Application of trichoderma in alleviating continuous cropping obstacles of ginseng and salvia miltiorrhiza

Abstract

The application provides a wood mould in relieving ginseng or salvia miltiorrhiza continuous cropping obstacles, and relates to the field of crop cultivation technology. The wood mould can effectively relieve soil chemical property deterioration and soil microbial change after ginseng or salvia miltiorrhiza continuous cropping, and then can improve the soil chemical property after continuous cropping of ginseng or salvia miltiorrhiza, increase the relative abundance of beneficial bacteria in the soil, inhibit the relative abundance of harmful bacteria, and simultaneously can increase the content of effective components of ginseng and salvia miltiorrhiza, and improve the quality of continuous cropping of ginseng or salvia miltiorrhiza.

Description

Technical Field

[0001] This invention relates to the field of crop cultivation technology, and in particular to the application of a Trichoderma in alleviating the obstacles of continuous cropping of ginseng or salvia miltiorrhiza. Background Technology

[0002] Continuous cropping disorder refers to abnormal crop growth and development caused by continuously cultivating the same or closely related crops in the same soil. Symptoms generally include poor growth and development, reduced yield and quality, and in extreme cases, localized seedling death, failure to sprout, or weak sprouting. Most affected plants exhibit browning of the root system, reduced branching, low vitality, and narrow distribution, leading to a decreased ability to absorb water and nutrients.

[0003] Currently, with the increase in the planting area of ​​ginseng and salvia miltiorrhiza and the pursuit of authentic medicinal materials, the problem of continuous cropping obstacles has gradually become prominent. Continuous cropping weakens plant growth, leads to the deterioration of soil physical and chemical properties, imbalance of microbial communities, and exacerbation of soil-borne diseases, ultimately resulting in a decline in crop yield and quality. In addition, in order to reduce the incidence of diseases and increase the yield of ginseng and salvia miltiorrhiza, some farmers have increased the use of chemical fertilizers and pesticides, which has led to soil degradation and excessive pesticide residues. Therefore, the problem of continuous cropping has become a major issue that urgently needs to be addressed in the cultivation of ginseng and salvia miltiorrhiza, seriously restricting its sustainable development.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Trichoderma in alleviating the continuous cropping obstacles of ginseng or salvia miltiorrhiza, to alleviate the deterioration of soil chemical properties and changes in soil microorganisms caused by continuous cropping obstacles of ginseng or salvia miltiorrhiza, while increasing the content of effective components of ginseng and salvia miltiorrhiza and improving the quality of continuously cropped ginseng or salvia miltiorrhiza.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] According to one aspect of the present invention, the present invention provides the application of *Trichoderma* in alleviating continuous cropping obstacles of ginseng or *Salvia miltiorrhiza*. The application of *Trichoderma* provided by the present invention in alleviating continuous cropping obstacles of ginseng or *Salvia miltiorrhiza* is achieved through in-depth research and creative labor. *Trichoderma* can effectively alleviate the deterioration of soil chemical properties and changes in soil microorganisms after continuous cropping of ginseng or *Salvia miltiorrhiza*, thereby improving the soil chemical properties after continuous cropping of ginseng or *Salvia miltiorrhiza*, increasing the relative abundance of beneficial bacteria in the soil, inhibiting the relative abundance of harmful bacteria, and simultaneously increasing the content of effective components in ginseng and *Salvia miltiorrhiza*, thus improving the quality of continuously cropped ginseng or *Salvia miltiorrhiza*.

[0008] In a preferred embodiment of the present invention, the ginseng or salvia miltiorrhiza continuous cropping obstacle refers to the deterioration of soil chemical properties and changes in soil microorganisms caused by continuous planting of ginseng or salvia miltiorrhiza for 4 to 6 years.

[0009] The deterioration of soil chemical properties includes: a decrease in the content of available soil nutrients and a decrease in the content of soil organic matter.

[0010] The changes in soil microorganisms include: a decrease in the relative abundance of beneficial Bacillus species and an increase in the relative abundance of harmful pathogenic Fusarium species.

[0011] In a preferred embodiment of the invention, the Trichoderma is applied in the form of an irrigation solution;

[0012] Preferably, the concentration of Trichoderma in the irrigation solution is 1×10⁻⁶. 7 cfu·mL -1 ~9×10 7 cfu·mL -1 Preferably 1×10 7 cfu·mL -1 .

[0013] In a preferred embodiment of the present invention, the application method includes: transplanting ginseng or salvia miltiorrhiza seedlings into ginseng or salvia miltiorrhiza continuous cropping obstacle land, and then irrigating the plant with the above-mentioned irrigation solution.

[0014] The irrigation method is as follows: apply twice during the entire growth period after transplanting ginseng or salvia miltiorrhiza seedlings, each time applying 500 ml / m². 2 The remaining water management is the same as that for field management.

[0015] Note: The method of applying the fertilizer twice during the entire growth period is as follows: the first irrigation application is carried out at the time of transplanting, and the second irrigation application is carried out 60 days after transplanting.

