CN113593642B - Identification method of microorganism influence mechanism by slope tillage ridge ditch layout - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 103
- 244000005700 microbiome Species 0.000 title claims abstract description 40
- 238000003971 tillage Methods 0.000 title claims abstract description 7
- 239000002689 soil Substances 0.000 claims abstract description 212
- 230000000813 microbial effect Effects 0.000 claims abstract description 85
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 84
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 claims abstract description 80
- 238000012417 linear regression Methods 0.000 claims abstract description 43
- 238000012360 testing method Methods 0.000 claims abstract description 24
- 238000012165 high-throughput sequencing Methods 0.000 claims abstract description 12
- 238000004172 nitrogen cycle Methods 0.000 claims description 22
- 239000006185 dispersion Substances 0.000 claims description 16
- 238000000540 analysis of variance Methods 0.000 claims description 11
- 238000005259 measurement Methods 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
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- 241001347978 Major minor Species 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
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- 230000009286 beneficial effect Effects 0.000 description 11
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Abstract
The invention provides a method for identifying a microorganism influence mechanism by slope cultivation ridge ditch layout, which belongs to the technical field of soil microbiology and comprises the following steps: determining a slope tillage ridge ditch layout parameter; an experimental cell and a control cell are arranged; performing high-throughput sequencing on soil microorganisms to obtain soil microbial community structure diversity related indexes and soil carbon nitrogen circulation process functional gene abundance data; determining the primary and secondary sequences of the slope cultivation ridge ditch layout parameters influencing the structural diversity related indexes of the soil microbial communities and the functional gene abundance data of the soil carbon nitrogen circulation process; constructing a linear regression equation; and (3) performing significance test on the linear regression equation to finish the identification of the microorganism influence mechanism of the slope tillage ridge ditch layout. According to the invention, four representative furrow layout parameters are selected, the diversity of soil microbial communities under different furrow layouts is analyzed through a high-throughput sequencing technology, the influence mechanism of the hillside furrow layout on soil microorganisms is identified, and a reference is provided for formulating a proper hillside furrow layout mode.
Description
Technical Field
The invention belongs to the technical field of soil microbiology, and particularly relates to a method for identifying a microorganism influence mechanism by slope cultivation land ridge ditch layout.
Background
The hillside fields are used as a common agricultural soil resource, and the special landform types severely restrict the agricultural production of dry lands, and the furrow cultivation is used as a common protective cultivation measure, so that the problems of water running, fertilizer running and the like caused by the hillside fields can be effectively solved under certain conditions. Microorganisms in soil participate in material circulation and energy flow among soil ecosystems, a promoting effect is provided for growth and development of crop root systems, at present, research on influence of ridge and ditch cultivation on soil microorganisms in hillside cultivation is mainly focused on the influence of ridge and ditch cultivation combined with film covering and straw covering on the microorganisms, and whether the mode of ridge and ditch cultivation affects the soil microorganisms cannot be known simply due to restriction of multiple factors of film covering and straw.
Disclosure of Invention
Aiming at the defects in the prior art, the identification method for the microbial influence mechanism of the slope cultivation ridge ditch layout solves the problem that whether the ridge ditch cultivation mode affects soil microorganisms cannot be known simply.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the scheme provides a method for identifying a microorganism influence mechanism by slope cultivation ridge ditch layout, which comprises the following steps:
s1, selecting a research area, and determining a planting type and a slope cultivation ridge ditch layout parameter, wherein the slope cultivation ridge ditch layout parameter comprises the following steps: ridge gradient, ridge height, furrow width ratio and furrow trend;
s2, arranging an experimental cell and a control cell according to a four-factor three-level orthogonal table by using a ridge gradient, a ridge height, a furrow width ratio and a furrow trend, and planting selected crops;
s3, respectively collecting soil samples of each experimental cell and each control cell before crop planting, during the middle growing period and during the harvesting period, and carrying out high-throughput sequencing on the soil microorganisms to obtain the diversity related indexes of soil microbial communities of different experimental cells and control cells and the functional gene abundance data of the soil carbon nitrogen circulation process;
s4, checking the influence of the ridge gradient, the ridge height, the furrow width ratio and the furrow trend on the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen cycle process by using an analysis of variance method, and determining the major and minor sequences of four factors affecting the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen cycle process;
s5, constructing a linear regression equation by the primary and secondary sequences and the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process respectively;
s6, performing significance test on the linear regression equation, and taking standard coefficients of ridge gradient, ridge height, furrow width ratio and furrow trend of the linear regression equation as evaluation slope furrow layout to influence soil microorganisms according to test results, so as to complete identification of a microorganism influence mechanism of slope tillage ridge furrow layout.
