Effects of Azospirillum brasilense and Pseudomonas fluorescens on ...

Effects of Azospirillum brasilense and Pseudomonas fluorescens on nitrogen transformation and enzyme activity in the rice rhizosphere Junhua Zhang, Sajid Hussain, Futao Zhao, Lianfeng Zhu, Xiaochuang Cao, Shengmiao Yu & Qianyu Jin Journal of Soils and Sediments ISSN 1439-0108 J Soils Sediments DOI 10.1007/s11368-017-1861-7

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Author's personal copy J Soils Sediments https://doi.org/10.1007/s11368-017-1861-7

SOILS, SEC 2 • GLOBAL CHANGE, ENVIRON RISK ASSESS, SUSTAINABLE LAND USE • RESEARCH ARTICLE

Effects of Azospirillum brasilense and Pseudomonas fluorescens on nitrogen transformation and enzyme activity in the rice rhizosphere Junhua Zhang 1 & Sajid Hussain 1 & Futao Zhao 2 & Lianfeng Zhu 1 & Xiaochuang Cao 1 & Shengmiao Yu 1 & Qianyu Jin 1

Received: 25 June 2017 / Accepted: 21 October 2017 # Springer-Verlag GmbH Germany 2017

Abstract Purpose Azospirillum brasilense (A. brasilense) and Pseudomonas fluorescens (P. fluorescens) have long been known to benefit inoculated rice plants, but their effects on nitrogen (N) transformations and enzyme activities in the rice rhizosphere are unclear. This study explored whether A. brasilense and P. fluorescens are involved in modifying N transformations, N-supplying capacity, and enzyme activities in the rice rhizosphere, and the performance of rice biomass after inoculation with A. brasilense and P. fluorescens was also evaluated. Materials and methods Rice inoculation was conducted under pot culture conditions in 2014 and 2015, and the experiment included four treatments: a control (CT), rice seedling roots inoculated with A. brasilense (Mb), rice seedling roots inoculated with P. fluorescens (Mp), and rice seedling roots inoculated with a mixture of A. brasilense and P. fluorescens (Mbp). The rice variety used in this trial was Zhongzheyou 1 (Indica). To explore the effects of A. brasilense and P. fluorescens strains on N transformations and enzyme activities in the rice rhizosphere, N fertilizer was not applied in this trial, while the full doses of P (150 mg P2O5 per pot) and K (320 mg K2O per pot) were applied. Responsible editor: Chengrong Chen * Junhua Zhang [email protected] * Qianyu Jin [email protected] 1

State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, Zhejiang 310006, China

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Industrial Technology Research Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China

Results and discussion Inoculating the rice rhizosphere with A. brasilense and P. fluorescens greatly improved the ammonification activities in the soil, and the effects were more favorable in the Mbp treatment. However, the contribution of inoculation to the nitrification activity in the rhizosphere was poor. Compared to CT, the average mineralized N content in the Mbp, Mb, and Mp treatments was increased by 165.7, 110.2, and 88.5%, respectively. Co-inoculation with A. brasilense and P. fluorescens in the rice rhizosphere greatly increased the nitrogenase activities in the soil, and inoculating with A. brasilense alone in the rice rhizosphere also showed good results. The microbial biomass N and enzyme activities were positively related to the N transformations in the soil. Finally, rice biomass increased greatly after A. brasilense and P. fluorescens inoculation in the rhizosphere. Conclusions Inoculating A. brasilense and P. fluorescens in the rice rhizosphere accelerated N transformations and improved the N-supplying capacity of the rhizosphere soil, and increased rice biomass. The most beneficial effects were observed with A. brasilense and P. fluorescens co-inoculation in the rice rhizosphere. Keywords A. brasilense . Enzyme activity . N transformation . P. fluorescens . Rice biomass . Rice rhizosphere

1 Introduction Rice (Oryza sativa L.) is one of the most globally important food crops, and it is a staple dietary for almost half of the human population, accounting for 23% of the world’s calorie intake (Prasanna et al. 2012). Nitrogen (N) is the most common limiting nutrient in rice production; 1 kg of N is required to produce 15–20 kg of grain (Ladha and Reddy 2003). As a

