Soil physical and chemical properties
The physical and chemical properties of the soil samples collected from the different locations of C. fungigraminus roots are shown in Table 1. The soil pH value, concentrations of total nitrogen, available nitrogen, organic matter and organic carbon gradually decreased with soil depth, and the concentrations of each index were lowest in G4. However, the water content increased gradually and highest in G4.
Table 1
Physicochemical characteristics of C. fungigraminus soil from different locations (n = 6, means ± SD)
Sources
|
Moisture content
(WC, %)
|
pH
|
Total nitrogen
(TN, g/kg)
|
Available nitrogen(AN, mg/kg)
|
Organic matter(OM, %)
|
Organic carbon
(OC, %)
|
G1
|
8.80 ± 0.04a
|
6.08 ± 0.02a
|
0.20 ± 0.12a
|
88.08 ± 3.64a
|
7.18 ± 0.39a
|
4.12 ± 0.22a
|
G2
|
8.28 ± 0.12b
|
6.18 ± 0.05b
|
0.12 ± 0.01b
|
53.67 ± 2.67b
|
4.61 ± 0.46b
|
2.65 ± 0.27b
|
G3
|
8.46 ± 0.18b
|
5.92 ± 0.01c
|
0.07 ± 0.02c
|
35.58 ± 1.01c
|
2.37 ± 0.11c
|
1.36 ± 0.06c
|
G4
|
8.92 ± 0.07a
|
5.84 ± 0.02d
|
0.06 ± 0.06c
|
32.08 ± 1.01d
|
1.93 ± 0.22d
|
1.11 ± 0.13d
|
Note: Values with different letters in the same column are significantly difference (P < 0.05), and values with the same letters are not significantly different (P > 0.05) |
DNA extraction and nifH gene copy determination
The results of the genomic DNA extracted from the 24 samples and the electrophoretic amplification of the nifH were detected by 1% agar gel electrophoresis (Fig. S1). The nifH PCR products were approximately 360 bp in size and the concentration and purity met the quality requirements for high-throughput sequencing. The Ct value was obtained by AQ-PCR amplification of the nifH gene and was taken as the ordinate (Y). There was a good linear relationship between X and Y, in which the standard curve equation was Y =-3.59X + 39.89, R2 = 0.999, and the amplification efficiency was 89.91% (Fig. S2). The melting curve had a single peak, indicating that there was no non-specific amplification or primer dimer. The copy number of the nifH gene in each group was highest in G2 (1.56 ± 0.17×107 copy/g) (Fig. S3).
Nitrogen-fixing bacteria population diversity
By prepossessing the sequences obtained from sequencing and removing low-quality and ambiguous sequences, a total of 3.28 million raw data were obtained from the 24 samples. After initial quality filtering, removal of noise and correction, 3.07 million valid sequences remained with the sequence length ranging between 33 to 444 bp, and the average sequence length of 319 bp. Based on a principle of similarity greater than 97%, the total OTUs number of the G1, G2, G3 and G4 samples was 1650, 1821, 1704 and 1729, respectively. The diversity of nitrogen-fixing bacteria in rhizosphere soil (G2) was higher than that in non rhizosphere soil (G1, G3, G4) (Fig. 1a). The 4 groups of sample dilution curves with Observed_species to be stable as the test depth deepened, which constructed by QIIME2 (v2.13.4). The non-metric multidimensional scaling (NMDS) revealed that the repeated samples in each group were clustered well and indicated that the amount of sequenced data was reliable and could reflect the diversity of nitrogen-fixing bacteria communities (Fig. 1b). Alpha diversity analysis can reveal the richness and diversity of microbial communities in each group of samples. Table 2 shows that the Chao 1 index, the number of Observed species, and the Shannon index of nitrogen-fixing bacteria species in each group following as G2 > G4 > G3 > G1. The diversity and abundance of nitrogen-fixing bacteria groups were the highest in C. fungigraminusr rhizosphere soil (Table 2).
