Flue-cured tobacco continuous cropping method based on earthworm compost and application of flue-cured tobacco continuous cropping method

Abstract

The invention belongs to the technical field of flue-cured tobacco continuous cropping, and particularly relates to a flue-cured tobacco continuous cropping method based on earthworm compost and application of the flue-cured tobacco continuous cropping method. The flue-cured tobacco continuous cropping method based on earthworm compost comprises the following steps that 1, earthworm compost fertilizer is obtained, the earthworm compost fertilizer is earthworm compost, or the earthworm compost fertilizer is prepared from, by weight, 88%-92% of earthworm compost, 1%-5% of biochar and 5%-9% of microbial fertilizer; and (2) applying fertilizers to the flue-cured tobaccos subjected to continuous cropping according to the application amount of 1000-2000kg / 667m < 2 >. According to the method, the wormcast generated by earthworm composting is applied to tobacco field soil which is continuously cropped for twenty years, and the excellent improvement effect on soil nutrients, soil enzyme activity, soil microflora and flue-cured tobacco aroma substances is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of continuous cropping of flue-cured tobacco, and particularly relates to a continuous cropping method of flue-cured tobacco based on vermicomposting and application thereof. Background Art

[0002] Flue-cured tobacco is an economic crop that cannot tolerate continuous cropping. However, in existing cultivation systems, due to limited arable land, the drive for high returns, and irrational planting methods, continuous cropping has become widespread both domestically and internationally. Long-term continuous cropping can lead to deterioration of the physical and chemical properties of tobacco-growing soils, reduced soil enzyme activity, accumulation of toxic substances (heavy metals, autotoxic substances, etc.), and imbalances in microbial communities. Even short-term continuous cropping can weaken tobacco's resistance to pests and diseases, resulting in reduced yield and quality, and a concomitant decline in cigarette quality. This causes significant economic losses and severely restricts the sustainable development of flue-cured tobacco production.

[0003] Aroma compounds, their types, and their composition are crucial factors in determining tobacco leaf aroma. Volatile aroma components are a crucial material foundation for tobacco leaf aroma characteristics. Although most volatile aroma compounds are present in low concentrations in tobacco leaves, they nonetheless have a significant impact on aroma. Appropriate organic fertilizers can harmonize tobacco leaf chemical composition, improving its inherent quality while also enhancing aroma, improving smoking quality, and reducing irritation. The addition of appropriate proportions of organic fertilizers can significantly increase the quality and quantity of key aroma compounds, primarily by increasing the levels of phenylalanine, browning products, cypermethrinoids, and carotenoid degradation products in the middle and lower leaves. It can also significantly reduce nicotine and nitrogen content in tobacco leaves and increase reducing sugar content.

[0004] Vermicompost is a naturally decomposed compost produced according to the living habits of earthworms. The resulting vermicompost can regulate soil properties and structure, significantly reducing soil erosion, increasing soil fertility, maintaining soil carbon balance, and regulating soil pH. Its soil-improving effects are significantly superior to those of conventional organic fertilizers. The main component of vermicompost is humus, formed through reactions between earthworm secretions. The humus content in vermicompost can be as high as 10% to 25%. Humus not only provides nutrients for crop growth and development but also continuously improves soil nutrient structure and cation exchange capacity. The high concentration of humus in vermicompost can also fix and replace exchangeable heavy metals, continuously reducing the availability of heavy metals in the soil. However, its application in the field of continuous flue-cured tobacco cropping is rare. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides a flue-cured tobacco continuous cropping method based on vermicomposting and its application. The method utilizes vermicompost produced by vermicomposting and applies it to the soil of tobacco fields that have been continuously cropped for 20 years. This method significantly improves soil nutrients, soil enzyme activity, soil microbial communities, and tobacco aroma compounds.

