i.e. in- et al. 2015). The positive results on specific plant growth- creased plant growth, nodulation and metal uptake) from co- promoting properties of P. brassicacearum Zy-2-1 in this inoculation of M. lupulina with Sinorhizobium and PGPB study suggest that this particular bacterium can promote plant P. brassicacearum Zy-2-1, which can be exploited for the growth under Cu stress conditions by more than one mecha- phytoremediation of copper-contaminated soils. nism, and that these properties could be better exploited as co- Nevertheless, these results need to be tested under field con- ditions, and further work is required to determine the precise inoculant. It was previously observed that an increase or decrease in role played by the PGPB P. brassicacearum Zy-2-1 in plant the amount of metal taken up by plant tissues is a consequence copper uptake. of the particular host plant involved, the bacterium, and the metal species and its concentration (Rajkumar et al. 2009). In Acknowledgments This work was supported by funds from the 863 this study, metal accumulation was considerably higher in Project of China (2012AA101402) and National Science Foundation of roots than in shoots in the presence of excess Cu. The value China (41601337, 31125007 and 31370142). of the translocation factor was considerably less than 1 for both single- and double-inoculated plants, suggesting that Cu accumulated mainly in roots, with a very low level of Cu References translocation to shoots. This may be explained by the fact that 2+ metal cations such as Cu bind quite tightly to organic li- Alexander DB, Zuberer DA (1991) Use of chrome azurol S reagents to gands within the root cell walls (Kochian 1991; Garau et al. evaluate siderophore production by rhizosphere bacteria. Biol Fertil 2015). 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A nodule endophytic plant growth-promoting Pseudomonas and its effects on growth, nodulation and metal uptake in Medicago lupulina under copper stress

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Springer Journals
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Copyright © 2016 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-016-1235-1
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Abstract

Ann Microbiol (2017) 67:49–58 DOI 10.1007/s13213-016-1235-1 ORIGINAL ARTICLE A nodule endophytic plant growth-promoting Pseudomonas and its effects on growth, nodulation and metal uptake in Medicago lupulina under copper stress 1,2,3 4 3 1 1 Zhaoyu Kong & Zhenshan Deng & Bernard R. Glick & Gehong Wei & Minxia Chou Received: 27 April 2016 /Accepted: 9 September 2016 /Published online: 22 September 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract The aim of this study was to determine the plant translocation to shoots were observed in co-inoculated plants. growth-promoting potential of the nodule endophytic These results demonstrate that co-inoculation of M. lupulina Pseudomonas brassicacearum strain Zy-2-1 when used as a with S. meliloti and P. brassicacearum Zy-2-1 improves plant co-inoculant of Medicago lupulina with Sinorhizobium growth, nitrogen nutrition and metal extraction potential. This meliloti under copper (Cu) stress conditions. Strain Zy-2-1 can be of practical importance in the remediation of heavy was capable of producing ACC deaminase activity, IAA and metal-contaminated soils. 2+ siderophores, and was able to grow in the presence of Cu up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 en- Keywords Pseudomonas brassicacearum Sinorhizobium . . . hanced M. lupulina root fresh weight, total plant dry weight, meliloti Copper stress Co-inoculation Phytoremediation number of nodules, nodule fresh weight and nitrogen content 2+ in the presence of 100 or 300 mg/kg Cu . In the presence of 2+ 500 mg/kg Cu , co-inoculation with S. meliloti and strain Zy- Introduction 2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoc- Copper is an essential redox-active micronutrient for normal ulation alone. Furthermore, a higher amount of Cu accumula- growth and development of plants, as it is directly involved in tion in both shoots and roots and a higher level of Cu a variety of metabolic activities, including photosynthesis, respiration, protein synthesis, cell wall lignification and