Effects of PGPR microbial inoculants on the growth and soil properties of Avena sativa, Medicago sativa, and Cucumis sativus seedlings

Soil & Tillage Research 199 (2020) 104577

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Effects of PGPR microbial inoculants on the growth and soil properties of Avena sativa, Medicago sativa, and Cucumis sativus seedlings

T

Haiyun Lia,b,1, Yizhi Qiuc,1, Tuo Yaoa,b,*, Yachun Maa,b, Huirong Zhanga,b, Xiaolei Yanga,b a

College of Grassland Science, Gansu Agricultural University, Lanzhou, China Key Laboratory of Grassland Ecosystem, Gansu Agricultural University, Ministry of Education, Lanzhou, China c School of Life Science, Lanzhou University, Lanzhou, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Nitrogen fixation PGPR microbial inoculants Phosphate solubilization Soil enzyme activity Soil physiochemical properties

Plant growth promoting rhizobacteria (PGPR) are an important bacterial resource for microbial fertilizers, which can promote plant growth and increase crop yields. In this study, multifunctional PGPR microbial inoculants with the ability to fix nitrogen, solubilize phosphate, secrete indole-3-acetic acid (IAA) and exhibit biological control were generated. These PGPR microbial inoculants contained four compatible strains of Providencia rettgeri P2, Advenella incenata P4, Acinetobacter calcoaceticus P19, and Serratia plymuthica P35. All four strains had the ability to solubilize inorganic and organic phosphate, and fix nitrogen. In addition, strains P2 and P4 had the ability to secrete IAA, and strain P35 exhibited inhibition of Helminthosporium tritici-vulgaris, Alternaria solani, Fusarium oxysporum, and Sclerotinias clerotiorum. The effects of the PGPR microbial inoculants on the growth, physiology, and soil properties of oat (Avena sativa), alfalfa (Medicago sativa), and cucumber (Cucumis sativus) seedlings were verified by a pot experiment. The PGPR microbial inoculants increased the dry weight (DW), plant height (PH), root length (RL), root average diameter (RAD), root surface area (RSA), root volume (RV), and chlorophyll content (PCH) of A. sativa, M. sativa, and C. sativus seedlings, and enhanced the activity of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in the seedlings. Furthermore, PGPR microbial inoculants increased soil urease (SURE), invertase (SIN), alkaline phosphatase (SAKP), catalase (SCAT) activity, available nitrogen (AN), available phosphorus (AP), available potassium (AK), and organic carbon (SOC) in the rhizosphere of A. sativa, M. sativa, and C. sativus. In summary, our results showed that the PGPR microbial inoculants had a promoting effect on A. sativa, M. sativa, and C. sativus seedlings by increasing soil enzyme activity and available nutrient content. These PGPR microbial inoculants can be used as an alternative method of environmentally friendly biocontrol of plant disease or to improve crop systems.

1. Introduction At present, 50 % of the nutrients necessary for plant growth in agricultural production are mainly supplied by chemical fertilization (Minaxi and Saxena, 2011; George et al., 2013). Extensive use of chemical fertilizers can lead to the deterioration of soil properties, imbalance of nutrient proportions, decline in quality of agricultural products, and destruction of ecological environments (Bhattacharyya and Jha, 2012; Mal et al., 2015). There are several ways to address this major problem, such as changing how we use resources, exploring

renewable resources, and transforming abiotic resources into infinite biological resources. Plant growth promoting rhizobacteria (PGPR) are a group of microorganisms that can colonize the rhizosphere of plants, and are capable of fixing nitrogen (Anand et al., 2013; Puri et al., 2016), dissolving organic and inorganic phosphorus (Yin et al., 2015), secreting auxin and antibiotics (Mohite, 2013), antagonizing pathogens, and promoting plant growth (Baig et al., 2012; Ahmad et al., 2012). PGPR can promote plant growth by several direct or indirect mechanisms (Egamberdieva et al., 2011; Zhang et al., 2014). PGPR directly convert elements, such

Abbreviations: PGPR, plant growth promoting rhizobacteria; IAA, indole-3-acetic acid; DW, dry weight; PH, plant height; RL, root length; RAD, root average diameter; RSA, root surface area; RV, root volume; PCH, plant chlorophyll; POD, peroxidase; CAT, catalase; SOD, superoxide dismutase; EC, electrical conductivity; AN, available nitrogen; AP, available phosphorus; AK, available potassium; SOC, soil organic carbon; SCAT, soil catalase; SURA, soil urease; SIN, soil invertase; SAKP, soil alkaline phosphatase ⁎ Corresponding author at: College of Grassland Science, Gansu Agricultural University, Lanzhou, China. E-mail address: [email protected] (T. Yao). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.still.2020.104577 Received 14 May 2019; Received in revised form 12 January 2020; Accepted 20 January 2020 0167-1987/ © 2020 Elsevier B.V. All rights reserved.

