Trichoderma, liquid fungicide, solid fungicide, method for repairing heavy metal cadmium pollution and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to trichoderma, a liquid fungicide, a solid fungicide, a method for repairing heavy metal cadmium pollution and application. The invention provides a Trichoderma sp. Strain JY22N4XM018, the preservation number of the Trichoderma sp. Strain JY22N4XM018 is CCTCC NO: M 20242327, the Trichoderma sp. Strain JY22N4XM018 provided by the invention is high in cadmium removal rate, and the adsorption mode of the Trichoderma sp. Strain JY22N4XM018 to heavy metal cadmium is mainly extracellular adsorption. The trichoderma JY22N4XM018 disclosed by the invention can reduce the cadmium content in a culture medium and also can reduce the cadmium content in plants, and the strain disclosed by the invention can be used for pollution treatment of heavy metal cadmium and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Trichoderma, a liquid bacterial agent, a solid bacterial agent, a method for repairing heavy metal cadmium pollution and applications. Background Art

[0002] Heavy metals are an important pollutant of the environment and agricultural products. Compared with other heavy metal elements, cadmium is highly mobile in the soil and is easily absorbed by crops and stored in the harvested parts. The biological half-life of cadmium is 10 to 30 years. It is very easy to accumulate in the human kidneys, causing great damage to human health. For non-smokers, 90% of cadmium in the human body comes from food intake. It is necessary to reduce the cadmium content in soil and food.

[0003] The main methods for remediation of heavy metal pollution are: physical remediation, chemical remediation and biological remediation. Although physical and chemical technologies have significant treatment effects, they are expensive and complex. In addition, such technologies have strong application limitations and are applicable to a single scenario. In contrast, biotechnology is not only low-cost and sustainable, but also has a variety of application scenarios, such as microbial remediation. The removal rate of cadmium by Trichoderma reported in the prior art is low. Summary of the invention

[0004] The purpose of the present invention is to provide a Trichoderma, a liquid bacterial agent, a solid bacterial agent, a method for repairing heavy metal cadmium pollution and an application thereof. The Trichoderma JY22N4XM018 provided by the present invention is used for removing heavy metal cadmium, and the cadmium removal rate is high.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a Trichoderma sp. JY22N4XM018, with the preservation number being CCTCC NO: M20242327.

[0007] The present invention provides a liquid bacterial agent, comprising mycelium and / or culture solution of Trichoderma JY22N4XM018 described in the above technical solution.

[0008] The present invention provides a method for preparing a liquid bacterial agent, comprising the following steps:

[0009] The Trichoderma JY22N4XM018 is inoculated into a culture medium for cultivation to obtain a culture solution; the culture solution is filtered to obtain the Trichoderma JY22N4XM018 mycelium; the culture temperature is 27-29° C., the culture speed is 100-150 rpm, and the culture time is 2-3 days.

[0010] The present invention provides a solid bacterial agent, comprising the Trichoderma JY22N4XM018 described in the above technical solution and a matrix; the matrix comprises millet.

[0011] Preferably, the method comprises: mixing millet and Trichoderma JY22N4XM018 to obtain a solid bacterial agent.

[0012] The present invention provides the use of the Trichoderma JY22N4XM018 described in the above technical scheme, or the liquid bacterial agent described in the above technical scheme, or the liquid bacterial agent prepared by the preparation method described in the above technical scheme, or the solid bacterial agent described in the above technical scheme, or the solid bacterial agent prepared by the preparation method described in the above technical scheme in repairing heavy metal cadmium pollution and / or reducing cadmium accumulation in plants.

[0013] Preferably, the repairing of heavy metal cadmium pollution includes repairing heavy metal cadmium pollution in water and / or soil.

[0014] Preferably, the plant comprises wheat.

[0015] The present invention provides a method for repairing heavy metal cadmium pollution, comprising: mixing the Trichoderma JY22N4XM018 described in the above technical scheme or the liquid bacterial agent described in the above technical scheme or the liquid bacterial agent prepared by the preparation method described in the above technical scheme or the solid bacterial agent described in the above technical scheme or the solid bacterial agent prepared by the preparation method described in the above technical scheme with an article to be repaired, and culturing them.

[0016] The present invention provides a method for reducing cadmium accumulation in plants, comprising: applying the Trichoderma JY22N4XM018 described in the above technical solution, or the liquid bacterial agent described in the above technical solution, or the liquid bacterial agent prepared by the preparation method described in the above technical solution, or the solid bacterial agent described in the above technical solution, or the solid bacterial agent prepared by the preparation method described in the above technical solution to plants for culturing.

[0017] Beneficial effects of the present invention: The present invention provides a Trichoderma sp. JY22N4XM018, the preservation number of which is CCTCC NO: M 20242327. The Trichoderma sp. JY22N4XM018 provided by the present invention has a high cadmium removal rate. It has been verified that the cadmium removal rate of the Trichoderma sp. 2+ At different concentrations, the extracellular accumulation of mycelium was significantly higher than the intracellular accumulation. It can be seen that extracellular adsorption is the main way for Trichoderma JY22N4XM018 strain to adsorb cadmium.