[0006] The technical solutions provided by the present invention are as follows: A flue-cured tobacco continuous cropping method based on vermicomposting comprises the following steps: 1) obtaining vermicompost fertilizer, wherein the vermicompost fertilizer comprises the following components in percentage by weight: 88-92% vermicompost, 1-5% biochar, and 5-9% microbial fertilizer; 2) According to 1000-2000kg / 667m 2 The application rate is used to fertilize continuously cropped flue-cured tobacco.

[0007] Based on the above technical solution: Applying earthworm compost to the soil of tobacco fields with continuous flue-cured tobacco cultivation has an excellent effect on improving soil nutrients, soil enzyme activity, soil microbial community and tobacco aroma substance indicators; Through earthworm compost compound fertilizer, the carbon activity, nutrient content and microbial content of the soil can be further improved, thereby further improving soil nutrients, soil enzyme activity, soil microbial community and tobacco aroma substance indicators.

[0008] Preferably, the fertilizer comprises the following components in percentage by weight: 90% earthworm compost, 3% biochar, and 7% microbial fertilizer.

[0009] Preferably, the vermicompost is processed vermicompost compound fertilizer, and the processing method comprises the following steps: a) using any one or more of garden waste, cow dung or chicken dung as food for earthworms, obtaining fertilizer secreted by the earthworms, and then naturally composting the fertilizer; b) adding biochar and microbial fertilizer to the decomposed fertilizer in proportion, and mixing to obtain the earthworm compost.

[0010] Based on the above technical solution: Through step a), the nutrient content of earthworm compost and the abundance of microbial colonies can be increased, thereby improving soil nutrients, soil enzyme activity, soil microbial community and tobacco aroma substance indicators; Through step b), the carbon activity, nutrient content and microbial content of the earthworm compost compound fertilizer can be improved, thereby further improving soil nutrients, soil enzyme activity, soil microbial community and flue-cured tobacco aroma substance indicators.

[0011] Specifically: the variety of the flue-cured tobacco is K326.

[0012] Compared with other varieties of flue-cured tobacco, the K326 variety is more sensitive to continuous cropping and has a higher incidence of diseases and insect pests.

[0013] Specifically: The fields to be fertilized are soils where flue-cured tobacco has been grown for 1 to 20 years.

[0014] The flue-cured tobacco continuous cropping method provided by the present invention can be applied to soil where flue-cured tobacco has been continuously cropped for up to 20 years.

[0015] The present invention also provides an application of a flue-cured tobacco continuous cropping method based on vermicomposting, which is used to increase the content of available nitrogen, available phosphorus, available potassium or organic matter in tobacco field soil.

[0016] The present invention also provides an application of a flue-cured tobacco continuous cropping method based on vermicomposting, which is used to increase the urease activity, sucrase activity, catalase activity or phosphatase activity of tobacco field soil.

[0017] The present invention also provides an application of a flue-cured tobacco continuous cropping method based on vermicomposting, which is used to increase the Ace, Chao1 or Shannon index of fungi and bacteria in tobacco field soil.

[0018] The present invention also provides an application of a flue-cured tobacco continuous cropping method based on vermicomposting, which is used to increase the content of lipid or aldehyde aroma substances in tobacco leaves.

[0019] The present invention also provides an application of a flue-cured tobacco continuous cropping method based on vermicomposting, which is used to increase the content of 3-mercaptohexanol hexanoate, n-pentyl acrylate, m-cresol acetate, neopentylbenzenesulfonate, and gamma-octanolactone in tobacco leaves.

[0020] The beneficial effects of the present invention are as follows: The present invention, after adding vermicompost and vermicompost compound fertilizer, improves soil pH and electrical conductivity (EC), significantly increases the content of soil available nitrogen, available phosphorus, available potassium, and organic matter, and also increases soil urease, phosphatase, sucrase, and catalase activities with the addition of vermicompost; and increases the Ace, Chao1, and Shannon indices of soil fungi and bacteria. The addition of vermicompost and vermicompost compound fertilizer significantly increases the content of lipid and aldehyde aroma substances in tobacco leaves. The results show that the addition of vermicompost and vermicompost compound fertilizer significantly changes the composition of soil nutrients and increases soil enzyme activity, thereby directly or indirectly improving the composition and abundance of rhizosphere microbial colonies and increasing the content of volatile aroma substances in tobacco leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the petal-level OTU map of tobacco rhizosphere soil bacteria under different treatments.