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Table 1 Growth promoting characteristics of four PGPR strains. Strains

P2 P4 P19 P35

Inorganic phosphorus

Organic phosphorus

pH

P solubilization capacity (μg mL−1)

pH

173.64 438.35 254.02 131.26

7.31 7.25 7.34 7.42

6.23 4.76 6.07 6.00

± ± ± ±

0.07 0.05 0.01 0.09

± ± ± ±

4.66 4.01 0.95 3.95

Nitrogenase activity (nmol mg−1 protein h−1)

IAA contents (μg mL−1)

25.38 ± 2.71 160.03 ± 10.68 864.97 ± 40.08 108.90 ± 11.77

41.04 ± 0.07 11.72 ± 0.06 － －

P solubilization capacity (μg mL−1) ± ± ± ±

0.07 0.04 0.02 0.03

25.50 28.66 35.46 13.36

± ± ± ±

0.73 1.29 1.70 0.97

pot experiment. These results will provide microbial resources and basic data for the development and applications of PGPR as biofertilizers.

as nitrogen and phosphorus, into nutrients that can be absorbed and utilized by plants, and they secrete indole-3-acetic acid (IAA), gibberellin (GA), and cytokinin (CTK) to directly promote plant growth (Mohite, 2013). Moreover, PGPR colonize plant rhizospheres and inhibit or reduce soil-borne diseases. PGPR also improve the plant’s own defense ability by inducing plant resistance, thereby reducing the adverse effects of disease on plant growth, development, and yield (Verma et al., 2015). Therefore, the application of PGPR in agricultural production plays an important role in reducing the use of chemical fertilizers and pesticides, inhibiting the occurrence of pests and diseases and ensuring the sustainable development of modern agriculture while achieving the goal of increasing production. In recent years, working on PGPR mainly focused on plant growth (Singh and Jha, 2017; Lally et al., 2017; Shahid et al., 2019), soil improvement (Turan et al., 2011), crop disease control (Brooke et al., 2017), and other aspects. For instance, Singh and Jha (2017) inoculated Stenotrophomonas maltophilia SBP-9 into wheat plants, which significantly increased plant growth parameters, such as shoot length, root length, fresh weight, dry weight, and chlorophyll content. Huang et al. (2015) found that the inoculation of Bacillus subtilis, Bacillus atrophaeus, and Bacillus pumilus significantly promoted the growth of corn and tomato. Abbasi et al. (2011) isolated the PGPR wpr 32, wpr 42, and wpr 51 from the rhizosphere of wheat and these were shown to promote plant growth, height and length, increase the fresh and dry weight of aerial parts by 25 %, 45 %, and 86 %, respectively, and increase the weight of dry and fresh root by 27 %, 102 %, and 76 %, respectively. Shahid et al. (2019) showed that inoculation of PGPR strain Enterobacter sp. Fs-11 enhanced sunflower yield and oil contents under diverse environmental conditions. In addition, some studies have shown that PGPR inoculation can improve the salt tolerance (Bharti et al., 2014) and disease resistance (Li et al., 2019) of plants. Although there are many reported strains of PGPR, there are few strains with broad-spectrum efficacy and strong adaptability. This is likely because the living environments of PGPR are very complex, and they are sensitive to external factors, such as soil fertility, soil physical properties, cultivation measures, crop varieties, and others (Huang et al., 2015). Given that a single strain and a single-function fertilizer can no longer meet the needs of modern agricultural development and provide the various nutrients for crop growth and development, it is particularly important to develop a combination of various PGPR. The combination of PGPR could potentially provide multiple functions and increase the adaptability compared to a single strain in the environment. In addition, they could rapidly propagate into dominant microbial flora in the rhizosphere of plants, improve the soil’s ecological environment, and enhance plant disease resistance (Molina-Romero et al., 2017). Moreover, the applications of PGPR combination are more in line with the natural law and allows for the interaction and synergy between various microorganisms in the natural ecological environment (Bharti et al., 2014). The main objectives of this study were: (1) to screen a formulated compatible PGPR combination that have the ability to fix nitrogen, solubilize phosphorus, secrete auxin, and act as biological control agents, (2) to verify the effects of inoculation with PGPR on oat (Avena sativa), alfalfa (Medicago sativa), and cucumber (Cucumis sativus) by a

2. Materials and methods 2.1. Identification of PGPR strains In this study, 36 strains were isolated from the rhizosphere soil of Trifolium pratense and Polygonum viviparum by national botanical research institute’s phosphate (NBRIP) (Nautiyal, 1999), Mongina and nitrogen-deficient malate (NFM) agar plates (Doebereiner, 1994), and strains were kept on Luria Bertani (LB) agar medium at 28 °C (See Table 1 and Table S1 for specific information). Four strains (P2, P4, P19 and P35) with excellent effect were selected from these strains. Four strains were inoculated in LB liquid medium, and incubated at 37 °C with shaking (160 rpm) for 24 h. Genomic DNA was then extracted from each strain using the bacterial genomic DNA isolation Kit (Tiangen Biochemical Technology., Beijing, China). The 16S rRNA gene was amplified using primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Galkiewicz and Kellogg, 2008). The polymerase chain reaction (PCR) was performed in 50 μL reactions containing 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 25 μL 2×Taq PCR MasterMix, 2 μL template DNA, and doubledistilled H2O. The PCR conditions were 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 57 °C for 60 s, and 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. The PCR products were qualified by 1 % agarose gel electrophoresis and then sequenced using the dideoxy chain-termination method at Sangon Biotech Co., Ltd. (Shanghai, China). The 16S rRNA gene sequences were verified with the EzBio Cloud database (http://www.ezbiocloud.net). The phylogenetic tree was constructed using the maximum likelihood method of the software package MEGA 6.0 and the topology of the tree was evaluated by bootstrap test with 1000 replicates (Tamura et al., 2013). The 16S rRNA gene sequences of the four strains were submitted and deposited in the GenBank database and their accession numbers were obtained (i.e., MN615664, MN615708, MN615709, and MN615712). 2.2. Determination of plant growth promotion characteristics of PGPR strains The ability of the strains to dissolve inorganic and organic phosphorus was measured by the molybdenum blue colorimetric method (Murphy and Riley, 1962). Specifically, the strains were incubated in liquid NBRIP medium (Nautiyal, 1999) and Mongina medium at 28 °C with shaking (180 rpm) for 12 d. The pH and soluble phosphorus content in culture solution was then measured. The strains were incubated in NFM agar medium and the nitrogenase activity was measured by the acetylene reduction method (Hardy et al., 1968). The bacterial cells were centrifuged and collected, and the Bradford assay (Toyama et al., 2007) was used to measure the protein content of the bacterial cells. The strains were inoculated into 50 mL of King's liquid media (Nomura et al., 2012) and incubated at 28 °C with shaking (180 rpm) 2

