Screening and compounding model of artemisia argyi rhizosphere efficient cadmium-tolerant growth-promoting flora and application of screening and compounding model

Abstract

The invention provides a screening and compounding model of artemisia argyi intergrassy high-efficiency cadmium-tolerant growth-promoting flora and application of the screening and compounding model. According to the model, firstly, wormwood root system samples with sorting potential are rapidly judged, then the wormwood root system samples are prepared into turbid liquid through a limit dilution method, the turbid liquid is transferred to a 96-hole culture plate for large-scale propagation, and the wormwood root system samples with sorting potential are obtained by combining the steps of function prediction, culture medium optimization, function verification, function screening and the like. Only 15 days are needed to obtain nearly hundreds of root system bacteria with cadmium-resistant and growth-promoting functions. On the basis of the excellent technical effect of the provided screening model, the argy wormwood rhizosphere cadmium-resistant growth-promoting bacteria are screened and are further compounded to form the efficient and stable multifunctional bacterial agent, so that the growth capability and the remediation effect of the argy wormwood under cadmium pollution stress can be effectively improved, and abundant strain resources and technical support are provided for ecological remediation of cadmium-polluted mining area soil.

Description

Technical Field

[0001] The invention relates to a screening model for a highly efficient cadmium-resistant growth-promoting bacterial community, and in particular to a screening and compounding model for a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of wormwood and an application thereof, and belongs to the technical field of separation and cultivation of environmental functional microorganisms. Background Art

[0002] Artemisia argyi, a perennial upright herb of the genus Artemisia in the Asteraceae family, is famous for its rich aroma. It is widely distributed in East Asia, such as China, Japan, and the Korean Peninsula, and is commonly found on hillsides, hills, and some wastelands. Artemisia argyi grows rapidly, has high biomass, a strong root system, and has good adaptability to a variety of climate and soil conditions. It has strong tolerance and a certain absorption capacity for a variety of heavy metals such as cadmium, so it has great application potential in the field of heavy metal contaminated soil remediation. At the same time, as an aromatic plant, wormwood essential oil is mainly used in non-food fields, which is safe, economical, and environmentally friendly. There is no accumulation of harmful metals in the synthesis and extraction of wormwood essential oil, and the risk of heavy metals spreading through the food chain is low. In addition, as the aerial part of an aromatic plant, wormwood has insect-avoiding properties and is suitable for large-scale planting. Therefore, using wormwood for ecological remediation of heavy metal-contaminated soil can simultaneously achieve good environmental and economic benefits.

[0003] The rhizosphere of wormwood is a very important area for wormwood metabolism, and it can carry out very active material exchange with microorganisms in the soil through the rhizosphere soil. Microorganisms in the rhizosphere of wormwood can directly or indirectly promote plant growth and improve plant resistance. Plant rhizosphere growth-promoting bacteria have always attracted widespread attention from scholars because of their advantages in promoting plant growth, increasing crop yields, reducing heavy metal toxicity, and reducing fertilizer costs. Among them, rhizosphere bacteria have the highest abundance in the rhizosphere, reaching 10 6 ~10 8 CFU / g. However, when obtaining the rhizosphere growth-promoting bacterial resources of mugwort, the traditional isolation and culture methods are time-consuming and complex in bacterial flora, which makes it difficult to meet the requirements for the sorting and synthesis of efficient cadmium-resistant growth-promoting bacterial flora in the rhizosphere of mugwort. Therefore, this patent discloses a simple and fast method for synthesizing efficient cadmium-resistant growth-promoting bacterial flora. First, the mugwort root samples with sorting potential are determined, and then they are prepared into a suspension by the limiting dilution method, and then transferred to a 96-well culture plate for large-scale expansion and reproduction. Combined with the steps of function prediction, culture medium optimization, function verification, and function screening, nearly 100 root bacteria with cadmium resistance and growth-promoting functions can be obtained in only 15 days. Further compounding it into an efficient and stable multifunctional bacterial agent can effectively improve the growth ability and restoration effect of mugwort under cadmium pollution stress, and provide rich bacterial resources and technical support for the ecological restoration of soil in cadmium-contaminated mining areas. Summary of the invention

