Phosphate solubilizing bacteria stimulate wheat rhizosphere and endosphere biological nitrogen fixation by improving phosphorus content

Bacteria strains and growth conditions

P. beijingensis BJ-18 (accession number: JN873136) was isolated from rhizosphere soil of wheat (Wang et al., 2013). Paenibacillus sp. B1 (accession number: KY111475), isolated from the maize rhizosphere, is a novel species with P solubilizing ability and produce IAA (Li et al., 2017). P. beijingensis BJ-18 and Paenibacillus sp. B1 were inoculated individually in Erlenmeyer flasks (250 mL) containing 100 mL of Luria Bertani broth and cultured at 30 °C overnight. After growth, the bacterial cells were harvested by centrifugation at 4,000×g for 5 min and diluted with deionized water to 10⁸ cells/mL.

Plant culture and collection

Seedling growth assays were performed in plastic pots (diameter of 9 cm; height of 12 cm) filled with 1 kg low nutrition-content soil. The collection and physicochemical properties of soil were described by Li et al. (2019b). The treatments contained: (1) wheat seedling inoculated with Paenibacillus sp. B1 (B1) (2) wheat seedling inoculated with P. beijingensis BJ-18 (BJ-18) (3) wheat seedling inoculated with Paenibacillus sp. B1 and P. beijingensis BJ-18 (v:v = 1:1; Mix) (4) non-inoculated wheat seedlings as a negative control (CK) and (5) wheat seedlings inoculated with P. beijingensis BJ-18 and watered with nutrient solution containing soluble P (BJ-18+P) as a positive control. Plants were irrigated with N-free nutrient solution (0.65 mM MgSO₄, 2 mM CaCl₂, 0.75 mM K₂SO₄, 0.1 mM KCl, 0.25 mM KH₂PO₄, 0.2 mM Fe-EDTA, 1 × 10⁻³ mM MnSO₄, 1 × 10⁻³ mM ZnSO₄, 1 × 10⁻⁴ mM CuSO₄, 6.2 × 10⁻⁶ mM Na₂MoO₄, 1 × 10⁻³ mM H₃BO₃) in the presence or absence of KH₂PO₄ where applicable (43 mL per pot).

Plump seeds of wheat “Jimai 22” (Shandong Runfeng Seed Industry Co., Ltd., Gaomi, Shandong, China) were soaked in 10% sodium hypochlorite for 10 min, and rinsed thrice with sterilized water. The sterilized wheat seeds were placed on the sterile plates containing moist filter papers and cultivated for 3–5 days at room temperature (25 °C). After germination, vigorous and homogeneous wheat seedlings were chosen. For inoculation, the wheat seedlings were soaked in bacteria suspension (10⁸ cells/mL) for 30 min to facilitate colonization. For CK treatment, the plant seedlings were soaked in sterilized deionized water. Then the inoculated and non-inoculated seedlings were respectively transplanted into pots (3 hills per pot and 15 seedlings per hill). Sixteen millilitres of the bacteria suspension were applied to pots containing inoculated seedlings and 16 mL of sterile water was applied to pots containing non-inoculated seedlings on day 7. All treatments had five replicates. Pots were placed in the greenhouse under optimum conditions (15 h light/9 h dark cycle, 25–30 °C day/15–20 °C night temperature and 40% day/60% night humidity). The seedlings were watered every 5 days.

On day 35 after transplanting, the samples of wheat were collected. For collection of rhizosphere soil, the wheat seedling was uprooted carefully, and the intact root was shaken gently to remove the loosely adhering soil. Then the tightly adhering soil was gathered as rhizosphere soil. Shoot and root samples were separated and the lengths of the roots and shoots measured immediately. Then other root and shoot samples were dried for dry weight analysis. Then, the samples were ground for plant total P and total N content. The remaining plant samples were kept in a freezer at −80 °C. Chlorophyll content of seedlings was determined at 8:30–9:30 AM on the harvest day using the SPAD-502 chlorophyl meter (Minolta Camera Co. Ltd., Tokyo, Japan).

Elemental analyses of soil and plant tissue

The oven-dried samples were weighed and ground into fine powders. An appropriate amount of soil and plant samples was digested by an H₂SO₄–H₂O₂ mixture at 370 °C and then total N was determined using the modified Kjeldahl method (Baker & Thompson, 1992). Plant P content was determined using the standard vanadomolybdate method (Hanson, 1950). Available P of soil was extracted with resin and measured following (Hedley & Stewart, 1982).