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result, rice production currently depends on high inputs of N fertilizers, which increase costs and can have negative environmental impacts (Zhang et al. 2013). Approximately 65% of the applied mineral N is lost from the plant-soil system through gaseous emissions, runoff, erosion, and leaching (Bgattacharjee et al. 2008). With increasing concern for food and environmental quality and the dependence of modern agriculture on the application of chemical inputs, it is necessary to search for viable alternative practices to promote sustainable agriculture (Wu et al. 2015). Plant growth-promoting rhizobacteria (PGPR) can colonize rice rhizospheres and promote rice growth and crop yield (Chauhan et al. 2015), so they have a potential role in the development of sustainable rice production systems (George et al. 2016). Bgattacharjee et al. (2008) discussed the biological nitrogen fixation (BNF) of nitrogen-fixing bacteria in rice, and their experiments conclusively showed that an increase in the host-plant N content, up to 30–45 mg of N per plant (6week-old seedlings), resulted from BNF. Prasanna et al. (2012) revealed the superior performance of PGPR in increasing rice growth and grain yield and improving soil health in addition to saving 40–80 kg N ha−1. For these reasons, there is great interest in exploring the diversity of PGPR as substitutes for some chemical agricultural inputs. Generally, the N-transformation processes in the rice rhizosphere include N mineralization (ammonification and nitrification), denitrification, N fixation, and ammonia volatilization. Microbial-mediated mineralization and BNF are very important to the level of available N content in the soil and the N uptake by rice (Richardson et al. 2009), so an increase in the abundance of microbial populations could result in a faster N mineralization rate and increased N availability for plants (Lu et al. 2009). The activities of invertase, urease (UT), and protease were found to be higher in the rhizosphere than the bulk soil, and the microbial biomass showed significant positive correlations with the amount of invertase and protease in the whole soil (Li et al. 2015). However, little information is available regarding the effects of PGPR on N transformation and the related enzyme activities in rice rhizospheres. Of the PGPR, the rhizobacteria Azospirillum brasilense (A. brasilense) and Pseudomonas fluorescens (P. fluorescens) have been used as model organisms to investigate associative plant growth promotion (Hayat et al. 2010; Sahoo et al. 2014), and both have long been known to benefit inoculated rice cultivars (Valverde et al. 2015). A study of the inoculation of paddy rice with A. brasilense and P. fluorescens suggested that the treatment increased the aerial biomass production, harvest index, and grain yield of rice by 4.7, 16.0, and 20.2%, respectively (Salamone et al. 2012). However, there has been little investigation into whether A. brasilense and P. fluorescens are involved in N transformation and enzyme activity in the soil of the rice rhizosphere. The purpose of this study was to explore the effects of A. brasilense and P. fluorescens inoculation on

mineralization, N fixation, and enzyme activities in the rice rhizosphere; to determine the resulting performance of rice plant growth and development; and to evaluate the influences of A. brasilense and P. fluorescens on N-supplying capacity in rice rhizospheres and the resulting rice plant growth.

2 Materials and methods 2.1 Bacterial strains and inoculant preparation Strains of A. brasilense (accession number 03125) and P. fluorescens (accession number 04138), which were isolated from the roots of field-grown rice, were kindly provided by the China Center of Agricultural Culture Collection. Azospirillum brasilense strains were cultured in modified Azospirillum medium with agitation (125 rpm) for 48 h at 28 °C, and pure bacterial cultures were centrifuged and diluted to a final concentration of 108 CFU ml−1 in a sterile saline solution (0.85% NaCl). The modified Azospirillum medium was composed of yeast extract (1 g L−1), peptone (3 g L−1), NH4Cl (0.5 g L−1), NaCl (1 g L−1), MgSO4.7H2O (0.2 g L−1), Na2MoO4.2H2O (0.002 g L−1), MnSO4.H2O (0.1 g L−1), CaCl 2 (0.0265 g L − 1 ), EDTA-Na 2 (0.0035 g L − 1 ), FeCl3.6H2O (0.0025 g L−1), KH2PO4 (0.43 g L−1), and K2HPO4 (0.56 g L−1), and it was adjusted to a pH of 7. The P. fluorescens strains were cultured in modified nutrient broth (NB) medium with agitation (125 rpm) for 24 h at 30 °C, and the pure bacterial cultures were centrifuged and diluted to a final concentration of 108 CFU ml−1 in a sterile saline solution (0.85% NaCl). The modified NB medium was composed of peptone (5 g L−1), beef extract (3 g L−1), NaCl (5 g L−1), and it was adjusted to a pH of 7. Both the A. brasilense and P. fluorescens strains were cultured separately, and the inoculants were prepared for a pot culture experiment. 2.2 Experimental design Paddy rice inoculation was conducted under pot culture conditions at the China National Rice Research Institute (39° 4′ 49″ N, 119° 56′ 11″ E), Zhejiang Province, China, in 2014 and 2015. The soil used in the experiment was loam clay containing 4.7% organic matter with available N-P-K at 342.1, 17.4, and 72.3 mg kg−1, respectively. The soil was finely ground and passed through a 2-mm sieve, and 6 kg of this soil was placed in 24-cm-diameter pots. The rice variety used in this trial was Indica, Zhongzheyou 1, for which the growth period ranges from 138 to 150 days from sowing to physiological maturity. The experiment consisted of four treatments: a control (CT), rice seedling roots inoculated with A. brasilense (Mb), rice seedling roots inoculated with P. fluorescens (Mp), and rice seedling roots inoculated with a mixture of A. brasilense


and P. fluorescens (Mbp). For CT, the rice seedling roots were treated with autoclaved saline solution (0.85% NaCl) without inoculants, and for the Mbp treatment, the inoculums were prepared using a 1:1 ratio (v/v) of A. brasilense and P. fluorescens strains. Twenty-five-day-old rice seedlings in bundles (100 seedlings) were inoculated with different strains by submerging their roots in 250 ml of bacterial suspension (108 CFU ml−1) for 2 h, thus ensuring that rice roots alone were immersed in the inoculums, and then immediately transplanted into the experimental pots. Two rice seedlings were evenly distributed in each pot. To explore the effects of A. brasilense and P. fluorescens strains on N transformation and enzyme activity in the rice rhizosphere, N fertilizer was not applied in this trial, while the full recommended doses of P (150 mg P2O5 per pot) and K (320 mg K2O per pot) were applied. The pots were arranged in a completely randomized design with four replications per treatment and three pots per replicate. Normal agronomic practices were applied.