[0018] The Trichoderma JY22N4XM018 of the present invention can reduce the cadmium content in the culture medium and can also reduce the cadmium content in plants. It can be seen that the strain of the present invention can be used in the pollution control of heavy metal cadmium and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The microscopic morphological observation diagram of Trichoderma JY22N4XM018 on a glass slide; a, b, c represent hyphae morphology, and d represents spore morphology;

[0020] Figure 2 This is a diagram showing the growth status of Trichoderma JY22N4XM018 on PDA medium;

[0021] Figure 3 is the phylogenetic tree of Trichoderma JY22N4XM018;

[0022] Figure 4 For different Cd 2+ Growth status of Trichoderma JY22N4XM018 in solid culture medium under concentration treatment, where A, B, C, D, E, F, G, and H represent 0 mg / L, 2.5 mg / L, 5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 400 mg / L, respectively;

[0023] Figure 5 For different Cd 2+ Diagram of the growth diameter of Trichoderma JY22N4XM018 hyphae under concentration treatment;

[0024] Figure 6 For Trichoderma JY22N4XM018 in different Cd 2+ Growth status diagram in liquid culture medium under concentration conditions; Figure 6 The first row from top to bottom is a picture of the culture bottle culture status, and the second row is an enlarged picture of the bottom of the culture bottle corresponding to the first row;

[0025] Figure 7 For different Cd 2+ The removal capacity of Trichoderma JY22N4XM018 on cadmium under the treatment of concentration (P<0.05);

[0026] Figure 8 For different Cd 2+ The extracellular adsorption and intracellular accumulation of Trichoderma JY22N4XM018 at different concentrations;

[0027] Fig. 9 The effect of Trichoderma JY22N4XM018 on the aboveground and root biomass of wheat seedlings;

[0028] Fig.10 The effect of Trichoderma JY22N4XM018 on the Shoot Cd concentration of wheat under cadmium stress;

[0029] Fig.11The effect of Trichoderma JY22N4XM018 on the cadmium content in the roots of wheat under cadmium stress (Root Cd concentration);

[0030] Fig.12 The effect of Trichoderma JY22N4XM018 on the Cdtransfer factor of wheat under cadmium stress;

[0031] Fig.13 The effect of Trichoderma JY22N4XM018 on the whole plant cadmium accumulation (Plant CdAccumulation) of wheat under cadmium stress;

[0032] Fig.14 The effect of Trichoderma JY22N4XM018 on root cadmium uptake per unit mass (Root Cduptake) of wheat under cadmium stress;

[0033] Fig.15 The effect of Trichoderma JY22N4XM018 on the biomass of the aboveground and roots of wheat under cadmium stress;

[0034] Fig.16 The effect of Trichoderma JY22N4XM018 on the shoot Cd content of wheat seedlings.

[0035] Fig.17 The effect of Trichoderma JY22N4XM018 on the root cadmium content of wheat seedlings.

[0036] Fig.18 The effect of Trichoderma JY22N4XM018 on TF (cadmium transport coefficient) of wheat seedlings;

[0037] Fig.19 The effect of Trichoderma JY22N4XM018 on the cadmium accumulation in wheat seedlings.

[0038] Fig. 20 The effect of Trichoderma JY22N4XM018 on root cadmium uptake per unit mass (Root Cduptake) of wheat seedlings;

[0039] Fig.21 The search and comparison results of the mitochondrial genome co1 and co2 sequences of strain JY22N4XM018 in NCBI;

[0040] Figures 10 to 14 "NANA" or "NANANA" in 16-20 means no cadmium added and no significance.

[0041] Biological Deposit Description

[0042] Trichoderma sp. JY22N4XM018 was deposited in the China Center for Type Culture Collection (CCTCC) on October 24, 2024, with the deposit number CCTCC NO:M 20242327. The address of the depository unit is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0043] The present invention provides a strain of Trichoderma sp. JY22N4XM018, with a deposit number of CCTCC NO: M20242327. The ITS gene sequence of the Trichoderma sp. JY22N4XM018 of the present invention is shown in SEQ ID NO.1.

[0044] SEQ ID NO.1:GGTTTGGAGTTTACACTCCCAACCCATGTGAACGTTACCAAACTGTT.

[0045] The Trichoderma JY22N4XM018 of the present invention is a fungus, which is separated and screened from wheat planted in a cadmium-contaminated area. It can adsorb heavy metal cadmium, reduce the amount of cadmium adsorbed by plants, and can be used for heavy metal cadmium pollution control. The colony morphology of Trichoderma JY22N4XM018 is that the colony is white and flocculent in the early stage and dark green in the later stage; the spore-producing structure often has 2 or 3 side branches, and the spore-producing stalk is bottle-shaped and has two-level branches; the spore morphology is spherical and the color is blue-green. The Trichoderma JY22N4XM018 provided by the present invention has a high cadmium removal rate. The adsorption mode of Trichoderma JY22N4XM018 on heavy metal cadmium is mainly extracellular adsorption. 2+ At different concentrations, the extracellular accumulation of mycelium was significantly higher than the intracellular accumulation, and extracellular adsorption was the main way for the JY22N4XM018 strain to adsorb cadmium.