oxi- dative stress protection. Indeed, these properties make copper ions indispensable for the life of plants; however, they are also Electronic supplementary material The online version of this article (doi:10.1007/s13213-016-1235-1) contains supplementary material, the reason that the copper ion could be strongly toxic for which is available to authorized users. plants when it is present at even slightly higher than optimal levels. Over the centuries, as a result of industrial production, * Minxia Chou sewage irrigation and extensive use of feed additives, organic minxia95@126.com fertilizer, fungicides and urban sewage-sludge compost, cop- per pollution of both soil and water has become a major envi- State Key Laboratory of Soil Erosion and Dryland Farming on the ronmental problem, as it poses a significant direct toxicity Loess Plateau, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China threat to plants, which in turn impacts negatively on both human and environmental health (Figueira et al. 2002;Lu Key Laboratory of Poyang Lake Environment and Resource, Ministry of Education, College of Life Science, Nanchang et al. 2009;Manusadžianas et al. 2012; Srinivasa Gowd University, Nanchang 330022, China et al. 2010). For example, the East China Sea and Pearl Department of Biology, University of Waterloo, 200 University River estuary were subjected to heavy copper pollution as a Avenue West, Waterloo, ON, Canada result of the rapid development of information technology 4 2 College of Life Sciences, Yan’an University, Yan’an 716000, China (IT). In Jiangxi Province, up to 163 hm of farmland along 50 Ann Microbiol (2017) 67:49–58 the Le’an River has been contaminated by wastewater from the selection of the appropriate co-inoculation partners copper mining, resulting in severe crop losses (Tang 2006). and the traits that they encode. Phytoremediation, as a cost-effective and environmentally According to a field survey, we found that M. lupulina was friendly biotechnological approach for remediation of heavy a dominant plant growing in lead-zinc mine tailings in metal contamination of soil, has been highly touted (Ali et al. Northwest China (Wei and Ma 2010). The Cu-resistant strain 2013; Brígido and Glick 2015; Ma et al. 2016). However, Sinorhizobium meliloti CCNWSX0020 was isolated from the many of the plants used in phytoremediation are characterized root nodules of M. lupulina, and this symbiosis was found to by slow growth rates and/or low biomass production, thus display potential for use in Cu phytostabilization (Kong et al. reducing their remediation potential and restricting their prac- 2015a, b). The aim of the present study was the characteriza- tical use in this technology (Baker et al. 1994; Komárek et al. tion of a plant growth-promoting bacterium, Pseudomonas 2007). Plant growth-promoting bacteria (PGPB) can act as brassicacearum strain Zy-2-1, and its effects when co- adjuncts in heavy metal phytoremediation and signifi- inoculated with Sinorhizobium meliloti on the symbiotic per- cantly facilitate the growth of plants in the presence of formance and metal uptake of Medicago lupulina plants under otherwise inhibitory levels of metals (Glick 2010; copper stress. Gamalero and Glick 2011; Kong et al. 2015b). The as- sociation of PGPB with plants may confer a number of advantages upon host plants, including the production of Materials and methods the phytohormone indole-3-acetic acid (IAA), solubiliza- tion of phosphate, secretion of siderophores to mobilize Bacterial strains and cultures iron, and synthesis of the enzyme ACC deaminase to lower stress ethylene levels in plants. Pseudomonas brassicacearum strain Zy-2-1 was originally Legumes are well known for their ability to form nodules on isolated from the root nodules of the leguminous weed roots and stems with compatible rhizobial strains, within which Sphaerophysa salsula growing on the Loess Plateau in China atmospheric nitrogen is reduced to ammonia. The legume– (Deng et al. 2011). This strain was deposited in the Agricultural rhizobia symbiosis is of great environmental and agricultural Culture Collection of China and named ACCC19944. Strain importance, and has been studied extensively (Hao et al. 2014; P. brassicacearum Zy-2-1 inoculants were grown for 2 days at