[0022] Figure 2This is the OTU level map of fungi in rhizosphere soil of flue-cured tobacco under different treatments.

[0023] Figure 3 This is an analysis of the Alpha diversity of bacteria in the rhizosphere soil of flue-cured tobacco under different treatments.

[0024] Figure 4 This is an analysis of the Alpha diversity of fungi in the rhizosphere soil of flue-cured tobacco under different treatments.

[0025] Figure 5 This is the relative distribution of bacterial species at the phylum level under different treatments.

[0026] Figure 6 This is the relative distribution of fungal species at the phylum level under different treatments.

[0027] Figure 7 This is a chart showing the proportion of volatile metabolites in tobacco leaves.

[0028] Figure 8 This is the PLS-DA analysis of aroma compounds in vermicomposting.

[0029] Figure 9 This is the Venn analysis diagram of the differential metabolites in different treatments.

[0030] Figure 10 This is the first volcano plot showing the differences in volatile metabolites of tobacco leaves among different treatments.

[0031] Figure 11 This is the second volcano plot showing the differences in volatile metabolites of tobacco leaves under different treatments.

[0032] Figure 12 This is the third volcano plot showing the differences in volatile metabolites of tobacco leaves under different treatments.

[0033] Figure 13 This is the fourth volcano plot showing the differences in volatile metabolites of tobacco leaves under different treatments.

[0034] Figure 14 This is a graph showing the differences in volatile metabolites in tobacco leaves treated with different methods. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0036] Unless otherwise specified, the test methods used in the examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0037] The test site is located in Xueping Town, Nanzhang County, Xiangyang City, Hubei Province. The test field is a continuous cropping soil for growing flue-cured tobacco for 20 years. The test flue-cured tobacco variety is K326, which is provided by Nanzhang County Tobacco Monopoly Bureau (Marketing Department); conventional organic fertilizer is ordinary organic fertilizer provided by Ningxia Wanhui Biotechnology Co., Ltd., with an organic matter content of ≥50%, beneficial bacteria ≥50 million, and N+P2O5+K2O ≥5%; vermicompost is provided by Ningxia Wanhui Biotechnology Co., Ltd. (vermicompost is made by composting cow dung and chicken manure digested by Eisenia fetida).

[0038] Microbial fertilizer was provided by Hubei Qiming Bioengineering Co., Ltd.

[0039] The preparation of vermicompost compound fertilizer is as follows: a) using a mixture of 10% garden waste, 80% cow dung, and 10% chicken manure as food for earthworms, obtaining fertilizer secreted by the earthworms, and then naturally composting the fertilizer; b) The vermicompost compound fertilizer includes the following components in percentage by weight: 90% vermicompost, 3% biochar, and 7% microbial fertilizer.

[0040] Example A single-factor randomized block design was used, with a total of six treatments, each with one acre of planting area. The specific experimental design is shown in Tables 1 and 2.

[0041] Table 1 Experimental design 1 Table 2 Experimental design 2 Test indicators and methods Determination of soil physical and chemical properties Soil samples were collected during the peak flue-cured tobacco season using a five-point sampling method from the 0-20 cm tillage layer of each treatment. After air-drying, soil samples were passed through a 2 mm sieve and then measured for physical and chemical parameters. Soil nutrient and enzyme activity parameters were determined according to the Soil Agrochemical Analysis standard.