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2.5. Dry weight (DW), plant height (PH), and root system assay

for 3 d. Extraction of IAA from culture solutions with ethyl acetate and each treatment process was done in triplicate. The IAA content was measured by high-performance liquid chromatography (Wang et al., 2016). The stationary phase was an Agilent ZORBAX Eclipse Plus C18 column (250 × 4.6 mm i.d., 5 μm, Agilent Technologies, USA) and the mobile phase was methanol-water (containing 0.2 % glacial acetic acid) with a volume ratio of 2:3. The detection wavelength was 210 nm, the column temperature was 30 °C, the flow velocity was 1.0 mL min−1, and the injection volume was 20 μL. The inhibition abilities of PGPR strains against four plant pathogens (Helminthosporium tritici-vulgaris, Alternaria solani, Fusarium oxysporum and Sclerotinias clerotiorum) were measured by the plate confrontation method (Weselowski et al., 2016). Three replicates were used and plants inoculated with pathogen only were used as a control. The inhibition rate was calculated according to the formula:

One seedling was randomly selected from each pot and it was removed for analysis. The height of the aerial part was measured as the plant height (PH). The dry weight (DW) was weighed after it was dried at 65 °C for 24 h. Root length (RL), root surface area (RSA), root volume (RV), and root average diameter (RAD) were measured using a root LA2400 Scanner (Jeili Electronics Trading Co., Ltd, Shanghai, China). 2.6. Plant enzyme activities and chlorophyll content assay The leaves of each seedling (0.3 g) were split into three equal parts and each part was ground with 0.05 M phosphate buffer at a pH of 5.5, 7.0, or 7.8, to extract the enzyme solution of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), respectively (Chance and Maehly, 1955; Nakano and Asada, 1981, and Giannopolitis and Ries, 1977). To calculate the activities of POD, CAT, and SOD, the absorbance values were measured at the wavelengths of 470, 240, and 560 nm, respectively. To determine the chlorophyll content, 0.1 g of freshly picked leaves of each seedling were weighed and a small amount of quartz sand was added to them. They were ground with 80 % acetone and centrifuged at 9000g for 10 min at 4 °C. The absorbance of the supernatant was measured at 663 nm and 645 nm to determine the content of chlorophyll a and b, respectively, and the total chlorophyll content was calculated by the Moran and Porath (1980) method.

Inhibition rate (%)=(D-d)/D × 100 % Where D is the colony diameter of plant pathogens in the control group and d is the colony diameter of plant pathogens in the treatment group.

2.3. The validation of the compatibility of PGPR strains The compatibility between pair-wise strains was verified by the plate confrontation method (Weselowski et al., 2016). Each strain was inoculated in LB liquid medium and incubated at 28 °C with (180 rpm) for 48 h. Bacterial culture (100 μL) was pipetted and spread as a lawn on LB agar plates, which were allowed to air-dry in a laminar-flow cabinet. Three small pieces of round filter paper were placed on the plate and 10 μL of three other bacterial suspensions were inoculated on the filter paper in triplicate. After inoculation, the plates were incubated at 28 °C for 3 d to observe whether there was an inhibition zone. The presence of an inhibition zone indicated that there was an inhibition effect between the two strains. If the two strains had no inhibition effect, they could be mixed in culture.

2.7. Soil enzyme activities and physiochemical properties assay Soil urease (SURE) activity was measured by sodium phenol-sodium hypochlorite colorimetry (Tabatabai, 1994), soil alkaline phosphatase (SAKP) activity was measured by the sodium phenylene phosphate colorimetric method (Tabatabai and Bremner, 1969), soil invertase (SIN) activity was measured by the 3,5-dinitrosalicylic acid colorimetric method (Frankeberger and Johanson, 1983), and soil catalase (SCAT) activity was measured by back-titrating residual H2O2 with KMnO4 (Johnson and Temple, 1964). Soil pH values (soil: H2O ratio 1:2.5) and electrical conductivity (EC, soil: H2O ratio 1:5) were measured with a pH-conductivity meter (Multi-Parameter PCTestrTM 35, Japan), soil organic carbon (SOC) was measured using the potassium dichromate sulfuric acid heating method, soil available nitrogen (AN) was measured using the alkali diffusion method (Xie et al., 2011), soil available phosphorus (AP) content was measured using the molybdenum blue method (Xiong et al., 2015), and soil available potassium (AK) content was measured by the methods of Zhang et al. (2013). There were six repeats for each index.