Naveed et al. 2015). However, environmental constraints such 30 °C with shaking at 150 rpm in tryptic soybean broth (TSB) as drought, freezing, high temperature, salinity and toxic metals medium (BD Difco, Detroit, MI, USA). can reduce or restrict the expected beneficial effects of rhizobial Sinorhizobium meliloti strain CCNWSX0020, which is re- 2+ symbiosis on plant growth, as both nodulation and nitrogen sistant to 1.4 mmol/L Cu , was isolated from Medicago fixation processes can be impaired (Tejera et al. 2005;Wani lupulina plants growing in lead-zinc mine tailings in China et al. 2008; Sánchez-Pardo et al. 2013). Enhancement of le- (Fan et al. 2010). This strain was deposited in the Agricultural gume nitrogen fixation by inoculation with both rhizobia and Culture Collection of China and named ACCC19736. The a PGPB is a way to overcome these environmental limitations S. meliloti CCNWSX0020 inoculant was grown for 2 days and improve plant growth (Yadegari et al. 2010; Fox et al. at 28 °C, with shaking at 150 rpm in tryptone yeast extract 2011; Hungria et al. 2013). In this regard, recent studies have (TY) liquid medium (5 g tryptone, 3 g yeast extract, and 0.7 g reported the use of legumes inoculated with rhizobia and metal- CaCl ·2H O per liter; pH 7.2). 2 2 resistant PGPBs for metal phytoremediation (Dary et al. 2010; Pseudomonas putida UW4 was originally isolated from the Fatnassi et al. 2013). Although most previous studies dealing rhizosphere of common reeds, based on its ability to utilize ACC with co-inoculation in legumes have reported plant growth pro- as a sole source of nitrogen (Glick et al. 1995;Duanet al. 2013). motion and enhancement of symbiotic parameters, contradic- Pseudomonas fluorescens 17400, obtained from the American tory results have also been observed, suggesting that co- Type Culture Collection, was previously reported to have inoculation might impair rhizobial colonization and interfere no plant growth-promoting activity (Shah et al. 1998). with the nodulation process (Lucas García et al. 2004a; Lucas The Pseudomonas spp. strains, growing in TSB medium García et al. 2004b; Berggren et al. 2005; Estévez et al. 2009). at 30 °C, were used as the positive and negative controls, For example, Estévez et al. (2009) reported that soybean respectively, for the measurement of plant growth- plants co-inoculated with Chryseobacterium balustinum promoting characteristics. and rhizobia did not always show better symbiotic per- formance under moderate saline conditions. These con- Presence and activity of ACC deaminase trary results indicate that in order to optimize the phytoremediation potential of the system under particular The presence of the ACC deaminase structural gene (acdS) in environmental conditions, close attention must be paid to P. brassicacearum Zy-2-1 was tested by polymerase chain Ann Microbiol (2017) 67:49–58 51 reaction (PCR). Genomic DNA from P. brassicacearum Zy-2- spotted onto solid seawater yeast extract (SWYE) medium 1 was extracted according to the method described by (Nieto et al. 1987). In this way, 20 cultures could be tested Terefework et al. (2001). The acdS DNA fragment was am- per plate. The medium was supplemented with a filter- plified from the genomic DNA of strain Zy-2-1 by PCR using sterilized CuSO solution at concentrations of 0.005, 0.01, the oligonucleotides 5’-GGCAAGGTCGACATCTATGC-3’ 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mmol/L to determine and 5’-GGCTTGCCATTCAGCTATG-3’ as primers. The the minimum inhibitory concentration (MIC) of each PCR product was electrophoresed at 100 V for 40 min in bacterial strain. The MIC was defined as the lowest con- 1 % w/v agarose gel, and the band corresponding to the ex- centration of metal that prevented bacterial growth. 