Introduction
Tillage is an indispensable and
important part of agricultural production system. Different methods of tillage
can affect soil structure (bulk density, porosity, aggregates, compactness)
(Sun et al. 2018a), soil nutrients
(nitrogen, phosphorus, potassium, organic matter) (Li et al. 2015) and micro-ecological environment (microbial biomass
carbon, nitrogen, microbial quantity, diversity and soil enzyme activity) (Sun et al.
2018b; Fu et al. 2019).
Yunnan, an important tobacco leaf production area in China, its
tobacco-growing soil is mainly mountainous along with red and yellow soil, with
acidity, viscosity, barrenness, and drought to be the mainly characteristics.
However, rotary and shallow tillage (REF) are the current tobacco farming
methods. The long-term single tillage mode has resulted in soil compaction,
thickening of the plow bottom and upward movement and shallow tillage. The
year-round continuous cropping with heavy application of chemical fertilizers,
and insufficient of organic fertilizers can eventually lead to the soil
degradation, reduced soil microbial activity and high incidence of soil-borne
diseases, which can seriously affect the growth, yield and quality of tobacco
leaves, because the loose and deep soil are the fundamental for high-quality
tobacco leaves production (She et al.
2017; Bai et al. 2019). Therefore,
seeking farming measures that are conducive to improving soil quality and
environment has theoretical and practical benefits for the above problems.
Reasonable farming measures can
improve the structure and quality of the soil layer, coordinate the
relationship among soil water, fertilizer, gas, heat and other factors, thereby
effectively improving physical and chemical characteristics of the soil and
micro-ecological environment (Sun et al.
2018a). Soil microbial activity is the core of the soil nutrient cycle (Wang et al. 2020), whereas soil enzyme
activity and soil fertility directly reflect the dynamics of soil nutrient
conversion (Tan et al. 2014). These
are important indicators of quality of soil microecological environment
(Pajares et al. 2011).
Deep tillage (DT) uses plow and other agricultural tools to shovel, loosen
and turn over the soil. Subsoiling tillage (ST) is a soil tillage measure that
loosens the soil through loose parts such as subsoiling tillage shovel or
chisel plow but without turning over the soil. A wealth of researches showed
that DT and ST measures can effectively break the bottom of the soil plow and
reduce the soil bulk density (Osunbitan et
al. 2005). In contrary, the stubble crops and other plant residues into
deeper soil layer are beneficial to increase the soil fertility, improve the
soil permeability and promote root growth of crops (Lampurlanés and
Cantero-Martínez 2003). In addition, DT and ST could improve physical
properties of soil such as bulk density, porosity and moisture in tobacco field
in mountainous areas, optimize the soil environment to some
extent, promote the growth and development and optimize the spatial
distribution and construction of root system (Liu et al. 2019).
However, the impacts of DT and ST measures on soil
micro-ecological environment of tobacco field in Yunnan mountainous areas and
its mechanism remain elusive. Therefore, this study aim to identify differences
in composition and variations in bacterial communities in flue-cured tobacco
rhizosphere soil under different tillage treatments, identify the
characteristics of enzyme activity and to analyze the effect mechanism of DT
and ST measures on soil quality and efficiency improvement. The findings of
this study are expected to provide reference for the application of DT and ST
measures in tobacco field in Yunnan mountainous areas.
Materials and Methods
Study area
The experiment was conducted from March to September 2018 in Gaocang
Street (24°30′N, 103°32′E) of Hongta District, Yuxi City, a typical
mountain tobacco growing area in Yunnan Dianzhong Tobacco area. The cultivated
land is mainly gentle slope or terrace, the soil type is red soil, with
previous crop of wheat. The test flue-cured tobacco variety is K326. The basic
soil properties of the 0–20 cm soil layer before plowing are listed in Table 1.
Experiment design and field management
A single-factor randomized block
arrangement was used with 4 treatments: rotary tillage 20 cm (RT20, control),
using RT blade to cut, break up soil block, loose mixed layer soil, with rotary
tillage depth 20 cm; deep tillage 30 cm (DT30), through a tractor (Dongfanghong
904) 3 points mounted moldboard plow for deep plowing tillage, with deep
tillage depth 30 cm; subsoiling tillage 30 cm (ST30), subsoiling tillage 40 cm
(ST40), through the tractor (Dongfanghong 904) 3 points mounted chisel
subsoiler to loose soil without turning over soil layer. The height of
subsoiling tillage machine and the depth of subsoiling tillage were controlled
by tractor hydraulic pressure at 30 and 40 cm, respectively.
