Depth-dependent Influence of Biochar Application on the Abundance and Community Structure of Diazotrophic Under Sugarcane Growth

doi:10.21203/rs.3.rs-148982/v1

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Depth-dependent Influence of Biochar Application on the Abundance and Community Structure of Diazotrophic Under Sugarcane Growth

https://doi.org/10.21203/rs.3.rs-148982/v1

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Despite progress in understanding diazotrophic distribution in surface soils, few studies have investigated the distribution of diazotrophic in dipper soil layers. Here, we leveraged high-throughput sequencing (HTS) of nifH genes obtained to assess the influence of biochar amended soil (BC) and control (CK), and soil depths (0-20, 20-40 and 40-60 cm) on diazotrophic abundance and community structures, and soil biochemical properties. Although soil physio-chemical properties, enzymes activities, diazotrophic genera and enriched OTUs were significantly influenced across the entire soil profiles, our results also revealed that BC amended soil significantly increased cane stalk height and weight, soil NO₃^-, NH₄⁺, OM, TC and AK, and enhanced diazotrophic genera in soil depth 0-20 cm compared to CK treatment. Soil TC, TN, OM and NH₄⁺ were the major impact factors shifting diazotrophic community structures in soil depth 0-20 cm. Moreover, multivariate ANOVA analysis revealed that changes in majority of the soil parameters measured were associated with soil depth rather than fertilization. More diazotrophic genera were to a greater extent significantly and positively associated with many soil environmental variables especially in the surface soil 0-20 cm than diazotrophic phyla. Overall, these results were more pronounced in 0-20 cm soil depth in BC than CK treatment.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

diazotrophic abundance and community structure

soil biochemical properties

soil depth

biochar

sugarcane growth

Sugarcane (Saccharum officinarum L.) is an economically vital crop planted in tropical and subtropical regions globally with an annual production of approximately 16 million tons. It contributes considerably to the sugar and biofuel-producing industries^1–2. Fertilization is a crucial agricultural approach that not only improves plant nutrient storage but also simultaneously alters soil attributes and microbial communities^3–4. In the past few decades, extensive fertilization, especially nitrogen fertilizer, has been used to raise sugarcane production to meet the growing sugar demand^5–6. Although inorganic fertilization has a positive effect on sugarcane yield, on the other hand, it has unfavorable indirect effects on soil quality by causing soil acidification, enhancing soil pathogens, intensifying nitrification and leaching of nitrates^7–8. In contrast, organic fertilization is an alternative approach to chemical fertilization to mitigate soil acidification and improve soil nutrient status, thus ensuring sugarcane productivity².

Biochar (BC) a soil supplement that is derived from pyrolysis of organic materials at a higher temperature under a limited oxygen environment is a stable organic material, dark in color, porous and carbonaceous^9–10. BC as a soil amendment is a promising approach to improve soil fertility because it plays an important role in establishing reliable carbon storage, facilitating environmental functions, and mitigating greenhouse gas emissions^11,12. In addition, adding BC to the soil can increase nutrient retention by absorbing carbon and reducing soil acidity¹³. Biochar induced changes may influence not only nutrients and organic materials cycling, but also plant growth and development ¹⁰. In addition, studies have shown that BC amendment to the soil can cause changes in soil bacterial abundance and community composition^12–14. However, our understanding of the BC amendment on soil fertility, soil bacterial community composition and their activities with a focus on soil depth as well as overall impact on sugarcane growth is limited.

Soil microbial community plays a pivotal role in nutrients recycling, by changing inorganic compounds and decaying organic materials^15–16, and maintaining quality underground water, thereby improving environmental functions¹⁷. Soil profiles serve as an ecological filter for many soil microorganisms¹⁸. Moreover, the existence of the microbial web in soil is very complex, as well as their compositions and diversity differ in soil layers¹⁹. Soil microbial are essential in N cycling, and they modulate soil available N that are required by plants¹⁵.