2+ pected size (approximately 1 kb) was excised from the gel, Duplicate plates were prepared for each Cu concentra- purified and sequenced directly. The sequence that was ob- tion and incubated at 28 °C for 10 days. The agar plates tained was deposited in the GenBank database (http://blast. without CuSO were used as controls. For the purpose of ncbi.nlm.nih.gov/Blast.cgi) and was aligned with related defining copper resistance, a strain that could grow in the 2+ sequences. presence of 1 mmol/L Cu was considered to be resis- ACC deaminase activity was determined by spectrophoto- tant (Nieto et al. 1987). metrically measuring the production of α-ketobutyrate as de- scribed by Penrose and Glick (2003), with a standard curve of α-ketobutyrate from 0.05 to 0.5 μ moles. The protein concen- Plant growth and treatments tration of the disrupted cell suspension was determined ac- cording Bradford (1976) using the Bio-Rad protein reagent Medicago lupulina seeds (provided by Gansu Agricultural (Bio-Rad Laboratories, Hercules, CA, USA), according to University, China) were surface-sterilized by treatment with the manufacturer’s instructions. 75 % v/v ethanol for 2 min, followed by 10 min in 20 % v/v NaClO (containing 8 % available chlorine). After the seeds Indoleacetic acid (IAA) production were thoroughly rinsed with several changes of sterile dis- tilled water, they were germinated on moist sterile filter paper IAA production was measured as described by Patten and in the dark at 25 °C for 3 days. The 3-day-old sterilized Glick (2002), with minor modifications. Aliquots of 20 μlof seedlings were planted in plastic pots (10 cm diameter) filled overnight bacterial cultures were used to inoculate 5 mL TSB with 100 g of a sterilized perlite–vermiculite (1:1) mixture. medium without and with tryptophan (100, 250 and 500 μg/ The seedling medium was supplemented with copper in the mL; Sigma-Aldrich, St. Louis, MO, USA) and incubated at form of CuSO . Copper stock solution (0.1 mol/L, pH 3.97) 30 °C for 24 h. When the cell cultures reached stationary was made in distilled water, sterilized by filtration through a phase, they were centrifuged (5,500×g, 10 min), and 1 mL 0.22-μm-pore membrane filter. The seedling medium was of supernatant was mixed with 4 mL Salkowski’s reagent thoroughly watered by properly diluting this stock solution (150 mL of concentrated H SO , 250 mL distilled H O, with distilled water to produce Cu (II) concentrations of 2 4 2 7.5 mL of 0.5 mol/L FeCl ·6H O) and incubated for 100 mg/kg (lightly polluted), 300 mg/kg (moderately pollut- 3 2 20 min at room temperature, after which the absorbance was ed) and 500 mg/kg (heavily polluted). The range of copper measured at 535 nm. The concentration of IAA in each culture concentrations was determined in preliminary experiments was determined by comparison with a standard curve of pure (data not shown). After it was thoroughly mixed with the IAA (Sigma-Aldrich) from 0.1 to 40.0 μg/mL. copper solution, the soil medium was packed into the plastic pots and allowed to equilibrate for 1 week. The seedlings Siderophore production were then maintained in a plant growth incubator at 25 °C at 200 μmol/(m /s) light for 16 h, and 21 °C in the dark for Siderophore levels in the bacterial culture were assayed ac- 8 h. Fåhraeus nitrogen-free mineral nutrient solution cordingto the universalchemicalassayofSchwynand (Fåhraeus 1957) was used to water the plants when necessary Neilands (1987). A 5-μL of aliquot of overnight bacterial (approximately 150 mL every 5–6 days). Six seedlings were culture in King’s B medium was spotted onto a chrome azurol planted in each pot, and four replicates were conducted for S (CAS) agar plate (Alexander and Zuberer 1991)in triplicate, each treatment. After 5 days, seedlings were inoculated with and incubated at 30 °C for 48 hours. cell suspensions of S. meliloti CCNWSX0020 or a co- inoculant cell suspension of S. meliloti CCNWSX0020 with Tolerance of bacteria to copper P. brassicacearum Zy-2-1, respectively. The bacterial cultures were standardized to an optical density of 0.8 at 600 nm, and Pseudomonas strains were grown overnight at 30 °C in TSB 1 mL of the bacterial cell suspension was inoculated onto medium at pH 7.3 ± 0.2, and 10 μL of each culture was then each seedling. 