2.4. Pot experiment Before planting, the four strains were separately inoculated into 250 mL Erlenmeyer flasks containing 100 mL of LB liquid medium and incubated at 28 °C with shaking (180 rpm) for 48 h. The bacterial cultures were centrifuged at 8000g for 10 min at 4 °C and the collected bacterial cells were repeatedly centrifuged and washed with sterilized water three times and then diluted with sterilized water to achieve an OD600 of 0.6, which was the final volume ratio used as the inoculum. Full and uniform sized seeds of oat (A. sativa), alfalfa (M. sativa), and cucumber (C. sativus) were selected, sterilized with 1 % NaClO for 5 min, and rinsed three times with sterilized water. Then, 500g of sterilized soil and 1 % (v/v) PGPR inoculum were mixed and added into a 750 mL plastic cup (95 × 85 × 175 mm). A cup was inoculated with an equal volume of sterile water as a control. Five A. sativa seeds were planted in each cup. There were 6 repeats of the treatment and control groups. M. sativa and C. sativus seeds were planted in the same way. The planted cups were placed in a random order in an artificial climate incubator (Ningbo Jiangnan Instrument Factory, Ningbo, China) at 25 °C with light (16 h)/dark (8 h) cycles (lights at 350 μE m−2 s−1) and a humidity of 80 %. Plant and soil indexes were measured until 45 days of plant growth. The culture soil was sierozem with a pH of 8.28 and the contents of soil organic matter, total nitrogen, phosphorus, and potassium were 45.36 g kg−1, 0.59 g kg−1, 0.36 g kg−1, and 42.29 g kg−1, respectively. The contents of available nitrogen, phosphorus and potassium were 86.48 mg kg−1, 11.25 mg kg−1, and 129.7 mg kg−1, respectively.

2.8. Statistical analysis Test data were analyzed using SPSS 19.0 software for the t-test. The data of gas chromatography and liquid chromatography were processed and analyzed by the external standard method. Detrend correspondence analysis of plant characteristics was carried out using the CANOCO 4.5 software. According to the lengths of gradients, the relationship between plant characteristics and soil properties were analyzed by redundancy analysis (RDA). 3. Results 3.1. Plant growth promoting characteristics and identification of PGPR strains The inorganic phosphate solubility of the four PGPR strains were between 131.26–438.35 μg mL−1 and strain P4 had the highest inorganic phosphate solubility, which was 438.35 μg mL−1. The organic 3

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3.2. Effects of inoculation with PGPR microbial inoculants on plant growth characteristics

Table 2 The inhibition abilities of four PGPR strains. Strains

Inhibition rate (%) Helminthosporium tritici-vulgaris

P2 P4 P19 P35

Alternaria solani

Fusarium oxysporum

Sclerotinias clerotiorum

27.92 ± 0.99 67.71 ± 1.99

52.98 ± 2.52

Inoculation with PGPR microbial inoculants influenced the growth characteristics of A. sativa, M. sativa and C. sativus seedlings (Fig. 2). Compared with the control, PGPR microbial inoculants significantly increased the DW, PH, and RL of A. sativa seedlings by 271.33 %, 81.19 %, and 28.92 %, respectively (p < 0.01). Furthermore, the RSA and RV increased by 22.71 % and 28.61 %, respectively (p < 0.05). The PGPR microbial inoculants increased the DW, PH, and RL of M. sativa seedlings by 45.13 %, 61.96 %, and 37.16 %, respectively (p < 0.01). In addition, the PGPR microbial inoculants significantly increased the DW, RL, RSA, and RV of C. sativus seedlings by 91.85 %, 20.47 %, 58.69 %, and 85.77 %, respectively (p < 0.01), the RAD increased by 16.74 % (p < 0.05).

52.15 ± 1.32 53.16 ± 0.97

52.61 ± 0.84

phosphate solubility of the four strains ranged from 13.36 to 35.46 μg mL−1 and strain P19 had the highest organic phosphate solubility, which was 35.46 μg mL−1. The nitrogenase activity of the four strains were between 25.38–864.97 nmol mg−1 protein h−1 and strain P19 exhibited the highest nitrogenase activity of 864.97 nmol mg−1 protein h−1. The strains P2 and P4 had the ability to secrete IAA and the IAA contents were 41.04 μg mL−1 and 11.72 μg mL−1, respectively (Table 1). The inhibition abilities of the four PGPR strains on different plant pathogens are shown in Table 2. Strain P35 effectively inhibited Helminthosporium tritici-vulgaris, Alternaria solani, Fusarium oxysporum and Sclerotinias clerotiorum, with inhibition rates of 53.16 %, 52.61 %, 67.71 %, and 52.98 %, respectively. The 16S rRNA gene sequences of the four strains were analyzed and phylogenetic trees were constructed (Fig. 1). The similarity between strain P2 and Providencia rettgeri DSM 4542 (AM0404492) was 99.09 %, which was preliminarily identified as Providencia rettgeri (MN615664). The similarity between strain P4 and Advenella incenata CCUG 45225 AM944734 was 99.33 %, which was preliminarily identified as Advenella incenata (MN615708). The similarity between strain P19 and Acinetobacter calcoaceticus DSM 30006 AIEC01000170 was 99.86 %, which was preliminarily identified as Acinetobacter calcoaceticus (MN615712) and the similarity between strain P35 and Serratia plymuthica DSM 4540(AJ233433) was 99.86 %, which was preliminarily identified as Serratia plymuthica (MN615709).