[0016] In a preferred embodiment of the present invention, the Trichoderma includes at least one of Trichoderma truncatella, Trichoderma breviculatum, Trichoderma viride, Trichoderma pellucida, and Trichoderma tuftum.

[0017] The *Trichoderma koningiopsis* strain is Tri112, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23210; the deposit date is August 19, 2021.

[0018] The *Trichoderma brevicompactum* strain is Tri502, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23213; deposited on August 19, 2021.

[0019] The *Trichoderma viridescens* strain is Tri403, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23212; deposited on August 19, 2021.

[0020] The *Trichoderma atroviride* strain is Tri802, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23214; deposit date: August 19, 2021.

[0021] The *Trichoderma velutinum* strain is Tri401, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23211; the deposit date is August 19, 2021.

[0022] In a preferred embodiment of the present invention, the application includes:

[0023] Application of Trichoderma truncatum Tri112 in alleviating ginseng continuous cropping obstacles;

[0024] Application of Trichoderma brevis 502 in alleviating ginseng continuous cropping obstacles;

[0025] Application of Trichoderma viride Tri403 in alleviating ginseng continuous cropping obstacles;

[0026] Application of Trichoderma viride Tri802 in alleviating ginseng continuous cropping obstacles;

[0027] Application of Trichoderma tuftedatum Tri401 in alleviating ginseng continuous cropping obstacles.

[0028] Application of Trichoderma tegmentans Tri112 in alleviating the continuous cropping obstacles of Salvia miltiorrhiza;

[0029] Application of Trichoderma brevis 502 in alleviating the continuous cropping obstacles of Salvia miltiorrhiza;

[0030] Application of Trichoderma chloroticum Tri403 in alleviating the continuous cropping obstacles of Salvia miltiorrhiza;

[0031] Application of Trichoderma harzianum Tri802 in alleviating the continuous cropping obstacles of Salvia miltiorrhiza;

[0032] Application of Trichoderma tuftedatum Tri401 in alleviating the continuous cropping obstacles of Salvia miltiorrhiza.

[0033] In a preferred embodiment of the present invention, the application further includes the combined application of Trichoderma and chitosan oligosaccharide to alleviate the deterioration of soil chemical properties and changes in soil microorganisms after continuous cropping of ginseng or salvia miltiorrhiza.

[0034] According to one aspect of the present invention, an irrigation solution for alleviating the obstacle of continuous cropping of ginseng or salvia miltiorrhiza is provided, the irrigation solution comprising the aforementioned Trichoderma;

[0035] The Trichoderma species include at least one of Trichoderma truncatum, Trichoderma breviculatum, Trichoderma viride, Trichoderma peltata, and Trichoderma tuftata.

[0036] The *Trichoderma koningiopsis* strain is Tri112, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23210; the deposit date is August 19, 2021.

[0037] The *Trichoderma brevicompactum* strain is Tri502, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23213; deposited on August 19, 2021.

[0038] The *Trichoderma viridescens* strain is Tri403, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23212; deposited on August 19, 2021.

[0039] The *Trichoderma atroviride* strain is Tri802, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23214; deposit date: August 19, 2021.

[0040] The *Trichoderma velutinum* strain is Tri401, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23211; the deposit date is August 19, 2021.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] This invention was developed through in-depth research and creative work. Trichoderma can effectively alleviate the deterioration of soil chemical properties and changes in soil microorganisms after continuous cropping of ginseng or salvia miltiorrhiza, thereby improving the soil chemical properties after continuous cropping of ginseng or salvia miltiorrhiza, increasing the relative abundance of beneficial bacteria in the soil, inhibiting the relative abundance of harmful bacteria, and increasing the content of effective components of ginseng and salvia miltiorrhiza, thus improving the quality of continuously cropped ginseng or salvia miltiorrhiza. Attached Figure Description

[0043] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is a diagram showing the relative abundance of bacteria at the phylum level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in Example 2 of the present invention.

[0045] Figure 2 This is a diagram showing the relative abundance of bacteria at the genus level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in Example 2 of the present invention.

[0046] Figure 3 This is a diagram showing the relative abundance of fungi at the phylum level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in Example 2 of the present invention.

[0047] Figure 4 This is a diagram showing the relative abundance of fungi at the genus level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in Example 2 of the present invention.

[0048] Figure 5 This is a diagram showing the relative abundance of bacteria at the phylum level in the soil after continuous cropping of Salvia miltiorrhiza in each Trichoderma treatment group provided in Example 3 of the present invention.

[0049] Figure 6 This is a diagram showing the relative abundance of bacteria at the genus level in the soil after continuous cropping of Salvia miltiorrhiza following treatment of each Trichoderma treatment group provided in Example 3 of the present invention.

[0050] Figure 7 This is a diagram showing the relative abundance of fungi at the phylum level in the soil of continuously cropped Salvia miltiorrhiza after each Trichoderma treatment group provided in Example 3 of the present invention.

[0051] Figure 8 This is a diagram showing the relative abundance of fungi at the genus level in the soil of continuously cropped Salvia miltiorrhiza after each Trichoderma treatment group provided in Example 3 of the present invention. Detailed Implementation

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] The technical solution of the present invention will be further described below with reference to the embodiments.

