Comparison of Paenibacillus polymyxa wild-type and Nif− mutant in colonization, plant-growth promotion and nitrogen xation contribution

This study aimed to compare the effect on colonization, plant-growth promotion and nitrogen xation contribution by inoculation with Paenibacillus polymyxa wild-type and Nif − mutant. Methods Paenibacillus polymyxa wild-type and Nif − mutant was labeled with GFP and then the GFP-labeled bacteria were used to inoculate cucumber. The colonization patterns of P. polymyxa WLY78 in these plants were observed under the confocal laser scanning microscope. The effects of plant-growth promotion were investigated by greenhouse experiments. The nitrogen xation contribution was estimated by 15 N isotope dilution experiments. effectively experiments that inoculation with P. polymyxa WLY78 can signicantly enhance the lengths and fresh wights of cucumber roots and shoots, but inoculation with ΔnifB-V mutant can not. 15 N isotope dilution experiments showed that cucumber plants derive 25.93% nitrogen from nitrogen xation performed by P. polymyxa WLY78, but the ΔnifB-V mutant nearly can not provide nitrogen for plant. 1.02%, respectively. These results indicate that the cucumber plant has incorporated the provided by BNF of P. polymyxa WLY78 under low N conditions and BNF is inhibited by high concentration of available in the environment. The ΔnifB-V strain-inoculated cucumbers nearly did not derive from BNF, consistent with the ΔnifB-V strain has no nitrogenase. The data also indicate that P. polymyxa WLY78 can be used to provide nitrogen nutrition to plants and reduce the use of


Introduction
Nitrogen is the most important nutrient in plant growth, but plants can not directly use nitrogen in the atmosphere. Biological nitrogen xation (BNF) is a process in which nitrogen-xing microorganisms reduce nitrogen in the air to ammonia through nitrogenases. Biological nitrogen xation is an important part of the natural nitrogen cycle (Dart 1986) and it plays an important role in the sustainable development of agriculture (Raymond et al. 2004). Nitrogen-xing microorganisms include symbiotic nitrogen-xing bacteria, autogenous nitrogen-xing bacteria and associative nitrogen-xing bacteria.
Associative nitrogen-xing bacteria can colonize root surface cells, invade plant roots, and form close contact with plants, thereby promoting plant growth (Baldani et al. 1997). Associative nitrogen-xing bacteria promote the absorption of nitrogen by non-legume plants (Geddes et al. 2015). Biological nitrogen xation can not only reduce the use of nitrogen fertilizer, but also improve soil fertility and the absorption of nutrients by crops (Farrar et al. 2014).
It has been reported that the associative nitrogen-xing bacteria play an important role in promoting growth of non-legumes by xing nitrogen and producing phytohormone (Chalk 1991). 15 N isotope and N balance studies have shown that several sugarcane varieties obtain over 60% of their nitrogen (< 150 kg N ha − 1 year − 1 ) from biological nitrogen xation performed mainly by Acetobacter diazotrophicus and Herbaspirillum spp. (Boddey et al. 1995). Diazotrophic bacteria present in the mucilage of aerial roots contribute 29-82% of the N nutrition of Sierra Mixe maize (Van Deynze et al. 2018). Inoculation with nitrogen-xing Klebsiella pneumoniae 342 (Kp342) increased total N and N concentration in the wheat plant (Iniguez et al. 2004). Inoculation of the rhizobacteria including Azospirillum brasilense and Azospirillum lipoferum contributed up to 20-50% of the total nitrogen requirement of the oil palm seedlings through nitrogen xation (Amir et al. 2003). Diazotrophic Paenibacillus beijingensis BJ-18 provides nitrogen for wheat, maize and cucumber plants and promotes plant growth, nitrogen uptake and metabolism . A recombinant nitrogen-xing Pseudomonas protegens Pf-5 X940 was constructed by introducing the nif genes of Pseudomonas stutzeri A1501 via the X940 cosmid to the bene cial rhizobacterium Pseudomonas protegens Pf-5, and inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-de cient conditions (Fox et al. 2016;Setten et al. 2013). Inoculation with Azospirillum brasilense Ab-V5 cells enriched with exopolysaccharides and polyhydroxybutyrate enhances the productivity of maize under low N fertilizer input (Oliveira et al. 2017;) Paenibacillus polymyxa WLY78 is a nitrogen-xing bacterium containing a compact nif gene cluster consisting of 9 genes (nifBHDKENXhesAnifV) (Wang et al. 2013;Xie et al. 2014). In addition to nitrogen xation, this bacterium has the ability of phosphate solubilization and IAA production (Xie et al. 2016).
Also, this bacterium can produce furaricidins that are a class of cyclic lipopeptide antibiotics to inhibit plant pathogenic fungi . These speci c traits suggest that P. polymyxa WLY78 is a member of plant growth-promoting bacteria (PGPB) and is of great usage as an inoculant in agriculture. However, the nitrogen contribution to plants derived from nitrogen xation of P. polymyxa WLY78 is unclear. In this study, the nif gene cluster deletion mutant (ΔnifB-V) of P. polymyxa WLY78 is constructed. Comparisons of P. polymyxa wild-type and ΔnifB-V mutant in colonization, plant-growth promotion and nitrogen xation contribution rate are investigated. Our study will provide foundation for application of P. polymyxa WLY78 as a biofertilzer in agriculture.