The beneficial effects of the invention are as follows: according to the invention, the experimental design is carried out on different furrow layout parameters by selecting orthogonal experiments, so that the experimental times are greatly reduced; the analysis of variance method is utilized to test and screen out furrow layout parameters with the greatest influence on soil microorganisms, the importance degree of each furrow layout parameter on experimental results can be accurately estimated, a linear regression equation is constructed between each furrow layout parameter estimated by the analysis of variance method and soil microorganism community structure and functional diversity, and a standard coefficient is used for evaluating the influence of furrow layout on soil microorganisms, so that theoretical reference can be provided for furrow layout of hillside lands, and the purpose of agricultural high yield is achieved.
Further, the step S3 includes the steps of:
s301, selecting two sampling areas of a ridge and a ditch in the upper, middle and lower sections of an experimental cell and a control cell before crop planting, in the middle of the growing period and in the harvesting period, removing surface layer floating soil, collecting soil samples, removing impurities of a soil sample mixture of the same cell, and filling the soil samples into a sterilizing packaging bag for preservation;
s302, performing high-throughput sequencing on soil microorganisms on a soil sample to obtain the sequence number of microorganism DNA or RNA, OTU abundance information and species classification information of the soil sample in each experimental cell and each control cell;
s303, calculating and obtaining the diversity related indexes of the soil microbial community structures of different experimental cells and control cells and the functional gene abundance data of the soil carbon and nitrogen circulation process according to the sequence number of the soil sample microbial DNA or RNA, the OTU abundance information and the species classification information.
The beneficial effects of the above-mentioned further scheme are: by sampling soil samples of the experimental plot in three periods, the influence on experimental errors on a time scale is avoided; the high-throughput sequencing is carried out on the soil sample, so that the abundance and the diversity of the microbial community structure in the soil sample can be judged more quickly and accurately, and the soil microbial community structure diversity and the functional gene abundance data in the soil carbon nitrogen circulation process are used as evaluation indexes for the influence of soil microorganisms, thereby being beneficial to comprehensively analyzing the influence of the furrow layout on the soil microbial community in structural and functional aspects.
Still further, the step S4 includes the steps of:
s401, calculating the related indexes of the microbial community structure diversity in soil samples of each experimental cell and each control cell, and the average value of the functional gene abundance data in the soil carbon nitrogen circulation process;
s402, calculating to obtain the dispersion square sum and the degree of freedom of the microbial community structure diversity related indexes and the soil carbon nitrogen circulation process functional gene abundance data in the soil samples of each experimental cell and the control cell according to the average value;
s403, according to the dispersion square sum and the degree of freedom, using an analysis of variance method to test the influence of different ridge gradients alpha, ridge heights h, furrow width ratios beta and furrow trend d on the diversity related indexes of the soil microbial community structure and the functional gene abundance data of the soil carbon nitrogen circulation process;
s404, performing significance test on the influence result obtained in the step S403;
s405, sorting the influence results obtained in the step S403 from large to small according to the significance test result to obtain a major-minor sequence of four factors of ridge gradient, ridge height, furrow width ratio and furrow trend, which influence the structural diversity related index of the soil microbial community and the functional gene abundance data in the soil carbon nitrogen circulation process.
The beneficial effects of the above-mentioned further scheme are: the influence of different furrow layout parameters on the soil microbial community structure diversity related index and the soil carbon nitrogen circulation process functional gene abundance is tested by calculating the average value, the dispersion square sum and the degree of freedom of the microbial community structure diversity related index and the soil carbon nitrogen circulation process functional gene abundance data in the soil samples of each experimental cell and the control cell, so that the importance degree of the influence of the experimental result of each factor can be accurately estimated.