[0054] Example 1: Preparation of Microbial Agent

[0055] The preparation method of the Trichoderma agent in this embodiment includes:

[0056] (1) Prepare nutrient solution: Add 10g potassium dihydrogen phosphate and 5g magnesium sulfate to 1000ml distilled water and adjust the pH value to 5.

[0057] (2) Culture medium preparation: Spread rice to the bottom of the culture bottle, add nutrient solution until it covers the rice, and seal with sealing film for later use. Place the prepared rice culture medium in an autoclave at 121°C for 20 minutes. Once the culture bottle temperature has cooled to room temperature, it is ready for inoculation.

[0058] The inoculation process involves inoculating the following 5 strains separately:

[0059] The fungus *Trichoderma koningiopsis* is Tri112, with accession number CGMCC No. 23210.

[0060] The *Trichoderma brevicompactum* fungus is Tri502, with accession number CGMCC No. 23213;

[0061] The *Trichoderma viridescens* fungus is Tri403, with accession number CGMCC No. 23212.

[0062] The *Trichoderma atroviride* fungus is Tri802, with accession number CGMCC No. 23214.

[0063] The fungus *Trichoderma velutinum* is Tri401, with accession number CGMCC No. 23211.

[0064] (3) Inoculation: Wash the Trichoderma on the petri dish with sterile water to obtain Trichoderma solution. Add the Trichoderma solution to the culture flask, seal with sealing film, and place in an incubator.

[0065] (4) Cultivation conditions: temperature at 25-28℃, humidity at 50%-70%, and cultivation time at 5-8 days.

[0066] (5) Drying and crushing: Cultivate until the spores fill the culture bottle, dry at 30℃ for 20h, crush and put into a sterile self-sealing bag to obtain the above five Trichoderma inoculants.

[0067] Example 2: Experiment on the relief of ginseng continuous cropping obstacles by Trichoderma inoculum

[0068] (I) The experiment was conducted in Choushui Township, Fusong City, Jilin Province. Ginseng has been continuously cultivated on this plot of land for four years. Six treatments were set up in the experiment:

[0069] Treatment Group A, Trichoderma koningiopsis: treated with the Trichoderma koningiopsis fungicide from Example 1;

[0070] Treatment group B, Trichoderma brevicompactum: treated with the Trichoderma brevicompactum fungicide from Example 1;

[0071] Treatment group C, Trichoderma viridescens: treated with the Trichoderma viridescens fungicide from Example 1;

[0072] Treatment group D, Trichoderma atroviride: treated with the Trichoderma atroviride fungicide from Example 1;

[0073] Treatment group E, Trichoderma velutinum: treated with the Trichoderma velutinum fungicide from Example 1;

[0074] The control group (CK group, untreated plants, control) had a plot area of ​​4 square meters for each treatment. Each treatment had 3 replicates.

[0075] Note: The plots selected for the above 6 treatments were all plots where ginseng had been continuously planted for 4 years, and the soil parameters of each plot were at the same level.

[0076] (II) Select ginseng seedlings of uniform growth for transplantation. After transplantation, each treatment group was irrigated with the above-mentioned Trichoderma agents A-E diluted with water at a concentration of 10. 7 The concentration of cfu / mL was measured, and the control group (CK group) was irrigated with the same amount of water. Soil and ginseng samples were collected after the ginseng growing season ended.

[0077] Rhizosphere and periroot soil were collected using sterile plastic bags. A portion of each soil sample was placed at -80°C for subsequent DNA extraction, while the remainder was air-dried for chemical analysis. Ginseng plant samples were air-dried for testing of active ingredient content.

[0078] (III) The effects of each Trichoderma treatment group on the soil chemical properties of continuously cropped ginseng after testing are shown in Table 1.

[0079] Table 1: Effects of Trichoderma treatment groups on soil chemical properties of continuously cropped ginseng.

[0080]

[0081] The results showed that, in terms of soil chemical properties, the soil pH value of all Trichoderma treatment groups was lower than that of the control group; the total nitrogen content of Trichoderma brevicompactum (B), Trichoderma viridescens (C), and Trichoderma atroviride (D) treatment groups was higher than that of the control group; the organic carbon content of all Trichoderma treatment groups was higher than that of the control group, with the highest content in treatment group B, which was 14.39% higher than that of the control group.

[0082] The ammonia nitrogen content of Trichoderma was lower in all treatment groups than in the control group, while the nitrate nitrogen content of Trichoderma was higher in all treatment groups than in the control group. The content of treatment group B was the highest, exceeding the control by 117.94%.

[0083] The content of available phosphorus in all Trichoderma treatment groups was higher than that in the control group, with the highest content in treatment group D, which was 24.42% higher than that in the control.

[0084] (iv) Soil microbial changes

[0085] Figure 1 The relative abundance of bacteria at the phylum level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in this embodiment.