Materials And Methods
Bacteria strains and culture conditions Paenibacillus polymyxa WLY78, isolated from the rhizosphere of bamboo in Beijing (Wang et al., 2013). This bacterium has multiple antagonistic activities against plant pathogens and produces IAA (Xie et al., 2016). The nitrogen-xing gene cluster deletion mutant ΔnifB-V of P. polymyxa WLY78 was constructed by a homologous recombination method. Primers 5′ CGGCCACGATGCGTCCGGCGTAGAGGATCCGCGTGGTGGATGTGGA CG 3′ and 5′ AACGCTTTTTCGGTTATCATTCCTTCACATCTATTTTCGTC 3′ were used to amplify a 950 bp-long DNA sequence located upstream of nifB. Primers 5′ GAAGGAATGATAACCGAAAAAGCGTTCCCGT C 3′ and 5′ GACTGCGCAAAAGACATAATCGATAAGCTTCCTGATAAGGCAGACAAGGCTC 3′ were used to amplify a 1107 bp-long sequence located downstream of nifV. The two fragments were then fused with BamH /Hind III digested pRN5101 vector using Gibson assembly master mix (New England Biolabs), generating the four recombinant plasmids. Then, the recombinant plasmid was transformed into P. polymyxa WLY78 as described by Wang et al., (2018), and the marker-free deletion mutant (the doublecrossover transformant) ΔnifB-V was selected for from the initial erythromycin resistance (Em r ) transformants after several rounds of nonselective growth at 39˚C and con rmed by PCR ampli cation and sequencing analysis. P. polymyxa WLY78 and ΔnifB-V were inoculated into Luria-Bertani (LB) liquid medium, cultured at 30°C and 180 rpm to logarithmic growth phase, and then centrifuged to collect the bacterial cells and suspend the bacterial cells with physiological saline. The cell concentration was set to 10 8 cells mL − 1 .

Preparation of soil and seeds
The soil was taken from the Shangzhuang Experimental Station of China Agricultural University. They were all 0-20 cm deep topsoil. The soil was low nitrogen sandy soil. After the soil was air-dried and crushed, the debris were removed with a 2 mm sieve to reduce heterogeneity, and then packed into plastic pots with a diameter of 35 cm and a height of 25 cm. Each pot was lled with 2 kg of soil to grow cucumbers. No trace elements were applied during plant growth.
Cucumber seeds ("Zhongnong 8" of Beijing Shengfeng Garden Agricultural Technology Co., Ltd.) were rst disinfected with 10% sodium hypochlorite for 10 minutes, then rinsed with sterile water three times, and spread the seeds in a sterile petri dish with damp lter paper. Leave it in the dark at room temperature (25℃) for 3-5 days until the seeds germinate. During the period, 1-2 mL of sterile water was added dropwise with a pipette to keep the lter paper moist.

