Effect of Pennisetum giganteum z.x.lin mixed nitrogen-fixing bacterial fertilizer on the growth, quality, soil fertility and bacterial community of pakchoi (Brassica chinensis L.).

Biofertilizer plays a significant role in crop cultivation that had reduced its inorganic fertilizer use. The effects of inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer on the growth, quality, soil nutrients and diversity of the soil bacterial community in the rhizosphere soil of pakchoi were studied. The experiment composed of 6 treatments, including CK (no fertilization), DL (10% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer), ZL (25% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer), SL (50% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer), FHF (100% inorganic fertilizer) and JZ (100% inorganic fertilizer combined with sterilized Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer). Compared with conventional fertilization, the 25% reduction in chemical fertilizer applied with the Pennisetum giganteum mixed nitrogen-fixing biofertilizer resulted in higher plant height, plant weight, chlorophyll content, soluble protein content, soluble sugar content, vitamin C content, alkali hydrolyzed nitrogen content, available phosphorus content, available potassium content and organic matter content in pakchoi, and these variables increased by 11.81%, 8.54%, 7.37%, 16.88%, 17.05%, 23.70%, 24.24%, 36.56%, 21.09% and 19.72%, respectively. In addition, the 25% reduction in chemical fertilizer applied with the Pennisetum giganteum mixed nitrogen-fixing biofertilizer also had the lowest nitrate content, which was 53.86% lower than that with conventional fertilization. Different fertilizer treatments had a significant effect on the soil bacterial community structure. Compared with conventional fertilization, the coapplication of Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer and inorganic fertilizer significantly increased the relative abundance of Proteobacteria and Actinobacteria in the soil. The results of the redundancy analysis (RDA) showed that soil organic matter, alkali-hydrolyzed nitrogen, available phosphorus, available potassium, pH and water content had a specific impact on the soil bacterial community. Among the factors, soil water content was the main factor affecting the soil bacterial community, followed by soil organic matter, soil pH, available potassium, soil available phosphorus and soil alkali-hydrolyzed nitrogen.

Introduction

Soil microorganisms are an important part of the soil ecosystem [1], participating in the decomposition of organic matter, nutrient element cycles and energy conversion [2-4]. These microorganisms play an important role in maintaining the productivity, function and stability of the ecosystem [5-6], which is a key indicator for measuring soil quality and productivity [7]. Bacteria are the most abundant and largest group of microorganisms, usually accounting for 70%~90% of soil microorganisms. Bacteria have the richest genetic diversity and can effectively promote the decomposition of organic matter and release of nutrients, participating in nutrient cycling processes such as carbon and nitrogen cycling and maintaining the energy flow and material cycles of the ecosystem [8-10].

With the development of modern agriculture in China, inorganic fertilizer plays a very important role in agricultural production. However, the long-term unsustainable application of inorganic fertilizers has resulted in serious problems to the ecological environment that occur daily. A large number of studies have shown that the amount of fertilizer applied in China has exceeded the optimal amount economically and has resulted in losses of economic benefits to farmers [11]. At the same time, excessive application of inorganic fertilizer has also caused critical environmental pollution. Scientific research has confirmed that the overuse of inorganic fertilizers has become the main source of agricultural pollution [12-14]. Biofertilizers are products containing living cells of different types of microorganisms that have the ability to convert nutritionally important elements from unavailable to available forms through biological processes [15]. Biofertilizer can not only promote plant growth and development and improve the stress resistance of crops and the quality of agricultural products [16-17] but also improve soil fertility, fertilizer utilization efficiency and soil microbial community structure [18-21].

Pakchoi (Brassica chinensis L.) is cultivated within a large northern to southern range in China [22]. Pennisetum giganteum z.x.lin belongs to the phylum angiospermae, class monocotyledons, family gramineae and genus Pennisetum. It is a typical C4 plant that is suitable for growing in tropical, subtropical and temperate zones [23]. Pennisetum giganteum z.x.lin has high nutritional value, good palatability and wide application. It can not only replace sawdust to cultivate edible and medicinal mushrooms, addressing the conflict between forest and mushroom industry development but also be used as animal forage, and it has wide application in ecological management [24]. An endophytic diazotroph is a kind of microorganism that colonizes healthy plants and combines with host plants for nitrogen fixation. It plays an important role in nitrogen fixation, biological control and plant growth [25]. Peng [26] et al. found that the light absorption and growth rate of rice leaves increased by 12% and the yield increased by 16% after inoculation with endophytic diazotrophs. Govindarajan [27] et al. inoculated Burkholderia MG43 into sugarcane and found that this approach could replace half of nitrogen fertilizer and save 70 kg ha-1 nitrogen fertilizer. At present, there are many studies on the endophytic nitrogen-fixing bacteria of Gramineae and crops, but there are few studies on the endophytic nitrogen-fixing bacteria of Pennisetum giganteum and its nitrogen-fixing bacteria fertilizer. To provide a theoretical basis for the scientific and appropriate application of Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer and partial replacement of chemical fertilizer, this experiment was carried out to study its effect on the growth and quality of pakchoi, the physical and chemical properties of soil and the diversity of the bacterial community.