[0046] The invention uses the ITS sequence of Trichoderma sp. JY22N4XM018 to perform BLAST homologous sequence search in NCBI, constructs a phylogenetic tree with sequences with high homology, and determines that the strain JY22N4XM018 is Trichoderma sp.

[0047] The present invention provides a liquid bacterial agent, comprising mycelium and / or culture solution of Trichoderma JY22N4XM018 of the above technical solution.

[0048] The present invention provides a method for preparing a liquid bacterial agent, comprising the following steps:

[0049] The Trichoderma JY22N4XM018 is inoculated into a culture medium for cultivation to obtain a culture solution; the culture solution is filtered to obtain mycelium; the culture temperature is 27-29° C., the culture speed is 100-150 rpm, and the culture time is 2-3 days. The present invention activates the Trichoderma strain by culture, and the activated mycelium is placed in a PDB culture medium for cultivation. The liquid microbial agent prepared by the present invention is a highly efficient cadmium adsorption fungal microbial agent, and the preparation process is simple and the cost is low.

[0050] As an optional embodiment, the present invention inoculates the Trichoderma JY22N4XM018 cake into a culture medium for cultivation to obtain a culture solution. As an optional embodiment, the present invention has no special limitation on the culture method of the cake, and a conventional method can be used. As an optional embodiment, 2 to 4 cakes are inoculated in every 100 mL of culture medium, or 3 cakes; in a specific embodiment of the present invention, the diameter of the cake is 5 mm. As an optional embodiment, the culture temperature of the present invention is 27°C to 29°C; in a specific embodiment of the present invention, the culture temperature is 27, 28 or 29°C. The culture time is 2 to 3 days. In a specific embodiment of the present invention, the culture time is 2 or 3 days. As an optional embodiment, the rotation speed of the culture of the present invention is 100 to 150 rpm. The culture medium used in the culture of the present invention includes liquid potato dextrose water culture medium, i.e., PDB culture medium. As an optional embodiment, the number of viable bacteria of Trichoderma JY22N4XM018 in the culture solution is ≥3×10 8 CFU / mL.

[0051] The method for preparing mycelium of the present invention comprises: filtering the culture solution of the above technical solution to obtain mycelium. As an optional embodiment, the filtering method comprises filtering with sterile cloth. As an optional embodiment, after filtering, the obtained mycelium is washed to obtain mycelium. As an optional embodiment, the washing is completed with ultrapure water, and the number of washing times is 3 to 5 times.

[0052] The present invention provides a solid bacterial agent, comprising the Trichoderma JY22N4XM018 of the above technical solution and a matrix; the matrix comprises millet.

[0053] The present invention provides a method for preparing the solid bacterial agent described in the above technical solution, comprising: mixing and culturing millet and Trichoderma JY22N4XM018 to obtain a solid bacterial agent.

[0054] As an optional embodiment, before mixing, the millet of the present invention is boiled, filtered, air-dried and sterilized to obtain sterilized millet; the boiling time is 2 to 3 minutes; the air-drying method is natural air-drying; the water content of the air-dried millet is 50%; the millet is boiled and air-dried as a culture medium, the culture medium is loose and has a large specific surface area, and can promote the growth of Trichoderma JY22N4XM018. As an optional embodiment, the sterilization is high-pressure sterilization.

[0055] As an optional embodiment, the culture temperature is 27-29° C. In a specific embodiment of the present invention, the culture temperature is 27, 28 or 29° C. The present invention is cultured until the mycelium grows fully in the millet. The advantages of the present invention in preparing solid bacterial bodies are that the mycelium grows fast, spores are easily produced, labor-saving and time-saving, and simple to make.

[0056] The present invention provides the use of the Trichoderma JY22N4XM018 described in the above technical scheme, or the liquid bacterial agent described in the above technical scheme, or the mycelium prepared by the preparation method described in the above technical scheme, or the solid bacterial agent described in the above technical scheme, or the solid bacterial agent prepared by the preparation method described in the above technical scheme in repairing heavy metal cadmium pollution and / or reducing cadmium accumulation in plants.

[0057] As an optional embodiment, the remediation of heavy metal cadmium pollution in the present invention includes remediation of heavy metal cadmium pollution in water and / or soil.

[0058] As an alternative embodiment, the plant of the present invention includes wheat.

[0059] The present invention provides a method for repairing heavy metal cadmium pollution, comprising: mixing the Trichoderma JY22N4XM018 described in the above technical scheme or the liquid bacterial agent described in the above technical scheme or the mycelium prepared by the preparation method described in the above technical scheme or the solid bacterial agent described in the above technical scheme or the solid bacterial agent prepared by the preparation method described in the above technical scheme with a product to be repaired, and culturing them.

[0060] As an optional implementation manner, the present invention has no special limitation on the mixing method, and a conventional method may be used.

[0061] As an optional embodiment, the object to be repaired in the present invention includes water containing cadmium and / or soil containing cadmium.