Colonization of P. polymyxa and ΔnifB-V on cucumber
The recombinant plasmid pGFP300 carrying the gfp gene (Hao and Chen, 2017) was transferred into P. polymyxa WLY78 and ΔnifB-V to prepare cell suspension of the GFP-tagged strains so that the cell concentration was 10 8 cells mL − 1 . The sterilized cucumber seeds were sown in sterile glass bottles containing 100 mL of 1/2 MS semi-solid agar medium, and one seed was placed in each bottle. After dark treatment for about a week, the seeds will grow into seedlings, which will be transferred to a light incubator for cultivation. After the seedlings grow 2-3 young leaves, inoculate the cell suspension of GFP-tagged strains at the root of cucumber. Three days later, the colonization of GFP-labeled strains in plant tissues were observed with a laser confocal scanning microscope (CLSM, Olympus FluoViewTM FV1000 confocal microscope), and images were collected with FV10-ASW software.

Greenhouse pot experiment
The research was conducted in the greenhouse of China Agricultural University using greenhouse potting. The experimental design was arranged by random factors, with three inoculation treatments and two nitrogen level treatments. Each treatment was repeated three times, for a total of 18 pots of cucumber plants. Nitrogen level treatment included high nitrogen and low nitrogen levels. Nitrogen fertilizer was applied in the form of 15 N labeled (NH 4 ) 2 SO 4 (10.16% 15 N atom, Shanghai Research Institute of Chemical Industry, China). The high nitrogen level was 250 mg N kg − 1 soil, and the low nitrogen level was 83 mg N kg − 1 soil. Nitrogen fertilizer was applied in three times, one-third each time, and the rst time was applied as a base fertilizer, followed by 7 and 14 days after transplantation.
The inoculation treatment included three treatments: inoculation of P. polymyxa WLY78 (WT), ΔnifB-V, and equal amount of deionized water (as a control). The germinated cucumber seeds with robust and consistent growth were picked and immersed in the bacterial suspension for 20 minutes. The seeds were immersed in deionized water for 20 minutes as a control group, and then transplanted into plastic pots.
Four seeds were planted in each pot, and three repetitions were set for each treatment. In the rst and second weeks after planting, the P. polymyxa WLY78 and ΔnifB-V bacterial suspensions were reinoculated into the roots of the plants, and the control group was added with the same amount of deionized water. Place the ower pots under the best conditions in the greenhouse to obtain suitable light conditions. The seedlings were regularly watered every 3 days until the relative humidity of the soil reached 40%.

Plant sample collection
On 30th day of cucumber planting, the plants were collected by destructive sampling. The whole seedling was rst uprooted, and rinsed with deionized water to remove the soil attached to the root system, then the root and shoot samples were separated, and the fresh weight and length of the root and shoot were weighed. The root and shoot samples were killed in an oven at 105°C for 30 minutes, and then dried at 65°C until constant weight for dry weight analysis. The dried sample was ground, sieved with a 1 mm sieve and placed in a bag, and the plant N content and 15 N enrichment determination were performed by an isotope mass spectrometer. The remaining samples were immediately frozen in liquid nitrogen for subsequent analysis.

Contribution of nitrogen by biological nitrogen xation
The 15 N isotope dilution technique was used to quantitatively determine the contribution of inoculated bacteria to plant biological nitrogen xation. After inoculation with P. polymyxa WLY78 and ΔnifB-V, the percentage of nitrogen from cucumber biological nitrogen xation to the nitrogen content in cucumber (% Ndfa) was calculated by the following formula: Among them, %NdfF is the 15 N enrichment of cucumber stems and leaves of inoculation treatment, and% NdfNF is the 15 N enrichment of cucumber stems and leaves of uninoculated treatment (control group).