[0086] Depend on Figure 1The results showed that at the phylum level, the top 10 bacterial groups remained unchanged across different treatment groups: Proteobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Actinobacteria, Firmicutes, Gemmatimonadetes, Rokubacteria, Bacteroidetes, and Patescibacteria. However, their relative abundance varied across treatments. The sum of the relative abundances of Proteobacteria and Acidobacteria reached 51.93-55.64%. Figure 1 ).

[0087] Compared with the control group, the relative abundance of Acidobacteria, Verrucomicrobia and Rokubacteria increased in all Trichoderma treatment groups, and the relative abundance of Chloroflexi increased in all treatments except for A and B.

[0088] It should be noted that Acidobacteria not only play an important role in soil ecosystems but also show great application potential in environmental protection and ecological restoration. Chloroflexi participate in the biogeochemical cycles of C, N, and S and are considered to play an active role in crop growth. Therefore, the increase in the relative abundance of Acidobacteria and Chloroflexi after treatment with various Trichoderma fungi in this application can effectively alleviate soil ecosystem problems following ginseng continuous cropping.

[0089] Figure 2 The relative abundance of bacteria at the genus level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in this embodiment.

[0090] Depend on Figure 2 The results showed that at the bacterial genus level, the top 10 bacterial population categories did not change in different treatment groups, but the relative abundance of different treatments varied.

[0091] Compared with the control group, the relative abundance of uncultured *Candidatus*, uncultured bacterium c subgroup 6, uncultured bacterium o Acidobacteriales, and uncultured bacterium o Rokubacteriales was increased in all *Trichoderma* treatment groups.

[0092] Figure 3 The relative abundance of fungi at the phylum level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in this embodiment.

[0093] At the phylum level, the top 10 fungal populations remained unchanged across the different treatment groups: Ascomycota, Basidiomycota, Mortierellomycota, Rozellomycota, Chytridiomycota, Zoopagomycota, Kickxellomycota, Glomeromycota, Calcisporiellomycota, and Mucoromycota. However, their relative abundance varied across the treatments. The combined relative abundance of Ascomycota and Basidiomycota reached 72.90–75.18%.

[0094] At the fungal genus level, the top 10 fungal population categories remained unchanged across the different treatment groups, but their relative abundances varied. Compared to the control group, the relative abundance of *Trichoderma* increased in *Trichoderma* treatment groups A, B, and E, while the relative abundance of *Fusarium* decreased in all treatment groups except for group A.

[0095] It should be noted that root rot is a major disease leading to decreased yield and quality of ginseng, and the main pathogen causing it is Fusarium. Fusarium is a common plant pathogen that can hinder the growth of many crops. The observed reduction in Fusarium in the soil indicates that the addition of Trichoderma inhibits Fusarium and can prevent ginseng root rot.

[0096] Figure 4 The relative abundance of fungi at the genus level in the soil of continuously cropped ginseng after each Trichoderma treatment group provided in this embodiment.

[0097] The changes in soil chemical properties and soil microorganisms after irrigation treatment with the above Trichoderma treatment groups indicate the following:

[0098] (1) Treatment Group A: After irrigation with Trichoderma tumefaciens Tri112 (CGMCC No. 23210), the pH, ammonia nitrogen, and active potassium content of the soil in the plot where ginseng was continuously planted for 4 years were significantly reduced, while the nitrate nitrogen content was significantly increased. However, the content of available nutrients in the soil, such as total nitrogen, organic carbon, and available phosphorus, did not increase.

[0099] At the bacterial phylum level of soil microorganisms, the relative abundance of Acidobacteria, Verrucous Microbes, and Corynebacteria all increased, but the relative abundance of Chlorconoidea did not increase. At the fungal genus level, the relative abundance of Trichoderma increased, but the relative abundance of Fusarium did not decrease.

[0100] Therefore, it can be concluded that the *Trichoderma tegmentans* Tri112 of this application has a certain effect on improving the soil chemical properties and soil microbial changes in ginseng-continuous cropping soil. However, it does not have the effect of increasing the total nitrogen content, organic carbon content, and available phosphorus content of the soil, nor does it have the effect of increasing the relative abundance of *Chlamydomonas* or decreasing the relative abundance of *Fusarium* in terms of soil microorganisms.

[0101] Therefore, this application provides an application of Trichoderma tumefaciens Tri112 in alleviating ginseng continuous cropping obstacles; the Trichoderma tumefaciens Tri112 can be widely used to increase the relative abundance of Trichoderma genus in the soil after ginseng continuous cropping.

[0102] (2) Treatment Group B: Irrigation with Trichoderma short-necked spores Tri502 (preservation number: CGMCC No. 23213) significantly reduced the pH, ammonia nitrogen, and active potassium content of the soil in plots where ginseng had been continuously planted for 4 years, while significantly increasing the total nitrogen and available phosphorus content. Simultaneously, the nitrate nitrogen and organic carbon content in the soil increased significantly, with organic carbon content 14.39% higher than the control and nitrate nitrogen content 117.94% higher than the control.

[0103] At the bacterial phylum level of soil microorganisms, the relative abundance of Acidobacteria, Verrucous Microbes, and Corynebacteria all increased, but the relative abundance of Chlorconoidea did not increase. At the fungal genus level, the relative abundance of Trichoderma increased, while the relative abundance of Fusarium decreased.