2.3 Sampling The plants and rhizosphere soils were sampled at the time of rice transplanting, the maximum tillering stage, the full heading stage, and maturity. Rhizosphere soil and plant samples were collected by inserting a cylindrical cutting ring, which was 10 cm in diameter and 10 cm in height with a sharp low edge, into the soils with the plant at the center of the ring. The ring was removed, and the rhizosphere soil was extracted from the rice roots by shaking off the loosely adhering soil and using a brush to remove the tightly adhering soil. These rhizosphere soil samples were used to determine the ammonification activity, nitrification activity, N mineralization, microbial biomass N (MBN), nitrogenase activity, UR activity, and catalase (CAT) activity. Rice plants were divided into roots and shoots to measure the root biomass and the aboveground biomass, respectively.

2.4 Ammonification activity The ammonification activity in the soil was determined by modifying the method described by Li et al. (2008). A 1-ml soil suspension (soil:water = 1:10) was inoculated with 10 ml of ammonifying bacteria medium, which was composed of peptone (10 g L −1 ), beef extract (3 g L −1 ), and NaCl (5 g L−1) and adjusted to a pH of 7. The soil suspension was then incubated at 28 °C for 48 h before being filtered to measure the ammonium nitrogen (NH4+-N) content. The NH4+-N was analyzed using an ultraviolet spectrophotometer (UV2600, Shimadzu, Japan), and the ammonification activity was expressed as mg NH4+-N per kg dry soil.

2.5 Nitrification activity The nitrification activity in the soil was determined according to Li et al. (2008) with modifications. A 1-ml soil suspension (soil:water = 1:10) was inoculated with 30 ml of nitrate bacteria medium, which was composed of NaNO2 (1 g L−1), K2HPO4 (0.75 g L−1), NaH2PO4 (0.25 g L−1), MgSO4.7H2O (0.03 g L −1 ), Na 2 CO 3 (1 g L −1 ), and MnSO 4 .4H 2 O (0.01 g L−1) and adjusted to a pH of 7. The soil suspension was then incubated at 28 °C for 15 days before being filtered to measure the nitrite (NO2-N) content. The NO2-N content was analyzed using an ultraviolet spectrophotometer (UV2600, Shimadzu, Japan), and the nitrification activity was expressed as the percent reduction in NO2-N after inoculation. 2.6 N mineralization The method described by Xue et al. (2006), with modifications, was used to determine the N mineralization in the soil. Soil samples that had been pre-incubated at room temperature for 3 days were placed in polyethyleneglycol bottles (250 ml); the moisture content was adjusted to 50% of the field capacity; and the samples were incubated under a constant temperature of 25 °C. The samples were covered with plastic wrap to minimize water loss but to allow CO2 and O2 gas exchange (Bremmer and Douglas 1971). Three replicate samples were extracted with 2 mol L−1 KCl at days 0, 7, 14, 21, 28, 42, 56, and 70 of the incubation period, and the KCl extracts were used in the chemical analysis for inorganic N (NH4+-N and NO3−-N). Soil inorganic N at day 0 was determined for preincubated soils. The NH4+-N and NO3−-N contents were analyzed using an ultraviolet spectrophotometer (UV-2600, Shimadzu, Japan). 2.7 Microbial biomass N The MBN was determined using the fumigation-extraction method as previously described (Kanerva and Smolande 2007). Soil samples were fumigated for 24 h at 28 °C with ethanol-free chloroform vapor, and the N flush from the microbial biomass was calculated by subtracting the K2SO4-extractable N in the unfumigated control samples from those in the fumigated samples. The N flush was converted to MBN using the formulas of Martikainen and Palojärvi (1990). 2.8 Nitrogenase activity The nitrogenase activity can represent the N-fixing activity of rhizosphere soil, and it was determined by an acetylene reduction assay (ARA) (Prasanna et al. 2003). Approximately 10 g of soil was placed in vessels stopped with Suba-seals, which were added to the incubation system. The incubation system was then tightly closed, and 10% of its gas volume was


replaced with acetylene. The system was incubated in the dark at 28 °C for 24 h, and the samples were assayed for ethylene (C2H4) after 24 h by injecting them into a gas chromatograph equipped with a GS-GASPRO column and a flame ionization detector (FID) (Agilent 7890A, Agilent Technologies Inc., USA).The nitrogenase activity in the rhizosphere soil was expressed as nmol C2H4 per g dry soil per h. 2.9 Urease activity The UR activity in the soil was assayed by a previously reported method (Sivapalan and Fernando 1983) with modifications. Five grams of air-dried soil (passed through a 2-mm sieve) was placed in a 100-ml Erlenmeyer flask, and 1 ml of toluene was added. The contents were mixed well and allowed to stand for 15 min; then 20 ml of 0.1 mol L−1 McIllvaine’s buffer (citric acid-disodium hydrogen phosphate, pH of 7) and 10 ml of 10% urea solution were added, and the contents were mixed and incubated at 37 °C for 24 h. Next, 35 ml of the inhibitor KC1-HgCl2 solution were added, and the solution was shaken for 30 min. The samples were filtered to determine the ammonium contents, and the UR activity was expressed as mg NH 4 + -N per g soil under the specified incubation conditions. 2.10 Catalase activity The CAT activity was determined by back-titrating residual H2O2 with KMnO4 (Li et al. 2008). Five grams of the soil samples was added to 40 ml of distilled water and 5 ml of 0.3% H2O2 solution, and the mixture was incubated at 37 °C for 20 min. After incubation, the reaction was stopped by adding 5 ml of 1.5 mol L−1 H2SO4, and the mixture was filtered and titrated using 0.02 mol L−1 KMnO4. The amount of 0.02 mol L−1 KMnO4 per gram of dried soil consumed was used to express the CAT activity. 2.11 Rice root biomass and aboveground biomass Rice plants were sampled at the time of rice transplanting, the maximum tillering stage, the full heading stage, and maturity, and the plants were divided into rice shoots and rice roots. The dry weights of the samples were determined after fixation at 105 °C for 30 min and drying to a constant weight at 75 °C. 2.12 Statistical analysis The data were statistically analyzed by a standard analysis of variance (ANOVA), and pairs of mean values were compared by a least significant difference (LSD) test at the 5% level using the SPSS17.0 software package.

