CN115232753B - Trichoderma harzianum ZL-811, microbial inoculum, preparation method and application thereof - Google Patents
Trichoderma harzianum ZL-811, microbial inoculum, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Trichoderma harzianum (Trichoderma harzianum) ZL-811, a microbial inoculum, a preparation method and application thereof, wherein the strain is preserved in China general microbiological culture collection center (CGMCC) No.23238 in the 9 th month 18 of 2021 and the preservation address is China academy of sciences microbiological culture collection center. The strain has stronger salt tolerance and heat resistance, and shows excellent petroleum hydrocarbon degradation performance, and is especially suitable for repairing petroleum polluted saline-alkali soil.
Description
Technical Field
The invention belongs to the technical field of soil improvement and restoration bacteria, and relates to trichoderma harzianum ZL-811, a microbial inoculum, a preparation method and application thereof.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
The saline-alkali soil is taken as an important land resource, and the coastal saline-alkali soil and inland saline-alkali soil generated by natural formation or artificial beach reclamation are important backup land resources in China, such as the saline-alkali soil in the high-efficiency ecological area of yellow river delta in the national-level agricultural high-area. In addition, yellow river delta reserves abundant oil gas resource, and in oil exploitation, oil testing, well flushing, oil well overhaul, water shutoff, pump loosening, oil gas gathering and transportation processes, crude oil is scattered on the ground, and oil pollution is caused. The petroleum pollution will destroy the soil structure, change the availability of nutrient components, influence the growth and reproduction of organisms, reduce the quality of the whole wetland ecosystem, seriously destroy the biodiversity and seriously threaten the safety of the yellow river delta ecosystem.
At present, the technology for improving the saline-alkali soil is changed from a physical and chemical means based on hydraulic engineering to biological repair, but the biological repair technology based on plants is limited by factors such as salt-tolerant plant types, plant growth periods and the like. In recent years, microbial remediation is increasingly paid attention to in saline-alkali soil remediation, and the microbial inoculum can improve plant rhizosphere environment, lighten the inhibition effect of salt on crop growth and achieve the aim of improving the saline-alkali soil. However, the salt tolerance and petroleum hydrocarbon degradation capability of the existing microorganisms are difficult to meet the requirements, and the restoration effect on the petroleum-polluted saline-alkali soil is poor.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide trichoderma harzianum (Trichoderma harzianum) ZL-811, a microbial inoculum, a preparation method and application thereof. The strain is separated from petroleum-polluted saline-alkali soil in yellow river delta area, has the characteristics of high colony growth speed, large chlamydospore yield, strong adaptability to saline-alkali environment, capability of degrading petroleum pollutants and the like, can remarkably promote the salt tolerance of plants, and has the functions of saline-alkali soil improvement and petroleum-polluted soil restoration.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a trichoderma harzianum (Trichoderma harzianum) ZL-811, which is preserved in the China general microbiological culture collection center (CGMCC) with the preservation number of 23238 and the preservation address of the institute of microorganisms, which is the national academy of sciences, at the 18 th year of 2021.
In a second aspect, the invention provides a microbial agent, which comprises the trichoderma harzianum ZL-811 spores and a carrier, wherein the trichoderma harzianum ZL-811 spores are loaded on the carrier.
In a third aspect, the invention provides a preparation method of the microbial agent, comprising the following steps: and uniformly mixing trichoderma harzianum ZL-811 spore liquid with a carrier, drying and crushing to obtain the microbial inoculum.
In a fourth aspect, the invention provides application of the trichoderma harzianum (Trichoderma harzianum) ZL-811 or the microbial agent in saline-alkali soil remediation or/and petroleum polluted soil remediation;
or, the trichoderma harzianum (Trichoderma harzianum) ZL-811 or the microbial agent is applied to promoting plant seed germination or promoting plant seedling growth under the condition of salt stress;
or application of the trichoderma harzianum (Trichoderma harzianum) ZL-811 or the microbial agent in field disease control.