Still further, the expression of the average value in step S401 is as follows:
j＝1,2,…,p；k＝1,2,…,r；i＝1,2,…,N
wherein ,the average value of the microbial community structure diversity related index and the soil carbon nitrogen circulation process functional gene abundance data of the soil samples of each experimental cell and the control cell is represented, p represents the number of the experimental cells, r represents the measurement times, j represents the jThe k represents the kth measurement of the experimental cell, x jk The index related to the structural diversity of the microbial community measured in the jth experimental community at the kth time is represented, N represents the number of functional genes related to the soil carbon nitrogen cycle process, i represents the number of functional genes related to the ith soil carbon nitrogen cycle process, and FCN ijk The functional gene abundance value of the ith soil carbon nitrogen circulation process measured in the kth experimental community is shown.
The beneficial effects of the above-mentioned further scheme are: and respectively calculating the related indexes of the structural diversity of the soil microbial community and the average value of the functional gene abundance data of the soil carbon nitrogen circulation process, and preparing for the subsequent analysis of variance.
Still further, the expression of the sum of squares of the dispersion and the degree of freedom in the step S402 is as follows:
f T ＝p-1
f A ＝q-1
A＝{α，β，h，d}
wherein ,ST The total dispersion square sum of the microbial community structure diversity related index and the soil carbon nitrogen cycle process functional gene abundance data in the soil samples of each experimental cell and the control cell is represented, S A Representing the sum of squares of dispersion caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, S e Representing error dispersionSum of squares, f T 、f A 、f e All represent S T 、S A and Se Corresponding degrees of freedom, y j The related index of the structural diversity of the microbial community in the soil sample of the j-th experimental plot and the functional gene abundance data of the soil carbon-nitrogen circulation process are expressed,the average value of the microbial community structure diversity related index and the functional gene abundance data in the soil carbon nitrogen circulation process in each experimental cell and control cell is represented, q represents the horizontal number of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, m represents the column number of an orthogonal table, u represents the u th column and k th column of the orthogonal table o Represents the arithmetic mean of the experimental results obtained when the four factors on any column were taken at level o.
The beneficial effects of the above-mentioned further scheme are: and respectively calculating the deviation square sum and the degree of freedom of the experimental and control district soil microbial community structure diversity related indexes and the soil carbon nitrogen circulation process functional gene abundance data caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, which are beneficial to comprehensively analyzing the soil microbial community structure and functional influence from different furrow layout parameters and preparing for analysis of variance calculation.
Still further, in the step S403, the expression for checking the influence of different ridge gradients α, ridge heights h, furrow width ratios β, furrow directions d on the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen cycle process is as follows:
wherein ,FA Soil microorganisms caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d are represented by the mean square of soil microbial community structure diversity related indexes and functional gene abundance data in the soil carbon-nitrogen circulation process, wherein the soil microbial community structure diversity related indexes are caused by ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend dThe ratio of the mean square of the data error of the functional gene abundance in the community structure diversity related index and the soil carbon nitrogen cycle process.
The beneficial effects of the above-mentioned further scheme are: the analysis of variance is carried out on the functional gene abundance data of the soil microorganism community structure diversity related index and the soil carbon nitrogen circulation process, which are affected by the ridge gradient alpha, the ridge height h, the furrow width ratio beta and the furrow trend d, respectively, the difference significance of the results of the influence of different furrow layout parameters on soil microorganisms is checked, the importance degree of the influence of each furrow layout parameter on the experimental results can be accurately estimated, and then the furrow layout parameters with the greatest influence on the soil microorganisms are screened out.
Still further, the expression for performing the significance test in step S404 is as follows:
wherein phi represents F of each ridge gradient alpha, ridge height h, ridge width ratio beta and ridge trend d α 、F h 、F β 、F d Ratio to F value at gamma=0.05 significance level, F A The degree of freedom of each experimental cell and the control cell caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d is shown as (f) A ，f e ) The ratio of the mean square of the soil microbial community structure diversity related index and the functional gene abundance data of the soil carbon nitrogen circulation process to the mean square of experimental errors caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, F 0.05 The degree of freedom of each experimental cell and the control cell, which is caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d, is expressed as (f A ，f e ) The ratio of the mean square of the soil microbial community structure diversity related index and the soil carbon nitrogen circulation process functional gene abundance data to the mean square of experimental errors caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d at the significance level of gamma=0.05.
The beneficial effects of the above-mentioned further scheme are: whether the furrow layout has obvious influence on the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process is checked from four influencing factors of the furrow gradient alpha, the furrow height h, the furrow width ratio beta and the furrow trend d, and the process analysis result is further improved.