3 Results 3.1 Effects of A. brasilense and P. fluorescens on N transformations in the rice rhizosphere 3.1.1 Ammonification activity The variations in the ammonification activity in the rhizosphere soil during the 2014 rice ontogenetic stages were similar to those in 2015 (Fig. 1). Compared with the CT treatment, the average ammonification activity at the rice maximum tillering stage, the full heading stage, and maturity was increased by 162.1, 268.9, and 171.3%, respectively. Generally, the ammonification activities under the inoculation treatments were higher than that for CT during the rice ontogenetic stages, especially those under the Mbp and Mp treatments. At the rice maximum tillering stage, the ammonification activities under the Mbp and Mp treatments were significantly higher than under CT, increasing by 72.0–79.8 and 63.3–69.8%, respectively, compared to CT. The ammonification activity under the inoculation treatments at the rice full heading stage and at maturity also showed the same variations as at the rice maximum tillering stage. On average, the ammonification activity under the Mbp, Mb, and Mp treatments at the rice full heading stage was higher than that under CT by 72.4, 29.9, and 60.7%, respectively, while it was higher than that of CT at rice maturity by 66.0, 25.3, and 38.2%, respectively. The ammonification activities of the Mbp and Mp treatments always exhibited better performance throughout the rice life cycle, and the ammonification activities under the Mp treatment were generally higher than under the Mb treatment. The results indicated that regulation by A. brasilense and P. fluorescens mixtures or individual P. fluorescens in rice rhizospheres improved the ammonification activity in the soil. 3.1.2 Nitrification activity The variations in the nitrification activity in the rice rhizosphere were similar to the variations in the ammonification activity in the rice rhizosphere (Fig. 2). The nitrification activity in all treatments at the rice maximum tillering stage ranged from 8.3 to 14.7% in 2014 and 2015, and there were no significant differences between inoculating treatments and CT in the nitrification activity in the rice rhizosphere. The nitrification activity from all treatments at the rice full heading stage ranged from 12.8 to 19.6% in 2014 and 2015, but only the nitrification activity under the Mbp treatment in 2015 was significantly higher than that in CT in 2015. The nitrification activity in the rhizosphere soil at rice maturity followed the same trends as at the full heading stage. The results indicated that inoculating rice roots with a mixture of A. brasilense and P. fluorescens enhanced the nitrification activity in the rhizosphere in general, but the contribution of the mixed strains to

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Fig. 1 Ammonification activity of control (CT), inoculated with Azospirillum brasilense (Mb), inoculated with Pseudomonas fluorescens (M p ), inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015.

Vertical bars represent mean ± SE (n = 3). Bars with the same letter(s) for each rice ontogenetic stage are not significantly different at P < 0.05 according to the LSD test

the nitrification activity in the soil was lower than to the ammonification activity. The regulatory effects of A. brasilense or P. fluorescens alone on nitrification activity in the rice rhizosphere were not beneficial.

+NO3−-N) increased linearly during the first 30 days of the incubation experiment (Fig. 3), and the cumulative mineralized N contents from all treatments were significantly positively related to the number of incubation days in 2014 and 2015 (r = 0.955*, p = 0.05). After 30 days of incubation, the average mineralized N content for the Mbp, Mb, and Mp treatments as well as CT reached 229.2, 171.4, 137.0, and 92.2 mg kg−1, respectively. The extreme rate of mineralization tended to slow and stabilize after 30 days. Additionally, the mineralized N contents showed a declining trend later in the incubation, and this effect may be ascribed to the N lost due to denitrification and ammonia volatilization.

3.2 Effects of A. brasilense and P. fluorescens on N-supplying capacity in the rice rhizosphere 3.2.1 N mineralization Rhizosphere soils were sampled at rice harvest to measure the N mineralization dynamics. The total mineralized N (NH4+-N 2014 a a

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Fig. 2 Nitrification activity of control (CT), inoculated with Azospirillum brasilense (Mb), inoculated with Pseudomonas fluorescens (Mp), inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015. Vertical bars

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and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015. Vertical bars represent mean ± SE (n = 3)

The mineralized N content under the Mbp treatment was significantly higher than under the Mb and Mp treatments and CT throughout the inoculation. The mineralized N content under the Mb treatment was similar to the mineralized N content under the Mp treatment, and both contents were obviously higher than under CT during inoculation, which was supported by the net N mineralization rate (data not shown). The highest N mineralization rate was observed on the 14th day of the inoculation experiment. The average net N mineralization rate for the Mbp treatment was 13.6 mg kg−1 day−1, which was 1.2, 1.7, and 2.4 times greater than that under the Mb treatment, the Mp treatment, and CT, respectively. The cumulative mineralized N contents under the inoculation treatments were also markedly higher than that under CT at the end of the inoculation time. The average mineralized N content with the Mbp treatment increased by 165.7% compared to that of CT; that of the Mb treatment increased by 110.2% compared to CT; and that of the Mp treatment increased by 88.5% compared to CT. To some extent, the N mineralization results can represent the N-supplying capacity of the soil, so the high mineralized N content could supply a large amount of inorganic N for rice uptake.