After DT and ST, the tobacco fields were plough until the soil was deemed
appropriate. Each treatment was repeated three times with routine single ridge
transplanting. The row spacing was 1.2 m with plant spacing of 0.6 m; the ridge
height was 25 cm after transplanting. The pest control and other field
management measures were carried out according to the local high-quality
tobacco production management measures.
Sample collection
Tobacco plants are selected
according to a five-point sampling method during the vigorous growth period.
After removing the topsoil, entire tobacco plant was pulled up, the roots were
carefully shaken to remove the loosely adhering soil, and the remaining
attached soil was carefully collected by using sterile brushes and considered
as the rhizosphere soil. The rhizosphere soil samples were thoroughly mixed and
transported from the field to the laboratory in an ice-cooled container; in the
laboratory, the samples were sieved (2 mm mesh) to remove plant debris and then
split into two parts: one was air-dried for soil enzyme activity analysis, the
second was stored in the refrigerator (MDF-U5386S, SANYO, JPN) at -80°C for DNA
extraction.
Determination of soil enzyme activity
The soil catalase activities,
urease activities, acid phosphatase activities, invertase activities, protease
activities and cellulase activities were determined as per instructions of
Enzyme Activity Kit (Suzhou Keming Biotechnology Co., Ltd.). The activity of
catalase was determined by measuring the solution absorbance at 240nm after
reaction with soil. Indophenol blue colorimetry was used to determine NH3-N
produced by urease hydrolyzing urea. The activity of acid phosphatase was
determined by measuring the content of phenol produced by hydrolysis of
disodium diphenylphosphate catalyzed by acid phosphatase. Invertase activity
was determined by measuring the content of reducing sugar produced by the
degradation of sucrose by invertase. The protease activity was determined by
measuring the tyrosine content produced by the soil acid protease catalyzing
the hydrolysis of casein. The anthrone colorimetric method was used to
determine the content of glucose produced by soil cellulase catalyzed cellulose
degradation to determine cellulase activity.
DNA extraction and PCR amplification
The DNA extraction of each
rhizosphere soil samples was performed as per instructions (FastDNA®SPIN Kit
for Soil, MP, USA). The concentration and purity of DNA were determined by
NanoDrop2000, and the quality of DNA extraction was detected by 1% agarose gel
electrophoresis. Using two primers namely 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and
806R (5'-GGACTACHVGGGTWTCTAAT -3'), PCR was performed to amplify the V3-V4
region of the 16S rDNA, according to the following thermoprofile; 95°C
pre-denaturation for 3 min, 27 cycles (denatured at 95°C for 30s, annealed at
55°C for 30s, extended at 72°C for 45s), and finally extended at 72°C for 10 min.
Amplification mixture as set with following reagents; 20 μL: 4 μL
5*FastPfu buffer; 2 μL 2.5 mmol·L-1 dNTPs; 0.8 μL
Forward Primer (5 μmol·L-1); 0.8 μL Reverse
Primer (5 μmol·L-1) ; 0.4 μL FastPfu
polymerase; 0.2 μL BSA; 10 ng DNA template; add ddH2O to
20 μL. The PCR products were recovered using 2% agarose gel, and
further purified and recovered using the AxyPrep DNA Gel Extraction Kit
(Axygen, U.S.A.) kit, and MiSeq sequencing by Shanghai Meiji Biomedical
Technology Co., Ltd, China.
Data quality control and analysis
The original sequencing sequences
were quality controlled using Trimmomatic software and FLASH (Caporaso et al. 2011) software was used for
sequence assembly. The sequences were filtered by Usearch (Edgar 2013) software
(v. 7.0) and the chimeric sequence was removed to obtain the effective sequence.
The operational classification unit was divided by Uparse software (v. 7.1) at
97% similarity level whereas the species annotation was performed by RDP
classifier (Wang et al. 2007)
software and SILVA (Altschul et al.