Still further, the expression of the linear regression equation in the step S5 is as follows:
or (b)
wherein ,b0 、b 1 、b 2 、b 3 、b 4 Coefficients, alpha, respectively representing linear regression equations j 、h j 、β j 、d j Respectively representing the data of the ridge gradient alpha, the ridge height h, the furrow width ratio beta and the furrow trend d, y of the jth experimental cell j The data of the related index of the structural diversity of the soil microbial community and the functional gene abundance in the soil carbon nitrogen circulation process of the jth experimental plot are represented, p represents the number of the experimental plot, and x jk The index related to the structural diversity of the microbial community, which represents the kth measurement of the jth experimental cell, FCN ijk The functional gene abundance value of the ith soil carbon nitrogen circulation process measured in the kth experimental community is shown.
The beneficial effects of the above-mentioned further scheme are: by constructing a linear regression equation, the linear relation between the furrow layout parameters and the structural diversity related indexes of the soil microbial communities and the functional gene abundance data in the carbon-nitrogen circulation process of the soil is more clearly expressed, and the statistical rule hidden behind the randomness is found.
Still further, the expression for performing the significance test on the linear regression equation in the step S6 is as follows:
wherein ,RJ 、R G Representing complex correlation coefficients of each linear regression equation, S RJ Regression square sum of linear regression equation representing ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and soil microbial community structure diversity correlation index of each experimental cell and control cell, S TJ Representing the total sum of squares of linear regression equations of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and soil microbial community structure diversity correlation index of each experimental cell and control cell, S RG Expressing regression square sum of linear regression equation of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and functional gene abundance data of soil carbon nitrogen circulation process of each experimental cell and control cell, and S TG And the total sum of squares of linear regression equations of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and functional gene abundance data in the soil carbon nitrogen circulation process of each experimental cell and the control cell are represented.
The beneficial effects of the above-mentioned further scheme are: the reliability of the equation is checked by performing a significance test on the solved linear regression equation.
Drawings
FIG. 1 is a flow chart of the method of the present invention.
Fig. 2 is a layout diagram of furrow parameters of each experimental cell in this embodiment.
Fig. 3 is a cross-sectional view of the distribution of the furrow parameters of each experimental cell in this embodiment.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Examples
As shown in fig. 1, the invention provides a method for identifying the microorganism influence mechanism of a slope cultivation ridge ditch layout, which comprises the following steps:
s1, selecting a research area, and determining a planting type and a slope cultivation ridge ditch layout parameter, wherein the slope cultivation ridge ditch layout parameter comprises the following steps: ridge gradient, ridge height, furrow width ratio and furrow trend;
in the embodiment, a research area is selected, and the experimental area furrow arrangement control parameters, the parameter variation range and the typical crop are determined by combining local cultivation habits and typical crop through field investigation.
S2, arranging experimental cells and comparison cells according to a four-factor three-level orthogonal table by using ridge gradients, ridge heights, furrow width ratios and furrow directions, and planting selected crops, wherein the experimental cells and the comparison cells are arranged as shown in fig. 2 and 3, alpha in fig. 3 represents the ridge gradients, h represents the ridge heights, c represents the furrow widths, e represents the furrow widths, beta represents the furrow width ratios (beta=c/e), and d represents the furrow directions;
in this embodiment, the orthogonal table selects four-factor three level L without consideration of interaction 9 (3 4 ) The orthogonal table is designed as shown in table 1.
TABLE 1
In the orthogonal table, alpha 1 、α 2 、α 3 Three levels representing the slope of the ridge; h is a 1 、h 2 、h 3 Three levels representing ridge heights; beta 1 、β 2 、β 3 Three levels representing furrow-to-width ratios; d, d 1 、d 2 、d 3 Three levels representing the trend of the furrows are distributed among the levels according to an arithmetic progression within the variation range of furrow control parameters in a study area.
S3, respectively collecting soil samples of each experimental cell and each control cell before crop planting, during the middle growing period and during the harvesting period, and carrying out high-throughput sequencing on the soil microorganisms to obtain the diversity related indexes of soil microbial communities of different experimental cells and control cells and the functional gene abundance data of the soil carbon nitrogen circulation process, wherein the implementation method comprises the following steps:
s301, selecting two sampling areas of a ridge and a ditch in the upper, middle and lower sections of an experimental cell and a control cell before crop planting, in the middle of the growing period and in the harvesting period, removing surface layer floating soil, collecting soil samples, removing impurities of a soil sample mixture of the same cell, and filling the soil samples into a sterilizing packaging bag for preservation;
s302, performing high-throughput sequencing on soil microorganisms on a soil sample to obtain the sequence number of microorganism DNA or RNA, OTU abundance information and species classification information of the soil sample in each experimental cell and each control cell;
s303, calculating and obtaining the diversity related indexes of the soil microbial community structures of different experimental cells and control cells and the functional gene abundance data of the soil carbon and nitrogen circulation process according to the sequence number of the soil sample microbial DNA or RNA, the OTU abundance information and the species classification information.