Generally, the MBN under the Mbp, Mb, and Mp treatments at the rice maximum tillering stage was higher than that of CT by 112.5, 69.9, and 100.0%, respectively, while that at the rice full heading stage was higher than that of CT by 111.1, 81.2, and 80.9%. The MBN for the Mbp, Mb, and Mp treatments at rice maturity was higher than that of CT by 102.1, 93.7, and 83.2%, respectively. On average, the differences in the MBN of the Mbp, Mp, and Mb treatments were more pronounced than compared to CT during the rice plant cycle, indicating favorable regulation of MBN by A. brasilense and P. fluorescens.

3.2.2 Microbial biomass N The MBN plays a positive role in N transformation, N supply, and N regulation in paddy rice. On the one hand, microbes are closely associated with the biochemical cycle and the transformation of N; on the other hand, MBN can be an important N reservoir, providing N to rice plants and maintaining the ecological function of the rice rhizosphere. The variations in MBN during the rice ontogenetic cycle in 2014 followed the same trends as the variations in 2015 (Fig. 4). The highest MBN was observed at the rice maximum tillering stage, which was greater than the MBN at the rice full heading stage and at rice maturity and significantly higher than the MBN at rice transplanting.

3.3 Effects of A. brasilense and P. fluorescens on enzyme activities in the rice rhizosphere 3.3.1 Nitrogenase activity The nitrogenase activity can represent the N-fixing activity of rhizosphere soil. Generally, the nitrogenase activity in the rhizosphere soil was higher at the rice maximum tillering and full heading stages than that at rice maturity and the rice transplanting stage (Fig. 5). On average, the nitrogenase activity under the Mbp, Mb, and Mp treatments at the rice maximum tillering stage was significantly higher than under CT, increasing by 394.6, 280.1, and 242.3%, respectively, compared to CT. The nitrogenase activity under the Mbp, Mb, and Mp treatments at the rice full heading stage was also significantly higher than under CT, increasing by 178.9, 180.6, and 104.7%, respectively, compared to CT. Inoculating the rice rhizospheres with both a mixture of A. brasilense and P. fluorescens or A. brasilense alone improved the nitrogenase activity in the rhizosphere soil, which increased the available N content. At the rice full heading stage, the nitrogenase activity under the Mb treatment was even higher than under the Mbp treatment. Therefore, the results suggested that the high nitrogenase activity after inoculation with A. brasilense was closely and positively related with N fixation activity.

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Fig. 4 Microbial biomass N of control (CT), inoculated with Azospirillum brasilense (Mb), inoculated with Pseudomonas fluorescens (M p ), inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015.

Vertical bars represent mean ± SE (n = 3). Bars with the same letter(s) for each rice ontogenetic stage are not significantly different at P < 0.05 according to the LSD test

Pseudomonas fluorescens played a positive role in improving the ammonification activity in rice rhizospheres (Fig. 1), while A. brasilense played a positive role in improving N fixation. However, inoculation with a mixture of A. brasilense and P. fluorescens most favorably modified ammonification activity, nitrification activity, and N fixation activity in the rice rhizosphere compared to inoculation with A. brasilense or P. fluorescens alone. Accordingly, the co-inoculation of A. brasilense and P. fluorescens positively modulated N transformation in the rice rhizosphere, improving the available N content in the soil and thus benefitting rice growth and development.

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In general, the UR activities in 2015 were higher than those in 2014, which may be ascribed to the differences in weather between 2014 and 2015 (data not shown). Generally, the average UR activity at the rice maximum tillering stage, the full heading stage, and maturity was 262.0, 304.8, and 191.0% higher, respectively, than at rice transplanting (Fig. 6). The UR activity at the rice full heading stage was slightly higher than that at the rice maximum tillering stage and rice maturity, but significantly higher than at rice transplanting.

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Fig. 5 Nitrogenase activity of control (CT), inoculated with Azospirillum brasilense (Mb), inoculated with Pseudomonas fluorescens (Mp), inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015. Vertical bars


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Fig. 6 Urease activity of control (CT), inoculated with Azospirillum brasilense (Mb), inoculated with Pseudomonas fluorescens (Mp), inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015. Vertical bars


At rice transplanting, the UR activity from the Mbp treatment increased by 33.3–58.3% compared to CT, the Mb treatment increased by 14.7–48.5%, and the Mp treatment increased by 13.2–34.8%. At the rice full heading stage, the UR activity under the Mbp treatment was 47.8–52.2% higher than under CT, the Mb treatment was 28.9–34.8% higher, and the Mp treatment was 21.1–34.0% higher. The UR activities under the Mbp, Mb, and Mp treatments were all higher than under CT at the rice maximum tillering and the full heading stages, and during these times, the UR activity for the Mbp treatment exhibited the highest value, which was significantly greater than that of CT. No significant difference was found between the inoculating treatments and CT at rice maturity, which may be associated with senescence of the rice plant. UR activity was significantly positively related to the MBN, ammonification activity, nitrification activity, and nitrogenase activity (Table 1). The results revealed that favorable modification of UR activity by A. brasilense and P. fluorescens is beneficial to N transformation and N-supplying capacity.