[0062] As an optional implementation, when the solid bacterial agent is applied to the object to be repaired, the application amount of the solid bacterial agent is 20 g / kg; when the liquid bacterial agent is applied to the object to be repaired, the application amount of the liquid bacterial agent is 20 mL / L.

[0063] The present invention provides a method for reducing cadmium accumulation in plants, which is characterized by comprising: applying the Trichoderma JY22N4XM018 described in the above technical solution, or the liquid bacterial agent described in the above technical solution, or the liquid bacterial agent prepared by the preparation method described in the above technical solution, or the solid bacterial agent described in the above technical solution, or the solid bacterial agent prepared by the preparation method described in the above technical solution to plants for culturing.

[0064] As an optional embodiment, the plant of the present invention includes wheat.

[0065] As an optional embodiment, the application method of the present invention includes root dipping.

[0066] As an optional embodiment, when the solid bacterial agent is applied to plants, the application amount of the solid bacterial agent is 20 g / kg; when the liquid bacterial agent is applied to plants, the application amount of the liquid bacterial agent is 20 mL / L. The Trichoderma JY22N4XM018 in the present invention can reduce the amount of cadmium adsorbed by plants.

[0067] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] The composition of the separation medium is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration (0~400mg / L).

[0069] The fermentation medium consisted of 200 g / L potato and 20 g / L glucose.

[0070] The composition of PDA medium is: 200g potato, 20g glucose, 15-20g agar and 1000mL distilled water. If agar is not added to PDA medium, it is PDB medium.

[0071] Example 1 Screening, Isolation and Identification of Strain JY22N4XM018

[0072] The roots, stems, leaves and ears of wheat were rinsed with distilled water and cut into 1 cm small segments. Each segment was first soaked in 75% ethanol for 30 seconds, then soaked in 10% sodium hypochlorite solution for 1 minute, washed with sterile water for 3 times, dried with sterile absorbent paper, and evenly placed in PDA culture medium. Cultured in a constant temperature incubator at 28°C, with 4 replicates for each treatment. After 3 days, the purified endophytic fungi were numbered in sequence.

[0073] When the purified endophytic fungi are growing in single colonies, they are stored in cryopreservation tubes and refrigerated in a refrigerator at 4°C.

[0074] At the same time, after the colonies grow, pick colonies of different shapes, sizes, colors, etc. and streak them on PDA medium plates until there are no foreign colonies. After cadmium concentration gradient acclimation and cultivation, a strain named JY22N4XM018 was obtained. The JY22N4XM018 strain was cultured in PDA medium at 28℃ for 6-7 days and the morphology was observed. The morphology of the JY22N4XM018 strain under the microscope was as follows: Figure 1The spore-producing structure often has 2 or 3 side branches, and the spore-producing stalk is bottle-shaped with two-level branches; the spores are spherical in shape and blue-green in color. The morphological characteristics of the JY22N4XM018 strain colony in the early stage: the colony is white and flocculent at first, then dark green, and Figure 2 The JY22N4XM018 strain was systematically identified by morphology with reference to the Manual of Fungal Identification, and it was found that the JY22N4XM018 strain belonged to the genus Trichoderma.

[0075] The ITS sequence of strain JY22N4XM018 was obtained by PCR. The sequence was determined and the NCBI database BLAST homology comparison and evolution analysis showed that the homology of strain JY22N4XM018 with Trichoderma afroharzianum GJS 04-02 and Trichoderma breve HMAS248844 reached 99%. The phylogenetic tree is shown in Figure 3 The ITS sequence was identified as Trichoderma afroharzianum. Beijing Novogene Biotechnology Co., Ltd. was commissioned to perform whole genome sequencing on strain JY22N4XM018. The mitochondrial genome co1 and co2 sequences of strain JY22N4XM018 are shown in SEQ ID NO.2. The alignment results of co1 and co2 sequences in NCBI are shown in Fig.21 , indicating that it is not Trichoderma afroharzianum, so it is temporarily classified as an unknown species of Trichoderma (Trichoderma sp.).

[0076]

[0077] Example 2 Heavy metal resistance of strain JY22N4XM018

[0078] The strain JY22N4XM018 was spread on the plates of separation medium 1 to 7 respectively.

[0079] The culture medium composition of the control group was: potato 200 g / L, glucose 20 g / L and agar 15 g / L; Cd 2+ Concentration is 0mg / L

[0080] The composition of separation medium 1 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration: 2.5 mg / L;

[0081] The composition of separation medium 2 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration 5mg / L;

[0082] The composition of separation medium 3 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration 25mg / L;

[0083] The composition of separation medium 4 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration 50mg / L;

[0084] The composition of separation medium 5 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration 100mg / L;

[0085] The composition of separation medium 6 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration: 200mg / L;

[0086] The composition of separation medium 7 is: potato 200g / L, glucose 20g / L, agar 15g / L and Cd 2+ Concentration 400mg / L;

[0087] Each culture medium was cultured at 28°C, and the growth diameter of the colony of strain JY22N4XM018 was measured. Figures 4-5 And Table 1. According to Table 1, Cd 2+ At lower concentrations, Cd had no significant effect on the growth of strain JY22N4XM018; 2+ When the concentration was ≥200 mg / L, the growth of strain JY22N4XM018 was significantly inhibited.