Biological nitrogen fixation (BNF) is an important N source which provides nutrient for crops¹⁶. Diazotrophic (N2 fixers) are diverse group of soil bacteria that are capable of transforming atmospheric N2 to plant-available ammonium (NH₄⁺)^20,21, by nitrogenase, a universal enzyme²⁰. This process serves as a major source of N, accounting for about 97% into the environment²²_’²³. Recently, a molecular marker of nifH gene family which encodes for the nitrogenase reductase subunit has been used to analyze diazotrophic community structures²¹. N2 fixer is sensitive to soil amendment practices. For instance, short-term application of biochar increased diazotrophic abundance and changed community compositions as well as increased the biological nitrogen fixation rate in an alkaline soil under soybean cultivation¹². Similarly, Wang et al.²⁰ found that BC amended soil revealed the highest abundance of nifH gene. Diazotrophic abundance and community structures decrease with increasing soil depth. Wang et al.²⁴ reported that soil depth was the main indicator that altered diazotrophic structures rather than fertilization or sampling time. In addition, Reardon et al.²² documented that soil depth was the influencing factor that altered nifH gene abundance, which was less in 10–20 cm as compared to 0–10 cm soil layer. However, our knowledge of the distribution pattern of soil microbe community structure and their related activities have been limited to topsoil 0–15 cm¹⁸, where soil bacterial and organic matters are abundant²⁵. Moreover, not much is known about diazotrophic relative abundance and community structure, as well as their relationship with soil environmental variables in dipper soil layer under biochar application during sugarcane growth, which is important for better understanding the role of N2-fixers distribution patterns in the soil environment. To address these knowledge gaps a field experiment was designed using biochar as soil amendment during sugarcane cultivation to assess the effect on diazotrophic, soil physio-chemical properties, soil enzyme activities, as well as sugarcane parameters. The study sought to: (a) investigate the variation in diazotrophic abundance and community structures in three soil profiles (0–20, 20–40 and 40–60 cm) under biochar application; (b) explore the relationships between the diazotrophic community structures and soil environmental variables using high-throughput sequencing (HTS).

Response of Sugarcane Parameters to Inorganic Fertilizer and Biochar Amended Soil

BC amended soil significantly increased sugarcane stalk number, stalk weight compared with CK treatment (P < 0.05) (Fig. 1A,C). However, compared to CK treatment, BC amended soil had no impact on sugar content (Fig. 1B).

Soil Environmental Variables

Soil physio-chemical properties varied considerable with increasing soil depth, and soil admendmets practices (Fig. 2). Soil NO₃⁻, NH₄⁺, TC, AP, AK and TN significantly decreased with soil depth (p < 0.05) (Fig. 2A,B,D,E,G,H). Moreover, soil OM decreased with increasing soil depth in BC treatment, while, in CK OM fluctuated (Fig. 2C,). In BC treatment, TC/TN fluctuated across the entire soil depth, but revealed no change in entire soil profile in CK, while soil pH in both CK and BC were significantly higher (p < 0.05) in 20–60 cm than 0–20 cm soil depth (Fig. 2F,I). Regarding various treatments,, BC amended soil significantly improved (p < 0.05) soil nitrate (NO₃⁻), NH₄⁺, OM, TC, and AK in one soil depth (0–20 cm) (Fig. 2A,B,C,D,) compared to CK. Moreover, BC treatment significantly enhanced (p < 0.05) TC/TN and AK at soil depths (20–40, 40–60 cm, and 0–20 and 20–40 cm, respectively) compared with CK treatment (Fig. 2F,G). However, in soil depth (40–60 cm) soil TN significantly diminished (p < 0.05) under BC amended soil compared with CK treatment (Fig. 2E). In the whole soil profile (0–60 cm), BC treatment did not change soil pH in comparison to that under CK treatment (Fig. 2I).

Soil Enzyme Activities

In both CK and BC treatments, cellulose decreased with soil depth (Fig. 3A). Phosphatase acid activity and β-glucosidase activity were significantly higher (p < 0.05) in topsoil (0–20 cm), than soil depth 20–60 cm in both CK and BC treatments (Fig. 3C,D). Additionally, urease activity was significantly higher (p < 0.05) in soil layer 20–40 cm than 0–20 and 40–60 cm soil profiles in BC treatment, whereas in 0–40 cm soil layer it urease activity was stable compared to 40–60 cm soil profile in CK (Fig. 3B). With respect the different treatments, cellulase and β-glucosidase activities in BC amended soil revealed no significant change in the entire soil layer (0–60 cm) compared to CK. In soil layer (20–40 cm), urease activity increased significantly under BC treatment compared with CK, while in one soil depth (0–20 cm) urease activity was significantly higher (p < 0.05) in CK treatment (Fig. 3B). In CK treatment, phosphatase acid activity was significantly higher (p < 0.05) in soil depth (0–20 cm) than BC amended soil and in 20–60 cm under both treatments (Fig. 3D).