Materials and methods

Experimental site description

The experiment was conducted in August 2018 at the greenhouse of the National Engineering Research Center of JUNCAO Technology base, Fujian Agriculture and Forestry University, Fuzhou, China. The soil organic matter content was 31.14 g/kg, the alkali-hydrolyzed nitrogen content was 36.0 mg/kg, the available phosphorus content was 63.1 mg/kg, the available potassium content was 115.5 mg/kg, and the pH value was 5.26.

Preparation of test materials

The Kosakonia radicincitans nitrogen-fixation strain was isolated from Pennisetum giganteum z.x.lin and preserved in the laboratory. The sterilized samples of macromycorrhizal roots were placed into a sterilized mortar and cut with sterile scissors, and then, the proper amount of PBS buffer to grind the roots was added. The extract (0.1 mL) was coated on an Ashby solid medium plate, and the culture was inverted at 28~30°C for 2–3 days to observe colony growth. Then, the single bacteria growing on the Ashby solid medium plate was inoculated to the Nfb solid medium again for rescreening. The strains that could grow on the Nfb medium were selected, the color of the medium changed from blue to green, and the strains were inoculated again on the Ashby solid medium. This process was repeated three times. Finally, the selected colonies were inoculated on the Nfb slant medium and saved for future use and identification [28]. Bacillus mucilaginosus was isolated from the rhizosphere soil of Pennisetum giganteum and preserved in the laboratory. After gradient dilution, the soil samples were coated on silicate bacteria culture medium and cultured in a constant temperature incubator at 30°C for 48 h. According to the growth speed and morphology of the colony, a clear colony with mucilaginous protuberance was removed from the separation plate and then separated on the solid plate until a pure culture was obtained [29]. The pakchoi variety was No.2 Brassica chinensis from New Zealand that was bought from the local market. Inorganic fertilizer (N:P:K = 18:6:6) was bought from Fujian AoLiGaoTa Fertilizer Co., Ltd. The stems and leaves of Pennisetum giganteum z.x.lin were selected from the jointing stage or mature stage, crushed, dried at a low temperature and passed through a 100 mesh sieve. The waste mushroom substrates of Ganoderma lucidum were selected and cultivated with Juncao grasses, dried and crushed at a low temperature and passed through a 100 mesh sieve. The nutrient solution formula consisted of brown sugar 30–50 g/L, MgSO4 0.5–1.5 g/L, and calcium superphosphate 10–20 g/L.

Preparation of Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer

The activated Kosakonia radicincitans and Bacillus mucilaginosus were inoculated into an Luria-Bertani (LB) liquid medium at a ratio of 1:1, the culture oscillated at 150–180 r/min at 30°C until logarithmic growth. In a polyethylene film fermentation bag with a breathing valve, 150–200 mL bacterial solution, 650–750 g of dry Pennisetum giganteum z.x.lin, 250–350 g waste mushroom substrates of Ganoderma lucidum, and 100–150 mL nutrient solution were mixed, and fermentation occurred at 25–32°C for 5–10 days. Fermentation ended if the material became soft and dark. Compound microbial fertilizer was tested according to NY411-2000 nitrogen-fixing bacteria fertilizer [28].

Experimental test design

The experiment of inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer was carried out. Six treatments were set up, and three plots with a replication of each treatment were arranged in completely random groups. The area of each plot was 12 m2, with a total of 18 plots. The amount of inorganic fertilizer applied was 525 kg ha-1 according to the recommended amount of fertilizer applied locally. The proportion of fertilizer applied is shown in Table 1.

Table 1

The proportion of fertilizer in the different fertilizer treatments.

Sample ID	Treatment groups	Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer application (kg ha-1)	Inorganic fertilizer application (kg ha-1)
CK	No fertilization	0	0
DL	10% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer	750	472.5
ZL	25% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer	750	393.75
SL	50% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer	750	262.5
FHF	Conventional fertilization	0	525
JZ	Conventional fertilization combined with sterilized Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer	750	525

Sampling and analysis

On the harvest day, the whole plant sample was collected. The root soil was washed, plant height was measured with a ruler, and the fresh weight was measured by electronic balance. Chlorophyll content was determined by a SPAD-502 Plus chlorophyll content analyzer. The soluble protein content was determined by Coomassie brilliant blue colorimetry [30]. The soluble sugar content was determined by anthrone colorimetry [31]. The vitamin C content was determined by the 2,6-dichlorophenol-indophenol method [32]. The nitrate content was determined by nitrate-nitrogen colorimetry [33].