The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:
(1) The Trichoderma harzianum ZL-811 strain with the preservation number of CGMCC No.23238 has the advantages of high colony growth speed and strong adaptability to saline-alkali environment, can obviously promote the germination and growth of plant seeds and seedlings in saline-alkali soil, promotes the salt tolerance of plants, and can be used for improving the saline alkalinity of soil.
(2) The Trichoderma harzianum ZL-811 strain can degrade petroleum hydrocarbon pollutant in high efficiency, and the total petroleum hydrocarbon degradation rate of 14d reaches 61.27%, and may be used in treating petroleum pollutant.
(3) The Trichoderma harzianum ZL-811 strain has excellent chlamydospore producing capacity, and chlamydospore yield after 10d culture in 200mL shaking bottle reaches 1-2×10 8 CFU/mL, which facilitates the preparation and preservation of trichoderma agents;
(4) The Trichoderma harzianum ZL-811 strain has good biological control capability, and after ZL-811 spore liquid is sprayed in field disease control, the morbidity of wheat powdery mildew, tobacco black shank and tomato fusarium wilt is obviously reduced, and the disease is lightened.
(5) The chlamydospores of the Trichoderma harzianum ZL-811 strain are mixed with a proper culture medium carrier to prepare the efficient trichoderma multifunctional microbial inoculum, and the microbial inoculum can remarkably increase the yield of plants and can be used for efficient ecological restoration of the petroleum-polluted saline-alkali soil.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram showing the whole genome sequencing result of Trichoderma harzianum ZL-811 of example 1;
FIG. 2 is a photograph (A) and a conidium micrograph (B and C) of the culture of Trichoderma harzianum ZL-811 by PDA in example 1;
FIG. 3 is a graph showing the chlamydospore production characteristics of Trichoderma harzianum ZL-811 induced by different inducers in example 2, wherein A is wheat straw, B is mushroom residue, C is mint residue, and D is a graph showing comparison of chlamydospore concentration in fermentation broth obtained by different inducers;
FIG. 4 is a graph showing the salt tolerance of Trichoderma harzianum ZL-811 of example 3;
FIG. 5 is a graph showing the heat resistance of Trichoderma harzianum ZL-811 of example 3, wherein A is a graph showing the comparison of the growth of Trichoderma harzianum ZL-811 at different culture temperatures, and B is a graph showing the comparison of the growth of Trichoderma harzianum ZL-811 after shifting from high temperature to low temperature;
FIG. 6 is a graph showing the comparison of the degradation rate of Trichoderma harzianum ZL-811 against petroleum hydrocarbon contaminants in example 4, wherein A is a graph showing the comparison of the degradation rate of Trichoderma harzianum ZL-811 against the degradation rate of 7d of total petroleum hydrocarbon in the control group, and B is a graph showing the comparison of the degradation rates of normal paraffins (n-alkine), isoparaffins (isoparaffin) and aromatics (aromatic hydrocarbon) in the crude oil of Trichoderma harzianum ZL-811;
FIG. 7 is a graph showing comparison of spore activities of Trichoderma harzianum ZL-811 inoculant stored for various times in example 7.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In a first aspect, the invention provides a trichoderma harzianum (Trichoderma harzianum) ZL-811, which is deposited in the China general microbiological culture collection center (ccm) for 9 and 18 days of 2021, with a deposit number of CGMCC No.23238.
Trichoderma strain ZL-811 has all of the identifying characteristics of Trichoderma harzianum (Trichoderma harzianum). The expression is as follows: culturing on PDA culture medium at 28deg.C, observing that conidiophore has long main axis when culturing for 4d, side branches are often contra-grown, and bottle peduncles are spirally arranged; observing when culturing for 7d, wherein the spore on the front side of the colony is yellow-green mycelium white, the back side of the colony is orange-yellow, and the matrix is distributed with orange-yellow water-soluble pigment; the surface of the bacterial colony is velvet-shaped, two circles of yellow-green concentric circular patterns are generated, hyphae are compact, no exudates are generated, a large amount of aerial hyphae are generated, and no spore cluster is generated; no obvious smell and no autolysis phenomenon; observing when culturing for 14d, the conidium is nearly spherical or oval, is yellowish green and has smooth surface; when cultured for 18d, the culture medium has intergrowth or acrogenesis chlamydospores, the intergrowth is elliptic, and the acrogenesis is spherical.