Relative to CT, there were no significant differences in CAT activity among the rice ontogenetic stages, but CAT activity was drastically enhanced across the ontogenetic stages by A. brasilense and P. fluorescens inoculation. Generally, the CAT activity under the Mbp, Mb, and Mp treatments was significantly higher than that under CT from the rice maximum tillering stage to rice maturity, and the CAT activity under the Mbp treatment was the most vigorous. The results suggested that inoculating A. brasilense and P. fluorescens positively affected CAT activity in the rice rhizosphere, and the most favorable performance was shown under co-inoculation with a mixture of A. brasilense and P. fluorescens, which is consistent with the N transformation and microbial activity results. The higher CAT activity during the rice vigorous growth stage could facilitate the higher microbial activity, which could accelerate the transformation of N from organic forms to inorganic forms. 3.4 Effects of A. brasilense and P. fluorescens on rice biomass

3.3.3 Catalase activity Different with the changes in UR activity in the rice rhizosphere, the CAT activity in the rice rhizosphere was highest at the rice maximum tillering stage, which was 116.1% higher than at rice transplanting (Fig. 7), while the CAT activity at the rice full heading stage and maturity was 41.2 and 59.9% higher, respectively, than at rice transplanting. The CAT activity in the soil was significantly positively related to the MBN, nitrogenase activity, and UR activity (Table 1), which demonstrated that microbial activity and N fixation were vigorous at the rice maximum tillering stage (Figs. 4 and 5).

Rice roots play an important role in nutrient uptake and mediate nutrient transportation and distribution in rice plants. Rice root biomass was increased with rice growth and development, and the rice root biomass growth rate from rice transplanting to the maximum tillering stage was drastically higher than the growth rate from the maximum tillering stage to maturity (Table 2). From the rice maximum tillering stage to maturity, the average root biomass under the Mbp treatment increased by 41.6–60.8% compared to CT; that under the Mb treatment increased by 29.0–48.3%; and that under the Mp treatment increased by 24.0–44.8%. The rice root biomass

Author's personal copy J Soils Sediments values followed by double asterisk are significantly correlated at P < 0.01 according to Pearson analysis

Table 1 The correlation analysis for N transformations and enzyme activities in 2014 and 2015. Values followed by single asterisk are significantly correlated at P < 0.05 according to Pearson analysis, and

Ammonification activity Nitrification activity

Nitrification activity

Nitrogenase activity

Microbial biomass N

Urease activity

Catalase activity

0.687**

0.598**

0.547**

0.474*

0.043

0.647**

0.514*

0.772**

0.055

0.864**

0.691**

0.562**

0.648**

0.616** 0.482*

Nitrogenase activity Microbial biomass N Urease activity

under the Mbp treatment from the rice maximum tillering stage to maturity was significantly higher than under CT, and the rice root biomass under the Mb and Mp treatments also presented favorable results. The rice aboveground biomass performed similarly to rice root biomass (Table 3); the rice aboveground biomass under the Mbp treatment was obviously higher than under CT from the maximum tillering stage to maturity. On average, the rice aboveground biomass under the Mbp treatment was 56.0, 66.9, and 35.8% higher than under CT at the maximum tillering stage, the full heading stage, and maturity, respectively. The Mb and Mp treatments also showed favorable mediation of the aboveground biomass of rice.

4 Discussion The ammonification process can occur under both aerobic and anaerobic conditions, and ammonification activity in the rice

a

a Cat alase act ivit y (ml (h.g)-1 )

CT Mb Mp Mbp

2014

6 a b a

4

a

a

a ab

a a a a

b

a

b

2

0

8

CT Mb Mp Mbp

2015 a ab

Cat alase act ivit y (ml (h.g)-1 )

8

rhizosphere plays a positive role in modifying the availability of N in the soil (Geßler et al. 2015; Chen et al. 2012). Soil microbes, especially ammonifying bacteria, are the major drivers of ammonification in soil (Cong et al. 2015), and there was a significant correlation (r = 0.9997, p = 0.05) between the number of microorganisms and ammonification activity (Drazkiewicz 1996). In the present study, the ammonification activities under the inoculated treatments were significantly higher than under CT during the rice ontogenetic stages, especially under the Mbp and Mp treatments. The ammonification activities under the Mp treatment were generally higher than those under the Mb treatment throughout the rice life cycle, suggesting that P. fluorescens strains thrive in the rice rhizosphere and may act as ammonifying bacteria. Rice root exudates involved in plant-microbe interactions are abundant from the maximum tillering stage to the grain filling stage (Aulakh et al. 2001; Huang et al. 2016), so plants with high concentrations of root solutes and rapid growth stimulate high microbial activity in the rhizosphere (Doornbos et al. 2012).