The soil samples were collected at depths of 0–20 cm. The "S" method was used in each plot to collect the rhizosphere soil samples of the pakchoi at 5 random points, and the samples were fully mixed as a soil sample. The method was repeated in each plot three times. Fresh soil samples were divided into two parts. One part was brought back to the laboratory for cryopreservation at -80 °C for soil microbial sequencing analysis. The second part was brought back to the laboratory for indoor air-drying to determine the soil physical and chemical properties. The soil organic matter was determined by the potassium dichromate volumetric dilution method. In a 500 mL triangular flask, 0.5000g of the soil sample was accurately weighed; then, 1 mol/L (1/6 K2Cr2O7) solution was added to the soil sample to ensure it mixed evenly; and finally, 20ml of H2SO4 was added, and the triangular flask was slowly rotated to ensure the reagent and soil fully mixed to oxidize the soil organic matter. The alkali-hydrolyzed nitrogen was determined by the alkali-hydrolyzed diffusion method. This method involved weighing 2.00 g of air-dried soil sample, passing it through a no. 18 sieve, putting the material into a diffusion dish, adding 1 mol/L NaOH solution to 10.0 ml of hydrolyzed soil, and converting the hydrolyzed nitrogen alkali hydrolyzed into NH3. The available phosphorus was determined by the molybdenum blue colorimetric method. This method involved weigh 2.5 g of the air-dried soil sample, passing the sample through a 20 mesh sieve into a 150 mL triangular flask and adding 50 mL of 0.5 mol/L NaHCO3 solution to ensure the sample was fully mixed with the soil to extract the available phosphorus in the soil. The available potassium was determined by the flame photometric method. This method involved weighing 5.00 g of the air-dried soil sample, passing it through a 1 mm sieve into a 100 mL triangular flask and adding 50 mL of a 1 mol/L neutral NH4OAc solution to ensure the sample was fully mixed with the soil for extraction of available potassium in the soil. The oil pH was determined by the potentiometric method [34].

Extraction of soil genomic DNA, amplification of the 16S rDNA v3-v4 region and high-throughput sequencing

Total genomic DNA from the soil was extracted using a Mobio PowerSoil® DNA Isolation Kit (Mobio, USA). After the extraction of genomic DNA, 1% agarose gel electrophoresis was used to detect the extracted genomic DNA. Using extracted soil genomic DNA as the template, the 16S rDNA v3-v4 region was selected as the amplified fragment by PCR amplification, followed by high-throughput sequencing. Primer sequences were 336F (5'—gtactcctacgggaggcagca-3 ') and 806R (5'—gtggactachvgggtwtctaat-3 ') [35]. The PCR system (25 μL) involved the following: 30 ng DNA samples, forward primer (5 μmol/L) 1 μL, reverse primer (5 μmol/L) 1 μL, BSA (2 ng/μL) 3 μL, 2 x Taq Plus Master Mix 12.5 μL, and dd H2O 7.5 μL. The PCR conditions were as follows: predenaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 60 s, and 30 cycles. Finally, the process was extended at 72°C for 7 min. The amplification results were subjected to 2% agarose gel electrophoresis, and the PCR products were recovered using an AxyPrepDNA gel recovery kit (AXYGEN). The PCR products were eluted by Tris_HCl, detected by 2% agarose electrophoresis and then sequenced by the Illumina Miseq sequencing platform from the Beijing Ollwegene Technology Co., LTD.

Processing of sequencing data

First, the double-ended sequence data obtained by the Illumina Miseq sequencing slice pairs of reads were joined together into a sequence according to the overlap relationship between the PE reads. At the same time, the quality of the reads and the effect of splicing were filtered by quality control, and the samples were effectively distinguished according to the sequence at the ends of the fore and aft barcode and primer sequence to calibrate sequence direction, namely, to optimize the data. Then, OTU clustering analysis was performed on the valid data in the samples at the 97% level. Based on the results of the OTU clustering analysis, a multiple diversity index analysis and sequencing depth detection were performed on the OTUs. Based on taxonomic information, statistical analysis of bacterial community structure was carried out at various classification levels.

The data were statistically analyzed by graphpad prism 5.01 software, Data were statistically evaluated using analysis of variance (ANOVA) tests. All statistical analyses were considered significant at the P <0.05 level. The results are presented as the mean ± standard deviation (SD). Different small letters are significantly different at the 0.05 level under different fertilization treatments.

Results

Growth and chlorophyll content of pakchoi under different fertilizer treatments

Compared with other treatments, in treatment ZL, the height of pakchoi significantly increased and was 11.81% higher than that in treatment FHF (Fig 1a). In terms of fresh weight per plant, there was no significant difference between treatment ZL and treatments SL and FHF. However, there was a significant difference among treatments DL, JZ and CK (Fig 1b). Among them, treatment ZL had the highest per plant fresh weight, which was 8.54% higher than that of treatment FHF (Fig 1b). In the chlorophyll fraction, there was no significant difference in chlorophyll content between treatment ZL and treatments DL and SL. However, compared with treatments FHF, JZ and CK, there were significant differences (Fig 1c). The highest chlorophyll content was found in treatment ZL, which was 7.37% higher than that in treatment FHF.