Relative Abundance of Dominant Diazotrophic Phyla and Genera

The relative abundance of dominant diazotrophic phyla and genera levels were examined under BC and CK treatments at three different soil depths (0–20, 20–40 and 40–60 cm). Our result revealed that soil depth and various soil amendment practices did not significantly altered diazotrophic phyla across the entire soil profiles (Fig. 4A). However, Proteobacteria (80.2–92.0%) was the highly dominant diazotrophic phylum followed by Cyanobacteria (0.0-8.6%) and Verrucomicrobia (0.2–0.6%) in both CK and BC tretments. (Fig. 4A).

Diazotrophic genera significantly changed with soil depth. At genera level of diazotrophic, the relative abundance of Geobacter (88.6–93.6%) was highly dominant, followed by Anaeromyxobacter (4.5–10.8%), Dechloromonas (0.4–1.4%), Burkholderia (0.1–1.3%), Desulfovibrio (0.2-1.0%) Methylomonas (0.1–0.8%) and Azotobacter (0.0–1.0%) (Fig. 4B) in all the samples. However, Anabaena and Burkholderia in 0–20 cm soil layer were significantly influenced in both CK and BC treatments compared to 20–60 cm soil profile (p < 0.05) (Fig. 4B). Moreover, Azotobacter were also significantly altered in 0–20 cm soil profile compared to 20–60 cm soil depth in BC treatment (p < 0.05), while they were slightly altered in the entire soil layer in CK (Fig. 4B).

With regard to various treatments, BC amended soil improved genus Anabaena, Stenotrophomonas and Burkholderia relative abundance in one soil layer (0–20 cm) compared to CK. However, compared to CK, BC amendment significantly diminished Anaeromyxobacter and Geobacter relative abundance in one soil layer (0–20 cm) (p < 0.05) (Fig. 4B).

Bacterial beta diversity

A nonmetric multidimensional scaling (NMDS) analysis employed clearly revealed the separation of the different soil depths (0–20, 20–40 and 40–60 cm) (Fig. 5). Moreover, an analysis of similarities (ANOSIM) reinforced further the significant differences (R² = 0.56, p < 0.001) among the different soil depths. From the results obtained from the (NMDS) analysis, a redundancy analysis (RDA) was employed in different soil depths to assess the effect of soil environmental variables on diazotrophic community structure. The analysis revealed that soil TC (R² = 1.1024, p < 0.001) (R² = 1.0984, p < 0.001), OM (R² = 1.0872, p < 0.001) and NH₄⁺ (R² = 1.0792, p < 0.001) were the minor impact factors that caused a shift in diazotrophic communities structure in soil depth 0–20 cm (Fig. 6A). At soil depths 20–40 and 40–60 cm, soil AP (R² = 1.2679, p < 0.001), (R² = 1.2168, p < 0.001) was the main driver shifting diazotrophic community structure, respectively (Fig. 6B,C).

Diazotrophic distribution patters in different soil depths

To better understand diazotrophic community distribution patter in the different soil profiles, we identified OTUs that were specifically enriched in various soil depths. Due to the fact that MNDS analysis on diazotrophic community structure did not show significant difference in different soil depths between CK and BC treatments (Fig. 5), we performed analysis using combined samples from diazotrophic OTUs (Fig. 7). Vann diagram analysis shows the similarity and overlap of species among various soil profile. Our results revealed that 59 (31.9%), 55 (29.7%) and 52 (28.1%) enriched diazotrophic OTUs were identified in 0–20, 20–40 and 40–60 cm soil depths, respectively. Thus indicating that diazotrophic OTUs community in the topsoil (0–20 cm) were higher compared to 20–40 and 40–60 cm soil depths (Fig. 7A). Next, we characterized enriched diazotrophic OTUs across the three soil depths, ternary plot analysis was employed to identify enriched diazotrophic OTUs using the combined samples. Enriched diazotrophic OTUs were identified in different soil depths (Fig. 7B). Unsurprisingly, subsequent boxplot analysis further revealed that these identified diazotrophic OTUs significantly depleted with soil depth (p < 0.05) (Fig. 7C).