[0088] Table 1 Colony diameter (cm) of strain JY22N4XM018 in different isolation media

[0089]

[0090] Note: Cd 2+ The unit of concentration is mg / L.

[0091] Example 3 Adsorption performance of strain JY22N4XM018 on heavy metal cadmium

[0092] JY22N4XM018 was inoculated in PDA medium to obtain bacterial cakes with a diameter of 5 mm. The bacterial cakes were inoculated in the fermentation medium, with 3 bacterial cakes inoculated per 100 mL of the medium. After inoculation, the culture was shaken at 28°C and a speed of 100-180 rpm for 3 days to obtain the fermentation liquid. The fermentation liquid was filtered with a sterile filter cloth, the medium was removed, and mycelium was obtained. 0.5 g of mycelium was placed in 100 mL of contaminated medium 1-6 for 3 days, and the cadmium content in the contaminated medium was detected.

[0093] The composition of contaminated medium 1 is: potato 200 g / L, glucose 20 g / L;

[0094] The composition of contaminated medium 2 is: potato 200g / L, glucose 20g / L and Cd 2+ Concentration 1mg / L;

[0095] The composition of contaminated medium 3 is: potato 200g / L, glucose 20g / L and Cd 2+ Concentration 10mg / L;

[0096] The composition of contaminated medium 4 is: potato 200g / L, glucose 20g / L and Cd 2+ Concentration 60mg / L;

[0097] The composition of contaminated medium 5 is: potato 200g / L, glucose 20g / L and Cd 2+ Concentration 100mg / L;

[0098] The composition of contaminated medium 6 is: potato 200g / L, glucose 20g / L and Cd 2+ Concentration 400mg / L;

[0099] The adsorption rate of cadmium by strain JY22N4XM018 was calculated according to the following formula.

[0100] Calculation method of adsorption rate (R):

[0101] R(%)=(C 0 -C e ) / C0 *100%

[0102] Among them, C 0 is the initial concentration of heavy metal cadmium ions in the solution, C e The concentration of heavy metal cadmium ions in the solution after t hours of bacterial adsorption.

[0103] Results Figures 6-7 , it can be seen that in Cd 2+ When the concentrations were 1, 10, 60, 100 and 400 mg / L, the cadmium removal rates were 87.66±0.28%, 85.75±0.30%, 79.19±0.78%, 75.57±0.69% and 71.95±0.11%, respectively. JY22N4XM018 had a significant effect on cadmium removal.

[0104] Example 4 Extracellular adsorption and intracellular accumulation of Cd by strain JY22N4XM018

[0105] Cd in PDB medium 2+ The concentrations were 1, 10, 60, 100, and 400 mg / L, respectively. After inoculating JY22N4XM018 mycelium into PDB medium and culturing for 3 days, the resulting culture solution was filtered with a sterile filter cloth to obtain mycelium. The mycelium was washed with ultrapure water 3 times, and then the harvested mycelium was resuspended in an EDTA solution with a molar concentration of 5 mM, the volume of the EDTA solution was 20 mL, and the EDTA solution was sterilized, and cultured at 25°C, 100-150 rpm for 30 minutes, and the mycelium and filtrate were collected.

[0106] The collected mycelium was dried to constant weight, and then acid digested and Cd was determined using an atomic absorption spectrophotometer. 2+ The concentration was used to calculate the intracellular accumulation of cadmium.

[0107] The collected filtrate was centrifuged at 10000g for 5 min, and the cadmium concentration was measured by atomic absorption spectrophotometer to calculate the extracellular adsorption of cadmium. The results are shown in Table 2 and Figure 8 .

[0108] According to Table 2 and Figure 8 It can be seen that within the range of 0-400 mg / L cadmium, the cadmium accumulation in Trichoderma cells first increased and then decreased, reaching a maximum of 0.752 mg / g when the cadmium concentration was 60 mg / L. 2+ The concentration increases gradually. 2+ The maximum concentration reached 18.8 mg / g when the concentration was 400 mg / L. Under different cadmium concentrations, the extracellular accumulation of mycelium was significantly higher than the intracellular accumulation. Extracellular adsorption was the main way for JY22N4XM018 strain to adsorb heavy metal cadmium.

[0109] Table 2 Determination results of intracellular accumulation and extracellular adsorption of cadmium by strain JY22N4XM018

[0110]

[0111] Note: Different letters represent P < 0.05. Intracellular bioaccumulation refers to intracellular accumulation; Extracellular biosorption refers to extracellular biosorption.

[0112] Example 5 Effect of strain JY22N4XM018 on cadmium absorption, transport and accumulation in wheat

[0113] Select plump and uniform wheat seeds, wash them with distilled water for 3 to 4 times, and soak them for 8 hours. Germinate them at room temperature until the seeds turn white, place them in a seedling tray, add water to the tray until it touches the bottom of the seeds, and transplant the uniformly plump wheat seedlings to the hydroponic pot after 3 to 4 days of growth. Change the nutrient solution every 3 days; use 1 / 4 concentration nutrient solution on the 1st to 3rd day of cultivation, 1 / 2 concentration nutrient solution on the 4th to 6th day of cultivation, and full nutrient solution with cadmium added on the 7th to 9th day of cultivation. The nutrient solution is Hoagland nutrient solution. The specific cadmium addition treatment is as follows:

[0114] Cd10: Treatment 1-1: Cadmium source is CdCl 2 2.5H 2 O was added into the complete nutrient solution, and the cadmium concentration was 10 μmol / L.