1990) database. The Mothur software (v. 1.30.1) was used to calculate the
Coverage, Shannon, Simpson, ACE and Chao1 index, and evaluate the diversity and
abundance index of the species; using the Bray-Curtis distance algorithm
established by Qiime software (v. 1.7.0) to make Principal Coordinate Analysis
(PCoA); using the Vegan software in R language to make RDA redundancy analysis
to detect the relationship among environmental factors, samples, and bacterial
communities or the relationship between two of them. The relationship among
different environmental factors and the composition of microbial species were
calculated by the Spearman correlation coefficient, and the correlation between
microbial classification and environmental variables was evaluated by the
correlation heatmap.
Data processing
Excel 2016 was used for data
processing, S.P.S.S. 22.0 was used for statistical analysis, and Duncan's new
multiple range test (MRT) was used for multiple comparison and analysis of
data.
Results
Changes of enzyme activity in rhizosphere soil under different tillage
methods
The activities of catalase, urease,
acid phosphatase and cellulase under different tillage treatments were
significantly different except that the changes in activities of sucrase and
protease which remain less evident (Table 2). The activity of catalase was the
highest in the rhizosphere soil of RT20 (control), and it was 47.83 and 68.16%
lower respectively in DT30 and ST40 treatment, with significant difference
(0.05) compared with RT20. The difference between DT30 and ST40 treatment was
non-significant. It appeared, DT and ST were beneficial to increase the
activity of urease, acid phosphatase and cellulase in the rhizosphere soil of
flue-cured tobacco. Compared to the RT20 (control), the activity of urease and
cellulase in rhizosphere soil treated by DT30 increased by 24.41 and 19.17%
respectively, and that of acid phosphatase in rhizosphere soil treated by ST30
and ST40 increased by 652.77 and 432.77% respectively. The difference was
significant (0.05) among these treatments.
There
was also significant difference in enzyme activity between DT and ST under the
same tillage depth. Compared to DT30, the soil, the activity of catalase and
acid phosphatase in rhizosphere soil treated by ST30 increased by 89.19 and
394.13% respectively (0.05), however, the activity of urease decreased by
18.37% (0.05). Under ST treatment, the impact of different soil depth on soil
enzyme activity can vary. The activity of catalase, urease and acid phosphatase
treated by ST40 was significantly lower than that of ST30, and decreased by
67.74, 18.14 and 29.23% (0.05). The activity of protease and cellulase also
showed ST40 < ST30, but the difference was not significant. Together, the
results showed that different tillage treatments and different tillage depth
can significantly affect the enzyme activity of flue-cured tobacco rhizosphere
soil.
Table 1: The basic soil properties of the 0-20 cm soil layer
before plowing
|
|
pH |
Organic matter (g·kg-1) |
Total N (g·kg-1) |
Total P (g·kg-1) |
Total K (g·kg-1) |
Dissolved inorganic K (mg·kg-1) |
Available P (mg·kg-1) |
Available K (mg·kg-1) |
|
Content / value |
6.72 |
17.80 |
0.75 |
1.28 |
7.70 |
79.10 |
37.40 |
204.00 |
Table
2: Changes of soil enzyme
activities in rhizosphere soil at different tillage treatments
|
Treatments |
Catalase activities (μmol·g·d-1) |
Urase activities (μg·g·d-1) |
Acid phosphatase activities (μmol·g·d-1) |
Invertase activities (mg·g·d-1) |
Protease
activities (mg·g·d-1) |
Cellulase activities (mg·g·d-1) |
|
RT20 |
17.02 ± 1.68a |
394.08 ± 39.58b |
2.35 ± 0.13c |
37.84 ± 1.79a |
3.34 ± 0.23a |
30.88 ± 1.62b |
|
DT30 |
8.88 ± 3.75b |
490.29 ± 27.86a |
3.58 ± 0.88c |
46.99 ± 5.63a |
2.99 ± 1.18a |
36.80 ± 5.73a |
|
ST30 |
16.80 ± 1.85a |
400.20 ± 18.99b |
17.69 ± 2.41a |
36.58 ± 10.43a |
2.95 ± 1.55a |
33.94 ± 2.70ab |
|
ST40 |
5.42 ± 0.30b |
327.60 ± 40.67c |
12.52 ± 1.63b |
39.06 ± 6.84a |
2.18 ± 0.38a |
28.36 ± 4.65b |
%20345-353_files/image002.png)
Fig. 1: Shannon index-based
rarefaction curves on bacterial in rhizosphere soils at different tillage
treatments
%20345-353_files/image004.png)
Fig.