[0004] In view of the problems existing in the existing problems, the first purpose of the present invention is to provide a screening and compounding model for efficient cadmium-resistant and growth-promoting bacteria in the rhizosphere of mugwort. The model first determines the mugwort root samples with sorting potential, and then prepares them into a suspension by limiting dilution method, and then transfers them to 96-well culture plates for large-scale expansion and reproduction. Combined with the steps of function prediction, culture medium optimization, function verification, function screening, etc., nearly 100 root bacteria with cadmium resistance and growth-promoting functions can be obtained in only 15 days. Further compounding it into an efficient and stable multifunctional bacterial agent can effectively improve the growth ability and restoration effect of mugwort under cadmium pollution stress, and provide rich bacterial resources and technical support for the ecological restoration of soil in cadmium-contaminated mining areas.

[0005] The second purpose of the present invention is to provide an application of a screening and compounding model for a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of mugwort, to screen cadmium-resistant growth-promoting bacteria in the rhizosphere of mugwort for the preparation of a composite bacterial agent, or a microecological preparation containing the bacterial agent, or a biological fertilizer containing the bacterial agent or the microecological preparation.

[0006] In order to achieve the above technical objectives, the present invention provides a screening and compounding model for a high-efficiency cadmium-resistant growth-promoting bacterial community in the rhizosphere of mugwort, comprising:

[0007] Step S1, collecting mugwort root samples from cadmium-contaminated sites for gene sequencing and functional gene analysis, and constructing a sample functional gene database;

[0008] Step S2, taking the cadmium resistance gene set as the training target, classifying and roughly selecting the data set obtained in step S1 through the random forest model to obtain a roughly selected sample;

[0009] Step S3, diluting the rough selected sample and transferring it to a culture plate for culture, extracting the bacterial genome of each turbid well in the culture plate, and performing gene sequencing and functional gene analysis, and classifying and selecting to obtain selected samples;

[0010] Step S4, the plant growth-promoting functions of the single bacteria and mixed bacteria in the selected samples are identified and separated and purified respectively, and the single bacteria obtained after the separation and purification of the single bacteria and / or mixed bacteria are compounded, the coexistence pattern between the strains is detected, the optimal compound bacterial community is screened out, and then the mugwort growth-promoting experiment is carried out.

[0011] As a preferred scheme, the construction process of the sample functional gene database is as follows: the genomic DNA of rhizospheric bacteria is extracted from the mugwort root samples collected from the cadmium-contaminated site as a template, PCR amplification is performed on the V5-V7 region with the best endophyte specificity using 799F as a forward primer and 1193R as a reverse primer, and all the amplified products are collected to obtain.

[0012] As a preferred solution, the 799F forward primer is 5'-barcode+AACMGGATTAGATACCCKG-3'.

[0013] As a preferred solution, the 1193R reverse primer is 5'-ACGTCATCCCCACCTTCC-3'.

[0014] As a preferred embodiment, the cadmium resistance gene set includes czcD, zipB, cmtR, cadC and ipdC.

[0015] As a preferred solution, in the random forest model, the number of decision trees is 300 to 800, and each time a decision is made, 30 to 40% of the features are randomly selected from all input features for node splitting; the random forest model also needs to be optimized, and the optimization of the model adopts 10-fold repeated cross validation.

[0016] The present invention uses 500 decision trees to build the model. During the construction process of each decision tree, one-third of the characteristic variables are selected by random sampling for node splitting to improve the generalization ability of the model. The training set and test set of the model are divided into 75% and 25%, and mtry is adjusted as a key parameter to obtain the optimal model performance. In addition, the Boruta algorithm is used to sort the input variables by importance to screen out the characteristic variables that have the greatest impact on the model prediction results, and these variables are used as input variables to build the final random forest model.