Trichoderma harzianum (Trichoderma harzianum) ZL-811 has good chlamydospore producing properties. The expression is as follows: in the spore-producing culture medium containing inducer, the chlamydospores are formed after about 40h, then the spore production is gradually increased, a large amount of chlamydospores can be produced after 70h of culture, and the chlamydospores yield reaches 1-2×10 after 10d of culture 9 CFU/mL。
Trichoderma harzianum (Trichoderma harzianum) ZL-811 has strong salt tolerance, ZL-811 can keep normal growth under the treatment of 3% NaCl, and ZL-811 can keep the growth rate close to 50% under the treatment of 5% NaCl.
Trichoderma harzianum (Trichoderma harzianum) ZL-811 has good heat resistance and can normally grow at 40 ℃; although the trichoderma harzianum ZL-811 cannot grow at 42 ℃ and 45 ℃, the trichoderma harzianum ZL-811 still can keep activity after being treated for 3 days, and the trichoderma harzianum ZL-811 can grow normally after being transferred to the optimal 28 ℃.
In addition, trichoderma harzianum (Trichoderma harzianum) ZL-811 can promote plant seed germination and plant seedling growth under salt stress conditions. For example, aiming at wheat (Jimai 22) and cucumber (Jinyan No. four), trichoderma harzianum ZL-811 improves the germination rate of seeds by 2% under NaCl stress, obviously promotes the growth of seedlings and increases the biomass of the seedlings.
Meanwhile, trichoderma harzianum ZL-811 shows excellent petroleum hydrocarbon degradation performance, and 7d degradation experiment results show that ZL-811 can effectively degrade the content of each component in total petroleum hydrocarbon.
The spore liquid of Trichoderma harzianum ZL-811 can also have better control effect on wheat powdery mildew, tobacco black shank, tomato fusarium wilt and other field diseases. After the trichoderma harzianum ZL-811 spore liquid is sprayed, the morbidity of wheat powdery mildew, tobacco black shank and tomato fusarium wilt is obviously reduced, and the diseases are relieved.
In a second aspect, the invention provides a microbial agent, which comprises the trichoderma harzianum ZL-811 spores and a carrier, wherein the trichoderma harzianum ZL-811 spores are loaded on the carrier.
In some embodiments, the microbial agent has a spore loading of 2 x 10 6 -2×10 8 CFU·g -1 。
Preferably, the carrier is a mixture of diatomaceous earth, turf and bran.
Preferably, the mass ratio of diatomite, turf and bran is 3-5:1.5-2.5:1, preferably 4:2:1.
The microbial inoculum can effectively improve the salinization degree of soil, improve the organic matter content of the soil, increase the quantity and diversity of microbial communities in the soil and increase the total quantity of bacteria and fungi; meanwhile, the application of the microbial inoculum effectively reduces the petroleum hydrocarbon content in the petroleum polluted saline-alkali soil.
In a third aspect, the invention provides a preparation method of the microbial agent, comprising the following steps: and uniformly mixing trichoderma harzianum ZL-811 spore liquid with a carrier, drying, crushing and sieving to obtain the microbial inoculum.
In some embodiments, the temperature of the drying is 35-40 ℃.
In some embodiments, the crushed material is passed through a 200 mesh screen.
In a fourth aspect, the invention provides application of the trichoderma harzianum (Trichoderma harzianum) ZL-811 or the microbial agent in saline-alkali soil remediation or/and petroleum polluted soil remediation;
or, the trichoderma harzianum (Trichoderma harzianum) ZL-811 or the microbial agent is applied to promoting plant seed germination or promoting plant seedling growth under the condition of salt stress;
or application of the trichoderma harzianum (Trichoderma harzianum) ZL-811 or the microbial agent in field disease control.
The invention is further illustrated below with reference to examples.