2:
Venn graph of bacterial OTUs distribution in rhizosphere soils at different
tillage treatments
Note: Different small letters
indicate significant difference among treatments at 0.05 level (P < 0.05), the same as below
Variation characteristics of bacterial communities in rhizosphere soil
under different tillage treatments
OTU richness and Alpha diversity of
bacteria: The OTU richness and diversity
index of the soil sample bacterial community at a similar level of 97% (Table
3). Fig. 1 showed that as the number of sequencing increases, the slope of the
dilution curve gradually decreases and tends to flat. This indicates that the
number of sequencing is sufficient. In this experiment, the coverage of the
four treatments was greater than 98% (Table 3), highlighting that the
sequencing read length was sufficient for this analysis.
The OTU richness of bacteria in the
rhizosphere soil samples of different tillage treatments was
ST40>RT20>ST30>DT30, however, the difference between four treatment
was not significant. The distribution of bacterial OTUs by Venn diagram (Fig.
2) showed that there were a total of 9690 OTUs in four rhizosphere soils, and
the same OTUs of the four treatments are 1887, accounting for only 19.47% of
the total number of OTUs; RT20 (control), DT30, ST30 and ST40 the number of
OTUs peculiar to rhizosphere bacteria accounted for 3.53, 3.57, 2.09 and 2.99%
of their respective total. Compared to the RT20 (control), number of peculiar
OTUs treated with rhizosphere bacteria in ST30 and ST40 decreased by 41.86 and
13.95%, respectively, indicating that there are significant differences in OTUs
level of bacterial communities in the rhizosphere of tobacco plants under
different Table 3: Bacterial OTU
abundance and alpha diversity index in rhizosphere soils at different tillage
treatments
|
Treatments |
Raw number |
Effective number |
OTU abundance |
Alpha
diversity |
Coverage/% |
|||
|
Shannon |
Simpson |
ACE |
Chao1 |
|||||
|
RT20 |
46739 |
26394 |
2439 |
5.86 |
0.0177 |
2266.10 |
2283.43 |
98.11% |
|
DT30 |
45083 |
24878 |
2380 |
6.07 |
0.0093 |
2259.36 |
2293.78 |
98.29% |
|
ST30 |
42843 |
26041 |
2392 |
5.82 |
0.0199 |
2215.41 |
2206.91 |
98.53% |
|
ST40 |
51677 |
30979 |
2479 |
5.85 |
0.0187 |
2328.87 |
2360.51 |
98.28% |
Table 4: The relative abundances of bacterial Phylum (%) in rhizosphere soils at
different tillage treatments
|
Taxonomic category (Phylum) |
% Bacterial abundance |
|||
|
RT20 |
DT30 |
ST30 |
ST40 |
|
|
Actinobacteria |
39.45 ± 2.69a |
37.31 ± 5.81a |
42.57 ± 6.66a |
39.32 ± 4.74a |
|
Proteobacteria |
29.24 ± 1.68a |
26.22 ± 2.11a |
30.35 ± 7.92a |
31.29 ± 7.98a |
|
Chloroflexi |
11.46 ± 1.18a |
11.30 ± 1.95a |
8.22 ± 1.27a |
10.01 ± 1.96a |
|
Acidobacteria |
8.39 ± 1.25a |
12.73 ± 4.92a |
7.14 ± 2.49a |
8.35 ± 3.04a |
|
Gemmatimonadetes |
4.72 ± 0.40b |
6.18 ± 0.53a |
5.71 ± 1.13ab |
5.26 ± 0.48ab |
|
Firmicutes |
1.69 ± 0.15a |
1.73 ± 0.05a |
1.96 ± 0.65a |
1.53 ± 0.58a |
|
Patescibacteria |
1.48 ± 0.10a |
0.53 ± 0.15b |
0.95 ± 0.54ab |
0.94 ± 0.49ab |
|
Bacteroidetes |
0.98 ± 0.32a |
0.75 ± 0.10a |
0.77 ± 0.35a |
0.99 ± 0.33a |
|
Cyanobacteria |
0.73 ± 0.35a |
0.71 ± 0.39a |
0.72 ± 0.32a |
0.51 ± 0.24a |
|
others |
1.41 ± 0.34a |
1.87 ± 0.68a |
1.07 ± 0.39a |
1.32 ± 0.52a |
Note: Different small letters
indicate significant difference among treatments at 0.05 level (P < 0.05), the same as below
tillage treatments.