Table 1

Multivariate ANOVA for the effects of soil depth, and different treatments on nifH OTUs, diversity, species richness, coverage and soil enzyme activities
	OUT	Shannon	Chao1	Coverage	Urease	Cellulase	Glucosidase	Phosphatase
Treatment	NS	NS	NS	NS	NS	NS	NS	NS
Depth	***	**	***	***	***	**	***	***
T X D	***	**	**	***	***	**	***	***

Depth stands for soil depth with 0–20 cm, 20–40 cm and 40–60 cm soil layers, treatment stands for control (CK); and biochar (BC).

Table 2

Multivariate ANOVA for the effects of soil depth, and different treatments on soil biochemical properties
	pH	EC	TN	TC	TC/TN	AP	OM	AK	NO₃⁻	NH₄⁺
Treatment	NS	NS	NS	NS	**	NS	NS	NS	NS	NS
Depth	***	***	***	***	NS	***	NS	***	***	***
T X D	***	***	***	***	**	***	*	***	***	***
Depth stands for soil depth with 0–20 cm, 20–40 cm and 40–60 cm soil layers, treatment stands for control (CK); and biochar (BC).

Multivariate ANOVA for the effects of soil depth, and different treatments on nifH OTUs, diversity, species richness, coverage and soil enzyme activities

Multivariate ANOVA analysis furthered revealed that soil depth significantly (p < 0.05) impacted diazotrophic OUTs, Shannon, chao1, coverage, as well as soil enzyme activities namely urease, cellulase, glucosidase and phosphatase compared to fertilization (Table 1). Furthermore, soil biochemical properties were significantly (p < 0.05) affected by soil depth than the different fertilizations used (Table 2).

Correlation between bacterial composition and soil properties

Pearson’s correlation coefficients were calculated to investigate the relationship between soil physio-chemical properties and the most abundant diazotrophic phyla and genera in three different soil depths. Our findings showed that diazotrophic phyla were to a lesser extent significantly and positively or negatively related to soil physiochemical properties compared diazotrophic genera across that entire soil depth gradient. Soil pH was significantly and positively associated with the relative abundance of Chlorobi at soil depth 0–20 cm. Furthermore, phyla Firmicutes and Proteobacteria showed a very strong and positive relationship with soil NH₄⁺ and AP at soil depths 20–40 cm, respectively. Whereas soil TC and Verrucomicrobia were positively and significantly correlated in soil depth 20–40 cm. However, Cyanobacteria and Chlorobi were significantly and negatively correlated with soil AP and pH at soil depth 20–40 cm, respectively. At soil depth 40–60 cm, phylum Firmicutes revealed a strong and positive relationship with soil pH, however, NO₃⁻ was significantly and negatively related to phylum Cyanobacteria (Fig. 8A).

Meanwhile, our analysis revealed that more diazotrophic genera were to greater extent significantly and positively associated with many soil environmental variables especially in the surface soil 0–20 cm than diazotrophic phyla. Anaeromyxobacter, Anabaena, Enterobacter genera were significantly and positively associated with soil TC, NH₄⁺ and AK, OM as well as TC and TN in soil surface soil 0–20 cm, respectively. Furthermore, in 20–40 cm soil layer, we observed that Burkholderia and Dechloromonas showed a strong and positive relationship with soil TC and NO₃⁻, respectively. However, in 40–60 cm soil depth, Anaeromyxobacter, Methylomonas and Dechloromonas were significantly and negatively correlated with soil OM and AK, respectively (Fig. 8B).