[0104] Therefore, the *Trichoderma spp.* Tri502 described in this application has the effect of improving the soil chemical properties and soil microbial changes in soil continuously cropped with ginseng. Compared with other *Trichoderma* species, it significantly increases nitrate nitrogen and organic carbon content, and effectively increases the relative abundance of Acidobacteria, Verrucous Microbes, and Corynebacteria at the bacterial level; and at the fungal level, it increases the relative abundance of *Trichoderma* and decreases the relative abundance of *Fusarium*. However, at the bacterial level, it does not have the effect of increasing the relative abundance of *Chlorophytum*.

[0105] Therefore, this application provides an application of Trichoderma short-density in alleviating ginseng continuous cropping obstacles; Trichoderma short-density can be widely used to increase the content of nitrate nitrogen and organic carbon in the soil after ginseng continuous cropping; compared with other Trichoderma species, it has the effect of increasing the relative abundance of Trichoderma species at the fungal level and decreasing the relative abundance of Fusarium species.

[0106] (3) Treatment Group C: Irrigation with *Trichoderma viride* Tri403 (CGMCC No. 23212) significantly reduced the pH, ammonia nitrogen, and active potassium content in the soil of ginseng-grown plots for four consecutive years, while significantly increasing the total nitrogen and available phosphorus content. Simultaneously, the nitrate nitrogen and organic carbon content in the soil also increased significantly. At the bacterial level, the relative abundance of Acidobacteria, Verrucous Microbes, and Corynebacteria all increased, and the relative abundance of *Vibrio parahaemolyticus* also increased significantly. At the fungal level, the relative abundance of *Fusarium* decreased.

[0107] Therefore, it can be concluded that *Trichoderma viride* Tri403 of this application has the effect of improving the soil chemical properties and soil microbial changes in ginseng continuous cropping. Compared with other *Trichoderma* fungi, it significantly increases the content of nitrate nitrogen and organic carbon, and effectively increases the relative abundance of *Vibrio lucida* at the bacterial level compared with treatment groups A and B. At the fungal level, it reduces the relative abundance of *Fusarium*.

[0108] Therefore, this application provides an application of *Trichoderma viride* Tri403 in alleviating ginseng continuous cropping obstacles; the *Trichoderma viride* Tri403 can be widely used to increase the content of nitrate nitrogen and organic carbon in the soil after ginseng continuous cropping; compared with treatment groups A and B, it can effectively increase the relative abundance of *Vibrio parahaemolyticus* at the bacterial level. At the fungal level, it can also reduce the relative abundance of *Fusarium*.

[0109] (4) Treatment Group D: After irrigation with Trichoderma viride Tri802 (preservation number: CGMCC No. 23214), the soil pH, ammonia nitrogen, and active potassium content of the plot where ginseng had been continuously planted for 4 years were significantly reduced, while the total nitrogen content and available phosphorus content were significantly increased. In particular, the available phosphorus content was significantly higher than that of the control by 24.42%.

[0110] At the bacterial phylum level of soil microorganisms, the relative abundance of Acidobacteria, Verrucous Microbes, and Corynebacteria all increased, and the relative abundance of Chlorconoidea also increased significantly. At the fungal genus level, the relative abundance of Fusarium decreased.

[0111] Therefore, the *Trichoderma viride* Tri802 strain described in this application has the effect of improving the soil chemical properties and soil microbial changes in soil continuously cropped with ginseng. Compared with other *Trichoderma* strains, it significantly increases the content of nitrate nitrogen and organic carbon, and effectively increases the relative abundance of *Vibrio lucida* at the bacterial level compared with treatments A and B. At the fungal level, it reduces the relative abundance of *Fusarium*.

[0112] Therefore, this application provides an application of Trichoderma viride Tri802 in alleviating ginseng continuous cropping obstacles; Trichoderma viride Tri802 can be widely used to increase the content of active phosphorus in the soil after ginseng continuous cropping; compared with treatment groups A and B, it can effectively increase the relative abundance of Chlorconiosis at the bacterial level. At the fungal level, it can also reduce the relative abundance of Fusarium.

[0113] (5) Treatment group E: After irrigation with *Trichoderma tuftedatum* Tri401 (preservation number: CGMCC No. 23211), the soil pH, ammonia nitrogen, and active potassium content of the plots where ginseng had been continuously planted for 4 years were significantly reduced. At the bacterial level of soil microorganisms, the relative abundance of Acidobacteria, Verrucous Microbes, and Corynebacteria all increased, and the relative abundance of Chlorconiosis also increased significantly. At the fungal level, compared with treatment groups C and D, *Trichoderma* not only reduced the relative abundance of Fusarium, but also increased the relative abundance of *Trichoderma*.

[0114] Therefore, it can be concluded that the *Trichoderma tuftedatus* Tri401 of this application has the effect of improving the soil chemical properties and soil microbial changes in ginseng continuous cropping. Compared with other *Trichoderma* species, the contents of nitrate nitrogen and organic carbon are significantly increased, and it can effectively increase the relative abundance of *Vibrio lucida* at the bacterial level compared with treatments A and B. Compared with treatments C and D, *Trichoderma* not only has a reduced relative abundance of *Fusarium*, but also has the effect of increasing the relative abundance of *Trichoderma*.