In addition, the diversity and richness of bacterial communities with
different tillage treatments showed obvious differences. The diversity and
richness index of ST30 treatment were lower than that of RT20 (control). However,
the Shannon index of DT30 treatment increased by 3.58%, whereas Simpson index decreased by 47.46%
compared to RT20 (control). The species richness ACE and Chao1 index of ST40
treatment increased by 2.77 and 3.38% compared to RT20 (control).
Bacterial community species composition and
relative abundance: According to the results of species annotations of
representative sequences in each OTU, the top 10 species with the highest
abundance of bacteria in the phylum classification level in each rhizosphere
soil sample were selected, and the species abundance of each sample gate level
was counted (Table 4). The analysis showed that the bacterial flora of
rhizosphere soil of tobacco plants under different tillage treatments is
similar at the phylum level, mainly composed of Actinobacteria, Proteobacteria,
Chloroflexi, Acidobacteria and Gemmatimonadetes. These five bacterial
communities accounted for more than 90% of the total treatment, but the
relative abundance of bacterial communities in different treatments were
different.
Among them, the relative abundance of Gemmatimonadetes and Nitrospirae
were higher than that of the control (RT20) in deep tillage (DT30) and
subsoiling tillage (ST30, ST40) treatments. Compared to the RT20, the relative
abundance of Gemmatimonadetes in DT30, ST30 and ST40 increased by 30.93, 20.97
and 11.44%, respectively, and the relative abundance of Nitrospirae increased
by 54.55, 22.73 and 11.36%. The difference between DT30 and RT20 reached a significant
level (0.05), however, the relative abundance of Patescibacteria in the deep
tillage (DT30) and subsoiling tillage (ST30, ST40) treatments was lower than
that of the control (RT20). The relative abundance of the Patescibacteria
phylum of the three treatments was reduced by 64.19, 35.81 and 36.49%,
respectively compared with the control (RT20). The difference between DT30 and
the control reached a significant level (0.05), however, for deep tillage
(DT30) and subsoiling tillage (ST30, ST40) there was no significant difference
in bacterial community composition at the gate level between treatments.
From the perspective of the classification level of
the genus (Fig. 3), there are also obvious differences in the genus of
rhizosphere soil bacterial flora under different tillage treatments, and the Sphingomonas treated by subsoiling
tillage (ST30, ST40) is more than the control (RT20) increased by 10.95 and
2.16%, respectively. The relative abundances of Gemmatimonas and Nocardioides
treated by deep tillage (DT30) and subsoiling tillage (ST30, ST40) were higher
than those of the control. The relative abundance of the three treatments of Gemmatimonas increased by 9.94, 16.96
and 13.45% respectively compared with the control (RT20), and the relative
abundance of Nocardioides increased
by 42.57, 66.34 and 53.47%. However, the relative abundances of Streptomyces and Bradyrhizobium treated by deep tillage (DT30) and subsoiling
tillage (ST30, ST40) were at %20345-353_files/image006.png)
Fig.
3: The
relative abundance of bacterial on genus level in rhizosphere soils at
different tillage treatments
%20345-353_files/image008.png)
Fig.
4:
PCoA cluster analysis of bacteria community in rhizosphere soils at different
tillage treatments
different
degrees of reduction compared with the control (RT20). The relative abundance
of three treatments of Streptomyces
decreased by 9.27, 7.58 and 8.99% compared to the control (RT20), and the
relative abundance of Bradyrhizobium
decreased by 22.77, 4.02 and 13.39%, respectively. In addition, the relative abundance
of Sphingomonas, Bradyrhizobium, Gemmatimonas,
Nocardioides, Terrabacter, Chujaibacter,
and sphingobium showed subsoiling tillage (ST30, ST40) > deep tillage
(DT30).