In the embodiment, two sampling areas of ridges and furrows (sampling areas at the same positions are selected in a flat comparison cell) are selected in an experiment cell and a comparison cell of 3 stages of a period of time of growing, a period of growing and a period of harvesting before crop planting, the period of growing and the period of harvesting, floating soil with 0-5cm on the surface layer is removed, 10g of soil sample is collected, and after the soil sample of the same cell is mixed, impurities such as animal and plant residues are removed, the mixture is filled into a sterilizing self-sealing bag and stored at the temperature of minus 80 ℃.
In this embodiment, soil samples are subjected to high-throughput sequencing of soil microorganisms, and the high-throughput sequencing platform is an Illumina Miseq platform. Clustering and flattening OTUs at a similar level of 97% on the data after high-throughput sequencing to obtain the number of sequences of microorganism DNA or RNA, the abundance information of the OTUs and the species classification information of samples of each experimental cell and the control cell.
In this example, the microbial community structure diversity related index Chao, shannon, PD and the functional gene abundance data of the soil carbon nitrogen cycle process in each soil sample were calculated and respectively recorded as CH jk ，S jk ，PD jk ，FCN ijk Where i=1, 2, …, N represents the number of functional genes involved in carbon nitrogen cycling, as determined based on actual experimental procedures; j=1, 2, …,10, j denotes the number of test cells, k denotes the kth measurement, k=1, 2,3.
S4, checking the influence of the ridge gradient, the ridge height, the furrow width ratio and the furrow trend on the diversity related index of the soil microbial community structure and the functional gene abundance data of the soil carbon nitrogen cycle process by using an analysis of variance method, and determining the major and minor sequences of the four factors influencing the diversity related index of the soil microbial community structure and the functional gene abundance data of the soil carbon nitrogen cycle process, wherein the implementation method is as follows:
s401, calculating the related indexes of the microbial community structure diversity in soil samples of each experimental cell and each control cell, and the average value of the functional gene abundance data in the soil carbon nitrogen circulation process:
j＝1,2,…,p；k＝1,2,…,r；i＝1,2,…,N
wherein ,the average value of the microbial community structure diversity related index and the soil carbon nitrogen circulation process functional gene abundance data of the soil samples of each experimental cell and the control cell is represented, p represents the number of the experimental cells, r represents the measurement times, j represents the j-th experimental cell, k represents the k-th measurement of the experimental cell, and x jk The index related to the structural diversity of the microbial community measured in the kth experimental community is represented, N represents the number of functional genes related to the carbon-nitrogen circulation of soil,i represents the number of functional genes related to the carbon nitrogen cycle of the ith soil, FCN ijk Expressing the functional gene abundance value of the ith soil carbon nitrogen circulation process measured by the kth experimental plot;
s402, calculating to obtain the dispersion square sum and the degree of freedom of the microbial community structure diversity related indexes and the soil carbon nitrogen circulation process functional gene abundance data in the soil samples of each experimental cell and the control cell according to the average value:
f T ＝p-1
f A ＝q-1
A＝{α，β，h，d}
wherein ,ST The total dispersion square sum of the microbial community structure diversity related index and the soil carbon nitrogen cycle process functional gene abundance data in the soil samples of each experimental cell and the control cell is represented, S A Representing the sum of squares of dispersion caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, S e Represents the sum of squares of error dispersion, f T 、f A 、f e All represent S T 、S A and Se Corresponding degrees of freedom, y j The related index of the structural diversity of the microbial community in the soil sample of the j-th experimental plot and the functional gene abundance data of the soil carbon-nitrogen circulation process are expressed,the average value of the microbial community structure diversity related index and the functional gene abundance data in the soil carbon nitrogen circulation process in each experimental cell and control cell is represented, q represents the horizontal number of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, m represents the column number of an orthogonal table, u represents the u th column and k th column of the orthogonal table o An arithmetic average value representing experimental results obtained when the four factors on any column are taken as level o;
s403, according to the dispersion square sum and the degree of freedom, using an analysis of variance method to test the influence of different ridge gradients alpha, ridge heights h, furrow width ratios beta and furrow trend d on the diversity related indexes of the soil microbial community structure and the functional gene abundance data of the soil carbon nitrogen circulation process:
wherein ,FA The method is characterized by comprising the steps of expressing the ratio of the mean square of the structural diversity related index of the soil microbial community and the functional gene abundance data in the carbon-nitrogen circulation process, which are caused by ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, to the mean square of the structural diversity related index of the soil microbial community and the functional gene abundance data error in the carbon-nitrogen circulation process, which are caused by the ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d;
s404, performing significance test on the influence result obtained in the step S403:
wherein phi represents F of each ridge gradient alpha, ridge height h, ridge width ratio beta and ridge trend d α 、F h 、F β 、F d Ratio to F value at gamma=0.05 significance level, F A The degree of freedom of each experimental cell and the control cell caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d is shownIs (f) A ，f e ) The ratio of the mean square of the soil microbial community structure diversity related index and the functional gene abundance data of the soil carbon nitrogen circulation process to the mean square of experimental errors caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d, F 0.05 The degree of freedom of each experimental cell and the control cell, which is caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d, is expressed as (f A ，f e ) The ratio of the mean square of the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon and nitrogen circulation process to the mean square of experimental errors caused by four factors of ridge gradient alpha, ridge height h, furrow width ratio beta and furrow trend d under the significance level of gamma=0.05;
s405, sorting the influence results obtained in the step S403 from large to small according to the significance test result to obtain a major-minor sequence of four factors of ridge gradient, ridge height, furrow width ratio and furrow trend, which influence the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process;
s5, constructing a linear regression equation by the primary and secondary sequences and the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process respectively;
in this embodiment, it is assumed that four factors of the ridge gradient α, the ridge height h, the furrow width ratio β, and the furrow trend d are F according to the arrangement order affecting the primary and secondary α >F h >F β >F d Let equation y=b 0 +αb 1 +hb 2 +βb 3 +db 4 The system of linear regression equations is:
or (b)
wherein ,b0 、b 1 、b 2 、b 3 、b 4 Coefficients, alpha, respectively representing linear regression equations j 、h j 、β j 、d j Respectively representing the data of the ridge gradient alpha, the ridge height h, the furrow width ratio beta and the furrow trend d, y of the jth experimental cell j The data of the related index of the structural diversity of the soil microbial community and the functional gene abundance in the soil carbon nitrogen circulation process of the jth experimental plot are represented, p represents the number of the experimental plot, and x jk The index related to the structural diversity of the microbial community, which represents the kth measurement of the jth experimental cell, FCN ijk The functional gene abundance value of the ith soil carbon nitrogen circulation process measured in the kth experimental community is shown.
S6, performing significance test on the linear regression equation, and taking standard coefficients of ridge gradient, ridge height, furrow width ratio and furrow trend of the linear regression equation as evaluation slope furrow layout to influence soil microorganisms according to test results, so as to complete identification of a microorganism influence mechanism of slope tillage ridge furrow layout.
In this embodiment, the linear regression equation is subjected to significance test, and α of each cell is determined j 、h j 、β j 、d j Substituting the values into the obtained linear regression equation, and calculating the substitution valueAnd then respectively calculating the regression square sum, the total square sum and the degree of freedom of the linear regression equation, and checking the significance of the regression equation, wherein the checking method is a correlation coefficient checking method, and the calculation formula is as follows:
wherein ,RJ 、R G Representing complex correlation coefficients of each linear regression equation, S RJ Representing the ridge gradient alpha and ridge of each experimental cell and the comparison cellRegression square sum of linear regression equation of high h, furrow width ratio beta, furrow trend d and soil microbial community structure diversity correlation index, S TJ Representing the total sum of squares of linear regression equations of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and soil microbial community structure diversity correlation index of each experimental cell and control cell, S RG Expressing regression square sum of linear regression equation of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and functional gene abundance data of soil carbon nitrogen circulation process of each experimental cell and control cell, and S TG And the total sum of squares of linear regression equations of ridge gradient alpha, ridge height h, furrow width ratio beta, furrow trend d and functional gene abundance data in the soil carbon nitrogen circulation process of each experimental cell and the control cell are represented.