Statistical Analysis
Graphs were prepared using GraphPad Prism software v. 8.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis was performed using SPSS software version 20 (SPSS Inc., Chicago, IL, United States). One-way analysis of variance (ANOVA) was employed to check the signi cant differences between treatments. Means of different treatments were compared using the least signi cant difference (LSD) at the 0.05 or 0.01 level of probability.

Results
Colonization of P. polymyxa WLY78 and ΔnifB-V mutant in cucumber P. polymyxa WLY78 contains a compact nif gene cluster consisting of 9 genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV) located in a 10. 5 kb. A nif gene cluster deletion mutant (ΔnifB-V) was constructed by recombination as described in Materials and Methods. The ΔnifB-V mutant does not have nitrogenase activity, no matter this mutant is cultivated in medium containing ammonium or no ammonium.
P. polymyxa WLY78 and the ΔnifB-V mutant were individually labelled with GFP and then the GFP-labelled bacteria were used to inoculate cucumber. After 3 days of inoculation, the samples of the cucumber roots, stems and leaves were observed under laser confocal microscopy. The cells of P. polymyxa WLY78 not only colonized on the surface of cucumber root, but also colonized interior of root, stem and leaf (Fig. 1ac). The presence of bacterial cells was observed in the vascular bundle of the stem and the leaves. Similarly, cells of the ΔnifB-V mutant were also observed in the primary root cortex of cucumber (Fig. 1d), stem (Fig. 1e) and leaf vein (Fig. 1f). The results indicated that P. polymyxa WLY78 and ΔnifB-V can colonize outside and inside of cucumber tissues. Deletion of the nif gene cluster did not affect the colonization of P. polymyxa WLY78.

Effects of P. polymyxa WLY78 and ΔnifB-V on the growth of cucumber
Cucumber samples were collected on the 30th day after plantation, and the length and fresh weight of plant shoots and roots were measured to evaluate the effects of inoculation with P. polymyxa WLY78 and ΔnifB-V mutant on plant growth under low and high nitrogen conditions. The un-inoculated plants served as a control. Compared to the un-inoculated control group, cucumber plants inoculated with P. polymyxa WLY78 under low nitrogen conditions showed increase of 63.03% in shoots fresh weight, of 71.20% in root fresh weight, of 53.31% in shoot length and of 97.51% in root length, but they showed a little increase in lengths and weights under high nitrogen conditions (Fig. 2a-d). Compared with the un-inoculated control group, the cucumber plants inoculated with the ΔnifB-V mutant showed a little increase in the fresh weight and length of the shoots and roots under both low and high nitrogen conditions (Fig. 2a-d).
Notably, the fresh weight and length of the shoots and roots of cucumbers inoculated with ΔnifB-V mutant under both conditions are similar to those of cucumbers inoculated with P. polymyxa WLY78 under high nitrogen conditions. Figure 3 is an experimental diagram of the greenhouse cultivation. The data indicate that the diazotrophic P. polymyxa WLY78 can effectively promote plant growth under low nitrogen conditions and disruption of nif genes encoding nitrogenase results in almost loss of the ability of promoting plant growth.

Quanti cation of BNF in P. polymyxa WLY78-and ΔnifB-V mutant-inoculated cucumbers
To estimate the contribution of BNF, 15 N isotope dilution technique was used to analyze the inoculated cucumber grown in soil contain 15 N-labeled (NH 4 ) 2 SO 4 as N fertilizer in greenhouse conditions (Table 1).
The nitrogen derived from gaseous nitrogen (%Ndfa) in the P. polymyxa WLY78 inoculated cucumber and the ΔnifB-V strain inoculated cucumber under low N conditions is 25.93% ± 2.32% and 2.93% ± 6.57%, respectively. Whereas, the nitrogen derived from gaseous nitrogen (%Ndfa) in the P. polymyxa WLY78 inoculated cucumber and the ΔnifB-V strain inoculated cucumber under high N conditions is 1.54 ± 0.66% and − 0.22 ± 1.02%, respectively. These results indicate that the cucumber plant has incorporated the nitrogen provided by BNF of P. polymyxa WLY78 under low N conditions and BNF is inhibited by high concentration of available nitrogen in the environment. The ΔnifB-V strain-inoculated cucumbers nearly did not derive nitrogen from BNF, consistent with the ΔnifB-V strain has no nitrogenase. The data also indicate that P. polymyxa WLY78 can be used to provide nitrogen nutrition to plants and reduce the use of nitrogen fertilizers. The results came from three biological replicates, the error represents SD, lowercase letters a and b indicate that there is a signi cant difference between the groups (P < 0.05), while the same letter indicates that there is no signi cant difference.