52 Ann Microbiol (2017) 67:49–58 Plant growth, nodulation, N content and Cu content Results Plants were harvested after 50 days, separated into above- Isolation and characterization of ACC deaminase gene ground plant tissues and roots, carefully rinsed with distilled water and dried at 65 °C for 48 hours before determining the The expected amplification product of approximately 1 kb dry weight. The fresh weight, dry weight, plant height, nodule representing the ACC deaminase structural gene (acdS) was fresh weight and the number of effective nodules (pink-red observed following PCR amplification of the genomic DNA colour) were recorded. The pink-red colour, because of the of P. brassicacearum Zy-2-1 (Supplemental, Fig. S1). The presence of leghemoglobin, was considered as an index of DNA sequence of this gene was deposited in the GenBank potential N fixation (Ott et al. 2005; Reichman 2007). database under accession number JN624298. Based on se- The N content of plant tissue samples was determined quence alignments, the acdSgeneof P. brassicacearum Zy- based on the Kjeldahl method using an automatic 2-1 had a high degree of similarity, 94.18 % and 94.65 %, to Kjeltec™ 8400 analyzer unit (FOSS A/S, Hilleroed, the acdS genes from P. brassicacearum strain Am3 Denmark). Nitrogenase activity in nodules was measured (AY604528) and P. fluorescens strain FY32 (FJ465155), by a acetylene reduction assay as described by Weaver respectively. and Danso (1994). Acetylene and ethylene were quanti- fiedthrough anHP-AL/M column(30m, I.D.0.53nm, Copper tolerance and plant growth-promoting 15 μm; J&W Scientific/Agilent Technologies, Folsom, characteristics of P. brassicacearum Zy-2-1 CA, USA) using a Shimadzu GC-17A gas chromato- graph (Shimadzu Corporation, Kyoto, Japan) and a flame 2+ The MIC of Cu for P. brassicacearum Zy-2-1 was ionization detector. Helium was used as the carrier gas, 2.0 mmol/L, which was higher than either P. putida UW4 or with a flow rate set at 6 mL/min and 36 kPa total pres- P. fluorescens 17400 (1.0 and 0.005 mmol/L, respectively) sure. The injector, column and detector temperatures (Table 1). Therefore, P. brassicacearum Zy-2-1 was consid- were 120 °C, 100 °C and 150 °C, respectively. ered to be resistant to CuSO . Ethylene elutes after 1.9 min, and acetylene elutes after P. brassicacearum Zy-2-1 was capable of producing ACC 3.0 min. The amount of ethylene produced by each nod- deaminase, IAA and siderophores, all to a greater extent than ule sample (0.20 g) was calculated using a standard either P. putida UW4 or P. fluorescens 17400 (Table 1). curve of ethylene. The above-ground plant tissues and roots were separated and rinsed three times with sterilized deionized distilled water Measurement of plant growth, nodulation and N content 2+ (ddH O) to remove any loosely bound Cu , and then dried at 65 °C for 48 hours. Aliquots of precisely 0.2 g powdered plant The total fresh and dry weights of plants decreased significant- tissue samples were digested with an acid mixture ly with an increased amount of Cu in the medium. No signif- (HNO :HClO = 3:1), and the copper content was determined icant differences were observed in plant biomass between the 3 4 by atomic absorption spectrophotometry (Z-5000; Hitachi, Sinorhizobium inoculation alone or in combination with Zy-2- Tokyo, Japan). To evaluate the transport behavior of Cu from 1 under control conditions (Fig. 1). However, in the presence 2+ plant roots to shoots under excess Cu conditions, the translo- of 100 or 300 mg/kg Cu , co-inoculation of plants with Zy-2- cation factor (Singh and Agrawal 2007) was calculated using 1 and Sinorhizobium produced a significantly greater plant the following formula: biomass than the single inoculation with Sinorhizobium.For the aerial parts, although the differences were not statistically Translocation factor ¼ Cu =Cu s r significant for the fresh weight (Fig. 1a), the dry weight of co- inoculated plants increased by 53.04 % and 78.3 % in the where Cu and Cu are Cu content in shoots and roots, s r 2+ presence of 100 and 300 mg/kg Cu , respectively, compared respectively. with the plants inoculated with Sinorhizobium alone (Fig. 1b). Similarly, the fresh weight of roots of co-inoculated plants Statistical analyses increased by 50.42 % and 65.99 % in the presence of 100 2+ and 300 mg/kg Cu , respectively, and the dry weight of roots All statistical analyses were performed with SPSS for was 39.97 % and 73.02 % greater in the co-inoculated plants Windows, version 16.0 (SPSS Inc., Chicago, IL, USA), sta- than in the plants with Sinorhizobium inoculation alone. The tistical software. Data were analyzed by one-way analysis of plant height was significantly increased, by 30.79 % and variance (ANOVA), followed by Duncan’stest (p <0.05). All 