Biological N₂ fixation assay

The acetylene reduction method was used to detect nitrogenase activity of rhizosphere soil (Jean et al., 2013; Perakis, Pett-Ridge & Catricala, 2017), with slight modification. Two grams of rhizosphere soil were added to a 26 mL glass jar fitted with septa and flooded with sterile deionized water to 240% moisture content. The jars were incubated at 28 °C in the dark for 48 h prior to replacing 10% (v/v) of the headspace with acetylene. After incubation for 2 h, 100 μL of headspace air were collected to measure ethylene production by gas chromatograph (GC-2010 Plus; Shimadzu, Kyoto, Japan). The nitrogenase activity was expressed as nmol ethylene g⁻¹ soil h⁻¹.

Quantitative real-time PCR analysis of nifH in plant roots and shoots

Transcript levels of nifH gene, one of nif genes coding Fe protein of nitrogenase, were quantified by RT-qPCR. RNAiso Plus reagent was used to extract plant total RNA according to the manufacturer’s instructions (RaKaRa, Kyoto, Japan). The RNA concentration was measured spectrophotometrically at 220 nm using a spectrophotometer (Nanodrop 1000; Thermo Scientific, Waltham, MA, USA). RNA was digested with DNase I (RaKaRa, Kyoto, Japan), and reversely transcribed into cDNA with using PrimeScript^™ RT reagent kit (RaKaRa, Kyoto, Japan). qRT-PCR was conducted in an Applied Biosystems 7500 Real-Time System (Bio-Rad, Hercules, CA, USA) using the SYBR^® Premix Ex TaqTM (Takara, Kyoto, Japan). The nifH primers were chosen for qRT-PCR (Roesch, Mergel & Bothe, 2002). The plant housekeeping gene actin was used as plant internal control (Padaria et al., 2014). The relative expression of the target genes was analyzed according to the standard comparative C(t) method (Livak & Schmittgen, 2001).

Statistical analysis

One-way analysis of variance (ANOVA) was conducted using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). The Pearson’s correlation coefficient was analyzed using GraphPad Prism 7.00 (GraphPad Software Inc., San Diego, CA, USA). Graphs were prepared using GraphPad Prism 7.00 and SigmaPlot software version 12.5 (Systat Software Inc., San Jose, CA, USA).

Plant growth parameters

Compared to CK treatment, B1, BJ-18, Mix and BJ-18+P treatments significantly increased wheat root length (Fig. 1A; 8%, 7%, 22% and 23%) and shoot length (Fig. 1B; 14%, 18%, 30% and 27%). However, Mix and BJ-18 treatments were not significantly different from each other in wheat length. Dry weight of wheat roots/shoots (Figs. 1C and 1D) in B1, BJ-18, Mix and BJ-18+P were significantly increased by 19%/25%, 16%/28%, 39%/49% and 42%/56%, respectively, compared to CK. For wheat dry weight, there was no significant difference between Mix and BJ-18+P treatments.

Plant biochemical parameters

Total N of wheat roots in BJ-18, Mix and BJ-18+P were significantly increased by 35%, 71% and 36%, respectively, compared to CK treatment (Fig. 2A). Compared to CK treatment, total N of wheat shoots in BJ-18, Mix and BJ-18+P were significantly increased by 38%, 79% and 70%, respectively (Fig. 2B). Total P of wheat roots in B1, Mix and BJ-18+P were significantly increased by 88%, 95% and 136%, respectively, compared to CK treatment (Fig. 2C). Total P of wheat shoots in B1, Mix and BJ-18+P were significantly increased by 70%, 42% and 46%, respectively, compared to CK treatment (Fig. 2D). Compared to BJ-18 treatment, root total N, shoot total N, root total P and shoot total P in Mix treatment were significantly increased by 27%, 30%, 63% and 30%, respectively. There were no significant differences in plant N and P uptake between Mix and BJ-18+P treatments.

Compared to CK treatment, BJ-18, Mix and BJ-18+P treatments significantly increased SPAD value. Mix and BJ-18 treatments were not significantly different from each other in chlorophyll content (Table 1).

Soil total N, available P and nitrogenase activity

Compared to CK treatment, treatments of BJ-18, Mix and BJ-18+P significantly increased total N of rhizosphere soil by 20%, 34% and 33%, respectively (Fig. 3A). B1, Mix and BJ-18 significantly increased available P in soils (Fig. 3B), by 6%, 8% and 12%, respectively, as compared to CK treatment. All treatments, B1, BJ-18, Mix and BJ-18+P, significantly increased rhizosphere soil nitrogenase activity by 26%, 52%, 158% and 163%, respectively, as compared to CK treatment (Fig. 3C). Soil nitrogenase activity of B1 did not differ significantly from the CK treatment. Mix treatment also showed much higher total N, available P and nitrogenase activity of rhizosphere soil than BJ-18 treatment. There were no significant differences in soil total N, available P and nitrogenase activity between Mix and BJ-18+P treatments.