[0017] As a preferred solution, the method of diluting the crude sample is the limiting dilution method, and the crude sample is prepared to a concentration of 1×10 6 ~1×10 8 CFU / mL of suspension.

[0018] As a preferred solution, the Cd 2+ The concentration of is 0.2~2mmol / L, and the concentration of tryptic soytone is 10~50%.

[0019] As a preferred solution, the classification and selection process includes:

[0020] Step S3-1, seal the culture plate with a sterile sealing film, and culture at room temperature until the number of turbid wells no longer increases, and retain the culture plate with 30% turbid wells;

[0021] Step S3-2, using the PICRUSt2 algorithm and KEGG database to predict the functional genes of the ASVs obtained by sequencing and annotating the turbid pores, and determining the rhizospheric bacterial pores with cadmium tolerance and growth-promoting potential and a purity of 1 to 5 genera (Genus) as selected samples for retention.

[0022] As a preferred solution, the plant growth-promoting function of the single bacteria in the selected sample is directly identified.

[0023] As a preferred solution, the mixed bacteria in the selected sample are first predicted by collaborative filtering using the transfer prediction model in KOMODO to obtain the optimal culture medium formula for each of the mixed bacteria, and then separated and purified to obtain single bacteria for plant growth promotion function identification.

[0024] As a preferred solution, the process of formulating the optimal culture medium for mixed bacteria is: using KOMODO in combination with NCBI microbial classification and microbial culture medium database DSMZ, based on the transfer prediction model and collaborative filtering prediction, according to the systematic evolutionary similarity of 16SrRNA and the species of known culture media, the culture medium of closely related species with high similarity is inferred for purifying turbid mixed bacteria pores.

[0025] As a preferred solution, the process of identifying the plant growth-promoting function is:

[0026] Step S4-1, inoculating the single bacteria obtained after separation and purification of the single bacteria or mixed bacteria into Ashby's medium, PVK medium and CAS medium respectively, culturing at 30-40° C. for 1-3 days, and observing the hydrolysis zone around the colony to determine whether the strain has the characteristics of nitrogen fixation, phosphorus solubilization and siderophore production;

[0027] Step S4-2, inoculate the single bacteria or the single bacteria obtained after separation and purification of mixed bacteria into LB liquid culture medium containing 0.1-1 g / L tryptophan, shake and culture in a shaker at a constant temperature of 30-40° C. for 3-5 days, and add a colorimetric solution to determine whether the strain has the characteristic of producing IAA.

[0028] As a preferred scheme, the screening process for the optimal composite bacterial community is as follows: single bacteria or mixed bacteria are separated and purified, and the single bacteria obtained are combined in pairs, and co-cultured in the same culture medium, and the type of interaction between each pair of strains is analyzed, and the strain combination with the best growth-promoting effect is selected, and then the final optimal composite ratio is determined under the optimal strain combination.

[0029] As a preferred solution, the process of the wormwood growth promotion experiment is as follows: the optimal composite bacterial flora is inoculated into LB liquid culture medium, and cultured in a shaker at 30-40°C and 150-200 r / min for 1-2 days until the concentration of the bacterial suspension reaches 1×10 6 ~1×10 8 CFU / mL, and the supernatant was removed by centrifugation and inoculated into potted plants to verify its growth-promoting effect on Artemisia argyi under cadmium stress.

[0030] The present invention also provides an application of a screening and compounding model for a high-efficiency, cadmium-resistant, growth-promoting bacterial community in the rhizosphere of mugwort, for screening cadmium-resistant, growth-promoting bacteria in the rhizosphere of mugwort to prepare a composite bacterial agent, or a microecological preparation containing the bacterial agent, or a biofertilizer containing the bacterial agent or the microecological preparation.