BC amended soil significantly increased the stalk weight and height of sugarcane. Similarly, Sarfraz et al.²⁶ and Ehsanullah et al.²⁷ mentioned that BC application significantly improved crop yields. Soil depth is an important environmental gradient, as well as soil amendmet practices affecting soil physio-chemical properties²⁸. In the current study, soil OM, TC, NH₄⁺ NO₃⁻, and AK significantly decreased with soil depth. BC is a promising alternative to improving soil fertility owing to the pivotal role it plays in building up reliable carbon storage⁹. Studies have revealed BC amendment had profound effect on soil environmental variable, especially N and C cycles²⁹. Similarly, our study revealed that biochar amended soil significantly increased soil OM, TC, NH₄⁺ NO₃⁻, and AK in one soil depth 0–20 cm compared to CK treatment, which we believed were resposible for the improved growth of sugarcane plant.

Soil enzyme activities are considered important indicators of fertile soil due to their important role they carry out in biochemical reaction and sustaining soil fertility²⁹. In our previous study, enzymes associated with C and N cycles were enhanced in the topsoil than in subsoil³⁰. In our study, numerous patterns in the activity of soil enzymes were observed. Soil cellulase activity decreased with soil depth, while β-Glucosidase activity and phosphatase activity were significantly enhanced in 0–20 cm than 20–60 cm soil profile in both CK and BC treatments. On the other hand, the application of BC may harm soil enzyme activities by impeding or enhancing soil organic matter contents³¹. In the present study, cellulase and β-glucosidase activity in BC in the entire soil profile revealed no significant change compared to CK. BC treatment significantly reduced urease and phosphatase acid activity in soil layer (0–20, 40–60 cm) and (0–20, 20–40 and 40–60 cm), respectively than CK. The diminishing trend of these enzymes could be attributed to the application of biochar.

Chu et al.³² revealed that soil microbial community structure is naturally sensitive to environmental fluctuations, and is an important indicators of fertile soil. Diazotrophic abundance decrease with soil depths, Reardon et al.²² revealed that diazotrophic abundance reduced in soil depth subsoil (10–20 cm) than upper soil layers (5–10 and 0–5 cm). Diazotrophic relative abundance was assessed at the phyla and genera level to measure the impact of soil depth. We observed that biochar significantly influenced diazotrophic genera in 0–20 cm soil layer, but did not affect the diazotrophic phyla.

The utilization of biochar to amend soil can have a significant influence on the biotic and abiotic components of soil, thus significantly altering soil microbial abundance and community composition ^30,33,34. In the current study, BC amendment had no significant influence on diazotrophic abundance at the phyla level. However, compared to CK treatment, BC amended soil had profound impact on diazotrophic abundance at the genera level.

Geobacter was highly abundant in both BC and CK treatments. However, genera Geobacter was improved in one soil depth (20–40) compared to CK treatment. This finding is roughly incontinence with studies conducted by Liao et al.³⁵ and Liu et al.¹², in which it was reported that in biochar amended soil, Geobacter were among the dominant bacterial. Our previous study also revealed that Geobacter in the subsoil was enhanced compared to the topsoil³⁰. This result further validates that Geobacter tends to grow in an anaerobic environment, which sequentially improves the lower soil with Deltaproteobacteria genera.

Information on free-living N-fixers in the bulk soil is hindered due to the diverse nature of soil microorganisms community and the challenges of linking N fixation activities to each genus of diazotrophic genera³⁶. However, Agnihotri³⁷ mentioned that Anabaena genus is known as a nitrogen fixer of a filamentous cyanobacteria genera. Chen et al.³⁸ reported that biochar amended soil increased soil microbial relative abundance at soil depth 0–15 cm. Similarly, relative abundance of Anabaena was significantly high in one soil layer (0–20 cm) in BC amended soil compared to CK treatment.

Azotobacter is a nitrogen fixation bacteria that can stimulate soil rhizosphere microorganisms, protects plant against phytopathogen, as well as boosts nutrient absorption which eventually enhancing biological N fixation. In the current study, Azotobacter was significantly in soil profile 0–20 cm. Our result agreed with Kalaigandhi et al.³⁹ and Abd-el-Malek et al.⁴⁰ who reported that Azotobacter abundance is depth-dependent, and was notably high in the rhizosphere soil⁴¹.