In the present embodiment, the complex correlation coefficient R for each linear regression equation J 、R G If the calculation result is between 0 and 1 or equal to 1, the linear regression equation passes the significance test, and if the calculation result is equal to 0, the linear regression equation has no significance.
Claims (2)
1. The method for identifying the microbial influence mechanism of the slope-cultivated land ridge ditch layout is characterized by comprising the following steps of:
s1, selecting a research area, and determining a planting type and a slope cultivation ridge ditch layout parameter, wherein the slope cultivation ridge ditch layout parameter comprises the following steps: ridge gradient, ridge height, furrow width ratio and furrow trend;
s2, arranging an experimental cell and a control cell according to a four-factor three-level orthogonal table by using a ridge gradient, a ridge height, a furrow width ratio and a furrow trend, and planting selected crops;
s3, respectively collecting soil samples of each experimental cell and each control cell before crop planting, during the middle growing period and during the harvesting period, and carrying out high-throughput sequencing on the soil microorganisms to obtain the diversity related indexes of soil microbial communities of different experimental cells and control cells and the functional gene abundance data of the soil carbon nitrogen circulation process;
s4, checking the influence of the ridge gradient, the ridge height, the furrow width ratio and the furrow trend on the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen cycle process by using an analysis of variance method, and determining the major and minor sequences of four factors affecting the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen cycle process;
the step S4 includes the steps of:
s401, calculating the related indexes of the microbial community structure diversity in soil samples of each experimental cell and each control cell, and the average value of the functional gene abundance data in the soil carbon nitrogen circulation process;
s402, calculating to obtain the dispersion square sum and the degree of freedom of the microbial community structure diversity related indexes and the soil carbon nitrogen circulation process functional gene abundance data in the soil samples of each experimental cell and the control cell according to the average value;
s403, checking different ridge gradients by using an analysis of variance method according to the dispersion square sum and the degree of freedomRidge height->Furrow width ratio->The trend of furrow->The influence on the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process;
s404, performing significance test on the influence result obtained in the step S403;
s405, sorting the influence results obtained in the step S403 from large to small according to the significance test result to obtain a major-minor sequence of four factors of ridge gradient, ridge height, furrow width ratio and furrow trend, which influence the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process;
the expression of the average value in step S401 is as follows:
wherein ,average value of microbial community structure diversity related index and soil carbon nitrogen cycle process functional gene abundance data of soil samples of each experimental cell and control cell is represented, and +.>Indicates the number of experimental cells, +.>Indicating the number of measurements>Indicate->Experimental cell>Represents +.>Secondary measurement(s)>Indicate->Personal entityCell verification no->Sub-measured microbial community structural diversity related index,/->Indicates the number of functional genes related to soil carbon nitrogen circulation, < + >>Indicate->Number of functional genes related to carbon nitrogen cycle of individual soil, < >>Indicate->Individual experimental cell>Second measurement>The abundance value of the functional genes in the carbon-nitrogen cycle process of the individual soil;
the expression of the sum of squares of the deviations and the degree of freedom in the step S402 is as follows:
wherein ,representing the total dispersion square sum of the microbial community structure diversity related index and the soil carbon nitrogen cycle process functional gene abundance data in the soil samples of each experimental cell and control cell>Indicating the gradient of the ridge>Ridge height->Furrow width ratio->The trend of furrow->Sum of squares of dispersion due to four factors, +.>Representing the sum of squares of the error dispersion,>all represent->、/> and />Corresponding degrees of freedom, < >>Indicate->Microbial community structure diversity related index and soil carbon nitrogen cycle process functional gene abundance data in soil samples of each experimental plot, and +_>Average value of microbial community structure diversity related index and soil carbon nitrogen circulation process functional gene abundance data in soil samples of each experimental cell and control cell is represented, and +.>Representing the gradient of ridgesRidge height->Furrow width ratio->The trend of furrow->Level number of->Representing the number of columns of the orthogonal table, +.>Representing the first of the orthogonal tablesuColumn (S)/(S)>Represents the level of four factors on any column +.>An arithmetic average of experimental results obtained at that time;