Table 2
Alpha diversity index of the nifH gene in C. fungigraminus soil from different locations (n = 6, means ± SD)
Group
|
Chao1
|
Goods_coverage(%)
|
Observed_species
|
Pielou_e
|
Shannon
|
Simpson
|
G1
|
2028.63 ± 237.27a
|
99.55 ± 0.05a
|
1647.05 ± 210.33a
|
0.72 ± 0.01a
|
7.69 ± 0.21a
|
0.98 ± 0.003a
|
G2
|
2366.97 ± 142.22b
|
99.46 ± 0.04b
|
1825.12 ± 152.10a
|
0.77 ± 0.02b
|
8.37 ± 0.12b
|
0.99 ± 0.003b
|
G3
|
2294.24 ± 148.45b
|
99.43 ± 0.03b
|
1702.62 ± 111.97a
|
0.74 ± 0.02c
|
7.98 ± 0.25c
|
0.98 ± 0.007b
|
G4
|
2307.76 ± 148.12b
|
99.44 ± 0.03b
|
1726.88 ± 134.63a
|
0.77 ± 0.01d
|
8.31 ± 0.19d
|
0.99 ± 0.001c
|
Note: There is significant difference between G1 and G2, G3 and G4 in Chao1 index and the Goods_coverage percentage (P < 0.05). There were significant differences in the Pielou_e index and the Shannon index between each group. There were no significant differences in the Simpson index between G2 and G3, and no significant differences in the Observed_species index between each groups (P > 0.05) |
Species abundance and community structure
Nitrogen-fixing bacteria comprised at the different levels of domain, phylum, order, family, genus and species have different Microbiota. At the phylum level, it was found that 90.93%-95.98% of the bacteria belong to Proteobacteria, with the greatest abundance occurring in G1 and the least abundance in G4. Then, followed by Actinobacteria with 0.12%-0.42% of the bacteria and with the greatest abundance in G2. Firmicutes, Cyanobacteria and Verrucomicrobia are present a small amounts in 4 groups, with abundances of 0.09%-0.34%, 0.05%-0.29% and 0.06%-0.14%, respectively. Particularly, Spirochaetes and Chlorobi are present in G2, with abundances of 0.02% and 0.01%, while do not appear in the other gropus. Unclassifiable clades are also present in 4 groups with abundances of 3.66%-5.35% (Fig. 2a).
At the genus level, the community composition and relative abundance of nitrogen-fixing bacteria in C. fungigraminus rhizosphere soils are significantly different among locations,. The genera with high abundance in 4 groups included Bradyrhizobium (29.64%-39.71%), Geobacter (9.35%-28.99%), Azospirillum (1.75%-15.15%), Desulfovium (1.75%-15.15%), and Azospirillum. 15.15%), Desulfovibrio (2.67%-5.58%), Burkholderia (1.42%-6.76%), Halorhodospira (0.68%-2.73%), Methylosinus (0.51%-4.30%), Azotobacter (1.07%-2.45%), Paraburkholderia (0.30%-1.70%), Frankia (0.11%-0.34%), Rhizobium (0.0023%-0.01%) and unidentified bacteria (23.26%-31.81%). Genera with high abundance in G1 included Proteobacteria (95.97%), Actinobacteria (1.16%), Verrucomicrobia (0.98%), Firmicutes (0.89%), Cyanobacteria (0.61%) and other unidentified genera (3.65%). In G2, high abundance bacteria in genera included Proteobacteria (93.91%), Actinobacteria (0.42%), Firmicutes (0.18%), Verrucomicrobia (0.06%), Cyanobacteria (0.04%), Rhizobium (0.0054%) and unidentified genera (5.35%). In group 3, Proteobacteria (90.93%), Actinobacteria (3.10%), Firmicutes (0.34%), Cyanobacteria (0.29%), and Verrucomicrobia (0.14%) are the main community components. In G4, Genera with high abundance included Proteobacteria (94.10%), Actinobacteria (0.38%), Firmicutes (0.32%), Cyanobacteria (0.16%), and Verrucomicrobia (0.09%) (Fig. 2b).
As shown in Fig. 3a, the number of OTUs in G1, G2, G3 and G4 are 1361, 976, 841 and 1076, respectively, and with common 445 OTUs in all groups. The unique OTUs in each group were 534 (G1), 399 (G2), 327 (G3) and 326 (G4). The overall classification of samples based on R language and pheatmap software were plotted as a heatmap, and the top 50 classification units in relative abundance are shown in Fig. 3b. The color change on the heatmap shows that the nitrogen-fixing bacteria community of G1 and G2 have analogous composition, while nitrogen-fixing bacteria community of G3 and G4 could clustered together. Meanwhile, the same result also shown in Fig. 1b. The relative abundance of Paraburkholderia, Ruficoccus, Azonexus, Azohydromonas, Azoarcus, Azospirillum, Burkholderia and Rhizobium are higher in G1 than in other groups, while the relative abundance of Azotobacter, Methylococcus, Rhizobium and Burkholderia are highest in G2. Azotobacter, Methylocystis, Methylosinus, Beijerinckia, and Geobacter have relatively high abundance in G3, while in G4, Ralstonia, Pelomonas, Desulfovibrio, Halorhodospira, Magnetospirillum, Frankia, and Bradyrhiz have higher relative abundance.