Example 1
Isolation and identification of Trichoderma strain ZL-811:
(1) Separating strains:
the Trichoderma strain ZL-811 is a salt-tolerant Trichoderma selective culture medium, and the culture medium comprises the following components: PDA, 0.4mol/L NaCl, 300mg/L chloramphenicol, 100mg/L streptomycin, 20mg/L rose bengal, and 0.05% Triton X-100.
Petroleum-contaminated saline-alkali soil (soil sample collection site characteristics: soil salinity of about 3.6%) collected from yellow river delta area of east ying, the mass fraction of petroleum contamination in the soil is 1032mg kg -1 . The vegetation in the soil layer is less and only distributed sporadically such as tamarix chinensis, suaeda salsa and the like), 10g of soil sample is weighed, crushed into fine powder, placed in a sterilization triangular flask containing 90mL of 0.9% NaCl salt water, and fully and uniformly mixed by shaking for 1h at the temperature of 28 ℃ and 160r/min to prepare soil suspension; sequentially carrying out gradient dilution, respectively taking 1mL of the mixture and adding the mixture into culture dishes with 90mm, pouring 20mL of a selective culture medium into each culture dish, rotating and uniformly mixing the mixture to uniformly disperse samples in the culture dishes in the culture medium, and placing the culture dishes in a culture box with the temperature of 28 ℃ for 3-4 days; screening suspected Trichoderma strains, selecting single colony, transferring to PDA plate, culturing to obtain colony, separating and purifying by single spore separation method, and storing. The PDA culture medium formula is as follows: 6g/L of potato soaked powder, 20g/L of glucose, 20g/L of agar and pH value of 5.6+/-0.2.
(2) Identification of strains:
the genomic DNA of Trichoderma was extracted by Fungi DNA Mini Kit (Omega, USA), amplified by primer ITS1-F/ITS4, and the PCR product was sequenced by Shanghai Biotechnology Co., ltd, and the result was aligned at NCBI and ISTH to find out that strain ZL-811 was Trichoderma harzianum (Trichoderma harzianum), designated Trichoderma harzianum (Trichoderma harzianum) ZL-811, which was deposited with China general microbiological culture Collection center with the accession number CGMCC No.23238.
Meanwhile, the whole genome sequencing work is carried out on Trichoderma harzianum (Trichoderma harzianum) ZL-811, the whole genome sequence of which is submitted to NCBI DDBJ/ENA/GenBank (Accession No. JAJOLQ 000000000000) (version JAJOLQ 01000000), bioProject: PRJNA785358, biosample: SAMN 23554013), and the genome map is shown in FIG. 1.
(3) Strain culture and observation:
the strain was grown in PDA medium for 12/12 h light-dark (FIG. 2). The strain grows rapidly on PDA culture medium: when culturing for 4d, the conidiophores have long main shafts, side branches are often opposite, and bottle peduncles are spirally arranged; when the culture is carried out for 7 days, the spore on the front side of the bacterial colony is yellow-green hypha and white, the bacterial colony is orange-yellow in back side, orange-yellow water-soluble pigment is distributed in a matrix, the surface of the bacterial colony is velvet-shaped, two circles of yellow-green concentric circular patterns are generated, the hypha is compact, no effusion is generated, a large amount of aerial hypha is generated, and no spore cluster is generated; when the culture is carried out for 14 days, the conidium is nearly spherical or oval, is yellowish green and has smooth surface; when cultured for 18d, they have intergrown or acrogenic chlamydospores, which are usually elliptical and acrogenic are usually spherical.
Example 2
The method for producing chlamydospores by fermenting Trichoderma harzianum ZL-811 comprises the following steps:
with MM medium (15 g/L KH 2 PO 4 ,5g/L(NH 4 ) 2 SO 4 ,0.6g/L MgSO 4 ,0.6g/L CaCl 2 ,0.005g/L FeSO 4 ·7H 2 O,0.0016g/L MnSO 4 ·H 2 O,0.0014g/L ZnSO 4 ·7H 2 O,0.0037g/L CoCl 2 ·6H 2 O,20g/L glucose1% inducer, natural pH value) as basic culture medium, 3% inducer for producing chlamydospore A (wheat straw), B (mushroom residue) and C (menthol residue) are respectively added, and no inducer is added as control. 5mL of the activated Trichoderma harzianum ZL-811 broth was inoculated into 200mL of the medium, and cultured in a constant temperature shaking incubator at 28℃and 180rpm for 10d. The growth condition of chlamydospores is observed by a microscope, and the chlamydospore yield of trichoderma harzianum ZL-811 is calculated by using a viable bacteria counting method.