[0115] When the seedlings have one leaf and one heart, the roots of the wheat are dipped in a centrifuge tube filled with 20 mL of JY22N4XM018 fermentation liquid for 1 hour. The volume of the centrifuge tube is 50 mL.

[0116] Treatment 1-2: Same as treatment 1-1, except that the roots were not dipped in JY22N4XM018 fermentation solution.

[0117] Cd30: Treatment 2-1: Cadmium source is CdCl 2 2.5H 2 O was added into the complete nutrient solution, and the cadmium concentration was 30 μmol / L.

[0118] When the seedlings have one leaf and one heart, the roots of the wheat are dipped in a centrifuge tube filled with 20 mL of JY22N4XM018 fermentation liquid for 1 hour. The volume of the centrifuge tube is 50 mL.

[0119] Treatment 2-2: Same as treatment 2-1, except that the roots were not dipped in JY22N4XM018 fermentation solution.

[0120] CK: Treatment CK-1: application of complete nutrient solution without adding cadmium source, cadmium concentration was 0 μmol / L.

[0121] When the seedlings have one leaf and one heart, the roots of the wheat are dipped in a centrifuge tube filled with 20 mL of JY22N4XM018 fermentation liquid for 1 hour. The volume of the centrifuge tube is 50 mL.

[0122] Treatment CK-2: Same as treatment CK-1, except that the roots were not dipped in JY22N4XM018 fermentation solution.

[0123] There were 6 treatments in total, each with 4 replicates, i.e. 4 pots, and 9 seedlings in each pot.

[0124] Wheat seedlings of each treatment were cultured in a light culture room under the following growth conditions: temperature 25°C, photoperiod of 14 h / d.

[0125] All treatments took 21 days from transplanting wheat seedlings to culturing in Hoagland nutrient solution. The composition of Hoagland nutrient solution is CaNO 3 ·4H 2 O 0.945g / L, KNO 3 0.607g / L, (NH 4 )H 2 PO 4 0.115g / L, MgSO 4 7H 2 O 0.493g / L, H 3 BO 3 2.86mg / L, MnCl 2 ·4H 2 O 1.81mg / L, ZnSO 4 7H 2 O 0.22mg / L, CuSO 4 ·5H 2 O 0.08mg / L, (NH 4 ) 2 Mo 7 O 24 ·4H 2 O 0.036mg / L, EDTANa 2 -Fe 36.71mg / L. The composition of the 1 / 2 concentration nutrient solution is the same as that of the Hoagland nutrient solution, and the concentration of each component is 1 / 2 of that of the Hoagland nutrient solution; the composition of the 1 / 4 concentration nutrient solution is the same as that of the Hoagland nutrient solution, and the concentration of each component is 1 / 4 of that of the Hoagland nutrient solution.

[0126] The wheat seedlings of each treatment were divided into the aboveground part and the root part. The aboveground part and the root part of the wheat seedlings were collected separately. The aboveground part was washed with deionized water, and the root system was soaked in the mixed solution for 30 minutes. The mixed solution was the morpholineethanesulfonic acid buffer solution, and the composition of the buffer solution was: 0.5mM CaCl 2 and 2mM MES (morpholineethanesulfonic acid); soak in the mixed solution to remove cadmium adsorbed on the root surface.

[0127] The aboveground and root samples were wiped dry with absorbent paper, sterilized at 105°C for 30 min, dried at 70°C to constant weight, and then acid digested to determine the cadmium content. At the same time, the cadmium accumulation of the whole plant, the transport coefficient, and the cadmium absorption capacity of the root per unit mass were calculated. The formula is as follows:

[0128] The cadmium accumulation of the whole plant = aboveground cadmium content × aboveground biomass + root cadmium content × root biomass;

[0129] Transport coefficient = cadmium content in the aboveground part / cadmium content in the root part;

[0130] Cadmium absorption capacity of root system per unit mass = Cadmium accumulation of the whole plant / root biomass;

[0131] Results Figures 9 to 14 and Table 3. Figures 9 to 14 In the table, the P values ​​of ANOVA for cadmium (Cd), Trichoderma (M10) and their interaction (Cd×M10) are as follows: * means P < 0.05, ** means P < 0.01, and ns means not significant. M10 represents Trichoderma JY22N4XM018.

[0132] Table 3 Effects of Trichoderma JY22N4XM018 on the growth of wheat seedlings

[0133]

[0134]

[0135] Note: -M10 (i.e. -JY22N4XM018) represents no root dipping treatment, +M10 (i.e. JY22N4XM018) represents root dipping treatment, and “NA” represents no cadmium addition, which is not significant.