in the step S403, different ridge gradients are checkedRidge height->Furrow width ratio->The trend of furrow->The expression of the influence on the soil microbial community structure diversity related index and the soil carbon nitrogen cycle process functional gene abundance data is as follows:
wherein ,indicating the gradient of the ridge>Ridge height->Furrow width ratio->The trend of furrow->The mean square and the gradient of each ridge of the induced soil microbial community structure diversity related index and the soil carbon nitrogen circulation process functional gene abundance data are +.>Ridge height->Furrow width ratio->The trend of furrow->The ratio of the mean square of the structural diversity related index of the soil microbial community and the functional gene abundance data error of the soil carbon nitrogen circulation process caused by the four factors together;
the expression for the significance test in step S404 is as follows:
wherein ,representing the gradient +.>Ridge height->Furrow width ratio->And furrow trend->Is->、/>、/>、/>Respectively and->The value is +.>Ratio at the level of significance, +.>Indicating that each experimental cell and control cell are formed by ridge gradient +.>Ridge height->Furrow width ratio->The trend of furrow->The four factors cause a degree of freedom of +.>Is related to the structural diversity of soil microbial communitiesMean square of index and soil carbon nitrogen cycle process functional gene abundance data and gradient of each ridge>Ridge height->Furrow width ratio->The trend of furrow->Ratio of mean square of experimental errors caused by four factors, < >>The representation shows that each experimental cell and the control cell are represented by ridge gradient +.>Ridge height->Furrow width ratio->The trend of furrow->The four factors cause a degree of freedom of +.>At->Mean square and gradient of soil microbial community structure diversity related index and soil carbon nitrogen circulation process functional gene abundance data under significance level>Ridge height->Furrow width ratio->The trend of furrow->The ratio of the mean square of experimental errors caused by the four factors together;
s5, constructing a linear regression equation by the primary and secondary sequences and the structural diversity related index of the soil microbial community and the functional gene abundance data of the soil carbon nitrogen circulation process respectively;
the expression of the linear regression equation in the step S5 is as follows:
or (b)
wherein ,coefficients representing the linear regression equation, respectively, +.>Respectively represent +.>Ridge gradient of each experimental plot>Ridge height->Furrow width ratio->And furrow trend->Data;
s6, performing significance test on the linear regression equation, and taking standard coefficients of ridge gradient, ridge height, furrow width ratio and furrow trend of the linear regression equation as evaluation slope furrow layout to influence soil microorganisms according to test results so as to finish identification of a microorganism influence mechanism of slope tillage ridge furrow layout;
the expression for performing the significance test on the linear regression equation in the step S6 is as follows:
wherein ,、/>complex correlation coefficients representing the respective linear regression equations, < +.>Ridge gradient ++representing each experimental cell and control cell>Ridge height->Furrow width ratio->Furrow walkingTo->Regression square sum of linear regression equation of diversity correlation index of soil microbial community structure, ++>Ridge gradient ++representing each experimental cell and control cell>Ridge height->Furrow width ratio->The trend of furrow->Total sum of squares of linear regression equations of structural diversity correlation index with soil microbial community,/->Ridge gradient ++representing each experimental cell and control cell>Ridge height->Furrow width ratio->The trend of furrow->Regression square sum of linear regression equation of functional gene abundance data of soil carbon nitrogen cycle process,/I>Representing the slope of the ridge of each experimental cell and the control cellRidge height->Furrow width ratio->The trend of furrow->And the total square sum of linear regression equation of the functional gene abundance data in the soil carbon nitrogen circulation process.
2. The method for identifying a mechanism of influence of a hillside furrow arrangement on microorganisms according to claim 1, wherein the step S3 includes the steps of:
s301, selecting two sampling areas of a ridge and a ditch in the upper, middle and lower sections of an experimental cell and a control cell before crop planting, in the middle of the growing period and in the harvesting period, removing surface layer floating soil, collecting soil samples, removing impurities of a soil sample mixture of the same cell, and filling the soil samples into a sterilizing packaging bag for preservation;
s302, performing high-throughput sequencing on soil microorganisms on a soil sample to obtain the sequence number of microorganism DNA or RNA, OTU abundance information and species classification information of the soil sample in each experimental cell and each control cell;
s303, calculating and obtaining the diversity related indexes of the soil microbial community structures of different experimental cells and control cells and the functional gene abundance data of the soil carbon and nitrogen circulation process according to the sequence number of the soil sample microbial DNA or RNA, the OTU abundance information and the species classification information.
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