[0042] Determination of rhizosphere microorganisms in flue-cured tobacco Collection and processing of rhizosphere soil samples When the flue-cured tobacco was in its vigorous growth period, five representative plants were selected from each treatment, placed in an ice box and brought back to the laboratory. The rhizosphere soil on the root surface was rinsed with an equal amount of PBS sterile buffer (pH 7.0). The roots were removed, and the PBS buffer containing the rhizosphere soil was centrifuged. The supernatant was discarded and the remaining rhizosphere soil was stored in a -20°C refrigerator.

[0043] DNA extraction from rhizosphere soil The main conditions and parameters are as follows: Used in the experiment Total DNA from rhizosphere soil was extracted using a DNA kit. Following the protocol and reagents provided in the kit, total DNA from the rhizosphere soil was extracted. After extraction, the DNA samples in the collection tubes were quality-checked using a Nanodrop 1000 spectrophotometer (A260 / A280 and A260 / A230). The samples were then stored at -20°C and used for rhizosphere microbial analysis, PCR amplification primer design and construction, PE library preparation, and Illumina sequencing.

[0044] The main conditions and parameters are as follows: The PCR amplification primer system adopts the existing technology, which is shown in Table 3 below.

[0045] Table 3 PCR reaction parameters: (1) pre-denaturation at 95°C for 3 min, for a total of 1 cycle; (2) 32 cycles at 95°C for 30 s, 55°C for 30 s, and 45°C for 30 s; (3) extension at 72°C for 10 min; (4) the products after the reaction were stored at 10°C.

[0046] PCR product identification: The products were detected by 2% agarose gel electrophoresis. The products of the same sample were mixed and repeated three times.

[0047] PCR product purification: PCR products were purified using the AxyPrep DNA Gel Extraction Kit.

[0048] PCR product quantification and normalization: PCR product quantification using Quantus TM Fluorometer detection. Products were mixed in appropriate proportions according to the required sequencing volume. Miseq libraries were constructed using the NEXTFLEX RapidDNA-Seq Kit.

[0049] Illumina sequencing: Shanghai Meiji Biotechnology Co., Ltd. provided the IlluminaMiseq PE300 platform for sequencing.

[0050] Determination of volatile aroma substances Sample preparation According to the flue-cured tobacco grading standard GB2635-1992, C3F grade tobacco leaf samples were selected, destemmed, dried, ground, and sieved. One gram of tobacco powder was accurately weighed and placed in a 20 mL headspace vial. A 2 μL internal standard solution of 105 mg / L phenylethyl acetate was injected using a microsyringe. The vial was immediately sealed and mixed thoroughly by oscillation. The sample vial was then equilibrated on a solid-phase microextraction (SPME) heating platform set at 60°C for 20 minutes. An activated SPME extraction tip was then used for 20 minutes of adsorption extraction. After extraction, the tip was quickly inserted into the gas chromatograph inlet and desorbed at 250°C for 10 minutes. Finally, analysis was performed using GC-MS.

[0051] GC-MS conditions The main conditions and parameters are as follows: Chromatographic column: HP-FFAP quartz capillary (50m×0.32mm×0.50μm); carrier gas: high-purity He, purity 99.999%; flow rate: 2.45mL / min; inlet temperature: 250℃; injection mode: splitless injection; heating program: initial temperature 45℃, hold for 1min, first increase to 230℃ at 5℃ / min, hold for 10min; ion source: EI source; ionization voltage: 70eV; ion source temperature: 230℃; transfer line temperature: 240℃; scanning mode: full scan; scanning range: 33-400amu.

[0052] Qualitative and quantitative analysis of aroma components Qualitative analysis The raw mass spectrometry data were post-processed using the Agilent MassHunter qualitative analysis platform. The following steps were used: First, the peak width in the deconvolution parameters was set to 20. The resolution, sensitivity, and chromatographic peak shape parameters were all adjusted to medium matching mode, with a matching threshold set to no less than 70. Metabolite structures were identified through dual alignment with the NIST (2020) standard library and the MWGC self-built database. After integration of the metabolite mass spectrum peaks, characteristic ions were selected for integration and correction, ultimately obtaining standardized metabolite qualitative and quantitative analysis results.