The results show that: after 10d fermentation, chlamydospores (figure 3) can be produced in 200mL fermentation liquid containing different inducers (A: wheat straw, B: mushroom residue, C: peppermint residue), the content reaches 1.4X10 respectively 8 (A: wheat straw), 0.9X10 8 (B: mushroom residue), 0.8X10 g 8 (C: mint residue).
Example 3
Identification of salt tolerance and heat resistance of Trichoderma harzianum ZL-811:
(1) Collection of sterile spores
Inoculating Trichoderma harzianum ZL-811 on a PDA plate, and culturing at 28 ℃ for 10 days to generate a large amount of fresh conidium; washing the mycelium surface with 10mL physiological saline (0.9% NaCl,0.05% Tween), filtering with glass wool paper, and removing mycelium to obtain spore suspension; suspending with 30% glycerol, mixing, packaging into 1.5mL centrifuge tube, marking name and time, and freezing at-80deg.C; and taking a tube of spore liquid for viable count, and determining the concentration of the spore liquid.
(2) Salt tolerance test
Activating Trichoderma harzianum ZL-811 on a PDA plate, culturing at 28deg.C in dark for 48-72 hr to make the original strain and mutant engineering strain grow uniformly, preparing bacterial blocks with uniform size by using a puncher, and transferring to the center of the PDA plate containing NaCl with different concentrations. The plate with the bacterial block is placed at 28 ℃ for culture, and the growth of bacterial colonies is observed.
As a result, it was found that Trichoderma harzianum ZL-811 had a strong salt tolerance, and ZL-811 was able to maintain normal growth under 3% NaCl treatment, and ZL-811 was able to maintain a growth rate close to 50% under 5% NaCl treatment (FIG. 4).
(4) Heat resistance test
Activating Trichoderma harzianum ZL-811 on a PDA plate, culturing at 28deg.C in dark for 48-72 hr to make the original strain and mutant engineering strain grow uniformly, preparing bacterial blocks with uniform size by using a puncher, and transferring to the center of the PDA plate containing NaCl with different concentrations. The plate with the fungus blocks is placed at 28-45 ℃ for culture, and the growth condition of the bacterial colony is observed. In addition, the plate with the fungus block is placed at 42 ℃ and 45 ℃ for 3d of culture, and then the plate is transferred to 28 ℃ for culture, and the colony growth condition is observed.
The result shows that: trichoderma harzianum ZL-811 has very strong heat resistance and can grow normally at 40 ℃. Although growth is not possible at 42 ℃ and 45 ℃, trichoderma harzianum ZL-811 still can keep activity after 3 days of treatment, and can normally grow under the condition of transferring to the optimal 28 ℃, as shown in figure 5.
Example 4
Degradation test of trichoderma harzianum ZL-811 on petroleum hydrocarbon contaminants:
200. Mu.L of spores of Trichoderma harzianum ZL-811 were inoculated into a PDB liquid medium, and after culturing at 28℃and 180rpm for 48 hours, the bacterial solution was inoculated into a MM medium (15 g/L KH) 2 PO 4 ,5g/L(NH 4 ) 2 SO 4 ,0.6g/L MgSO 4 ,0.6g/L CaCl 2 ,0.005g/L FeSO 4 ·7H 2 O,0.0016g/L MnSO 4 ·H 2 O,0.0014g/L ZnSO 4 ·7H 2 O,0.0037g/L CoCl 2 ·6H 2 O,20g/L glucose, 1% inducer, natural pH) at 28deg.C under shaking at 180rpm for 7d, and MM medium containing 0.5% crude oil without inoculating bacteria solution is used as control.