Transcript levels of nifH gene

Transcripts of nifH were significantly up-regulated in wheat roots of BJ-18, Mix and BJ-18+P treatments by 18, 63 and 54 fold, respectively, and in wheat shoots of BJ-18, Mix and BJ-18+P treatments by 9, 31 and 17 fold, respectively, compared to those in the CK treatment (Fig. 4). Furthermore, treatment with Mix doubled transcription of nifH in wheat roots relative to treatment with BJ-18 alone.

Relationships between biological nitrogen fixation and phosphorus content

In the Mix treatment, higher soil available P, soil nitrogenase activity, plant total P and nifH transcript levels were observed than in the BJ-18 treatment. For the data as a whole, the nifH transcript levels were strongly and positively correlated with P content in wheat biomass (Fig. 5A). For the data of wheat roots (Fig. 5B) and shoots (Fig. 5C), the nifH transcript levels were significantly positively correlated with plant total P content. In the rhizosphere soil, nitrogenase activity was strongly and positively correlated with soil available P content (Fig. 5D).

Plant growth promotion

Co-inoculation with this particular P-solubilizing strain improved the efficacy of a diazotrophic PGPR, in terms of plant growth and nutrition. Inoculation with P solubilizing bacteria can significantly promote plant growth, and allows for reduced fertilizer application rates without sacrificing yield (Mehta et al., 2015; Jilani et al., 2007). Diazotrophic bacteria inoculation can increase plant biomass and nutrient status in potato, bamboo and maize through BNF (Naqqash et al., 2016; Xie et al., 2016; Ke et al., 2019). In both greenhouse and field conditions, co-inoculation of P solubilizing bacteria and diazotrophic bacteria can significantly promote wheat growth and nutrient uptake (Kumar et al., 2017; Sogut & Cig, 2019). Increased plant growth corresponded to increased SPAD values (Table 1). SPAD values positively correlated with leaf chlorophyll content, which is a crucial indicator of plant internal N nutritional status (Sibley et al., 1996; Chapman & Baretto, 1997).

Are wheat rhizosphere BNF and soil available P related?

In this study, only one pair of P solubilizing bacterium and diazotrophic bacterium was studied, and since the soil used in greenhouse study was not sterile, soil nitrogenase activity observed in this study could have been in part due to indigenous diazotrophic bacteria in that soil. This increase of nitrogenase activity is comparable to the effects observed under other settings. P application doubled soil microbial N and soil N fixation rate in a clover-dominated sward and a restored prairie, respectively (Edwards, McCaffery & Evans, 2006; Reed et al., 2007). Yelenik, Perakis & Hibbs (2013) also reported that P limits BNF in the Douglas-fir series of the Klamath Region. In both greenhouse and field conditions, co-inoculation diazotrophic Enterobacter sp. and P solubilizing bacterium Serratia marcescens significantly promoted N and P uptake of wheat compared to single inoculation with diazotrophic bacteria or P solubilizing bacteria (Kumar et al., 2017).

Are wheat endosphere BNF and plant P nutrition related?

The ability of PGPR strains B1 and BJ-18 to form close associations with plant roots, as established with observation of GFP-tagged constructs (Li et al., 2017, 2019a), could explain the synergy reported herein. Endophytes interact closely with plant hosts and readily exert beneficial traits (Santoyo et al., 2016). Endophytic diazotrophic Klebsiella was isolated from various plants (Govindarajan, Kwon & Weon, 2007; Liu et al., 2017). Endophytic diazotroph Paenibacillus polymyxa P2b-2R was isolated form pine stem, reduced seedling mortality, provided plant N by BNF, and increased plant dry weight (Anand, Grayston & Chanway, 2013). Pseudomonas stutzeri A15, isolated from rice rhizosphere and endosphere, promotes the growth and development of rice seedlings through BNF (Pham et al., 2017). Divito & Sadras (2014) reported that high plant P content was required for symbiotic BNF in legumes. In the legume–rhizobial symbiosis, arbuscular mycorrhiza stimulated BNF of symbiotic diazotrophic bacteria by improving plant P nutrition (Pueschel et al., 2017). In our study there was a significant positive correlation between asymbiotic nifH expression of endophytic diazotrophic bacteria and plant total P, both in the plant as a whole and in plant roots and shoots. Although there are no other reports on the effects of non-leguminous plant P nutrition on nifH expression by endophytes, our data showed that inoculation with the P solubilizing bacterium Paenibacillus sp. B1 provided positive BNF-feedback to endophytic diazotrophic bacteria by improving plant P nutrition. Therefore, we infer that P. beijingensis cells in plant tissues sensed the P signal and then regulated nif expression according to change of P levels.