Burkholderia is Gram-negative bacteria mainly compose of various soil-dwelling bacteria that exhibit different environmental functions namely fixing nitrogen mutualists, pathogen and saprophyte³⁶. Its distribution pattern in the soil is not clearly understood⁴², however in three different agriculture management regimes namely crop rotation, maize monoculture, and grassland. It was established that the area under maize monoculture and grassland cultivation was dominated by Burkholderia strains. In the current study, Burkholderia relative abundance was improved in the topsoil (0–20 cm) in BC amended soil compared to CK treatment. This finding partially agreed with Lin et al.³³, in which it was reported that Burkholderia relative abundance was significantly increased under organic fertilizers.

Stenotrophomonas genus has been classified as a disease suppression bacteria⁴³, as well as a plant growth promoter and a biocontrol agent⁴⁴. A study found that Stenotrophomonas was significantly higher under no-mulching treatment at different soil depths. Similarly, our result revealed that Stenotrophomona’s relative abundance was higher at one soil depth (20–40 cm) in BC amendments soil; and was evident in the current study, where sugarcane growth parameters were enhanced.

NMDS analysis was conducted to assess the impact of CK and BC treatments on diazotrophic community in different soil depths. The analysis revealed no significant difference in different soil depths between CK and BC treatments. However, using the combine samples, Venn clearly established that diazotrophic is depth-dependent, while ternary plot was able to distinctly identify enriched diazotrophic OTUs across the different soil profiles. Boxplot analysis further clearly exhibited significant difference in enriched OUTs in among the soil depths. Our finding roughly corroborated with the results of Zgadzaj et al.⁴⁵, in which it was reported that bacterial community was significantly enriched in the soil.

Furthermore, multivariate ANOVA analysis further confirmed that soil depth is one of the important environment gradients that greatly influence soil bacterial and soil physiochemical as well as soil enzyme activities⁴⁶. We observed that soil depth significantly influenced diazotrophic OUTs, Shannon, chao1, coverage, nifH gene copies, as well as soil enzyme activities namely urease, cellulase, glucosidase and phosphatase and soil biochemical properties rather than fertilization. These results conformed with the findings of Fischer et al.⁴⁷, Lipson et al.²⁸ and Zhang et al.³⁰, in which they reported that soil bacterial, soil enzyme activities and soil physiochemical properties change were depth dependent.

Soil environmental variables play an important in influencing bacterial community structures ^6,12. Likewise, diazotrophic community structures are very responsive to soil environmental variables ⁴⁹. Zhang et al.³⁰ documented that soil pH and AK were the main factors affecting bacterial community compositions in the topsoil compared to AP and TC/TN, while in the subsoil, soil pH, AK and TC were the main factors in terms of changing bacterial community compositions. Similarly, in the current study, redundancy analysis (RDA) revealed that diazotrophic community structures were very sensitive to soil environmental namely TC, TN, OM, NH₄⁺ and AP. Soil TC was the major impact factor, whereas soil TN, OM and NH₄⁺ were the minor impact factor that caused a shift in diazotrophic community structures in soil depth 0–20 cm. While at soil depths 20–40 and 40–60 cm, soil AP was the main driver shifting diazotrophic community structures.

Pearson’s correlation coefficients analysis revealed that more diazotrophic genera were to greater extent significantly and positively related to many soil physiochemical properties mainly in the surface soil 0–20 cm than diazotrophic phyla

This study explored the distribution patters of diazotrophic genera and phyla, soil enzyme activities and soil physio-chemical properties in different soil depths under control, (CK) and biochar, (BC) amendments. We also assessed the response of sugarcane to these different soil management practices. Our findings revealed that soil depth had profound impact on soil parameters measured. We also observed that diazotrophic relative abundance at the genera level, and enriched diazotrophic OTUs were significantly improved with soil depth. Diazotrophic genera were to greater extent considerably and positively related to many soil environmental variables mainly in the soil surface horizon (0–20 cm) than diazotrophic phyla. These results are likely to enhance our understanding of how diazotrophic bacterial response different soil management practices, as well as and their relationship with soil physio-chemical properties in different soil profiles.