PCoA cluster analysis of bacterial community
composition: The PCoA was performed by Bray-Curtis distance
algorithm (Fig. 4). The degree of interpretation of principal component 1 (PC1)
and principal component 2 (PC2) on sample differences were 23.26 and 21.52%,
respectively. The total account for the 44.78% of all soil samples. As shown in
Fig. 4, both bacterial communities of rhizosphere soil treated by deep tillage
(DT30) were distributed in the 2nd quadrant, and the bacterial
communities of rhizosphere soil treated by subsoiling tillage (ST30, ST40) were
mainly distributed in the 1st and 3rd quadrant, while
that of control (RT20) are mainly distributed in the 1st and 4th
quadrants. It is obvious that the species composition of rhizosphere soil
bacterial community under different tillage methods was significantly
different. In addition, the bacterial communities treated by DT30 was the
furthest from the control (RT20), followed by ST40 and ST30, which indicated
that the larger disturbance to the soil during tillage would have a greater
effect on the composition of the soil bacterial community. The relatively
closer distance between ST30 and ST40 treatments indicated that the composition
of rhizosphere soil of two bacterial communities treated by subsoiling tillage
was highly similar.
Correlation analysis of bacterial community and environmental
factors in rhizosphere soil under different %20345-353_files/image010.png)
Fig.
5:
Redundancy analysis of bacterial composition and environmental factors (soil
enzyme activity) in rhizosphere soil at different tillage treatments
%20345-353_files/image012.png)
Fig.
6:
Correlation analysis of different soil environmental factors and bacterial
community composition on Genus level
tillage treatments
Redundancy
analysis (RDA) is the PCA of environment factor constraint, which can reflect
the sample and environment factor on the same 2-D sequence graph, and directly
reflecting the relationship among the sample environment factor, the sample,
the bacterial community, or the relationship between two of them.
The degree of interpretation of two axes of RDA 1 and RDA 2 was 42.93 and
16.38%, respectively. The cumulative degree of interpretation of the two axes
was 59.31% (Fig. 5). It indicated that the correlation may vary at different
soil enzyme activities (Fig. 5). There was positive correlation between the
activity of urease (UA), cellulase (XA) and sucrase (SA), whereas a negative correlation
between the activity of urease (UA), protease (PA), acid phosphatase (APA) and
catalase (CA) was observed. There was a negative correlation between the
activity of cellulase (XA), sucrase (SA) and catalase (CA) whereas there was
positive correlation between the activity of protease (PA), acid phosphatase
(APA) and catalase (CA). In addition, the change in soil enzyme activity had a
certain impact on the distribution of bacterial community, of which urease (UA)
and cellulase (XA) have the greatest impact, followed by protease (PA),
catalase (CA) was the least.
The
relationship between different environmental factors (soil enzyme activity) and
microbial community was further calculated by using Spearman correlation
coefficient. The correlation heat map between environmental factors and
bacterial community composition showed that among the top 50 species for
relative abundance at genus level, Nitrospira was significantly and
positively correlated with sucrase activity (SA) (Fig. 6) with a correlation
coefficient of 0.678. The Marmoricola, Nocardioides and Sphingobium were significantly and
positively correlated with acid phosphatase activity (APA) with a correlation
coefficient of 0.601, 0.580 and 0.587, respectively. Bradyrhizobium, Pseudolabrys, Ellin6067, Reyranella,
Streptomyces, Mesorhizobium, Mycobacterium
were significantly and positively correlated with urease activity (UA) and the
correlation coefficient was noticed to be -0.587, -0.629, -0.622, -0.685,
-0.741, -0.909, -0.609, respectively. In addition, other bacterial communities
which have not been accurately classified and named such as norank_o_Elsterales,
norank_c_TK10, norank_f_Gemmatimonadaceae, unclassified_f_ Micrococcaceae,
unclassified_f_Burkholderiaceae, have different correlations with different
soil enzyme activities. Taken together, the results showed that there was a
significant correlation between soil enzyme activity and bacterial community
distribution in the rhizosphere of flue-cured tobacco under different tillage
treatments.
Discussion
Many researches showed that soil
tillage can cause great disturbance to soil, cause changes in soil structure,
affect soil water conservation, ventilation and heat conduction, and change the
vertical distribution of soil organic matter and soil nutrients in the surface
layer of soil. Furthermore, it may have a certain impact on soil enzyme
activity and microbial biomass (Curci et
al. 1997; Munkholm et al. 2013).