3.3. Effects of inoculation with PGPR microbial inoculants on plant enzyme activities and chlorophyll content The effect PGPR microbial inoculants on the enzyme activities of A. sativa, M. sativa and C. sativus seedlings are shown in Fig. 3. Compared with the control, PGPR microbial inoculants significantly increased the POD, SOD, and CAT activity in A. sativa seedlings (p < 0.01), which increased by 71.38 %, 20.31 %, and 6.06 %, respectively. The PGPR microbial inoculants increased the activity of SOD and CAT in M. sativa seedlings (P < 0.01) by 105.37 % and 10.83 %, respectively. PGPR microbial inoculants increased the POD (p < 0.05), SOD (p < 0.05), and CAT (p < 0.01) activity in C. sativus seedlings by 21.80 %, 23.66 %, and 5.99 %, respectively. In addition, the PGPR microbial inoculants significantly increased the chlorophyll content of A. sativa, M. sativa, and C. sativus seedlings (p < 0.01) (Fig. 3d). Compared with the control, the chlorophyll content of A. sativa, M. sativa, and C. sativus seedlings increased by 10.94 %, 19.16 %, and 13.85 %, respectively.

Fig. 1. Phylogenetic tree of bacterial 16S rRNA gene sequences revealing four PGPR strains evolutionary divergence. Manually refined 16S rRNA sequences were aligned using MEGA 6.0 and the tree was constructed using the Maximum Likelihood method (approximately 1400 bp length fragments after refinement). Bootstrap values are indicated at tree branching points.

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Fig. 2. Effects of inoculation with PGPR microbial inoculants on growth characteristics of plants (n = 6). Control: no-inoculation with PGPR microbial inoculants, Treatment: inoculation with PGPR microbial inoculants. * means p < 0.05, ** means p < 0.01, ns means no significant difference.

Fig. 3. Effects of inoculation with PGPR microbial inoculants on plant enzyme activities and chlorophyll content (n = 6). Control: no-inoculation with PGPR microbial inoculants, Treatment: inoculation with PGPR microbial inoculants. * means p < 0.05, ** means p < 0.01, ns means no significant difference.

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Fig. 4. Effects of inoculation with PGPR microbial inoculants on soil enzyme activities (n = 6). Control: no-inoculation with PGPR microbial inoculants, Treatment: inoculation with PGPR microbial inoculants. * means p < 0.05, ** means p < 0.01, ns means no significant difference.

physiological characteristics of A. sativa seedlings (Fig. 6a, Table S2). Among them, soil properties (EC, AN, AP, AK, SOC, SCAT, SURE, SIN, and SAKP) exhibited a significant positive correlation with the growth and physiological characteristics of A. sativa seedlings (p < 0.05) (Table S3). RDA analysis of the relationship between the growth, physiological characteristics, and rhizosphere properties of M. sativa seedlings showed that the cumulative interpretation rates of the first and second sorting axes were 88.3 % and 9.8 %, respectively. EC, AN, AK, SOC, SCAT, SIN, and SAKP were the main factors affecting the growth and physiological characteristics of M. sativa seedlings (Fig. 6b, Table S2). Among them, soil properties (EC, AN, AP, AK, SOC, SCAT, SIN and SAKP) were significantly positively correlated with the growth and physiological characteristics of M. sativa seedlings (p < 0.05), whereas the pH was negatively correlated with the growth and physiological characteristics of M. sativa seedlings (Table S3). RDA analysis of the relationship between growth, physiological characteristics, and rhizosphere soil properties of C. sativus seedlings showed that the cumulative interpretation rates of the first and second sorting axes were 89.1 % and 6.8 %, respectively. AP, SOC, SCAT, SURE and SAKP were the main factors affecting the growth and physiological characteristics of C. sativus seedlings (Fig. 6c, Table S2). There was a significant positive correlation between soil properties (EC, AN, AP, AK, SOC, SCAT, SIN and SAKP) and the growth and physiological characteristics of C. sativus seedlings (p < 0.05), whereas the pH was negatively correlated with the growth and physiological characteristics of C. sativus seedlings (Table S3).