46 %, in co-inoculated plants compared to the plants inoculat- 2+ data were analyzed using OriginPro v8.0 (OriginLab ed with Sinorhizobium alone in the 300 and 500 mg/kg Cu Corporation, Northampton, MA, USA) to create figures. treatments, respectively (Fig. 2). Ann Microbiol (2017) 67:49–58 53 Table 1 Plant growth-promoting characteristics of P. brassicacearum Zy-2-1, P. putida UW4 and P. fluorescens 17400. Values indicate the mean ± SE of three replicates Strains ACCD activity IAA production (μg/mL) Siderophore MIC (mmol/L) 2+ [μmol α-keto/ production of Cu (mg Tryptophan Tryptophan Tryptophan Tryptophan protein · hr)] conc. 0 μg/mL conc. conc. conc. 100 μg/mL 250 μg/mL 500 μg/mL P. brassicacearum Zy-2-1 5.29 ± 0.50 5.18 ± 0.41 5.97 ± 0.05 7.51 ± 0.34 10.97 ± 0.79 ++ 2.0 P. putida UW4 3.38 ± 0.19 4.62 ± 0.14 6.49 ± 0.31 6.56 ± 0.20 8.90 ± 0.24 + 1.0 P. fluorescens 17400 — 0.52 ± 0.32 1.46 ± 0.51 4.58 ± 0.19 5.87 ± 0.33 + 0.005 2+ The pink-red colour of nodules could be observed, 500 mg/kg Cu (Fig. 3). Sinorhizobium inoculation alone or indicting the establishment of effective symbioses. The num- in combination with Zy-2-1 had little effect on the number of ber of effective nodules, nodule fresh weight and nitrogenase effective nodules, nodule fresh weight or nitrogenase activity activity were significantly reduced by treatment with 300 or under control conditions. However, dual inoculation of Sinorhizobium with Zy-2-1 produced 50 %, 100 % and 100 % more effective nodules per plant in the presence of 2+ 100, 300 and 500 mg/kg Cu , respectively, compared with Sinorhizobium inoculation alone (Fig. 3a). Similarly, nodule fresh weight of co-inoculated plants also showed significant 2+ increases in the presence of 100, 300 and 500 mg/kg Cu ,in comparison to the single inoculation with Sinorhizobium (Fig. 3b). No significant alterations were observed in the ni- trogenase activity of co-inoculated plants in either the absence or presence of Cu compared with the plants inoculated with Sinorhizobium alone under the same conditions (Fig. 3c). However, significant positive effects on N content were ob- served in both shoots and roots of co-inoculated plants under Cu stress conditions (Fig. 4). Cu content The Cu content in both shoots and roots of plants inoculated with either Sinorhizobium or Sinorhizobium + Zy-2-1 was sig- nificantly elevated with the increased level of Cu in the medi- um, an effect that was more pronounced in roots than in shoots (Fig. 5a). Furthermore, co-inoculation with Zy-2-1 dramati- cally increased the Cu content in both shoots and roots in the presence of excess Cu. The Cu content increased by 145.99 %, 209.56 % and 289.08 % in shoots, and by 108.29 %, 102.35 % and 89.40 % in roots of the co- inoculated plants in the presence of 100, 300 and 500 mg/kg 2+ Cu , respectively, compared with the plants inoculated with Sinorhizobium alone. Interestingly, co-inoculation with Zy-2- 1 also increased the Cu content in both shoots and roots under 2+ control (with no Cu in the medium) conditions. Plant dry weight and Cu content were calculated to obtain the total Fig. 1 Fresh weight (a) and dry weight (b)of shoots (white bar)and roots (grey bar)of Medicago lupulina plants with increasing amount of Cu uptake in each plant. The total Cu uptake in concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti both shoots and roots of co-inoculated plants were significant- CCNWSX0020; SM + Zy-2-1 = Co-inoculated with S. meliloti ly increased in comparison to the single-inoculated plants in CCNWSX0020 and P. brassicacearum Zy-2-1. The values indicate the 2+ the presence of 100 or 300 mg/kg Cu , while a significant mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test increase occurred only in the shoots of co-inoculated plants in 54 Ann Microbiol (2017) 67:49–58 P. putida UW4, also displayed a higher level of IAA produc- tion and positive siderophore activity. This can be understood in terms of the observation that stressful growth conditions Fig. 2 Plant height of Medicago lupulina plants with increasing concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = Co-inoculated with S. meliloti CCNWSX0020 and P. brassicacearum Zy-2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test 2+ the presence of 500 mg/kg Cu (Fig. 