[0115] Therefore, this application provides an application of *Trichoderma tuftedatus* Tri401 in alleviating ginseng continuous cropping obstacles; the *Trichoderma tuftedatus* Tri401 can be widely used to improve soil microorganisms in soil after ginseng continuous cropping, and can effectively increase the relative abundance of *Vibrio parahaemolyticus* at the bacterial phylum level compared to treatment groups A and B. Compared to treatment groups C and D, *Trichoderma* not only reduced the relative abundance of *Fusarium*, but also increased the relative abundance of *Trichoderma*.

[0116] (v) The effect of each Trichoderma treatment group on the content of effective components in the ginseng grown in this embodiment is shown in Table 2.

[0117] Table 2. Effects of each Trichoderma treatment group on the content of effective components in ginseng grown in this example.

[0118]

[0119] The effects of *Trichoderma* fungi on the content of ginsenoside monomers in ginseng are shown in Table 2. The contents of Rg1, Ro, Rc, Rb3, and Rd in all *Trichoderma* treatment groups were higher than those in the control group. *Trichoderma truncatella*, *Trichoderma viride*, and *Trichoderma tuftedum* increased the Re content; *Trichoderma truncatella*, *Trichoderma brevicaulis*, and *Trichoderma tuftedum* increased the Rf content; *Trichoderma brevicaulis*, *Trichoderma viride*, and *Trichoderma tuftedum* increased the Rg2 content; *Trichoderma truncatella*, *Trichoderma pellucida*, and *Trichoderma tuftedum* increased the Rb1 content; and except for *Trichoderma viride*, all *Trichoderma* treatment groups increased the Rb2 content. Therefore, overall, the application of *Trichoderma* fungi can increase the content of effective components in continuously cropped ginseng, effectively improving its quality.

[0120] Example 3: Experiment on the relief of Danshen continuous cropping obstacles by Trichoderma inoculum

[0121] (I) The experiment was conducted in Pingyi County, Linyi City, Shandong Province, where Salvia miltiorrhiza has been continuously cultivated for 6 years. Six treatment groups were set up in the experiment:

[0122] D1, Trichoderma koningiopsis: treated with the Trichoderma koningiopsis fungicide from Example 1;

[0123] D2, Trichoderma atroviride: treated with the Trichoderma atroviride agent from Example 1;

[0124] D3, Trichoderma brevicompactum: treated with the Trichoderma brevicompactum fungicide from Example 1;

[0125] D4. Trichoderma viridescens: Treated with the Trichoderma viridescens fungicide from Example 1;

[0126] D5, Trichoderma velutinum: treated with the Trichoderma velutinum fungicide from Example 1; and CK (untreated plants, control), with each treatment plot covering 6 square meters. Each treatment had 3 replicates, with 150 seedlings per plot.

[0127] Note: The plots selected for the above 6 treatments were all plots where Salvia miltiorrhiza had been continuously planted for 6 years, and the soil parameters of each plot were at the same level.

[0128] (II) Select Salvia miltiorrhiza seedlings with uniform growth and height for transplanting. After transplanting, the treatment group was irrigated with a Trichoderma fungicide diluted in water at a concentration of 10. 7 The cfu / mL concentration was used to irrigate the control group with the same amount of water.

[0129] Soil samples were collected after the growth period of *Salvia miltiorrhiza* was completed. Rhizosphere and periroot soil were collected using sterile plastic bags. A portion of each soil sample was placed at -80°C for subsequent DNA extraction, and the remainder was air-dried for chemical analysis. *Salvia miltiorrhiza* plant samples were air-dried for the determination of active ingredient content.

[0130] (III) The effects of each Trichoderma treatment group on the soil chemical properties of continuously cropped Salvia miltiorrhiza, see Table 3.

[0131] Table 3: Effects of Trichoderma treatment groups on soil chemical properties of continuously cropped Salvia miltiorrhiza

[0132]

[0133] As shown in the table above, in terms of soil chemical properties, except for D3, the soil pH value of all Trichoderma treatment groups was lower than that of the control group; the total nitrogen content of all Trichoderma treatment groups was higher than that of the control group, with the D1 treatment group having the highest content, which was 284.21% higher than the control.

[0134] Organic carbon content was higher in all Trichoderma treatment groups than in the control group, with the highest content in treatment group D2, exceeding the control by 82.06%. Ammonia nitrogen content was higher in all Trichoderma treatment groups except D4, and nitrate nitrogen content was also higher in all Trichoderma treatment groups than in the control group, with the highest content in treatment group D3, exceeding the control by 318.97%. Available phosphorus content was higher in treatment groups D3, D4, and D5 than in the control; available phosphorus content was higher in all Trichoderma treatment groups except D1. Therefore, overall, the application of Trichoderma helps improve soil chemical properties in continuously cropped Salvia miltiorrhiza, especially in terms of total nitrogen and nitrate nitrogen.