For the nitrogen-fixing bacterial community in different location of in C. fungigraminus riophere. total nitrogen (TN), available nitrogen (AN) and depth (DP) explained > 80% of the bacterial community variation. In C. fungigraminus growth surface soil (G1), total nitrogen, available nitrogen, organic matter (OM) and organic carbon (OC) are the main factors and have positively affecte with Azospirillum, Burkholderia and Rhizobium etc.; Compared to G1, depth (DP) is the only and most significant main factor and contributed nearly 40% of the total variance in nitrogen-fixing bacterial community variation in G2,G3 and G4 (Fig. 3c). LEfSe analysis shows an abundance rank tree which is plotted based on the overall classification of the samples. With a LDA value of 3 (species diversity and richness, P < 0.001), Actinobacteria and Proteobacteria have higher abundance at the phylum level, and at the genus level Bejerinckia, Bradyrhizobium, Methylocystis, Rhizobium and Azospirillum have higher abundance. The distribution of Bejerinckia and Methylocystis in G2 are significantly higher than other genera and these genera are the most abundant in this group, which could be as marker species (Fig. 3d). These results are consistent with the composition taxonomic statistics in Fig. 2.
Isolation, purification, identification and performance of nitrogen-fixing bacteria
The diluted bacterial solution is coated on the Ashby's nitrogen-free medium petri dish and typical colonies with high colony number and vigorous growth were selected. Through microscopic examination and nitrogenase activity determination, 28 typical colonies were selected for further physiological and biochemical tests (Table 3). Out of these, 11 representative strains classes were divided by SDS-PAGE electrophoresis analysis (Fig. 4).
Table 3
Colonies characteristics and nitrogenase activity (n = 3, means ± SD) of nitrogen-fixing bacteria in C. fungigraminus soil from different locations
Source
|
Stain number
|
Colony characteristic
|
Nitrogenase activity(U/g)
|
Stain number
|
Colony characteristic
|
Nitrogenase activity(U/g)
|
Stain number
|
Colony characteristic
|
Nitrogenase activity(U/g)
|
G1
|
1
|
Purple,
rod shaped
|
1.44 ± 0.04
|
2
|
Purple,
rod shaped
|
1.56 ± 0.06
|
3
|
Purple,
rod shaped
|
1.44 ± 0.06
|
4
|
Purple,
rod shaped
|
1.58 ± 0.03
|
5
|
Purple,
rod shaped
|
1.30 ± 0.08
|
6
|
Red,
rod shaped
|
1.74 ± 0.02
|
7
|
Red,
rod shaped
|
1.41 ± 0.07
|
8
|
Red,
rod shaped
|
1.45 ± 0.04
|
9
|
Red,
rod shaped
|
1.17 ± 0.05
|
G2
|
10
|
Purple,
rod shaped
|
1.19 ± 0.12
|
11
|
Purple,
rod shaped
|
1.08 ± 0.10
|
12
|
Red,
rod shaped
|
1.41 ± 0.06
|
13
|
Purple,
rod shaped
|
1.41 ± 0.06
|
14
|
Purple,
short rod-shaped
|
1.00 ± 0.06
|
15
|
Red,
rod shaped
|
1.05 ± 0.06
|
16
|
Purple,
short rod-shaped
|
1.22 ± 0.04
|
17
|
Purple,
short rod-shaped
|
0.95 ± 0.06
|
18
|
Purple,
globular
|
1.46 ± 0.07
|
19
|
Red,
rod shaped
|
1.34 ± 0.01
|
20
|
Red,
short rod-shaped
|
1.42 ± 0.02
|
|
|
|
G3
|
21
|
Red,
short rod-shaped
|
1.64 ± 0.06
|
22
|
Red,
long rod
|
1.64 ± 0.08
|
23
|
Purple,
rod shaped
|
2.00 ± 0.07
|
24
|
Purple,
rod shaped
|
1.88 ± 0.04
|
25
|
Purple,
short rod-shaped
|
1.72 ± 0.04
|
|
|
|
G4
|
26
|
Purple,
short rod-shaped
|
1.80 ± 0.04
|
27
|
Red,
rod shaped
|
1.92 ± 0.05
|
28
|
Red,
rod shaped
|
2.00 ± 0.07
|
11 representative strains’ determination experiment includes indole test, catalase test, methyl red test, NO3− reduction test. Ammonia production capacity, phosphorus solubility activity and iron producing carrier were also measured for the basic function of representative strains. Table 4 shows that none of the 11 selected strains had the ability to decompose glucose and tryptophan, which indicated there are not intestinal pathogens. Stain No.8 and No.28 had the ability to convert the nitrogen fixed into ammonia. Both strain also had a positive ability to catalyse hydrogen peroxide and produce H2O and O2−, and the ability to reduce NO3− to NO2− and even convert to nitrogen compounds for the plants growth. Both stain No.8 and No.28 could produce iron carriers, and had the ability to dissolve the ineffective calcium phosphate salts into effective phosphate salts for the plant absorption. In all, these capabilities hint that stain No.8 and No.28 may have a good promotion to the plant growth as the PGPR.