[0136] It can be seen that cadmium treatment has a significant effect on the aboveground and root biomass of wheat. Under cadmium-free conditions, the addition of Trichoderma significantly increased the aboveground biomass and root biomass of wheat compared with the treatment without Trichoderma addition.

[0137] According to the test results, under the condition of cadmium concentration of 30μmol / L, compared with the treatment without adding Trichoderma, the addition of Trichoderma reduced the aboveground biomass and root biomass of wheat. As the cadmium stress concentration increased, the wheat biomass gradually decreased. Under cadmium stress conditions, the addition of Trichoderma had no significant effect on the aboveground and root biomass of wheat.

[0138] With the increase of cadmium stress concentration, the cadmium content in the aboveground and roots of wheat increased significantly. Compared with the treatment without adding Trichoderma, the addition of Trichoderma significantly reduced the cadmium content in the roots of wheat. However, the addition of Trichoderma had no significant effect on the cadmium content in the aboveground part of wheat.

[0139] With the increase of cadmium stress concentration, the cadmium accumulation of wheat plants increased significantly. Compared with the treatment without adding Trichoderma, the addition of Trichoderma significantly reduced the cadmium accumulation of wheat plants.

[0140] With the increase of cadmium stress concentration, the cadmium absorption capacity of wheat root per unit mass significantly increased. Compared with the treatment without adding Trichoderma, the addition of Trichoderma significantly reduced the cadmium absorption capacity of wheat root per unit mass.

[0141] Compared with the treatment without adding Trichoderma, adding Trichoderma significantly increased the wheat transport coefficient.

[0142] Example 6 Effect of strain JY22N4XM018 on cadmium absorption, transport and accumulation in wheat

[0143] Millet matrix is ​​as follows: boil millet for 2-3 minutes, filter and air-dry naturally until the moisture content is 50%, and then sterilize at 121°C for 24 minutes before use.

[0144] The preparation method of Trichoderma agent is as follows: boil millet for 2-3 minutes, filter and air-dry naturally until the moisture content is 50%, sterilize and inoculate 5 blocks of Trichoderma JY22N4XM018, and culture until the mycelium is full to obtain Trichoderma agent. The number of viable bacteria of Trichoderma agent is 3×10 8 ~5×10 8 CFU / g; The method for determining the number of viable bacteria is: the number of viable bacteria per unit mass of Trichoderma eluted spores is 3×10 8 ~5×10 8 CFU / g.

[0145] Treatment 1 (Cd10): Treatment 1-1: (1) The wheat variety used for the test was Bainong 307. The test soil was collected from the Science and Education Park of Henan Agricultural University in Yuanyang County, Henan Province. It was a loamy tidal soil. First, the soil sample was air-dried and passed through a 2 mm sieve, sterilized at 121 °C for 30 min, and air-dried. Then, CdCl 2 2.5H 2 O aqueous solution was sprayed evenly into the soil and mixed to keep the soil moisture content at 60% of the maximum field water holding capacity. The cadmium concentration in the soil was 10 mg / kg.

[0146] Urea, potassium dihydrogen phosphate and potassium chloride are used as N, P and K fertilizer sources. 2 O 5 , K2 O was applied into the soil two days before wheat planting at the dosages of 0.20 g / kg, 0.15 g / kg and 0.20 g / kg, respectively.

[0147] (2) Wheat seeds were sterilized with 2% sodium hypochlorite solution for 5 min and then rinsed three times with sterile water. When planting wheat, 12 g of millet substrate was inoculated, 600 g of soil was added to each seedling pot, and 10 wheat plants were planted.

[0148] Treatment 1-2: Same as treatment 1-1, the only difference being that 12 g of Trichoderma was inoculated when wheat was planted.

[0149] Treatment 1-3 (Control): Same as treatment 1-1, except that no millet substrate was added and no Trichoderma inoculation was performed.

[0150] Treatment 2 (Cd30): Treatment 2-1: Same as treatment 1-1, the only difference being that the cadmium concentration in the soil in step (1) was 30 mg / kg;

[0151] Treatment 2-2: Same as treatment 1-2, except that the cadmium concentration in the soil in step (1) was 30 mg / kg;

[0152] Treatment 2-3 (Control): Same as treatment 1-3, the only difference being that the cadmium concentration in the soil in step (1) was 30 mg / kg;

[0153] Control group (Cd0): Control-1: Same as treatment 1-1, the only difference is that CdCl was not sprayed in the soil in step (1) 2 2.5H 2 O aqueous solution, cadmium concentration is 0 mg / kg;

[0154] Control-2: Same as treatment 1-2, except that CdCl was not sprayed on the soil in step (1). 2 2.5H 2 O aqueous solution, cadmium concentration is 0 mg / kg;

[0155] Control-3: Same as treatment 1-3, except that CdCl was not sprayed on the soil in step (1). 2 2.5H 2 O aqueous solution, cadmium concentration is 0 mg / kg;

[0156] There were 9 treatments in total, including treatment 1, treatment 2 and the control group, with 4 parallel experiments in each treatment. Wheat samples were collected 30 days after seed planting. The aboveground biomass, root biomass, aboveground cadmium content and transport coefficient of wheat seedlings in each treatment were measured using the same method as in Example 5. The results are shown in Table 1. Figures 15 to 20and Table 4, Shootbiomass is the aboveground biomass, Rootbiomass is the root biomass, Shoot Cd content is the aboveground cadmium content, Root Cd content is the root cadmium content, TF is the cadmium transport coefficient, Cdaccumulation inpot is the cadmium accumulation, Root Cduptake is the root cadmium absorption per unit mass, Figures 15 to 20 The P values ​​of variance analysis for cadmium (Cd), three inoculation treatments (Treatment) and their interaction (Cd×Treatment) are as follows: * means P < 0.05, ** means P < 0.01, and ns means not significant.