[0053] Quantitative analysis The internal standard semi-quantitative method was used for quantification, and the content of each volatile component was calculated as follows: X i =[(Vs×Cs) / M[×[Ii / Is]×10 -3 Wherein: Xi is the content of compound i in the sample to be tested / (μg / g); Vs is the volume of the internal standard 3-hexanone added / μL; Cs is the mass concentration of the internal standard / (μg / mL); M is the mass of the sample to be tested / g; Is is the peak area of ​​the internal standard; Ii is the peak area of ​​compound i in the sample to be tested.

[0054] Data Analysis Volatile metabolite data were preprocessed using a database built by Shanghai Meiji Biotechnology Co., Ltd. The raw data, after mass spectrometry analysis using MassHunter software, were used for qualitative and quantitative analysis and integral correction. Metabolite content was analyzed using a semi-internal standard quantification method, with isotopic standards selected as references for the content of the measured components. The peak area of ​​each metabolite represented its corresponding relative content. Data were summarized and organized using Microsoft Excel 2020 and IBM SPSS Statistics 25 software. After UV scaling of the raw data, multivariate statistical analyses, including partial least squares regression, and plotting were performed using SIMCA-P13.0 metrology software. Cluster heatmaps and correlation heatmaps were generated using Origin 2024 software.

[0055] Results and Analysis Effects of vermicomposting on physical and chemical properties of soil in continuous tobacco cropping fields Effects of different treatments on soil nutrients in tobacco fields As shown in Table 4, the application of vermicompost produced by vermicomposting had varying degrees of impact on the soil in tobacco fields. The pH and EC values ​​significantly increased, indicating that the application of vermicompost can significantly alleviate the acidification problem in the continuously cropped soil. After the application of earthworm manure and earthworm compost compound fertilizer, the contents of available nitrogen, available phosphorus, available potassium and organic matter in tobacco field soil increased significantly. Compared with T1 treatment, the available nitrogen content of T2-T6 increased by 59.38%, 42.12%, 163.96%, 191.14% and 194.07%, respectively; the available phosphorus content increased by 33.65%, 51.54%, 74.31%, 103.30% and 109.29%, respectively; the available potassium content increased by 20.93%, 10.71%, 43.34%, 50.97% and 58.07%, respectively; and the organic matter content increased by 61.15%, 36.44%, 115.74%, 109.35% and 124.01%, respectively. It can be seen that the application of earthworm castings and earthworm compost compound fertilizer significantly improved the soil physical and chemical environment and increased the soil nutrient content.

[0056] Table 4 Effects of different treatments on the physical and chemical properties of tobacco field soil Note: The letters after the data indicate significant differences at the P<0.05 (n=3) level. The same below.

[0057] Note:Letters following data indicate significant differences at the P<0.05(n=3)level.Same below. Effects of different treatments on soil enzyme activities in tobacco fields The addition of vermicompost and vermicompost compound fertilizer significantly activated soil microorganisms, thereby directly or indirectly increasing soil enzyme activity. As shown in Table 5, soil enzyme activities significantly increased after vermicompost and vermicompost compound fertilizer treatments compared to treatment T1. Urease activity increased by 37.35%, 45.09%, 106.49%, 140.59%, and 148.73%, respectively; sucrase activity increased by 22.10%, 31.91%, 74.90%, 83.08%, and 89.43%, respectively; catalase activity increased by 33.33%, 46.42%, 72.61%, 80.95%, and 94.04%, respectively; and phosphatase activity increased by 20.87%, 34.74%, 70.06%, 80.50%, and 88.18%, respectively.

[0058] Table 5 Effects of different treatments on soil enzyme activities in tobacco fields Effects of vermicomposting on rhizosphere microbial communities in flue-cured tobacco Analysis of OTU numbers in different treatments Depend on Figure 1 、 2 It can be seen that according to the species OTU petal map, the number of bacterial OTUs shared by the five treatments (T1 to T5) is 1530, among which the number of OTUs unique to the T1 treatment is the least, which is 355. The numbers of unique bacterial OTUs in the T2-T5 treatments are 381, 366, 400, and 411, respectively, which are 7.32%, 3.09%, 12.67%, and 15.77% higher than those in the control (T1), respectively.