Fig 1

Effect of different fertilizer treatments on pakchoi: (a) plant height, (b) fresh weight per plant, and (c) chlorophyll content.

Nutrient concentration

Compared with treatment FHF, treatments DL, ZL, and SL had higher soluble protein, soluble sugar, and vitamin C contents in pakchoi. Among them, treatment ZL had the highest soluble protein, soluble sugar, and vitamin C contents, and they increased by 16.88%, 17.05% and 23.70%, respectively, compared with those of treatment FHF. However, the nitrate content under treatment ZL was the lowest, which was 53.86% lower than that of treatment FHF (Table 2).

Table 2

Effect of different treatments on pakchoi nutrients.

Treatments	Soluble protein (mg/g)	Soluble sugar (mg/g)	Vitamin C (μg/g)	Nitrate (μg/g)
CK	25.57±0.77d	16.60±1.43c	180.98±14.03c	175.06±16.35ab
DL	29.84±0.94abc	20.45±0.75ab	232.96±22.47ab	147.71±18.89b
ZL	32.41±0.83a	22.11±1.34a	244.66±14.29a	143.12±16.37b
SL	30.90±1.16ab	21.02±0.78ab	225.21±16.80abc	163.46±14.49b
FHF	27.73±1.32cd	18.89±0.75bc	197.79±17.61bc	220.20±20.34a
JZ	29.11±0.73bc	19.38±1.47abc	204.03±16.83abc	214.69±17.62a

The data are presented as the mean ± standard deviation (SD). Different letters in the same column mean significant difference at 0.05 level.

Physicochemical properties of pakchoi rhizosphere soil

The physical and chemical properties of the soil are shown in Table 3. The results show that the content of organic matter, alkali-hydrolyzed nitrogen, available phosphorus and available potassium in the soil increased due to conventional fertilization and application of Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer in the six treatments. The content of alkali-hydrolyzed nitrogen in treatment ZL was the highest, which was not significantly different from that in treatments DL, SL, FHF and JZ but significantly different from that in treatment CK. The soil available phosphorus content in treatment ZL was the highest, which was not significantly different from that in treatment SL and was significantly different from that in treatments DL, FHF, JZ and CK. The available potassium content in the soil in treatment ZL was the highest; there was no significant difference between treatments DL, SL, FHF and JZ, but there was a significant difference between CK. The soil organic matter content in treatment ZL was the highest, which was not significantly different from that in treatments DL, SL, FHF and JZ and significantly different from that in treatment CK. The soil pH value of treatment DL was the highest, which was not significantly different from that of treatments ZL and CK but significantly different from that of the other treatments. There was no significant difference in soil moisture content between treatments ZL, SL, FHF and CK, while there was a significant difference between treatments DL and JZ.

Table 3

Soil basic properties under different treatments.

Treatments	AN (mg/kg)	AP (mg/kg)	AK (mg/kg)	OM (g/kg)	pH	WC (%)
CK	48.6±5.3b	75.8±8.6c	137.1±10.0b	40.1±5.3b	5.17±0.05ab	27.50±0.66a
DL	56.4±5.9ab	93.2±9.2bc	168.5±11.7ab	50.1±4.8a	5.36±0.08a	25.23±0.73b
ZL	65.1±6.5a	112.8±7.3a	176.3±13.5a	51.6±5.8a	5.31±0.03ab	27.25±0.22a
SL	58.6±3.4ab	102.6±8.2ab	160.1±15.1ab	48.6±6.7ab	4.78±0.07c	27.63±0.11a
FHF	52.4±5.2ab	82.6±5.6bc	145.6±11.3ab	43.1±6.4ab	5.13±0.12b	27.44±0.73a
JZ	54.6±5.3ab	87.9±5.9bc	148.9±7.6ab	44.2±5.0ab	4.82±0.04c	25.72±0.25b

OM, soil organic matter. pH, soil pH. AN, soil Alkali-hydrolyzed nitrogen. AP, soil available phosphorus. AK, soil available potassium. WC, soil water content. Note. The data are presented as the mean ± standard deviation (SD). Different letters in the same column mean significant difference at 0.05 level.

Alpha diversity of the soil bacterial community

In this study, Illumina Miseq high-throughput sequencing was carried out in 6 different fertilization treatments of pakchoi. The results in Table 4 show that the OTU bacterial communities in each sample were treatment CK > FHF > DL > JZ > ZL > SL. There was no significant difference in the Chao1 index between treatment CK and treatments DL, ZL, FHF, JZ, but there was a significant difference between treatment CK and treatment SL. There was no significant difference in the Shannon indexes of the bacterial communities among treatments. The sequence depth index of the nifH gene in each sample ranged from 92.22% to 94.17%, which indicated that the coverage rate of each sample library was very high. The high probability of sequencing in each sample indicated that the sequencing result can reflect the actual situation of microorganisms in each sample. According to the Shannon-Wiener curve (Fig 2), the curves of all the samples tended to be flat, indicating that the amount of sequencing data is large enough to reflect the vast majority of microbial information in the samples.