3.4. Effects of inoculation with PGPR microbial inoculants on soil enzyme activities and physicochemical properties In the presence of PGPR microbial inoculants there was an increase in enzyme activities in the rhizosphere of A. sativa, M. sativa, and C. sativus seedlings (Fig. 4). Compared with the control, PGPR microbial inoculants increased the SURE, SIN, and SAKP in the rhizosphere of A. sativa (p < 0.01) by 109.16 %, 18.04 %, and 34.17 %, respectively. PGPR microbial inoculants significantly increased the SIN, SAKP, and SCAT in the M. sativa rhizosphere (p < 0.01) by 14.81 %, 125.26 %, and 8.34 %, respectively. Compared with the control, PGPR microbial inoculants increased the SAKP (p < 0.01), SURE (p < 0.05), and SCAT (p < 0.05) in C. sativus rhizosphere by 57.66 %, 16.25 %, and 4.42 %, respectively. PGPR microbial inoculants influenced the physiochemical characteristics in the rhizosphere of A. sativa, M. sativa, and C. sativus seedlings (Fig. 5). Compared with the control, PGPR microbial inoculants increased the EC (p < 0.01), AK (p < 0.05), SOC (p < 0.01), AP (p < 0.01), and AN (p < 0.05) of the A. sativa rhizosphere by 20.44 %, 7.28 %, 6.57 %, 33.81 %, and 9.51 %, respectively. PGPR microbial inoculants increased the EC (p < 0.01), AK (p < 0.01), SOC (p < 0.01), and AN (p < 0.01) in the rhizosphere of M. sativa by 26.81 % and 5.08 %, 8.06 %, and 22.94 %, respectively, and the soil pH was lowered (p < 0.05). Compared with the control, PGPR microbial inoculants increased the AK (p < 0.05), SOC (p < 0.01), and AP (p < 0.01) in the rhizosphere of C. sativus by 3.01 %, 8.88 %, and 52.07 %, respectively. Further, they increased the soil’s EC and AN content and decreased the soil’s pH but these results were not significant.

4. Discussion In this study, we designed a multispecies PGPR inoculum containing Providencia rettgeri, Advenella incenata, Acinetobacter calcoaceticus, and Serratia plymuthica. Our multispecies PGRP had the ability to fix nitrogen, solubilize phosphorus, and produce auxin. The four strains of PGPR screened in this study produced inorganic and organic phosphorus amounts between 131.26–438.35 μg mL−1 and 13.36－ 35.46 μg mL−1, their nitrogenase activities were between 25.38–864.97 nmol mg−1 protein h−1, and the amount of IAA they

3.5. Relationship between plant and soil characteristics The relationship between the growth, physiological characteristics, and rhizosphere physicochemical properties of A. sativa seedlings was analyzed by RDA. The cumulative interpretation rates of the first and second sorting axes were 96.5 % and 2.3 %, respectively. EC, AP, SOC, SURE, and SAKP were the main factors affecting the growth and 6

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Fig. 5. Effects of inoculation with PGPR microbial inoculants on soil physicochemical properties (n = 6). Control: no-inoculation with PGPR microbial inoculants, Treatment: inoculation with PGPR microbial inoculants. * means p < 0.05, ** means p < 0.01, ns means no significant difference.

Fig. 6. The relationship between growth, physiological characteristics and soil properties of plant seedlings were analyzed by RDA. pH, soil pH; EC, electrical conductivity; AN, soil available nitrogen; AP, soil available phosphorus; AK, soil available potassium; SOC, soil organic carbon; SCAT, soil catalase activity; SURA, soil urease activity; SIN, soil invertase activity; SPHO, soil alkaline phosphatase activity; RSA, root surface area; RAD, root average diameter; RV, root volume; RL, root length; HE, plant height; DW, dry weight; SOD, superoxide dismutase; PCAT, plant catalase; POD, peroxidase; PCH, plant chlorophyll content.

secreted was between 11.7241.04 μg mL−1 (Table 1). There are many published studies on PGPR but most of them examine only a single PGPR strain. However, a single strain has the disadvantages of being able to carry out only one function and poor environmental adaptability. Compound PGPR microbial inoculants can compensate for these shortcomings to a certain extent. Gómez-Godínez (2019) evaluated the effect of a group of bacteria on the growth of one-month-old maize

plants and the results showed that the multispecies inoculum exerted a beneficial effect on maize plants that was greater than that obtained with a single bacteria. This suggests that in future research, the study of multispecies PGPR microbial inoculants is more applicable. In our study, the PH, DW, RL, RAD, RSA, and RV of A. sativa, M. sativa, and C. sativus seedlings were significantly increased in the presence of PGPR microbial inoculants (Fig. 2). This may be related to the 7