[0031] Compared with the prior art, the beneficial technical effects of the technical solution of the present invention are:

[0032] The present invention provides a simple and fast model for screening and compounding of efficient cadmium-resistant growth-promoting bacteria. The model first determines the wormwood root samples with sorting potential, then prepares them into suspensions by limiting dilution method, and then transfers them to 96-well culture plates for large-scale expansion and reproduction. Combined with the steps of function prediction, culture medium optimization, function verification, function screening, etc., nearly 100 root bacteria with cadmium resistance and growth-promoting functions can be obtained in only 15 days. Further compounding it into an efficient and stable multifunctional bacterial agent can effectively improve the growth ability and restoration effect of wormwood under cadmium pollution stress, and provide rich bacterial species resources and technical support for the ecological restoration of soil in cadmium-contaminated mining areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a flow chart of the model provided in Example 1 of the present invention;

[0034] Figure 2 This is a heat map of species abundance after high-throughput sorting provided in Example 1 of the present invention;

[0035] Figure 3 The bacterial genus quantity graph obtained for the TSB culture medium with different dilution concentrations provided in Example 1 of the present invention;

[0036] Figure 4 This is a functional verification diagram of the growth-promoting bacteria provided in Example 1 of the present invention;

[0037] Figure 5 This is a diagram showing the effect of the compounding ratio of the growth-promoting bacteria provided in Example 1 of the present invention;

[0038] Figure 6 This is a verification diagram of the inoculation of a potted mugwort plant with the composite bacterial flora provided in Example 1 of the present invention;

[0039] Figure 7 This is a picture of the wormwood root after harvest of the composite bacterial flora provided in Example 1 of the present invention;

[0040] Figure 8 This is a graph of physiological indicators of chlorophyll of mugwort of the composite bacterial community provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0042] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are conventional biochemical reagents and can be obtained from commercial sources unless otherwise specified.

[0043] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0044] Example 1

[0045] Flow chart of the synthesis method of the rhizosphere efficient cadmium-resistant growth-promoting bacteria of Artemisia argyi of the present invention ( Figure 1 ) to carry out this embodiment.

[0046] In 2024, this embodiment was carried out on mugwort in Changsha.

[0047] S1 After washing and sterilizing the root surface of wormwood collected from the cadmium contaminated site, the root system of wormwood was further ground under sterile conditions to obtain root slurry. The genomic DNA of rhizospheric bacteria was extracted, and the V5-V7 fragment of the 16S rRNA gene was amplified using the obtained DNA as a template, and all amplified products were collected. The resulting library was used for sequencing, bacterial annotation, and functional gene analysis. The sample functional gene database was constructed using PICRUSt2, and the random forest model was used to classify and predict the screening potential of the samples. The cadmium resistance and growth-promoting gene set (czcD, zipB, cmtR, cadC, ipdC) was used as the training target to screen samples with high content of cadmium resistance and growth-promoting genes for sorting rhizospheric bacteria.

[0048] S1.1 Take the rinsed mugwort roots and soak them in 75% ethanol solution for 1 min on a sterile workbench, then transfer them to 2.5% sodium hypochlorite solution and soak for 5-8 min. Rinse with sterile water for 3-5 times, collect the last rinse solution, spread it on a plate, and the surface is completely sterilized when aseptic growth occurs. Add 10mmol / L MgCl 2 The solution is ground to obtain a slurry of mugwort roots.

[0049] S1.2 The total genomic DNA samples of rhizospheric bacteria of mugwort were extracted using the OMEGA Soil DNA Kit, and the quantity and quality of the DNA were measured respectively; the V5-V7 region of the bacterial 16S rRNA gene with a length of about 394 bp was selected for sequencing, and specific primers 799F and 1193R were used for PCR amplification. The barcode in the front primer was used to distinguish different samples in the same library. PFU enzyme amplification was used for PCR, and the amplification system was a 25 μL system; the mugwort rhizospheric bacteria identification library was constructed using Illumina's TruSeq Nano DNALT Library Prep Kit, and the qualified library was subjected to 2×250 bp double-end sequencing, and the library quantity was controlled between 15-18 pM.

[0050] The specific primers 799F: 5'-barcode+AACMGGATTAGATACCCKG-3'; 1193R: 5'-ACGTCATCCCCACCTTCC-3'.