Soil enzyme is an important component of the soil ecosystem, which is
directly related to the dynamic transformation of soil nutrients and soil
fertility (Benítez et al. 2000),
wherein urease and phosphatase are the main hydrolases in soil. Urease can
directly transform nitrogen-containing organic compounds in soil, promote the
hydrolysis of amide peptide bond of nitrogen-containing organic compounds in
soil, and reflect the level of nitrogen supply in soil to some extent.
Phosphatase can promote the conversion of organic and inorganic phosphorus in
soil and play an important role in the accumulation of available phosphorus in
the soil. Cellulase and sucrase are mainly related to transformation of
carbohydrate in soil and are positively correlated with soil fertility and the
content of organic matter. Protease can catalyze the hydrolysis of protein into
amino acid and the transformation of nitrogen nutrition in soil, and provide
nitrogen source for crop growth. The catalases can decompose peroxide which is
toxic to organism, so as to mitigate the damage to plant organism caused by the
accumulation of peroxides in soil. These facts highlight that the soil enzyme
activity is an important index to evaluate soil micro-environment quality.
In
this study, both tillage method and tillage depth had significant impact on the
enzyme activity of flue-cured tobacco rhizosphere soil in mountain area tobacco
field. Compared with conventional rotary tillage (RT20), DT and ST could
improve the activity of urease, acid phosphatase and cellulase in flue-cured
tobacco rhizosphere soil. The activity of urease and cellulase treated by DT30
were increased by 24.41 and 19.17%, respectively. The activity of acid
phosphatase treated by ST30 and ST40 was increased by 652.77 and 432.77%,
respectively. At the same tillage depth (30 cm), the activity of catalase and
acid phosphatase treated by DT was significantly lower than that of ST, but the
activity of urease was significantly higher than that of ST. The results show
that DT could break the bottom of the soil plough and turn the stubble crops
and other plant residues into deeper soil layer, which is beneficial to
increase the content of organic matter and the soil fertility, while ST could
reduce the soil bulk density, increase soil porosity, facilitate soil gas
exchange (Javed et al. 2013), promote
the activation of aerobic microorganisms and the decomposition of minerals.
Therefore, it was more beneficial to the improvement of soil enzyme activity.
However, the effect of tillage on soil enzyme activity was influenced by soil
depth, and the activities of catalase, urease, acid phosphatase, protease and
cellulase in soil at different tillage depth under the same tillage treatment
(ST) showed ST40 <ST30. Kheyrodin et al. (2012) have also found that
urease activity decreased significantly with the increase of soil depth. Deng
also reported that phosphatase activity decreased with the increase in soil
depth (Deng and Tabatabai 1997). They believed the decrease may be related to a
drop in organic carbon content. In conclusion, in addition to directly changing
the physical properties of soil and regulating the factors such as water,
fertilizer, gas and heat, DT and ST can significantly affect the enzyme
activity related to soil organic matter conversion and soil nutrient cycling,
and have a great impact on the formation of soil fertility.
Soil microorganism is the most active organism in soil and is sensitive to
the change of soil environment (Diosma et
al. 2006). It can represent the change of soil quality and ecological
function in a timely manner. The composition and distribution of soil microbial
community are easily influenced by agricultural tillage measures (Mueller et al. 2015; Sun et al. 2016). The results showed that the soil bacterial community
composition and variety of flue-cured tobacco in mountain area tobacco field
were affected by DT and ST treatment. The OTU abundance of bacteria in
flue-cured tobacco rhizosphere soil treated by different tillage methods was
ST40> RT20> ST30> DT30. The specific OTUs number of bacteria treated
by different tillage methods was RT20>DT30>ST40>ST30. The decrease of
OTU abundance and the decrease of specific OTUs number in soil treated by DT and
ST may be due to different ecological niche created by different tillage
methods. The selection and adaptation of different microorganisms to the ecological
niche may result in great changes in microbial species composition and
community structure (Degrune et al.
2017).