5b). With respect to the transport behavior of Cu from the roots to shoots of plants, the translocation factor of co-inoculated plants increased in the 2+ presence of 300 and 500 mg/kg Cu , although the difference was not statistically significant for the co-inoculated plants 2+ treated with 100 mg/kg Cu (Fig. 5c). A significant increase was similarly observed in the translocation factor of co- inoculated plants under control conditions. Discussion Most of the bacterial endophytes that have been isolated from various plants are also capable of living outside plant tissues as rhizospheric bacteria, and thus the beneficial effects on their host plants appear to occur through mechanisms similar to those described for other PGPB (Zablotowicz et al. 1991; Höflich et al. 1994;DiFiore andDel Gallo 1995). P. brassicacearum Zy-2-1 was isolated as an endophytic bac- terium from root nodules of the wild legume Sphaerophysa salsula growing on the Loess Plateau in China, which has a dry monsoonal climate with sandy loam and saline/alkaline soil. The results presented here show that P. brassicacearum Zy-2-1, which has a greater level of ACC deaminase activity than the well-studied plant growth-promoting bacterium Fig. 3 Number of effective nodules (a), nodule fresh weight (b)and nitrogenase activity (c)of Medicago lupulina plants with increasing concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = Co-inoculated with S. meliloti CCNWSX0020 and P. brassicacearum Zy-2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test Ann Microbiol (2017) 67:49–58 55 Fig. 4 N content of shoots (white bar) and roots (grey bar)of Medicago lupulina plants with increasing concentrations of Cu. SM = Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = Co- inoculated with S. meliloti CCNWSX0020 and P. brassicacearum Zy- 2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test may select for plant-associated bacteria that possess an in- creased level of ACC deaminase, thereby protecting plants and facilitating both bacterial and plant survival (Belimov et al. 2001; Timmusk et al. 2011). In this regard, both siderophores and IAA produced by bacteria in the rhizosphere enhance plant growth and mineral uptake, resulting in in- creased plant nutrition (Wani et al. 2007;Nimnoi etal. 2014). Moreover, the IAA released by PGPB may be involved at different levels in plant–bacteria interactions. In particular, root development and nodulation are both significantly influ- encedbyIAA (Wanietal. 2007). The present study also revealed that P. brassicacearum strain Zy-2-1 has a high level of copper resistance, thereby facilitating the use of this bacte- 2+ rium for co-inoculation with Cu -resistant rhizobia in con- taminated environments. The present study shows that plant biomass and nodulation parameters under light or moderate Cu stress conditions were enhanced in co-inoculated plants compared to plants inoculat- ed with Sinorhizobium alone. Even though in some cases there were no significant differences between co-inoculated and single-inoculated plants under severe Cu stress conditions, co-inoculation of Sinorhizobium and P. brassicacearum Zy- 2-1 significantly increased plant height, number of effective Fig. 5 Cu content (a), total Cu uptake (= dry weight × Cu content) (b) and translocation factor (c)ofshoots(white bar)and roots(grey bar)of nodules and nodule fresh weight compared to single inocula- Medicago lupulina plants with increasing concentrations of Cu. SM = tion. Similarly, many previous studies working on co- Inoculated with Sinorhizobium meliloti CCNWSX0020; SM + Zy-2-1 = inoculation with PGPBs in legumes have reported increased Co-inoculated with S. meliloti CCNWSX0020 and P. brassicacearum plant growth and enhanced symbiotic performance (Hungria Zy-2-1. The values indicate the mean ± SE of four replicates. Bars with different letters are significantly different at p < 0.05 by the Duncan test et al. 2013;Nimnoi et al. 2014; Sánchez et al. 2014). On the other hand, plant growth and nodulation parameters in the 2+ absence of Cu showed no significant differences whether plants were inoculated with Sinorhizobium or with the co- growth-promoting characteristics of S. meliloti strain inoculation of Sinorhizobium and P. brassicacearum Zy-2-1. CCNWSX0020, including