[0051] The 25 μL amplification system: 5×buffer 5 μL; dNTP (2.5 mM) 2 μL; forward primer (10 μM) 1 μL; reverse primer (10 μL) 1 μL; DNA Template 1 μL; ddH 2 O 14.75μL; Fast pfu DNA Polymerase 0.25μL.

[0052] S1.3 The sequence obtained by sequencing was compared with the V5V7 fragment of the 16S rRNA gene in the Silva database, and the ASV of rhizospheric bacteria of wormwood was annotated. PICRUSt2 was used to predict the functional genes of the obtained rhizospheric bacteria of wormwood. The functional genes were referenced to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The cadmium tolerance-related genes such as czcD (K16264), zipB (K16267), cmtR (K21885), cadC (K21903) and the plant growth-promoting-related gene ipdC (K04103) were used as screening conditions to preliminarily determine the bacterial samples with higher content of cadmium tolerance genes and growth-promoting genes for further sorting.

[0053] S1.4 Organize the functional gene database data generated by PICRUSt2, select the functional genes related to the target variable (metadata) as features, and standardize the data to ensure that the data range of different features is the same; check the data type of the target variable (such as cadmium tolerance or growth promotion effect). If it is numerical, such as relative cadmium tolerance score, select regression analysis; if it is categorical, such as high, medium, and low cadmium tolerance classification, select classification analysis. A random forest model was constructed using 500 decision trees. At each decision, one-third of the features were randomly selected from all input features for the optimal decision (mtry), and the performance of the preliminary model was evaluated by the mean squared residual (MSE) and the explained variation (% Var explained). The caret package was used to divide the data set into a training set and a test set in a ratio of 75:25 to ensure the generalization ability of the model. The Boruta package was used for feature selection to identify which features are most important for model prediction. The Boruta algorithm can evaluate the relative importance of each input feature and help you screen out key cadmium-resistant genes (such as czcD, zipB, cmtR, cadC, ipdC) or other related features. Use the caret package to perform 10-fold 5-fold repeated cross-validation to optimize the mtry parameter and find the parameter combination that optimizes the model performance. Through cross-validation, the performance of the model under different parameter combinations is evaluated, and finally the parameter combination with the smallest model error and the strongest explanatory power is selected. The training effect of the model is evaluated through the prediction results on the training set, and the RMSE and R-squared values ​​are calculated to measure the degree of fit of the model. The model is evaluated using the test set data, and the prediction error and accuracy of the model are calculated to verify the generalization ability of the model. It can be seen from the calculation that the RMSE of the random Senli model provided by the present invention is 6.1564, and R 2 It is 0.805, which can achieve the screening effect of rhizospheric bacteria.

[0054] The S2 rhizobacterial eluate samples were prepared into a suspension of appropriate concentration using the limiting dilution method and transferred in batches to 100 slides containing 2 mmol / L CdCl 2 The cells were cultured in 96-well TSB (10% to 50% concentration) cell culture plates. The cells were cultured at room temperature and away from light for 15 days to rapidly separate and obtain a large number of candidate bacterial wells. The bacterial genomic DNA from each turbid well was extracted for high-throughput sequencing of the 16S rRNA gene and functional gene analysis of rhizosphere endophytes. Figure 2 is the relative abundance of candidate pores sequenced after high-throughput sorting; Figure 3 The difference in the number of bacterial genera obtained from TSB culture media with different dilution concentrations under cadmium stress directly reflects the effects of different culture medium concentrations and cadmium stress on the diversity of rhizospheric bacteria.

[0055] S2.1 The samples of cadmium-resistant growth-promoting bacteria from the rhizosphere of Artemisia selengensis that were initially screened were prepared into a concentration of 1×10 6 ~1×10 8 CFU / mL suspension; the culture medium was prepared by the limiting dilution method, with the dilution multiple ranging from 500,000 to 5 million times.