Table 4

Analysis of soil bacterial community diversity under different fertilization treatments.

Treatment	Chao1 index	Shannon index	Good coverage (%)
DL	3189.81±210.26ab	9.38±0.02a	93.77±0.36ab
ZL	3252.57±443.93ab	9.10±0.48a	93.37±0.88ab
SL	2864.32±211.32b	9.03±0.36a	94.17±0.46a
FHF	3276.36±393.68ab	9.13±0.63a	93.15±0.80ab
JZ	3225.52±268.01ab	8.99±0.22a	93.34±0.55ab
CK	3752.93±245.23a	9.74±0.26a	92.22±0.46b

The data are presented as the mean ± standard deviation (SD). Different letters in the same column mean significant difference at 0.05 level.

Fig 2

Shannon-Wiener curves of soil bacterial communities treated with different fertilizers.

PCA analysis of community composition structure at the generic level was carried out by R software. The results of the PCA community diversity analysis (Fig 3) showed that PC1 and PC2 explained 20.32% and 15.20% of the variation in the bacterial communities in pakchoi soil, respectively, and the cumulative total explained 35.52% of the total variables. The close distance between points DL and SL and ZL and FHF indicates the high similarity of the bacterial flora between the treatment points of DL and SL, ZL and FHF. The distance between all fertilization treatments and the control points was great, indicating that different fertilization treatments significantly changed the soil bacterial community structure.

Fig 3

PCA analysis of soil bacterial communities under different fertilization treatments.

Analysis of bacterial community composition at the phylum and genus levels

From the level of the middle phylum (Fig 4), Proteobacteria had the highest relative abundance of bacteria in the different fertilization treatments, with a relative abundance ranging from 27.42% to 37.70%, followed by that of Chloroflexi (10.63%-24.07%), Acidobacteria (9.67%-20.27%), Actinobacteria (10.81%-20.09), Gemmatimonadetes (5.27%-7.99%), Firmicutes (2.84%-11.29%), and Bacteroidetes.1.83%-3.4%). The relative abundance of Acidobacteria in treatment CK was significantly higher than that in the other five treatments. The amount of Proteobacteria was higher in treatment DL than in the other five treatments. The relative abundance of Gemmatimonadetes in treatment ZL was obviously higher than that in the other five treatments. The relative abundance of Actinomycetes and Bacteroidetes in treatment SL was obviously higher than that in the other five treatments. The relative abundance of Firmicutes in treatment FHF was obviously higher than that in the other five treatments. The relative abundance of Chlorophora in treatment JZ was higher than that in the other 5 treatments.

Fig 4

Community composition of soil bacteria at the phylum level.

At the genus level (Fig 5), the dominant bacteria genera under different treatments were Acidothermus (1.74%~4.78%), Rhizomicrobium (1.03%~2.39%), Gemmatimonas (1.21%~1.92%), Candidatus_Solibacter (1.09%~2.49%), Bryobacter (1.01%~1.43%), Sphingomonas (1.04%~2.11%), H16 (1.07%~2.16%), Rhodanobacter (1.42%~1.68%), Mizugakiibacter (1.76%~2.61%), Dyella (1.23%), Luteibacter (2.71%), Haliangium (1%~1.84%), Acidibacter (2.15%), Actinospica (1.27%), Bryzomicrobium (1.39%). The results showed that different fertilization treatments had a great influence on the species and relative abundance of soil bacteria.

Fig 5

Community composition of soil bacteria at the genus level.

Relationship between soil bacterial communities and soil physical and chemical factors

Soil physical and chemical factors and bacterial communities were analyzed using RDA correlation analysis. RDA1 and RDA2 explained 56.46% and 26.07% (Fig 6), respectively, of the changes in bacterial communities in each sample. That is, all soil physical and chemical factors explained 82.53% of the changes in the bacterial communities. The correlation between soil moisture content and bacterial community was the highest, followed by that between soil pH, organic matter, available potassium and bacterial community, and the correlation between available phosphorus, alkali-hydrolyzed nitrogen and bacterial community was the smallest. The angle between the vector arrows of soil pH, organic matter, available potassium, available phosphorus and alkali-hydrolyzed nitrogen environmental factors was small and may have a synergistic effect.

Fig 6

RDA analysis of the soil bacterial community and soil properties.

Discussion

We studied the effects of inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin nitrogen-fixing biofertilizer on Brassica chinensis L. The results showed that the treatments had different effects on plant height, fresh weight per plant, chlorophyll content, soluble protein, soluble sugar, vitamin C and nitrate content. Among them, the 25% inorganic fertilizer reduction combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer had the best effect on plant height, fresh weight per plant, chlorophyll content, soluble protein, soluble sugar and vitamin C content, and these factors increased by 11.81%, 8.54%, 7.37%, 16.88%, 17.05%, 23.70%, respectively, and the nitrate content decreased by 53.85% in this treatment compared with that in the conventional fertilization treatment. Studies have shown that biofertilizer has a great effect on maize yield [36]. Biofertilizer treatment can improve the chlorophyll content of oat leaves and promote the accumulation of total nitrogen in stems, leaves and ears of grain [37].