positive siderophore activity, a This may be due to the presence of the endogenous plant high level of IAA production and a moderate level of ACC 56 Ann Microbiol (2017) 67:49–58 deaminase activity (Kong et al. 2015a). Although an increase consistent with those of previous studies in which inoculation was found in the effective nodule number and nodule fresh with PGPB increased metal uptake by plant organs compared weight, the co-inoculation pattern did not promote nitrogenase with non-inoculated plants (Mastretta et al. 2009; Prapagdee activity of Medicago plants under either control or Cu stress et al. 2013). In addition to plant growth-promoting activities, conditions. However, significant increases were found in the certain metal-resistant soil microorganisms have been shown N content of both shoots and roots of co-inoculated plants to possess other traits that can alter heavy metal mobility and under Cu stress conditions. This means that the copper- availability to the plants. For instance, the inoculation of ca- resistant strains P. brassicacearum Zy-2-1 and S. meliloti nola (Brassica napus) with Pb-resistant endophytic bacteria CCNWSX0020 are able to survive under the Cu concentra- enhanced the availability of Pb to B. napus by bacterial tions used in this study, and thus the co-inoculation pattern siderophores or by solubilization of Pb (Sheng et al. 2008). promotes an increased level of plant nitrogen. Moreover, the Although an increased heavy metal concentration in plant tis- co-inoculated plants had a healthy green colour, indicting the sues has toxic effects on plants, increased levels are valuable establishment of effective symbioses, as supported by the ob- for effective phytoremediation of soils (Fan et al. 2010). These servation of pink-red colour of nodules. The observed benefits results demonstrate that the application of selected metal- to Medicago plants from the combined inoculation of resistant PGPBs can improve the metal extraction potential Sinorhizobium and P. brassicacearum Zy-2-1 may be due to of plants, which can be useful for the remediation of heavy improved N nutrition in addition to growth-promoting sub- metal-contaminated soils. Similarly, a higher translocation stances. Similar positive effects of bacterial co-inoculation factor was also observed for the co-inoculated plants under on the N content of associated legumes have been reported control conditions. Since Cu is also an essential micronutrient in previous studies (Tilak et al. 2006; Fox et al. 2011;Nimnoi for plants when it is present at optimal level, it is assumed that et al. 2014). These findings indicate that multiple plant plants grown under the growth-limiting conditions used in the growth-promoting properties, such as nitrogen fixation, experiments might eventually take advantage (i.e. a more ef- siderophore activity and IAA biosynthesis, together with ficient micronutrition uptake) of the root apparatus in the pres- ACC deaminase activity, are responsible for the observed ence of bacterial plant growth-promoting activities. plant growth promotion and yield increases (Sarathambal In summary, our results suggest positive effects (i.e. in- et al. 2015). The positive results on specific plant growth- creased plant growth, nodulation and metal uptake) from co- promoting properties of P. brassicacearum Zy-2-1 in this inoculation of M. lupulina with Sinorhizobium and PGPB study suggest that this particular bacterium can promote plant P. brassicacearum Zy-2-1, which can be exploited for the growth under Cu stress conditions by more than one mecha- phytoremediation of copper-contaminated soils. nism, and that these properties could be better exploited as co- Nevertheless, these results need to be tested under field con- ditions, and further work is required to determine the precise inoculant. It was previously observed that an increase or decrease in role played by the PGPB P. brassicacearum Zy-2-1 in plant the amount of metal taken up by plant tissues is a consequence copper uptake. of the particular host plant involved, the bacterium, and the metal species and its concentration (Rajkumar et al. 2009). 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Published: Sep 22, 2016

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