[0056] S2.2 The samples of cadmium-resistant growth-promoting bacteria from the rhizosphere of Artemisia argyi with different dilutions were transferred into 96-well cell culture plates containing TSB liquid medium with different concentrations (10% to 50%). 2 ) for high-throughput culture; the volume of each well was 160 μL, which was sealed with a sterile sealing film and cultured at room temperature;

[0057] S2.3 Select the culture plate with 30% turbid wells and no more turbid wells after 15 days.

[0058] TSB liquid culture medium composition: 17.0 g / L tryptone; 3.0 g / L soy peptone; 5.0 g / L sodium chloride; 2.5 g / L potassium dihydrogen phosphate; 2.5 g / L glucose; water as solvent; pH = 7.3 ± 0.2.

[0059] The cadmium stress refers to adding a cadmium chloride solution into the TSB liquid culture medium so that the cadmium concentration in the culture medium reaches 2 mmol / L.

[0060] S2.4 Take out a portion of the bacterial solution from the turbid wells of the culture plate for bacterial identification, add 80% (v / v) glycerol to the remaining bacterial solution at a volume ratio of 1:1, and store it in a -80℃ refrigerator for later use.

[0061] S2.5 Extract the total genomic DNA samples of the bacterial solution from the turbid bacterial wells retained in the culture plate, and measure the quantity and quality of the bacterial solution DNA respectively; select the V5V7 region of the bacterial 16S rRNA gene with a length of about 394bp for sequencing; compare the sequenced sequence with the V5-V7 fragment of the 16S rRNA gene in the Silva database, and annotate the cadmium-resistant growth-promoting rhizospheric bacteria ASV of mugwort.

[0062] S2.6 PICRUSt2 was used to predict the functional genes of the obtained ASV of mugwort rhizosphere bacteria. The functional genes were referenced to the KEGG database, including but not limited to plant growth-promoting related genes such as pstB (K02036), budC (K18009), gabD (K00135), ppdC (K02681), etc., carbon metabolism related genes such as cbbL (K01601), aclA (K15230), acsA (K00198), accA (K01962), etc., nitrogen metabolism related genes Related genes such as nasB (K00360), norC (K02305), nifH (K02588), sulfur metabolism related genes such as cysC (K00860), aprA (K00394), soxX (K17223), cadmium tolerance related genes such as czcD (K16264), zipB (K16267), cmtR (K21885), cadC (K21903), etc.; culture wells containing target bacteria with high purity (only 1 to 5 genera) were screened and retained.

[0063] S3 combined the NCBI microbial classification and microbial culture medium database DSMZ through the KOMODO website, based on the transmission prediction model and collaborative filtering prediction, input the classification information obtained from the 16S rRNA gene alignment of cadmium-resistant growth-promoting rhizospheric bacteria of mugwort, retrieved the strain list corresponding to the classification information, and obtained the optimal culture medium formula and composition corresponding to these strains.

[0064] The transfer prediction model means that if microorganisms A and B can both use culture medium 1, microorganisms B and C can use culture medium 2, and microorganism C can use culture medium 3, then by sequential transfer reasoning, microorganism A should also be able to use culture medium 3; the collaborative filtering prediction means inferring the culture medium of closely related species with high similarity based on the systematic evolutionary similarity of 16S rRNA and the species of known culture media.

[0065] The single bacteria in the turbid wells and the purified strains were inoculated into Ashby's medium, PVK medium and CAS medium respectively. After culturing at 37°C for 3 days, the hydrolysis zone around the colony was observed to determine whether the strain had the characteristics of nitrogen fixation, phosphorus solubilization and iron carrier production ( Figure 4 ).

[0066] The single bacteria in the turbid wells and the purified strains were inoculated into 100 ml LB liquid medium containing 0.5 g / L tryptophan, placed in a 30°C shaker, and cultured at 180 r / min for 4 days. The bacterial suspension was centrifuged at 10000 r / min for 5 min, 1 ml of the supernatant after centrifugation was taken into a 10 mL EP tube, and 4 mL of colorimetric solution was added at the same time. For the standard control, 1 mL of 50 mg·L -1The blank control was added with 1 mL LB liquid culture medium and 4 mL colorimetric solution (30 mL concentrated sulfuric acid; 1.5 mL 0.5 mol·L -1 FeCl 3 , 50 mL distilled water), placed at room temperature away from light, to determine whether the strain has the IAA production characteristic ( Figure 4 ).