Soil microorganisms are an important factor affecting soil ecological processes. These microorganisms play an important role in soil formation, the biogeochemical cycle of ecosystems, the degradation of pollutants and the maintenance of groundwater quality [38-39]. Soil microbial diversity is considered to be an important factor in maintaining soil health [40]. The results of this study showed that the dominant phyla of rhizosphere soil bacteria were Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Bacteroidetes. The results are similar to those obtained in previous studies on different types of farmland soil. The difference between those studies and this study is that the relative abundance of each dominant group varies greatly because the species and abundance of the dominant groups are influenced by soil type, texture and crop varieties [41-42]. Compared with no fertilization and only the application of inorganic fertilizer, the application of Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer combined with inorganic fertilizer significantly improved the abundance of Proteobacteria and Actinomycetes in the soil. Zhang et al. [43] found that microbial organic fertilizer increased the abundance of soil Proteobacteria. Wang et al. [44] found that fertilization and straw mulching significantly increased the relative abundance of Actinomyces. Our results are consistent with those of these authors. Previous studies have shown that Proteobacteria are gram-negative bacteria and are eutrophic bacteria. These bacteria have a positive correlation with nutrient content [45-46], which is critical to the global carbon, nitrogen and sulfur cycles [47], and they play a very important role in biological nitrogen fixation, biological control and plant growth promotion [48]. Actinomycetes can produce a variety of secondary metabolites (antibiotics) and extracellular enzymes, which play an important role in the defense of plant diseases [49]. The increase in the abundance of these two types of bacteria plays an important role in improving soil quality, increasing soil nutrient content, enhancing stress resistance and promoting plant growth and is conducive to the sustainable development of soil microecology. The increase in nutrient content in soil was assumed to be due to stimulation of the growth of Proteobacteria and Actinomycetes by applying the Pennisetum giganteum z.x.lin mixed nitrogen-fixing bacterial fertilizer combined with inorganic fertilizer, which led to their increase in abundance in the soil.

With the development of modern agriculture in China, the inappropriate application of inorganic fertilizers has caused increasingly serious harm to the agricultural ecological environment. In recent years, with the increasing attention of society on the protection of agricultural ecological environments, research on replacing inorganic fertilizers with biofertilizer has attracted much attention [50]. Han et al. [51] showed that the content of soil organic matter, total nitrogen, total phosphorus, total potassium, available phosphorus and available potassium increased by 42.2%, 58.8%, 8%, 12.6%, 37.2% and 40.2%, respectively, when treated with rhizobia and PGPR bacterial fertilizer. Pang et al. [52] showed that soil organic matter and quick-effect N, P and K contents were significantly higher than those in a control treatment without adding microbial agents. The results of this study showed that compared with no fertilization and only the application of inorganic fertilizer, the combined application of inorganic fertilizer and Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer could improve the physical and chemical properties of the soil to a certain extent. When a 25% reduction in chemical fertilizer was combined with Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer, the content of alkali-hydrolyzed nitrogen, available phosphorus, available potassium and organic matter in the soil increased most obviously, and the soil pH also improved to a certain extent. Changes in soil nutrients change the structure and functional diversity of microbial communities [53]. The growth, activity and functional diversity of the soil microbial community were affected by various soil physical and chemical properties, including soil pH, total nitrogen (TN), soil organic carbon (SOC) and soil enzyme activity. Under fertilization, pH is the main driving factor changing microbial communities such as bacteria, fungi, archaea and protozoa. TN, SOC, enzyme activity and other factors are important contributors to the composition of different microbial communities [54-57]. Soil bacteria are essential for maintaining soil fertility and ecosystem functions and are often sensitive to fertilizer inputs [58]. Many studies have shown that fertilization changes the soil fertility and nutrient content, such as organic carbon [59] and total nitrogen [60-61], directly driving the transformation of soil microbial communities and increasing or decreasing the diversity of the microbial community. Fertilization also indirectly affects soil microorganisms by changing soil properties [62]. Soil microorganisms can secrete active growth substances such as auxin, cytokinin, and zeatin to promote plant growth [63]. Therefore, under fertilization, soil physical and chemical properties and microorganisms interact to jointly promote the growth of plants, improving the yield of plants. The results of this study showed that soil organic matter, alkali-hydrolyzed nitrogen, available phosphorus, available potassium, pH and water content all had certain effects on the changes in the soil bacterial community. Among the factors, soil water content was the main factor affecting the change of bacterial community. Soil pH is also an important index affecting bacterial community structure [64-65]. However, pH was not the main factor affecting the structure of the bacterial community in this study. This result may have been due to the differences in the soil environments between different research areas or the relatively small differences in the pH values of the soil samples among the different fertilization treatments, which did not meet the threshold of changing bacterial communities and thus did not have enough impact on the soil bacterial community. The effects of fertilizer application on soil bacterial diversity and community composition were different in the ecosystems [66-70]. At present, there is contradictory information on the impact of biofertilizer on soil microbial diversity, which may be related to the type of fertilizer, the amount and duration of application, soil type and utilization mode. Therefore, the influence of other factors on soil microbial diversity needs to be further studied.