A further analysis of bacterial community composition in the rhizosphere
soil of flue-cured tobacco showed that the dominant bacterial community and
relative abundance under different tillage methods may significantly vary at
the phylum and genus levels. The relative abundance of Gemmatimonadetes and
Nitrospirae treated by DT30 and ST (ST30, ST40) was significantly higher than that
by conventional rotary tillage, and a group of beneficial soil microorganisms,
such as the relative abundance of Nocardioides
related to plant growth and biological protection, the genus Gemmatimonas which guide the phosphorus metabolism, and the genus Sphingomonas which has the ability of
degrading aromatic compounds, were found in samples treated by DT30 and ST
(ST30, ST40)>RT (RT20). The bacterial community diversity analysis showed
that the species diversity Shannon index treated by DT30, the species abundance
ACE and Chao1 index treated by ST40 were increased by 3.58, 2.77 and 3.38% than
those treated by RT20, respectively. This aligns with previous studies on the
positive effects of DT on soil microbial abundance (Ji et al. 2014).
Microbial diversity in rhizosphere soil is closely related to plant
health, and high microbial diversity and activity are beneficial to create
healthy and stable rhizosphere micro-ecological environment and promote plant
growth (Mendes et al. 2015; Jaiswal et al. 2017). The promotion of soil
beneficial microorganism growth by DT and ST measures in this study, and the
improvement of soil microbial diversity, show that DT and ST have positive
impact on improving the micro-ecological environment of flue-cured tobacco in
mountain area tobacco field. However, it has caused great disturbance to soil
in tillage, and destroyed the microbial community structure under traditional
rotary tillage and shallow tillage, and the soil microbial growth and community
structure reconstruction need a certain time. Therefore, the impact of
long-term DT and ST treatments on bacterial community composition and diversity
of flue-cured tobacco rhizosphere soil in mountain area tobacco fields warrant
future research.
The
microbial activity and soil enzyme activity are the strongest physiological
activities in the microecological environment of rhizosphere soil. Correlation
analysis shows that the bacterial community composition of flue-cured tobacco
rhizosphere soil under different tillage treatments has a significant
correlation with soil enzyme activity. Marmoricola,
Nocardioides and Sphingobium were positively correlated with acid phosphatase
activity (APA) whereas Bradyrhizobium,
Streptomyces, Mesorhizobium, and Mycobacterium
were all significantly negatively correlated with urease activity (UA). In
addition, the phenomenon that the relative abundance of Nocardioides increased in the deep tillage and subsoiling tillage
treatment is consistent with the phenomenon of the enhanced acid phosphatase
activity in deep and subsoiling tillage
treatment. At the same time, the decrease in
the relative abundance of Bradyrhizobium
and Streptomyces was also consistent
with the significantly enhanced urease activity in the rhizosphere soil at deep
and subsoiling tillage treatment. These results indicate that the biologically
active factors in flue-cured tobacco rhizosphere soil together affect the
micro-ecological environment in the rhizosphere of flue-cured tobacco. The
positive effects of different tillage treatments on the soil of mountain
tobacco fields are comprehensively reflected in many aspects.
Conclusion
Compared with the traditional
rotary tillage mode in mountain tobacco fields, the soil structure under the
traditional rotary tillage mode was broken during deep tillage and subsoiling,
which had a great impact on the structure of soil plough layer. Deep tillage
and subsoiling can not only improve soil permeability, but also create a micro
ecological environment conducive to the growth of soil bacteria, promote the
reconstruction of soil bacterial community structure, and play a positive role
in improving the diversity and richness of soil bacteria. At the same time,
deep tillage and subsoiling were conducive to the increase of relative
abundance of beneficial bacteria (Nocardioides and Sphingomonas),
and the improvement of soil enzyme activities (Urease, Acid Phosphatase and
Cellulase). The stable and diversified bacterial community structure of
rhizosphere soil was remolded under the joint action of tobacco plants and
environment, and a healthy rhizosphere micro ecological environment was
created. We concluded that the different ecological niche may create by great
disturbance to soil in deep tillage and subsoiling tillage, the selection and
adaptation of different microorganisms to the ecological niche may result in
great changes in microbial species composition and community structure.
Author Contributions
Xiaopeng Deng and Lei Yu conceived
and designed the experiments; Wenjie Tong wrote the paper; Min Yang, Hao Wang,
and Qingbing Feng performed the experiments; Liuchen Zhang, Bin Zhou and Feng
Chen collected the data; Feiyan Huang analyzed the data; Xiaolong Chen and
Yongzhan Cai contributed experimental materials.
Acknowledgements
This study was supported by the
Project of Yunnan Branch Company of China Tobacco Corporation (No.
2018530000241016, 2019530000241011, 2018530000241020, 2020530000242010).
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