[0135] (iv) Soil microbial changes

[0136] Figure 5 The relative abundance of bacteria at the phylum level in the soil after continuous cropping of Salvia miltiorrhiza following treatment of each Trichoderma treatment group provided in this embodiment.

[0137] Depend on Figure 5It was found that at the phylum level, the top 10 bacterial groups remained unchanged across different treatment groups, namely Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadets, Nitrospirae, Planctomycetes, Verrucomicrobia, Bacteroidetes, and Firmicutes. However, their relative abundance varied across treatments. The sum of the relative abundances of Proteobacteria, Acidobacteria, and Actinobacteria reached 74.16-74.38%. Figure 5 ).

[0138] Compared with the control group, the relative abundance of Actinobacteria, Chloroflexi, and Gemmatimonadets was increased in all five Trichoderma treatment groups.

[0139] It should be noted that Actinobacteria are widely distributed in soil and play an important role in nature. They not only decompose organic matter in the soil and produce antibiotics to inhibit various plant diseases, but also have symbiotic nitrogen fixation and phosphorus solubilization functions. Chloroflexi participate in the biogeochemical cycles of C, N, and S elements and are considered to play an active role in crop growth.

[0140] Figure 6 The relative abundance of bacteria at the genus level in the soil after continuous cropping of Salvia miltiorrhiza following treatment of each Trichoderma treatment group provided in this embodiment.

[0141] Depend on Figure 6At the bacterial genus level, the top 10 bacterial population categories remained unchanged across the different treatment groups, but their relative abundance varied. Compared to the control group, the relative abundance of *Gemmatimonas* increased in all five *Trichoderma* treatment groups. Members of the *Gemmatimonas* genus have multiple functions. *Gemmatimonas* are involved in nitrogen metabolism and transformation. They are autophotosynthetic genus rich in bacterial chlorophyll and can reduce N2O in the soil to nitrate. *Gemmatimonas* can enhance soil nutrients and improve nutrient uptake by converting insoluble phosphorus to soluble phosphorus. In our study, the addition of *Trichoderma* increased AP content compared to the control group, supporting this argument. Furthermore, *Gemmatimonas* can induce plant stress resistance or produce antifungal antibiotics to combat plant pathogens, inhibit soil-borne diseases, and promote plant growth.

[0142] Figure 7 The relative abundance of fungi at the phylum level in the soil of continuously cropped Salvia miltiorrhiza after each Trichoderma treatment group provided in this embodiment.

[0143] Depend on Figure 7 At the phylum level, the top 10 fungal populations remained unchanged across the different treatment groups: Ascomycota, Mortierellomycota, Basidiomycota, Chytridiomycota, Rozellomycota, Zoopagomycota, Kickxellomycota, Glomeromycota, Olpidiomycota, and Mucoromycota. However, the relative abundance varied across the treatments. The sum of the relative abundances of Ascomycota, Mortierellomycota, and Basidiomycota reached 93.72-95.93%.

[0144] Compared with the control group, the relative abundance of Ascomycota increased in all five Trichoderma treatment groups, the relative abundance of Morphozoa increased in all groups except for the D5 treatment group, and the relative abundance of Insectivora increased in all groups except for the D4 treatment group.

[0145] Figure 8 The relative abundance of fungi at the genus level in the soil after continuous cropping of Salvia miltiorrhiza following treatment of each Trichoderma treatment group provided in this embodiment.

[0146] Depend on Figure 8It was found that at the fungal genus level, the top 10 fungal populations remained unchanged across different treatment groups, but their relative abundance varied. Compared to the control group, the relative abundance of *Humicola* and *Botryotrichum* increased in all five *Trichoderma* treatment groups, while the relative abundance of *Fusarium* decreased in all groups except for the D3 treatment group. *Fusarium* is a major pathogen causing root rot in *Salvia miltiorrhiza*, leading to decreased yield and quality. *Fusarium* continues to cause significant diseases in many crops, such as tomatoes, corn, and cotton. Therefore, it is hypothesized that *Trichoderma* can reduce the incidence of diseases in *Salvia miltiorrhiza* by suppressing the abundance of pathogens such as *Fusarium*, thereby increasing the yield of *Salvia miltiorrhiza*.

[0147] The changes in soil chemical properties and soil microorganisms after irrigation treatment with the above Trichoderma treatment groups for continuously cropped Salvia miltiorrhiza can be summarized as follows:

[0148] (1) Group D1: After irrigation with Trichoderma tumefaciens Tri112 (CGMCC No. 23210), the total nitrogen content of the soil in the plots where Salvia miltiorrhiza was continuously planted for 4 years was significantly increased, exceeding the control by 284.21%. At the bacterial level of soil microorganisms, it increased the relative abundance of Actinobacteria, Chlorobacteria, and Bacillus; at the fungal level, it increased the relative abundance of Humus and Botrytis, and decreased the relative abundance of Fusarium.