Conclusions

In conclusion, when the ratio of inorganic fertilizer and Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer is appropriate, it can promote the growth of pakchoi, improve the nutritional quality, soil fertility and bacterial community of pakchoi. When the reduction ratio of chemical fertilizer is large, it can also improve the quality of pakchoi. However, the reduced ratio hinders the growth of pakchoi due to the lack of nutrient supply. When the reduction ratio of chemical fertilizer is small, the nutrient supply is sufficient, but the change in the soil environment leads to a reduction in beneficial bacteria in the soil, thus hindering the growth of pakchoi and reducing its quality. Therefore, considering factors such as the growth and quality of pakchoi and its soil environment, the effect of a 25% reduction of chemical fertilizer and the application of Pennisetum giganteum mixed nitrogen-fixing bacterial manure is the best. The results of this study can provide an important theoretical basis for the rational reduction of chemical fertilizer and the scientific and reasonable application of Pennisetum giganteum mixed nitrogen-fixing bacterial manure.

(DOCX)

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22 Oct 2019

PONE-D-19-26117

Effect of chemical fertilizer reduction and co-application with Pennisetum sp. mixed nitrogen-fixing bacterial fertilizer on growth, quality, soil fertility and bacterial community of pakchoi (Brassica chinensis L.)

PLOS ONE

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Reviewer #1: The authors investigated the effects of chemical fertilizer reduction and co-application with Pennisetum sp. Mixed nitrogen-fixing bacterial fertilizer on growth, quality, soil fertility and bacterial community of pakchoi. They found that only 25% chemical fertilizer reduction combined with Pennisetum sp. mixed nitrogen-fixing bacterial manure have the best effect. However, in its current state the authors spend too much time describing the results and too little time discussing their implications. Another issue is the language. Given the large number of writing issues, the author are strongly encouraged to seek further help from a native English speaker to improve the writing.

Below are minor, but more specific comments.

You need to simplify and modify your title to make it concise and clear.

Abstract:

L2-5, is it a sentence?

L21 most obviously ?

Introduction:

L23: improve soil ? please modify it.

Discussion

L19-20: presumably due to the application of chemical fertilizer will cause soil

acidification??? There are many mistakes like this. Please check you sentences carefully.

What is the relationship between soil physical and chemical properties and yield of pakchoi (Brassica chinensis L.)? Please discuss it.

How did you draw the conclusion that 25% chemical fertilizer reduction combined with Pennisetum sp. mixed nitrogen-fixing bacterial manure was the best practice to improve the growth and quality of pakchoi (Brassica chinensis L.)? To the extent, the conclusions section is a complete repetition of the main results. Please compare the difference of treatments and discuss it.

Please discuss how the soil properties affect the bacterial community structure and the growth of pakchoi (Brassica chinensis L.). Add latest references to enrich your paper.

Reviewer #2: Comments to authors

I have reviewed the manuscript titled “Effect of chemical fertilizer reduction and co-application with Pennisetum sp. Mixed nitrogen-fixing bacterial fertilizer on growth, quality, soil fertility and bacterial community of pakchoi (Brassica chinensis L.).”

From the beginning, I failed to grasp what the title was looking into, Pennisetum sp mixed nitrogen fixing bactierial fertilizer to me is meaningless. Pennisetum is a type of millet and how its used to prepare a fertilizer was not at all clear from the title to the manuscript contents itself. Overally, the experimental design does not allow for the repetition of this study at all, as there are a lot of variables involved and a lot of unclear treatment compositions. I do not feel this manuscript can be accepted in its form, but maybe with careful major revision it can be considered. The English is very bad and this needs to be edited by a profession English editor. Lack of continuous line numbers and page numbers also make it a challenge to make specific comments to the manuscript.

I have made specific comments below.

Title

I cannot get the meaning of what the title is looking into, what is Pennisetum sp. Mixed what what. How do you mix a millet with N fixing bacteria to create a fertilizer?

Abstract

What is hm2, what unit is this?

Line 5, including CK, what is this CK?

The 1st sentence reading “to explore the effects… is not complete, what was done to explore these. At least give one background statement.

Abstract is just too long, and the last sentence “the results of this study provide theoretical and scientific….what does this mean.

Introduction

The section fails to justify why this study was undertaken, what other researchers have done and what this study will contribute. Its common knowledge that inorganic fertilizers are detrimental to the soil, so, what does this study seek to address, apart from presenting an alternative organic fertilizer.