[0059] According to the species OTU petal diagram, the number of fungal OTUs shared by the five treatments (T1 to T5) was 353. Among them, the number of unique fungal OTUs in the T4 treatment was the least, at 448, while the number of unique fungal OTUs in the T3 treatment was the largest, at 565, which was 26.11% more than that in T4. Relatively speaking, the number of unique OTUs in the T1 treatment was larger, indicating that the application of vermicomposting could inhibit the production of fungi.

[0060] Alpha diversity analysis of soil microbial communities under different treatments Depend on Figure 3 、 4 It can be seen that after Alpha diversity analysis of microbial colonies, different treatments of bacteria ( Figure 3 ) and fungi ( Figure 4 ))'s goods-coverage reached 99%, indicating that the measured soil fungal and bacterial sequences were sufficient to reflect their true situation.

[0061] In the bacterial colony, the Richness index, Chao1 index, Shannon index, Simpson index, Invsimpson index, Pielou index, Ace index, and PD-whole-tree index of the T5 treatment were significantly higher than those of the T1 treatment, which were 5.07%, 5.01%, 2.98%, 0.54%, 63.41%, 7.28%, 5.79%, and 20.09% higher, respectively. The results showed that the application of earthworm compost to the soil of continuously cropped tobacco fields could increase the microbial diversity and richness of bacterial colonies. In the fungal colonies, compared with the T1 treatment, except for the Richness index, Chao1 index, Invsimpson index, Ace index, and PD-whole-tree index, the indicators of the T5 treatment were lower than those of T1. The other indicators, Shannon index, Simpson index, and Pielou index were significantly higher than those of T1, which were 10.23%, 13.17%, and 13.79% higher, respectively. This shows that earthworm compost can increase the diversity and richness of fungal microorganisms to a certain extent.

[0062] Beta diversity analysis of soil microbial communities under different treatments like Figure 5As shown, the dominant bacterial phyla in different soil treatments were Proteobacteria, Acidobacteria, Gemmatimonadetes, Actinobacteria, Chloroflexi, and Bacteroidetes. Other phyla with relative abundances greater than 1% included Methanogens, Nitrospira, and Myxomycetes. The results showed that compared with T1, the relative abundances of Proteobacteria and Acidiobacteria increased among all treatments. The relative abundance of Proteobacteria increased the most in T2, reaching 38.12%, and the relative abundance of Acidiobacteria increased the most in T3, reaching 42.34%. The relative abundance of Gemmatimonadetes did not differ significantly among the treatments, while the relative abundance of Actinobacteria increased the most in T2, reaching 25.96%. The main dominant bacterial communities with the largest relative abundance increases in the T4 treatment were Bacteroidetes and Proteobacteria, which increased by 20.34% and 14.57%, respectively, while the relative abundance of Acidobacteria decreased significantly, by 28.95%; the main dominant bacterial communities with the largest relative abundance increases in the T5 treatment were Bacteroidetes and Proteobacteria, which increased by 27.92% and 9.37%, respectively, while the relative abundance of Acidobacteria and Gemmatimonadetes decreased significantly, by 18.74% and 16.57%, respectively.