6

ab b a a

4

a

a

b

a a

b

a a a a 2

0 Transplanting Max tillering Full heading

Maturity

Growth Stage

Fig. 7 Catalase activity of control (CT), inoculated with Azospirillum brasilense (Mb), inoculated with Pseudomonas fluorescens (Mp), inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp) in rice rhizosphere in 2014 and 2015. Vertical bars


Maturity

Growth Stage


Author's personal copy J Soils Sediments Table 2 Effects of Azospirillum brasilense and Pseudomonas fluorescens inoculation on rice root biomass in 2014 and 2015. The experiment included four treatments: a control (CT), rice seedling roots inoculated with Azospirillum brasilense (Mb), rice seedling roots inoculated with Pseudomonas fluorescens (Mp), and rice seedling roots

inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp). Data are expressed as mean ± SE (n = 3). Values followed by different letters are significantly different (P < 0.05) according to LSD test

Year

Treatment

Transplanting g plant−1

Max tillering g plant−1

Full heading g plant−1

Maturity g plant−1

2014

CT

0.05 ± 0.01a 0.05 ± 0.01a 0.05 ± 0.01a 0.05 ± 0.01a

2.71 ± 0.36a 3.23 ± 0.35a 3.09 ± 0.38a 3.78 ± 0.45a

3.02 ± 0.69b 4.53 ± 0.33a 4.19 ± 0.71a 5.13 ± 0.43a

4.21 ± 0.24b 5.75 ± 0.19a 5.38 ± 0.50ab 6.35 ± 0.43a

2015

Mb Mp Mbp CT Mb Mp Mbp

0.08 ± 0.01a 0.08 ± 0.01a 0.08 ± 0.01a 0.08 ± 0.01a

2.83 ± 0.23b 4.03 ± 0.15ab 4.27 ± 0.59a 5.16 ± 0.29a

4.49 ± 0.33a 6.58 ± 0.42a 6.78 ± 0.45a 7.28 ± 0.28a

5.48 ± 0.24a 6.65 ± 0.39a 6.60 ± 0.69a 7.23 ± 0.30a

The results of this trial suggested that higher ammonification activity at the rice maximum tillering stage and the full heading stage may be associated with a greater release of compounds from rice roots and increased microbial activity. The nitrification activity in the rice rhizosphere was not significantly different between the inoculated treatments and CT, which may be because nitrification under anaerobic conditions is generally low, and the aeration conditions under paddy rice conditions are poorer than in an upland field (Huang et al. 2014; Wang et al. 2015). The biological fixation of atmospheric N, especially non-symbiotic soil N2-fixation, has been of increasing interest in recent decades, especially as it applies to low-input agriculture. Under controlled conditions and using strains of Azospirillum amazonense as inoculants, it was showed that the contribution of the BNF to rice productivity could range from 9.2 to 27.7% of the accumulated N in the plant, improving the BNF potential in the inoculated plants up to 4-fold relative to controls (Salamone et al.

2010). The results of this trial showed that the nitrogenase activity in the rhizosphere soil under the inoculated treatments was obviously higher than that of CT, especially under the Mbp and Mb treatments. The higher nitrogenase activity under the Mbp and Mb treatments demonstrated the higher Nfixing ability of A. brasilense, which increased the available N content in the rice rhizosphere. Islam et al. (2012) also suggested that a wide diversity of free-living N-fixing bacteria could be used as a feasible alternative to N fertilizers in rice paddy ecosystems. Paddy rice yield is fundamentally affected by the total amount of N absorbed (Inamura et al. 2009), and inorganic N, which can be directly absorbed by paddy rice, is mainly derived from fertilizer N and the mineralized organic N in paddy soil (Chen et al. 2010; Lim et al. 2015). Therefore, enhancing the N mineralization process and increasing the amount of inorganic N content in paddy soil may play a significant role in maintaining soil quality and reducing N

Table 3 Effects of Azospirillum brasilense and Pseudomonas fluorescens inoculation on rice aboveground biomass in 2014 and 2015. The experiment included four treatments: a control (CT), rice seedling roots inoculated with Azospirillum brasilense (Mb), rice seedling roots inoculated with Pseudomonas fluorescens (Mp), and rice seedling roots

inoculated with a mixture of Azospirillum brasilense and Pseudomonas fluorescens (Mbp). Data are expressed as mean ± SE (n = 3). Values followed by different letters are significantly different (P < 0.05) according to LSD test

Year

Treatment

Transplanting g plant−1

Max tillering g plant−1

Full heading g plant−1

Maturity g plant−1

2014

CT Mb Mp Mbp CT Mb Mp Mbp

0.16 ± 0.02a 0.16 ± 0.02a 0.16 ± 0.02a 0.16 ± 0.02a 0.47 ± 0.05a 0.47 ± 0.05a 0.47 ± 0.05a 0.47 ± 0.05a

22.06 ± 1.40a 26.86 ± 0.81a 22.52 ± 1.81a 29.14 ± 1.44a 16.82 ± 0.86b 28.79 ± 3.04a 29.64 ± 4.46a 30.26 ± 1.97a

42.49 ± 5.60b 53.58 ± 2.06a 50.53 ± 0.55ab 58.29 ± 4.17a 31.02 ± 1.96c 50.88 ± 1.77b 56.50 ± 1.04ab 61.01 ± 3.47a

45.08 ± 3.96c 55.78 ± 2.08a 51.77 ± 0.29ab 60.86 ± 2.86a 47.09 ± 3.43b 57.06 ± 1.59ab 58.94 ± 1.09a 64.32 ± 4.30a