A field experiment was conducted in March 2017 at the Sugarcane Research Center of Fujian Agriculture and Forestry University in Fuzhou, Fujian Province (latitude 26°5′0′′ east longitude 119°13′47′′). The local climate of the area is subtropical monsoon with an annual average temperature of 20°C and a rainfall of 1369 mm. The experiment was set in a randomized block design with two different treatments and each treatment was replicated three times; with each replicate covering an area of 25 m² (5 m x 5 m). The two treatments used were: control, (CK), biochar amended soil (BC). Biochar was applied at the rate of 20 t ha-¹. The biochar used in this study was bought from Nanjing Qinfeng Crop Straw Technology Company, China. The biochar was produced from sugarcane straw at the 550–650°C. The properties of the biochar were: TC = 3.08 g/kg; TN = 4.27 g/kg; content pH (H₂O) = 8.01; AP = 315.21 mg/kg and AK = 217.03 mg/kg. Whereas the basic properties for soil were: TC = 4.11 g/kg; TN = 5.07 g/kg; content pH (H₂O) = 6.99; AP = 205.91 mg/kg and AK = 45.02 mg/kg, NH₄⁺ = 4.01 g/kg and NO₃⁻. 3.90 = g/kg. The biochar was uniformly spread to the soil surface and instantly mixed into the plowed soil at the depth of 0–60 cm before cultivating the sugarcane. Sugarcane stems were cut at about 10–12 cm in length, maintaining two buds on each sett ⁵⁰, and 10 setts were planted on each row with 0.3 m plant-to-plant spacing and 0.5 m row-to-row spacing.

Plant Cane Growth Parameters

Sugarcane height was measured in centimeter (cm) with the help of a meter rod from the soil surface to the top of the sugarcane. Mean plant height was calculated by taking the average of three replications. Twenty sugarcane stalks were randomly sampled from each row, milled and analyzed the juice for pol and Brox using the method of Legendre⁵¹. Cane fresh weights were used to determine individual stalk weight (kg stalk-¹).

Soil Sampling

Soils collection at different depths (0–20, 20–40 and 40–60 cm) were carried out in December 2019. For every plot, a sample was collected at five different spots, homogenized and mixed accordingly forming one sample. Sieving of soils was done using 2-mm mesh and plant residues and stones were removed. Portion of the soil was stored at -20°C for the extraction of DNA and enzyme activities, and the remaining soil samples were stored at 4°C for analyzing soil environmental variables.

Analysis of Soil Physio-chemical Properties

Air-dried soils were used to analyze soil pH, available phosphorus (AP), available potassium (AK), total carbon (TC), total nitrogen (TN) and organic matter (OM). Soil pH was determined using glass electrode pH meter. AP was estimated using the Molybdenum Blue method. The Flash Smart elemental analyzer (Thermo ScientificTM, Waltham, MA, USA) was used to measure the TN and TC. Soil AK was extracted by ammonium acetate and determined by atomic absorption spectrophotometry. Soil OM was measured using Nelson and Sommers methods⁵². A fresh soil sample was used for the extraction of NH₄⁺ and NO₃⁻ with 2.0 M KCl and measured using the continuous flow analyzer (San++, Skalar, Holland).

Measurement of Soil Enzyme Activities

Soil enzyme activities were determined as described by Sun et al.⁵³. Incubation of soil was carried out using buffer sodium carboxymethylcellulose solution, cellulose, (glucose, mg/g 24 h, 37°C) activity to measured colorimetrically by quantifying a decrease in 3,5-dinitrosalicylic acid from reducing sugar. Soil urea activity (NH₃-N, mg/g 24 h, 37°C) was measured using improved sodium phenolate and sodium hypochlorite colorimetry. While a nitrophenyl phosphate disodium substrate was used to determined acid phosphatase activity (phenol, ug/g, 1 h, 37°C). After buffering the soil with p-nitrophenyl- β-Glucosidase activity (p-nitrophenyl, ug/g, 1 h, 37°C) was determined using a colorimeteric p-nitrophenol assay.