[0063] Through database comparison, the soil fungi under different treatments were annotated at the taxonomic level, and the colony structure of fungi classified at the phylum level was constructed, such as Figure 6 As shown in the figure, the dominant phyla in different soil treatments were Ascomycota, Basidiomycota, Mortierella, Unknown Fungi, Chytridiomycota, and Oleobacteria. The results showed that the different treatments did not change the composition of fungal colonies at the phylum level, but changed their colony abundance. The results showed that compared with T1, the relative abundance of Ascomycota decreased significantly among all treatments after the addition of vermicomposting, while the proportions of other dominant phyla, such as Basidiomycota, Mortierella and Unknown Fungi, increased significantly. Among them, the relative abundance of Basidiomycota increased the most in T2 treatment, increasing by 89.72%; the relative abundance of Basidiomycota, Mortierella and Oleobacteria increased the most in T3 treatment, increasing by 63.55%, 17.63% and 319.48% respectively; the relative abundance of Basidiomycota, Unknown Fungi and Oleobacteria increased the most in T4 treatment, increasing by 64.32% and 310.64% respectively; the relative abundance of Basidiomycota and Oleobacteria increased the most in T5 treatment, increasing by 78.64% and 189.36% respectively. In addition, the relative abundance of Chytridiomycota in T3, T4 and T5 treatments decreased significantly by 18.65%, 25.76% and 54.63%, respectively.

[0064] Analysis of the composition of volatile metabolites in tobacco leaves after vermicomposting In order to further analyze the effect of vermicomposting on the aroma substances of tobacco leaves, samples with different treatments were taken and the composition of volatile metabolites in tobacco leaves was analyzed. It was found that there were 1047 volatile metabolites in tobacco leaves. Figure 7 As shown, lipids have the highest number of species, with 247 species, accounting for 23.59%, followed by ketones, with 153 species, accounting for 14.61%. In addition, there are 100 alcohols (9.55%), 99 alkanes (9.46%), 73 hybrid compounds (6.97%), 53 aldehydes (5.06%), 35 terpenes (3.34%), 32 ethers (3.06%), 26 amides (2.48%) and other substances.

[0065] PLS-DA analysis of aroma compounds The partial least squares-discrimination analysis (PLS-DA) model has the characteristics of high accuracy and good stability, and can better judge the differences in volatile substances between samples. In order to further determine the mechanism of action of vermicomposting on tobacco aroma substances, PLS-DA analysis was performed on the aroma substances detected in tobacco leaves, as follows Figure 8 As shown in the figure, the first principal coordinate axis and the second principal coordinate axis can explain 38.72% and 29.33% of all variables respectively, and the variance contribution rate of the two principal components reaches 68.05% in total. The biological replicates among the samples are well clustered together, indicating that the repeatability of the samples is good. It can be seen from the figure that the T4 and T5 samples are scattered from the other samples, indicating that vermicomposting has a significant effect on volatile aroma substances.

[0066] Differential analysis of the effects of vermicomposting on volatile metabolites in tobacco leaves The 610 metabolites in the sample tobacco leaves were screened for differential metabolites. The results are shown in the Venn diagram of metabolite differences between the groups ( Figure 9 ) and 4 volcano maps ( Figure 10-13 ). According to the screening conditions of VIP+Fold Change (FC)+P-value, a total of 97 volatile metabolites were found to be significantly different between the T1 and T2 treatments, of which 59 were significantly up-regulated and 38 were significantly down-regulated ( Figure 10 ); Comparison of volatile metabolites between T1 and T3 treatments revealed 101 significant differences, of which 58 were significantly upregulated and 43 were significantly downregulated ( Figure 11 ); Comparison of volatile metabolites between T1 and T4 treatments revealed 149 significant differences, of which 105 were significantly upregulated and 44 were significantly downregulated ( Figure 12 ); Comparison of volatile metabolites between T1 and T5 treatments revealed 263 significant differences, of which 148 were significantly upregulated and 115 were significantly downregulated ( Figure 13It can be found that the most significant difference in volatile metabolites between tobacco leaves treated with organic fertilizer and tobacco leaves treated with vermicomposting occurred in the T5 treatment, indicating that vermicomposting treatment has a more significant effect on the volatile metabolites of tobacco leaves.