Avoid using plurals of words, such as, Indicators (line 5); promotes (line 8); researches (line 18) among others.

Line 7, how does genetic diversity promote decomposition, this is not clear.

Line 11, chemical fertilizer, do you mean inorganic, rather use this term.

Line 19, what is a microbial fertilizer, this is a new term.

Line 29, small and large, meaning?

Line 35, Pennisetum sp. Mixed… I can’t understand this part.

Line 42, you need a clear Objective, it has to be SMART.

Materials and methods

This section is poorly written and does not allow the repetition of this study. Some of the sticking points are: which Pennisetum species, nutrient solution was brown sugar (meaning); how do u isolate bacteria from Pennisetum?; fermentation was end if the material became soft and dark (this is not a quantifiable character, how do you repeat this?).

250-350 g of mushroom waste, why mushroom, and which species.

Line 16, Material, use a better term.

Line 17, 2 tested N fixing strains, give their full names, and how do you isolate these from a grass, Pennisetum.

Line 18, preserved in the laboratory, not authors lab.

Which buy from local market, correct gramma

Line 19-25; Chemical fertilizer….its not clear, I cant follow what was done here.

Line 31, how was the isolation done.

What is hm2

Table 1m give the actual names of the species used.

On soil chemical analysis, give the actual methods used to extract. Eg, available P was measured by calorimetry, how was the P extracted in the 1st place and provide references.

Data analysis, Factors with P < 0.05; what do you mean, which factors. Write this section clearly.

Results

Morphology, what is this, I see nothing relating to plant morphology. A parameter like number of leaves is very useless to measure as this is a GxE interaction.

Avoid referring to your treatments as A, BC.

Table 2, +/- what value.

Table 3, that footnote is a repletion of stuff presented before.

Discussion fails to link the results and explain what could have caused the responses. Avoid repeating results.

What is Naked oat leaves.

Conclusion

Best effect, what effect are you referring to.

Enhancing its quality, what is this, be specific to the parameter used to measure this.

…………………………………..END……………………….

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11 Dec 2019

Reviewer #1:

Abstract

L2-5, is it a sentence?

Yes, editor. It is a sentence. I use the Pennisetum giganteum z.x.lin mixed nitrogen-fixing biofertilizer. I test the growth, quality, physical and chemical properties of the soil and the bacterial community of the soil. I have revised this sentence in my paper. Please check it.

L21 most obviously ?

Dear editor, my most obviously is mean the

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

23 Jan 2020

Effect of Pennisetumgiganteum z.x.lin mixed nitrogen-fixing bacterial fertilizer on the growth, quality, soil fertility and bacterial community of pakchoi (Brassicachinensis L.)

PONE-D-19-26117R1

Dear Dr. Yulei Jia,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors generally did a good job in addressing the comments, concerns and suggestions of the editor and both reviewers. Specifically, the English has been corrected. I think the manuscript will be acceptable after minor revision.

Given the large number of writing issues identified, the authors are strongly encouraged to seek further help from a native English speaker to improve the writing and make it more concise. Why not change you FIg.1 to color?

Reviewer #2: Abstract

The experiment was divided into 6 treatments, rather, the experiment composed of 6 treatments.

I do not understand the reason for using terms like CK, DL, ZL to define treatments, what is the link, I understand the treatments are described in brackets but what is the use of using these letters.

Introduction

Line 42: participating in material cycling processes, Rather, participating in Nutrient cycling.

Pennisetum giganteum z.x.lin belongs to Angiospermae, Monocotyledon, Gramineae and Pennisetum. This statement is not complete, if it belongs to this, and so what?

Materials and methods

Experimental design: what instructed the application rate of the organic fertilizer in Table 1.

Sampling and analysis, this section is too crowded, rather use sub sections and indicate references for each method used. Plant height of each plant…this is grammatically not correct, just rather say plant height was measured.

Indicate the package used for data analysis.

Results

Table 3, indicate with a footprint what the small letters indicate, what does AN, AP stand for, these need to be described also in the Table. What does the +/- indicate also. Indicate in all tables.

Bacterial community analysis: Line 26 onwards, can this information not be presented in anoher way, its too crowded to follow.

Conclusion

This statement is not making sense to me.

In conclusion, because the ratio of reduced fertilizer and combined Pennisetum

32 giganteum z.x.lin mixed nitrogen-fixing biofertilizer is suitable, it can promote the

33 growth and quality of pakchoi, soil quality and soil bacterial community structure.

**********

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Reviewer #1: No

Reviewer #2: Yes: Hupenyu Allan Mupambwa

27 Jan 2020

PONE-D-19-26117R1

Effect of Pennisetum giganteum z.x.lin mixed nitrogen-fixing bacterial fertilizer on the growth, quality, soil fertility and bacterial community of pakchoi (Brassica chinensis L.)

Dear Dr. Jia:

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