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)
Identification and plant promotion test of nitrogen-fixing bacteria
PGPR strains NO.8 and NO.28 with high nitrogenase activity and strong phosphorus solubilization ability were identified by their 16S rDNA sequences. The homologous sequences were compared and downloaded from NCBI, which the likehood with known strains is greater than 97% and belong to the Rhizobium. The 2 typical strains cluster with MK241857.1, ON358080.1, NR169377.1, NR144599.1 and bootstrap value reached more than 97%, which indicated the 2 strains belong to Rhizobium pusense (Fig. 5). Rhizobium pusense NO.8 and Rhizobium pusense NO.28 clustered together and screened in G1 and G2 respectively (Fig. 3b, Table 3).
The plant height, leaf length, number of leaves, root length, and tiller number of C. fungigramminus after watering with R. pusense NO.8 bacterial solution (T1), NO.28 bacterial solution (T2), and mixed bacterial solution (T3) are shown in Table 5 and Fig. 6. Compare to the CK, the root length, height, leaf length of C. fungigramminus in T3 are significantly increased 56.79%, 76.99% and 55.71% (P < 0.05), respectively. The available nitrogen, organic matter and organic carbon of C. fungigramminus soil in T2 are significantly increased 3.09 times, 5.77 times and 5.77 times, respectively. The T1, T2 and T3 are significantly increased the water content and total nitrogen content in soil.
Table 5
Effects of R. pusense NO.8 (T1), R. pusense NO.28 (T2), and the mixture (T3) on C. fungigramminus seedlings and soil (n = 5, means ± SD)
Group
|
C. fungigramminus seedlings
|
C. fungigraminus soil
|
Root length (cm)
|
Number of roots
|
Plant height (cm)
|
Leaf length (cm)
|
Number of leaves
|
water content(%)
|
pH
|
total nitrogen content (g/kg)
|
Available nitrogen(mg/kg)
|
organic matter
(%)
|
organic carbon (%)
|
CK
|
537.55 ± 55.12a
|
8a
|
22.25 ± 4.29a
|
18.31 ± 2.60a
|
3a
|
15.59 ± 0.26a
|
5.93 ± 0.27a
|
0.26 ± 0.18a
|
38.97 ± 17.62a
|
0.48 ± 0.27a
|
2.78 ± 1.57a
|
T1
|
786.74 ± 57.83b
|
14bc
|
37.35 ± 8.61b
|
24.12 ± 4.82b
|
4b
|
25.02 ± 0.94b
|
6.22 ± 0.17a
|
0.69 ± 0.52b
|
120.63 ± 8.08b
|
2.77 ± 0.99b
|
16.05 ± 5.76b
|
T2
|
801.57 ± 59.48c
|
13b
|
34.09 ± 7.51b
|
22.29 ± 2.42b
|
4b
|
26.91 ± 0.82b
|
7.04 ± 0.07b
|
0.38 ± 0.03c
|
92.63 ± 16.16b
|
0.94 ± 0.28c
|
5.47 ± 1.66c
|
T3
|
842.80 ± 66.54d
|
15c
|
39.38 ± 4.72c
|
28.51 ± 2.79c
|
4b
|
21.48 ± 0.66b
|
6.91 ± 0.18b
|
0.86 ± 0.60d
|
108.97 ± 26.50b
|
1.85 ± 0.79c
|
10.73 ± 4.60c
|
Note: Values within columns with different letters in common are significantly difference at P < 0.05