[0067] To further analyze the specific effects of vermicomposting on tobacco leaf volatile metabolites, the five treated samples were combined for analysis, and the top 15 differential volatile metabolites were screened using the Kruskal-Wallis rank sum test (Kruskal-Wallis H test). Figure 14 ), from the composition of differential volatile metabolites, lipids accounted for the highest proportion, with 5 differential volatile substances, reaching 33.33%, namely 3-mercaptohexanol hexanoate, n-pentyl acrylate, m-cresol acetate, neopentylbenzenesulfonate, and gamma-octanolactone, followed by aldehydes and alcohols, with 3 differential volatile substances, reaching 20%, of which aldehydes included 4-(dimethylamino)-3-methylbenzaldehyde, 5-(1-piperidinyl)-2-furaldehyde, and cinnamaldehyde, and alcohols included 2-ethylhexanol, (1,2,4-trimethylcyclohexyl)methanol, and 2-(1-methylindolyn-5-yl)ethanol. Acids, ketones, and pyridines were relatively few, with only 2, 1, and 1 differential volatile metabolites, respectively. Acids included 2-methylheptanedioic acid and cyclohexanecarboxylic acid-D-11 acid, ketones included 3,4-hexanedione, and pyridines included 3,4-lutidine. It can be seen that the application of vermicompost can significantly increase volatile metabolites, among which lipids have the greatest impact on the aroma components of tobacco leaves.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A flue-cured tobacco continuous cropping method based on vermicomposting, characterized in that: The following steps are involved: 1) obtaining vermicompost fertilizer, wherein the vermicompost fertilizer is vermicompost, or the vermicompost fertilizer comprises the following components in percentage by weight: 88-92% vermicompost, 1-5% biochar, and 5-9% microbial fertilizer; 2) According to 1000-2000kg / 667m 2 The application rate is used to fertilize continuously cropped flue-cured tobacco.

2. The method for continuous tobacco cropping based on vermicomposting according to claim 1, characterized in that: The fertilizer comprises the following components in percentage by weight: 90% of earthworm compost, 3% of biochar, and 7% of microbial fertilizer.

3. The method for continuous tobacco cropping based on vermicomposting according to claim 1, characterized in that: The vermicompost is processed vermicompost, and the processing method comprises the following steps: a) using any one or more of garden waste, cow dung or chicken dung as food for earthworms, obtaining fertilizer secreted by the earthworms, and then naturally composting the fertilizer; b) adding biochar and microbial fertilizer to the decomposed fertilizer in proportion, and mixing to obtain the earthworm compost.

4. The method for continuous tobacco cropping based on vermicomposting according to claim 1, characterized in that: The variety of the flue-cured tobacco is K326.

5. The method for continuous tobacco cropping based on vermicomposting according to any one of claims 1 to 4, characterized in that: The fields to be fertilized are continuously cropped soils where flue-cured tobacco has been grown for 1 to 20 years.

6. An application of the flue-cured tobacco continuous cropping method based on vermicomposting according to any one of claims 1 to 5, characterized in that: Used to increase the content of available nitrogen, available phosphorus, available potassium or organic matter in tobacco field soil.

7. An application of the flue-cured tobacco continuous cropping method based on vermicomposting according to any one of claims 1 to 5, characterized in that: Used to increase the urease activity, sucrase activity, catalase activity or phosphatase activity in tobacco field soil.

8. An application of the flue-cured tobacco continuous cropping method based on vermicomposting according to any one of claims 1 to 5, characterized in that: Used to increase the Ace, Chao1 or Shannon index of fungi or bacteria in tobacco field soil.

9. An application of the tobacco continuous cropping method based on vermicomposting according to any one of claims 1 to 5, characterized in that: Used to increase the content of lipid or aldehyde aroma substances in tobacco leaves.

10. An application of the flue-cured tobacco continuous cropping method based on vermicomposting according to any one of claims 1 to 5, characterized in that: Used to increase the content of 3-mercaptohexanol hexanoate, n-pentyl acrylate, m-cresyl acetate, neopentylbenzenesulfonate or gamma-octanolactone in tobacco leaves.

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