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the activity of SOD, POD, and CAT was used as an important indicator of PGPR-induced plant resistance (UdDin et al., 2015; Israr et al., 2016; Hahm et al., 2017; Paredes-Páliz et al., 2018). In this study, the activity of SOD, POD, and CAT of A. sativa, M. sativa, and C. sativus seedlings significantly increased in the presence of PGPR microbial inoculants by 20.31–105.37 %, 12.52–71.38 %, and 6.06–10.83 %, respectively (p < 0.01) (Fig. 3). This indicates that the PGPR inoculation not only promoted the accumulation of plant biomass but also increased the antioxidant enzyme activities and resistance of A. sativa, M. sativa, and C. sativus seedlings. Jha and Subramanian (2013) showed that the rice biomass and antioxidant enzyme activity increased after inoculation of PGPR, which also increased the plant’s resistance to salt stress. A study was conducted on Curcuma longa to examine the effect of seed treatments with native PGPR on seedling growth and antioxidant enzyme activities. The activities of antioxidant enzymes (such as SOD, POD, and CAT) were significantly increased following the treatments with PGPR (Boominathan and Sivakumar, 2012). PGPR can not only promote plant growth but can also improve plant’s rhizosphere environment. Khati et al. (2017) evaluated the effect of PGPR on maize growth. They found that there was variability in different physiological parameters and that the activities of soil health indicator enzymes were enhanced 2–3 fold. Ju et al. (2019) found that co-inoculation of PGPR and rhizobium significantly increased soil microbial biomass, enzymatic activities, total nitrogen, available phosphorus, and soil organic matter contents compared with the uninoculated control. The same trend was observed in our study that showed PGPR inoculation significantly increased the activities of catalase, invertase, urease, and phosphatase in the rhizosphere of A. sativa, M. sativa, and C. sativus seedlings (Fig. 4), which increased by 4.42–8.34 %, 7.03–18.04 %, 5.62–109.16 %, and 34.17–125.26 % compared with the non-inoculated treatment. Similarly, PGPR inoculation increased the content of EC, AN, AP, and SOC in the rhizosphere of A. sativa, M. sativa, and C. sativus seedlings (Fig. 5) by 6.18–26.81 %, 9.51–22.94 %, 12.33–52.07 %, 3.01–7.28 %, 6.57–8.88 %, respectively. RDA was used to analyze the relationship between growth, physiological characteristics, and rhizosphere characteristics of A. sativa, M. sativa and C. sativus seedlings. The results showed that soil enzyme activity and soil properties were significantly correlated with the growth of A. sativa, M. sativa, and C. sativus seedlings (Fig. 6). Considering the above results, we suggest that inoculated PGPR can transform elements, such as nitrogen and phosphorus, into nutrients that can be absorbed and utilized by plants through nitrogen fixation and phosphorus solubilization (Tagore et al., 2014). Furthermore, we suggest that PGPR can secrete auxins, such as IAA, to directly promote plant growth (Selvakumar et al., 2012). Specifically, the increase in plant root activity promotes the secretion of organic acids and reduces the pH of the rhizosphere. This decrease of soil pH has a certain degradation effect on phosphorus and potassium in the soil, which increases the concentration of available nutrients in the rhizosphere leading to an increase of the EC value and absorption of nutrients by plant roots (Khan et al., 2009). On the other hand, the forms and contents of nutrients, such as nitrogen, phosphorus, and potassium, in soils are related to the changes in soil enzyme activities, and the enhancement of soil enzyme activity is closely related to the increase of soil nutrient content (Cusack et al., 2011; You et al., 2014). PGPR inoculation can accelerate the decomposition of soil organic compounds, provide substrates for enzymatic reactions, and promote microbial growth, thereby improving soil enzyme activity, increasing soil nutrient content, and providing a thriving soil ecological environment for the growth of plants (Piromyou et al., 2011; Saia et al., 2015). Therefore, PGPR inoculation can improve plant habitats by producing antimicrobial substances, enhancing plant resistance, and improving soil fertility, which indirectly promotes plant growth and increases plant yields (Khadeejath et al., 2017). Nadeem et al. (2014) showed that the application of microbial fertilizers was beneficial to improve the soil physiochemical properties and microflora by increasing the activities of

secretion of IAA by PGPR. Chandra et al. (2018) found that the ability to synthesize IAA is widely associated with PGPR and the PGPR strains CA1001 and CA2004 can be used as bioinoculants for plant crops to enhance root and shoot biomass. Some studies showed that the effects of PGPR on root development were related to IAA production, which can promote root growth and development, increase root surface area, and promote root metabolism (Glick, 2014; Agarwal et al., 2019). Inoculation of rapeseed seeds with Pseudomonas putida GR12-2, which produces low levels of IAA, resulted in a 20–30 % increase in primary root elongation. Another variant of the strain that produces higher levels of IAA promoted the development of a large number of secondary roots. Regardless of the rapid growth and development of the primary roots or secondary roots and adventitious roots, the root system of plant seedlings would be rapidly established (Defez et al., 2019). Monk et al., 2009 found that 10 % of bacteria in New Zealand fescue roots can produce auxin to promote plant growth and some PGPR can synthesize IAA and its analogs to promote plant root growth. After the inoculation of auxin producing PGPR on Brassica napus, which grows in oily soil, the number of stem branches significantly increased (Asghar et al., 2004). Therefore, it is likely that the enhanced growth of A. sativa, M. sativa, and C. sativus seedlings that was observed in the presence of PGPR microbial inoculants might be primarily attributed to the secretion of auxin. Inoculating PGPR can effectively make the invalid form of soil nutrients available and promote the efficient absorption and utilization of nutrients by plants (Zhang et al., 2014; Damodharan et al., 2018). Alam et al. (2001) found that after inoculation with nitrogen-fixing bacteria (Azotobacter sp.), the dry matter quality, nitrogen accumulation, and yield of rice increased by 6–24 %. Chauhan et al. (2017) isolated the PGPR strain CKMV1 that exhibited many of the same functions as our strains. Inoculation of CKMV1 remarkably increased seed germination, shoot length, root length, shoot dry weight and root dry weight of tomato under net house conditions. Gómez-Godínez et al. (2019) evaluated the effects of a group of PGPR on the growth of one-month-old maize plants and found that these bacteria had an active nitrogen fixation ability and promoted maize growth. Therefore, it is reasonable to suggest that the nitrogen fixation and phosphorus solubilization capacities of PGPR might be other possible mechanisms underlying their ability to promote the plant growth of A. sativa, M. sativa, and C. sativus seedlings. Chlorophyll is the main photosynthetic pigment of plants and its content directly affects the photosynthesis capacity of plants (Su et al., 2017). It is well known that the growth of plants requires the accumulation of photochemical products and that plant growth is closely related to the rate of photosynthesis. Decreased chlorophyll content causes reduced light energy absorption and utilization capacity of plants, resulting in a decrease in photosynthesis (Ansari and Ahmad, 2019). del Rosario Cappellari et al. (2015) measured plant growth parameters and chlorophyll content in peppermint (Mentha piperita) seedlings inoculated with PGPR strains. Compared to the controls, the treated plants showed increases in the above parameters. Huang et al. (2016) found that the Pseudomonas aeruginosa Pse-w-MT strain isolated from Cd-contaminated oil fields increased the biomass and chlorophyll content of pea roots. Similar results were observed in our study that showed that the chlorophyll content of A. sativa, M. sativa, and C. sativus seedlings was higher than that of the control after the addition of PGPR microbial inoculants (Fig. 3). This indicates that PGPR microbial inoculants had a certain promoting effect on photosynthesis of A. sativa, M. sativa, and C. sativus seedlings. This result was similar to the increase in growth parameters observed in A. sativa, M. sativa, and C. sativus seedlings. Therefore, PGPR microbial inoculants promote seedling growth and improve seedling photosynthetic capacity. The antioxidant enzymes (SOD, POD, and CAT) in plants can remove intracellular reactive oxygen species, reduce membrane peroxidation, stabilize membrane permeability, and improve plant photosynthesis (Gururani et al., 2013; Habib et al., 2016). In some reports, 8