[0157] Table 4 Effect of Trichoderma JY22N4XM018 on the growth of potted wheat seedlings

[0158]

[0159] Note: “NA” means no cadmium added, no significance.

[0160] It can be seen that:

[0161] Under 1-2 cadmium stress conditions, compared with the control group, the inoculation of millet matrix and Trichoderma alone could significantly increase the aboveground biomass of wheat; compared with the control group, the inoculation of millet matrix and Trichoderma alone had no significant effect on the root biomass of wheat.

[0162] In treatment 2, under high cadmium conditions, compared with the control group, inoculation with millet matrix and Trichoderma can effectively reduce the cadmium content in the aboveground part of wheat. In the control treatment, the cadmium content in the aboveground part and the root was the highest, followed by the millet matrix, and the cadmium content inoculated with Trichoderma was the lowest. Compared with the control group, the cadmium content in the aboveground part and the root of the millet matrix decreased; while the cadmium content in the aboveground part and the root of the Trichoderma decreased. The results showed that the effect of inoculation with Trichoderma in alleviating cadmium stress in wheat under high cadmium conditions was more significant than that of inoculation with millet matrix.

[0163] In treatment 1, under low cadmium conditions, compared with the control group, inoculation with millet substrate could effectively reduce the cadmium content in the aboveground part of wheat, but inoculation with Trichoderma had no significant effect on the cadmium content in the aboveground part of wheat. Compared with the control group, inoculation with millet substrate and Trichoderma had no significant effect on the cadmium content in the roots of wheat.

[0164] Under the high cadmium condition in treatment 2, the addition of millet substrate and Trichoderma significantly reduced the cadmium absorption capacity and cadmium accumulation of wheat roots, and the addition of Trichoderma had a more significant effect on the cadmium absorption capacity of the roots.

[0165] Under low cadmium conditions, adding millet substrate and adding Trichoderma significantly reduced the transport coefficient; but under high cadmium conditions, there was no significant difference in the transport coefficient.

[0166] In summary, the present invention provides a Trichoderma JY22N4XM018, which can reduce the cadmium content in plants, soil and culture medium.

[0167] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Trichoderma sp. JY22N4XM018, with a deposit number of CCTCC NO: M 20242327.

2. A liquid bacterial agent, characterized in that: It comprises mycelium and / or culture solution of Trichoderma JY22N4XM018 according to claim 1.

3. A method for preparing a liquid bacterial agent, characterized in that: The steps include: The Trichoderma JY22N4XM018 is inoculated into a culture medium for cultivation to obtain a culture solution; the culture solution is filtered to obtain the Trichoderma JY22N4XM018 mycelium; the culture temperature is 27-29° C., the culture speed is 100-150 rpm, and the culture time is 2-3 days.

4. A solid bacterial agent, characterized in that: The invention comprises the Trichoderma JY22N4XM018 of claim 1 and a substrate; the substrate comprises millet.

5. The method for preparing the solid bacterial agent according to claim 4, characterized in that: include: Millet was mixed and cultured with Trichoderma JY22N4XM018 to obtain a solid bacterial agent.

6. Use of the Trichoderma JY22N4XM018 described in claim 1, the liquid bacterial agent described in claim 2, the liquid bacterial agent prepared by the preparation method described in claim 3, the solid bacterial agent described in claim 4, or the solid bacterial agent prepared by the preparation method described in claim 5 in repairing heavy metal cadmium pollution and / or reducing cadmium accumulation in plants.

7. The use according to claim 6, characterized in that: The repairing of heavy metal cadmium pollution includes repairing heavy metal cadmium pollution in water bodies and / or soil.

8. The use according to claim 6, characterized in that: The plants include wheat.

9. A method for repairing heavy metal cadmium pollution, characterized in that: include: The Trichoderma JY22N4XM018 of claim 1 or the liquid bacterial agent of claim 2 or the liquid bacterial agent prepared by the preparation method of claim 3 or the solid bacterial agent of claim 4 or the solid bacterial agent prepared by the preparation method of claim 5 is mixed with the product to be repaired and cultured.

10. A method for reducing cadmium accumulation in plants, characterized in that: include: The Trichoderma JY22N4XM018 of claim 1, the liquid bacterial agent of claim 2, the liquid bacterial agent prepared by the preparation method of claim 3, the solid bacterial agent of claim 4, or the solid bacterial agent prepared by the preparation method of claim 5 is applied to plants for cultivation.

Images