Discussion
In this study, both GFP-tagged P. polymyxa WLY78 and GFP-tagged ΔnifB-V mutant are able to colonize the roots, stems, and leaves of cucumber. This result shows that the deletion of the nif gene cluster does not affect the colonization. Similarly, both of wild-type Klebsiella pneumoniae 342 and the nifH − mutant can colonize wheat (Iniguez et al. 2004) and both wild-type Pseudomonas stutzeri A1501 and its nifH − mutant can colonize maize root (Ke et al. 2019). Wild-type Acetobacter diazotrophicus PAl5 and the nifD − mutant have the same ability of colonization in sugarcane (Sevilla et al. 2001). A difference between our study with other's is that the ΔnifB-V mutant of P. polymyxa WLY78 has a deletion of a compact nif gene cluster comprising 9 genes (nifBHDKENXhesAnifV) and the nifH − mutant or nifK − mutant or nifD − mutant has a deletion of a single nif gene.
The effects of P. polymyxa WLY78 and ΔnifB-V on cucumber growth under different nitrogen concentrations were further studied through greenhouse cultivation experiments. Compared to the uninoculated control and the inoculation with the ΔnifB-V mutant, inoculation with P. polymyxa WLY78 signi cantly increased the fresh weights and lengths of cucumber shoots and roots under low nitrogen conditions, but this effect is not found in the cucumbers inoculated with the ΔnifB-V mutant. Notably, inoculation with P. polymyxa WLY78 under low nitrogen conditions led to the increased levels of root length and wight were much higher than those of shoot length and wight, consistent with the recent results obtained by inoculation with diazotrophic P. beijingensis BJ-18 in maize, wheat and cucumber . It was observed that copy numbers of P. beijingensis BJ-18 are much higher in roots than in shoot, suggesting that the densities of diazotrophs are positively corelated to plant growth traits ). These results have revealed that the nitrogen xation of P. polymyxa WLY78 plays an important role in promoting plant growth. Phosphate solubilization and IAA production of P. polymyxa WLY78 may exhibit a minor role in promoting plant growth. Similarly, inoculation with Pseudomonas stutzeri A1501 strain can increase the root and shoot dry weight of maize, but this effect is not found in the maize inoculated with nifH − mutant (Ke et al. 2019). In N 2 -de cient conditions, Kallar grass inoculated with Azoarcus sp. BH72 grew better and accumulated more nitrogen than plants inoculated with the nifK − mutant strain (Hurek, et al. 2002.). Inoculation with K. pneumoniae 342 resulted in increased dry weight, chlorophyll content, total N, and N concentration of wheat in comparison with the uninoculated and nifH − mutant-inoculated controls (Iniguez et al. 2004).
The 15 N isotope dilution technique is commonly used to determine the contribution of nitrogen-xing bacteria to plant nitrogen. In this study, the 15 N isotope dilution technique was used to determine the biological nitrogen xation of P. polymyxa WLY78 and ΔnifB-V mutant. Cucumber plants derived 25.93% ± 2.32% from the nitrogen xation of P. polymyxa WLY78 under low nitrogen xation, but the ΔnifB-V mutant hardly xed nitrogen. Similar reports are found that nitrogen xation of P. beijingensis BJ-18 provided 27.8% nitrogen for cucumber under low nitrogen conditions  and Paenibacillus polymyxa P2b-2R provided 15% nitrogen in maize by nitrogen xation (Padda et al. 2017 2019YFA0904700).
Con ict of interest The authors have no con icts of interest to declare.
Availability of data and material All data generated or analysed during this study are included in this published article.