2015


fertilizer use in rice cropping systems. After 30 days of incubation, the mineralized NH4+-N in the soil at rice maturity ranged from 60 to 80 mg kg−1 (Cheng et al. 2001), and generally, the mineralized N peaked from 28 to 45 days of incubation (Xue et al. 2006; Tian et al. 2010). The present study demonstrated that after 30 days of incubation, the cumulative mineralized N content under the Mbp, Mb, and Mp treatments as well as CT reached up to 229.2, 171.4, 137.0, and 92.2 mg kg−1, respectively. The cumulative mineralized N content under the Mbp treatment was significantly higher than under CT during the incubation experiment, and the cumulative mineralized N contents under the Mb and Mp treatment also showed favorable results during the incubation experiment. The results suggested that inoculating rice rhizospheres with A. brasilense and P. fluorescens increased the cumulative mineralized N content and the net N mineralization rate, thus improving the N-supplying capacity of the soil. Therefore, microbial activity was closely associated with N transformations in the soil (Wang et al. 2017). The present study showed that the performance of the ammonification activity, nitrification activity, and N-fixing activity was consistent with the performance of MBN, and the higher MBN under the inoculation treatments also suggested that microbial activity was enhanced by inoculation with A. brasilense and P. fluorescens (Salamone et al. 2012). In other words, the thriving A. brasilense and P. fluorescens strains in the rice rhizosphere positively regulated the N transformations in the soil, improved the N-supplying capacity, and increased the available N content for the rice plant. Thus, inoculating rice rhizospheres with A. brasilense and P. fluorescens separately or especially with a mixture of A. brasilense and P. fluorescens could facilitate a reduction in N fertilizer use and the development of sustainable rice production. Soil enzyme activities are closely related to soil microbial activities and are part of nutrient (especially C, N, and P) cycles (Fu et al. 2012; Zhang et al. 2015). Teng et al. (2016) reported that the activities of UR and CAT in paddy fields generally increase with rice root growth; the UR activity was highest on Aug. 29 (approximately the rice heading stage) while the CAT activity was highest on Aug. 19 (approximately the rice initial heading stage). Pandey et al. (2014) suggested that the UR activity in paddy soil was low during the initial stages of rice development but gradually increased, reaching its maximum by 35–40 DAT (days after transplantation), which corresponds to the early elongation phase. In the present study, the UR activity at the rice full heading stage was higher than that at the other rice growth stages, while the CAT activity at the maximum tillering stage was higher than at the other growth stages (Figs. 6 and 7). The biochemical characteristics of N-fixing bacteria isolated from paddy field soils has demonstrated that many N-fixing isolates positively influence UR activity and CAT production (Islam et al. 2012); AMF colonization was shown to clearly increase the

abundance of soil microorganisms and enzymes (excluding invertase) and their activities, thereby enhancing N and P uptake by ryegrass (Ye et al. 2015). The results of this trial showed that the UR activity and CAT activity under the Mbp, Mb, and Mp treatments were higher than under CT at the rice maximum tillering and the full heading stages, and the Mbp treatment yielded the most favorable results in enhancing the UR activity and CAT activity in the rice rhizosphere (Figs. 6 and 7). Bhattacharyya and Jha (2012) observed that microbial biomass was positively correlated with UR (r = 0.99, p < 0.01) and acid phosphatase (r = 0.97, p < 0.01). Generally, the activities of UR and CAT were also significantly positively related to ammonification activity, nitrification activity, nitrogenase activity, and MBN (Table 1), suggesting that inoculating the rice rhizosphere with A. brasilense and P. fluorescens greatly enhanced the enzyme activities in the rhizosphere soil and positively mediated the N transformations. Rice plant-microbe interactions determine the N transformations in the rhizosphere and rice nutrient absorption (Salamone et al. 2010). A combined inoculation of Azospirillum armeniacus and Azospirillum nigricans, as opposed to inoculation with either strain alone, significantly increased total N uptake and the rice yield (by 23%) (Piao et al. 2005). Relative to the control, significant biomass increases were also observed under A. brasilense inoculation treatments with percent increases of 15–35% at the rice tillering stage, 28–50% at the grain filling stage, and 4–8% in grain yield (Salamone et al. 2010). Generally, co-inoculation of the rice rhizosphere with A. brasilense and P. fluorescens greatly increased rice root biomass and aboveground biomass from the maximum tillering stage to maturity, but individual inoculation with A. brasilense or P. fluorescens also increased rice root biomass and aboveground biomass (Tables 2 and 3). The higher available N-supplying capacity and enzyme activities in the rice rhizosphere may explain the beneficial effects of A. brasilense and P. fluorescens on rice plants.

5 Conclusions Co-inoculation of the rice rhizosphere with A. brasilense and P. fluorescens or inoculation with P. fluorescens alone greatly enhanced the ammonification activity in the soil. However, the contribution of A. brasilense and P. fluorescens to the nitrification activity in the rhizosphere soil was not satisfactory. Inoculating rice rhizospheres with a mixture of A. brasilense and P. fluorescens favorably increased the cumulative mineralized N contents in the soil when compared to CT or individual inoculations. Co-inoculation with A. brasilense and P. fluorescens or inoculation with A. brasilense alone significantly increased the nitrogenase activity in the rice rhizosphere. The involvement of A. brasilense and P. fluorescens also


improved the performances of the MBN, UR activity, and CAT activity in the soil. Enzyme activities in soil were significantly positively related to the MBN, ammonification activity, and nitrification activity. Generally, inoculation with A. brasilense or P. fluorescens strains or a mixture of A. brasilense and P. fluorescens positively enhanced the N-supplying potential of the rice rhizosphere, thus increasing the rice root biomass and aboveground biomass. Co-inoculation with A. brasilense and P. fluorescens yielded the most favorable modifications to the N transformations in the rice rhizosphere and rice plant growth and development. Funding information This work was supported by the National Natural Science Foundation of China (31201174), Zhejiang Provincial Natural Science Foundation of China (LY16C130007), National Key Research and Development Program of China (2016YFD0200801), and Basic Research Foundation of National Commonweal Research Institute (2014RG004-5). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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