Soil DNA Extraction, Amplification, Sequencing, and Data Processing

The extraction of soil metagenomics DNA from 0.5 g fresh soil was carried out using Fast DNATM Spin Kit (MP Biomedicals, LLC, Santa Ana, USA) following the manufacturer guidelines. Assessment of DNA quality and quantity were carried out by calculating their absorbance (A260 and 280nm) using a spectrophotometer, and extracts were then stored at -20°C awaiting sequencing.

High throughput sequencing was used to determine diazotrophic community structures using the Illumina Miseq platform, and nifH amplification was carried out using primer pair PolF and PolR ⁵⁴ combined with Illumina adaptor sequences and barcode sequences⁵⁵. Sample libraries were generated from the purified PCR products. The Miseq 300 cycle Kit was used for paired-end sequencing on a Miseq benchtop sequencer (Illumina, San Diego, CA, United States). The raw nifH gene sequences were separated using sample based on their barcodes and permitting up to one mismatch. Btrim⁵⁶ was used to do quality trimming. Forward and reverse reads were merged into full-length sequences using FLASH ⁵⁷, and sequences with short or contained ambiguous bases were removed. Resampling was done with 10,000 sequences per sample randomly. UCLUST was used to classify the operational taxonomic units (OTUs) at the 97% similarity level, and singletons were removed. The frameshift caused by insertions and deletion in DNA sequences were checked and corrected by RDP FrameBox. Valid nifH gene sequences (300–320 pb) were translated in proteins sequences and taxonomic assignment was performed using RDP FrameBox tool⁵⁸, after the processing.

Statistical Analysis

A nonmetric multidimensional scaling (NMDS) analysis employed to assess the differences in diazotrophic community structures in different soil depths. Bray-Curtis dissimilarities were used to analyze similarities (ANOSIM) to estimate the dissimilarity in diazotrophic community structures in 0–20, 20–40 and 40–60 cm depths. Redundancy analysis (RDA) was further performed in the different soil profiles to determine the relationship between diazotrophic community structures and environmental variables. Vann diagrams were plotted to visualize unique and overlap diazotrophic genera in different soil depths (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Ternary plot analysis was conducted using R language based package153 ggtern an extension of package ggplot2 to determine the enriched abundant OTUs of diazotrophic bacterial in different soil depths of the combined samples. Pearson’s correlation coefficients were employed separately for the different soil depths to test the relationship between soil properties and diazotrophic community structures both at the phyla and genera levels, using R-software⁵⁹. The test data were calculated by ANOVA using DPS software (version 7.05, www.dpssoftware.co.uk), and the differences between the mean values of each treatment were matched by Tukey’s method at a 5% level.

Author Contributions: All authors contributed to intellectual input and provided assistance for this study and for manuscript preparation. N.F., M.T, Z.Y., C.Z., A.Y.A., Z.P., H.Z. and Z.L. designed the research and conducted the experiments. N.F., C.Z and A.A. analyzed the data. N.F. wrote the manuscript. M.T, H.Z and C.Z. reviewed the manuscript. H.Z. supervised the work and approved the manuscript for publication.

Funding: This research was funded by the Earmarked Fund for China Agriculture Research System (CARS-17).

Acknowledgments: We acknowledge the research was funded by the Earmarked Fund for China Agriculture Research System via grant number CARS-17. We want to praise the donor agencies of China for supporting this project.

Conflicts of Interest: The authors declare no conflicts of interest.

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You are reading this latest preprint version

Depth-dependent Influence of Biochar Application on the Abundance and Community Structure of Diazotrophic Under Sugarcane Growth

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Version 1

Abstract

Figures

Introduction

Results

Response of Sugarcane Parameters to Inorganic Fertilizer and Biochar Amended Soil

Soil Environmental Variables

Soil Enzyme Activities

Relative Abundance of Dominant Diazotrophic Phyla and Genera

Bacterial beta diversity

Diazotrophic distribution patters in different soil depths

Correlation between bacterial composition and soil properties

Discussion

Conclusions

Materials And Methods

Plant Cane Growth Parameters

Soil Sampling

Analysis of Soil Physio-chemical Properties

Measurement of Soil Enzyme Activities

Soil DNA Extraction, Amplification, Sequencing, and Data Processing

Statistical Analysis

Declarations

References

Status:

Version 1