[0149] (2) Group D2: After irrigation with Trichoderma viride Tri802 (preservation number: CGMCC No. 23214), the organic carbon content of the soil in the plots where Salvia miltiorrhiza was continuously planted for 4 years was significantly increased, exceeding that of the control by 82.06%. At the bacterial level of soil microorganisms, it can increase the relative abundance of Actinobacteria, Chlorobacteria, and Bacillus; at the fungal level, it can increase the relative abundance of Humus and Botrytis, and decrease the relative abundance of Fusarium.

[0150] (3) Group D3: After irrigation with Trichoderma brevistrix Tri502 (preservation number: CGMCC No. 23213), the nitrate nitrogen content in the soil of the plot where Salvia miltiorrhiza was continuously planted for 4 years was significantly increased, exceeding the control by 318.97%. At the bacterial level of soil microorganisms, it can increase the relative abundance of Actinobacteria, Chlorobacteria, and Bacillus; at the fungal level, it can increase the relative abundance of Humus and Botrytis.

[0151] (4) Group D4: After irrigation with Trichoderma viride Tri403 (preservation number: CGMCC No. 23212), the chemical properties of the soil in the plot where Salvia miltiorrhiza was continuously planted for 4 years were significantly improved. At the bacterial level of soil microorganisms, it increased the relative abundance of Actinobacteria, Chlorobacteria, and Bacillus; at the fungal level, it increased the relative abundance of Humus and Botrytis, and decreased the relative abundance of Fusarium.

[0152] (5) Group D5: After irrigation with Trichoderma tuftedum Tri401 (preservation number: CGMCC No. 23211), the chemical properties of the soil in the plot where Salvia miltiorrhiza was continuously planted for 4 years were significantly improved. At the bacterial level of soil microorganisms, it increased the relative abundance of Actinobacteria, Chlorobacteria, and Bacillus; at the fungal level, it increased the relative abundance of Humus and Botrytis, and decreased the relative abundance of Fusarium.

[0153] (V) Wash the soil off the roots of the whole Salvia miltiorrhiza plant and measure its fresh weight. Then dry it at 105℃ to a constant temperature and measure the dry weight of the roots. The results are shown in Table 4 below.

[0154] Table 4. Effects of different Trichoderma treatments on the fresh and dry weight of single plants of continuously cropped Salvia miltiorrhiza:

[0155]

[0156]

[0157] As shown in the table above, after one growth cycle of *Salvia miltiorrhiza*, the fresh weight and dry weight of *Salvia miltiorrhiza* in the D3, D4, and D5 treatment groups increased compared to the control (CK). The D3 treatment group, treated with *Trichoderma brevicornu*, showed the largest increase, with a 39.61% increase in fresh weight and a 31.49% increase in dry weight compared to the CK. This indicates that the application of *Trichoderma brevicornu*, *Trichoderma viride*, and *Trichoderma tuftedum* is beneficial for increasing the yield and dry matter accumulation of *Salvia miltiorrhiza*, and has a significant growth-promoting effect.

[0158] Table 5. Effects of different Trichoderma treatments on the content of effective components in continuously cropped Salvia miltiorrhiza.

[0159]

[0160] The effects of Trichoderma on the content of ginsenoside monomers are shown in Table 5. The contents of tanshinone IIA and salvianolic acid B in all Trichoderma treatment groups were higher than those in the control group. Except for Trichoderma viride, all Trichoderma treatment groups increased the contents of cryptotanshinone and tanshinone I. Therefore, in general, the application of Trichoderma can increase the content of effective components in continuously cropped ginseng and improve the quality of continuously cropped ginseng.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Trichoderma in relieving continuous cropping obstacles of Salvia miltiorrhiza; The Trichoderma is Trichoderma brevicompactum strain Tri502, which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Xibei Road, Beichen, Beijing, with a preservation number of CGMCC No. 23213 and a preservation date of August 19, 2021.

2. Use according to claim 1, characterized in that, The continuous cropping obstacles of Salvia miltiorrhiza refer to the deterioration of soil chemical properties and changes in soil microorganisms caused by continuous planting of Salvia miltiorrhiza for 4-6 years.

3. Use according to claim 2, characterized in that, The deterioration of soil chemical properties includes a decrease in the content of available nutrients in the soil and a decrease in the content of soil organic matter. The changes in soil microorganisms include a decrease in the relative abundance of beneficial microorganisms Bacillus and an increase in the relative abundance of harmful microorganisms Fusarium.

4. Use according to claim 1, characterized in that, The Trichoderma is applied in the form of irrigation liquid.

5. Use according to claim 4, characterized in that, The concentration of the Trichoderma in the irrigation liquid is 1 x 10 7 cfu.mL -1 ~9 x 10 7 cfu.mL -1 .

6. Use according to claim 4, characterized in that, The concentration of the Trichoderma in the irrigation liquid is 1 x 10 7 cfu·mL -1 .

7. The use according to any one of claims 4 to 6, characterized in that, The method of application comprises: transplanting the salvia miltiorrhiza seedlings to the salvia miltiorrhiza continuous cropping obstacle land, then irrigating and planting by using the irrigation liquid according to any one of claims 4-6, applying 2 times during the whole growth period, and 500ml / m 2 The rest of the water management is consistent with the field management.

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