[0067] S4 combined the sorted strains into pairs and co-cultured them in the same culture medium to analyze the interaction type between each pair of strains. The results are shown in Table 1.

[0068] Table 1 Types of interactions between different strains

[0069]

[0070]

[0071] The strain combination with the best growth-promoting effect, namely the Rahnella+Pseudomonas combination, was selected from Table 1, and multiple experiments were conducted on the combination to determine the effects under different compounding ratios. Figure 5 It can be seen that the best compound ratio was finally determined to be 1:1 Rahnella+Pseudomonas.

[0072] Furthermore, the present invention further verifies the growth-promoting and cadmium-resistant effect of the composite bacterial community through experiments. The candidate strains Rahnella and Pseudomonas are cultured to a fixed concentration of 1×10 8 CFU / mL, mixed in equal proportions, and compounded into bacterial flora.

[0073] The composite synthetic bacterial flora was inoculated into LB liquid medium and cultured in a shaker at 30°C and 180 rpm for 1 to 2 days until the concentration of the bacterial suspension reached 1×10 6 ~1×10 8 CFU / mL, centrifuged and removed the supernatant and added to the rhizosphere of potted wormwood test. Its growth was as follows Figure 6 As shown in the figure, in order to verify its growth-promoting effect on mugwort under cadmium pollution stress, the main measured indicators include plant root length, chlorophyll content and other plant physiological indicators, and the results are shown in the figure Figure 7 and Figure 8As shown in the figure, it can be seen that the wormwood inoculated with the composite bacteria of the present invention is superior to the control group and the single strain treatment group in terms of root length and leaf number. The wormwood inoculated with the composite bacteria has the highest chlorophyll content, which can effectively improve the photosynthesis efficiency of wormwood and promote plant growth in a cadmium-polluted environment. Combining these images and measurement results, the composite bacterial community screened by the present invention shows a more significant cadmium-resistant growth-promoting effect under cadmium pollution stress, verifying the excellence of the model of the present invention. These results show that the composite bacterial community has practical application value and can effectively improve the growth performance of plants in heavy metal pollution environments.

Claims

1. A screening and compounding model for efficient cadmium-resistant growth-promoting bacteria in the rhizosphere of Artemisia argyi, characterized in that: include: Step S1, collecting mugwort root samples from cadmium-contaminated sites for gene sequencing and functional gene analysis, and constructing a sample functional gene database; Step S2, taking the cadmium resistance gene set as the training target, classifying and roughly selecting the data set obtained in step S1 through the random forest model to obtain a roughly selected sample; Step S3, diluting the rough selected sample and transferring it to a culture plate for culture, extracting the bacterial genome of each turbid well in the culture plate, and performing gene sequencing and functional gene analysis, and classifying and selecting to obtain selected samples; Step S4, the plant growth-promoting functions of the single bacteria and mixed bacteria in the selected samples are identified and separated and purified respectively, and the single bacteria obtained after the separation and purification of the single bacteria and / or mixed bacteria are compounded, the coexistence pattern between the strains is detected, the optimal compound bacterial community is screened out, and then the mugwort growth-promoting experiment is carried out.

2. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 1 is characterized in that: The construction process of the sample functional gene database is as follows: extracting genomic DNA of rhizosphere bacteria from mugwort root samples collected from cadmium-contaminated sites as a template, using 799F as a forward primer and 1193R as a reverse primer to perform PCR amplification on the V5-V7 region with the best endophyte specificity, and collecting all amplified products to obtain; The 799F forward primer is 5'-barcode+AACMGGATTAGATACCCKG-3'; the 1193R reverse primer is 5'-ACGTCATCCCCACCTTCC-3'.

3. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 1 is characterized in that: The cadmium resistance gene set includes czcD, zipB, cmtR, cadC and ipdC; in the random forest model, the number of decision trees is 300 to 800, and each time a decision is made, 30 to 40% of the features are randomly selected from all input features for node splitting; the random forest model needs to be optimized, and the optimization of the model adopts 10-fold repeated cross validation.

4. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 1 is characterized in that: The method of diluting the crude sample is the limiting dilution method, and the crude sample is prepared to a concentration of 1×10 6 ~1×10 8 CFU / mL suspension; Cd in the liquid culture medium in the culture plate 2+ The concentration of is 0.2~2mmol / L, and the concentration of tryptic soytone is 10~50%.

5. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 1 is characterized in that: The classification and selection process includes: Step S3-1, seal the culture plate with a sterile sealing film, and culture at room temperature until the number of turbid wells no longer increases, and retain the culture plate with 30% turbid wells; Step S3-2, using the PICRUSt2 algorithm and KEGG database to predict the functional genes of the ASVs obtained by sequencing and annotating the turbid pores, and determining the rhizospheric bacterial pores with cadmium tolerance and growth-promoting potential and a purity of 1 to 5 genera (Genus) as selected samples for retention.

6. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of wormwood according to claim 1 is characterized in that: The single bacteria in the selected samples are directly identified for their plant growth-promoting functions; the mixed bacteria in the selected samples are first predicted through collaborative filtering using the transfer prediction model in KOMODO to obtain the optimal culture medium formula for each mixed bacteria, and then separated and purified to obtain single bacteria for identification of their plant growth-promoting functions.

7. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 6, characterized in that: The process of formulating the optimal culture medium for mixed bacteria is as follows: combining KOMODO with NCBI microbial classification and microbial culture medium database DSMZ, based on the transfer prediction model and collaborative filtering prediction, and inferring the culture medium of closely related species with high similarity according to the phylogenetic similarity of 16S rRNA and the species of known culture medium to purify turbid mixed bacteria pores.

8. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 6, characterized in that: The process of identifying the plant growth-promoting function is as follows: Step S4-1, inoculating the single bacteria obtained after separation and purification of the single bacteria or mixed bacteria into Ashby's medium, PVK medium and CAS medium respectively, culturing at 30-40° C. for 1-3 days, and observing the hydrolysis zone around the colony to determine whether the strain has the characteristics of nitrogen fixation, phosphorus solubilization and siderophore production; Step S4-2, inoculate the single bacteria or the single bacteria obtained after separation and purification of mixed bacteria into LB liquid culture medium containing 0.1-1 g / L tryptophan, shake and culture in a shaker at a constant temperature of 30-40° C. for 3-5 days, and add a colorimetric solution to determine whether the strain has the characteristic of producing IAA.

9. The screening and compounding model of a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to claim 1, characterized in that: The screening process of the optimal composite bacterial community is as follows: the single bacteria obtained after separation and purification of single bacteria or mixed bacteria are combined in pairs, co-cultured in the same culture medium, the interaction type between each pair of strains is analyzed, the strain combination with the best growth promotion effect is selected, and then the final optimal composite ratio is determined under the optimal strain combination; the process of the wormwood growth promotion experiment is as follows: the optimal composite bacterial community is inoculated into LB liquid culture medium, and shaken and cultured in a shaker at 30-40°C and 150-200r / min for 1-2 days until the concentration of the bacterial suspension reaches 1×10 6 ~1×10 8 CFU / mL, centrifuged and removed the supernatant and inoculated into potted plants to verify its growth-promoting effect on Artemisia argyi under cadmium stress 10. The use of a screening and compounding model for a highly efficient cadmium-resistant growth-promoting bacterial community in the rhizosphere of Artemisia argyi according to any one of claims 1 to 9, characterized in that: The cadmium-resistant growth-promoting bacteria in the rhizosphere of mugwort are screened to prepare a composite bacterial agent, or a microecological preparation containing the bacterial agent, or a biofertilizer containing the bacterial agent or the microecological preparation.

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