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soil invertase, phosphatase, peroxidase, and urease, which ultimately promotes plant growth. These findings are also consistent with the results of our study. Four strains of PGPR with the ability to fix nitrogen, solubilize phosphate, produce IAA, and biocontrol were screened in our study. These four PGPR strains effectively promoted plant growth in pot experiments. Thus, our study has provided novel strain resources for the development of microbial fertilizers. However, our study had a few limitations. Since the competition and utilization ability of PGPR strains for root exudates is only indirect evidence that it can colonize in the rhizosphere of plants, it is necessary to study the colonization ability of PGPR strains in different plant rhizospheres using the rhizosphere colonization test. In addition, the screening of strains in this study only considered the relationship between the PGPR strains and the plant and soil characteristics. It is also necessary to take into account the changes in the structure of indigenous microbial communities in the soil and analyze the relationship between the screened strains, plants and soil characteristics and soil microorganisms to further enhance the potential uses of PGPR.

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5. Conclusions In the present work, multifunctional PGPR microbial inoculants with the ability to fix nitrogen, solubilize phosphate, secrete IAA, and biocontrol were generated. These contained the four compatible strains Providencia rettgeri P2, Advenella incenata P4, Acinetobacter calcoaceticus P19, and Serratia plymuthica P35. Pot experiments showed that PGPR microbial inoculants could promote plant growth and increase the antioxidant enzyme activity and chlorophyll content of A. sativa, M. sativa, and C. sativus seedlings. Additionally, PGPR microbial inoculants could also increase the enzyme activity and available nutrient content in rhizosphere soil of plants. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 31660688) and the opening foundation of the Key Laboratory of Grassland Ecosystem, Gansu Agricultural University, Ministry of Education (No. GAU-XKJS-2018-007). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.still.2020.104577. References Abbasi, M.K., Sharif, S., Kazmi, M., Sultan, T., Aslam, M., 2011. Isolation of plant growth promoting rhizobacteria from wheat rhizosphere and their effect on improving growth, yield and nutrient uptake of plants. Plant Biosyst. 145, 159–168. https://doi. org/10.1080/11263504.2010.542318. Agarwal, P., Singh, P.C., Chaudhry, V., Shirke, P.A., Chakrabarty, D., Farooqui, A., Nautiyal, C.S., Sane, A.P., Sane, V.A., 2019. PGPR-induced OsASR6 improves plant growth and yield by altering root auxin sensitivity and the xylem structure in transgenic Arabidopsis thaliana. J. Plant Physiol. 240, 153010,. https://doi.org/10. 1016/j.jplph.2019.153010. Ahmad, M., Zahir, Z.A., Asghar, H.N., Arshad, M., 2012. The combined application of rhizobial strains and plant growth promoting rhizobacteria improves growth and productivity of mung bean (Vigna radiata L.) under salt-stressed conditions. Ann. Microbiol. 62, 1321–1330. https://doi.org/10.1007/s13213-011-0380-9. Alam, S., Cui, Z., Yamagishi, T., Ishii, R., 2001. Grain yield and related physiological characteristics of rice plants (Oryza sativa L.) inoculated with free-living rhizobacteria. Plant Prod. Sci. 4, 126–130. https://doi.org/10.1626/pps.4.126. Anand, R., Grayston, S., Chanway, C., 2013. N2-fixation and seedling growth promotion

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