(1) Crude oil degradation rate determination
The degradation rate of crude oil is measured by a gravimetric method. Extracting the residual oil on the culture solution and the bottle wall after 7d with carbon tetrachloride, placing the organic phase in a fume hood after liquid-liquid separation, naturally drying, weighing the residual oil after carbon tetrachloride is volatilized completely, and calculating the degradation rate of crude oil.
(2) Determination of Petroleum hydrocarbon Components
The petroleum solution after extraction and dehydration is concentrated to about 1mL by rotary evaporation, transferred to a magnesium silicate packed column for purification, 12mL of normal hexane is used for eluting the purification column, the eluent is collected, and the petroleum solution is concentrated by rotary evaporation again and the volume is fixed to 1.0mL. GC-MS (Agilent 7890A, USA) analyzed the extracts for petroleum hydrocarbon components. Analysis and calculation of the results were performed according to the methods of the institute of ecological standards in gas chromatography for determination of petroleum hydrocarbons (C10-C40) for soil and sediments (HJ 1021-2019).
(3) Analysis of results: the degradation rate of the Trichoderma harzianum ZL-811 after 7d to the total petroleum hydrocarbon was 69.33% compared to the control group, as shown in FIG. 6, panel A. Experiments on degradation characteristics of different components of crude oil show that the strain ZL-811 has obvious degradation effects on degradation rates of normal alkane (n-alcaine), isoparaffin (isoparaffin) and aromatic hydrocarbon (aromatic hydrocarbon) in the crude oil, wherein the degradation rates are 83.65%, 75.11% and 51.68%, respectively, as shown in a graph B in FIG. 6.
Example 5
Test of plant growth promotion by Trichoderma harzianum ZL-811 spore liquid under salt stress:
test material: trichoderma harzianum ZL-811 spore liquid, wheat (Jimai 22) and cucumber (Jinyan No. four)
Germination tests were performed in the laboratory. Selecting healthy and full seeds, sterilizing with 75% alcohol for 30s, sterilizing with 2% NaClO surface for 3min, washing with sterile water for 4-5 times, and air-drying in an ultra-clean workbench until the surface of the seeds is free of water. A culture dish with the diameter of 13cm, 3 layers of sterilization filter paper and 10mL of corresponding germination solution are selected for 20 seeds in each dish. Under the treatment of 2% NaCl, sterile water is used as germination solution as control group, and Trichoderma spore solution (final concentration 10 5 cfu/mL) was set for treatment groups, 5 replicates per treatment. The germination of the seeds was counted and the radicle length was measured.
The results show that: the effect of Trichoderma harzianum ZL-811 on seed germination in 2% NaCl treatment is shown in Table 1 (wheat) and Table 2 (cucumber). After the Trichoderma harzianum ZL-811 is treated, the germination rate and the radicle growth index of the wheat and cucumber seeds are obviously improved under the salt stress, which indicates that the Trichoderma harzianum ZL-811 can obviously improve the seed germination rate under the salt stress and promote the growth of seed embryos.
TABLE 1 influence of Trichoderma harzianum ZL-811 on wheat seed germination under salt stress
TABLE 2 influence of Trichoderma harzianum ZL-811 on cucumber seed germination under salt stress
The seedling growth effect test was performed in a laboratory. Selecting full and consistent seeds for disinfection treatment, shading and accelerating germination in a 25 ℃ incubator until the seeds are exposed to white, then carrying out 16h illumination/8 h darkness, and after a certain period of cultivation, selecting seedlings with consistent growth vigor, and transferring the seedlings to a hydroponic device for treatment. 1/2Hoagland nutrient solution containing 2% NaCl is used as control group, and Trichoderma spore solution (final concentration 10) 5 ) For the treatment group, 5 replicates were set for each treatment, and plant height and fresh weight were measured 7d after treatment.
The results show that: the effect of Trichoderma harzianum ZL-811 on seedling growth in 2% NaCl treatment is shown in Table 3 (wheat) and Table 4 (cucumber). After the Trichoderma harzianum ZL-811 is treated, the seedlings of the wheat and the cucumber grow under the salt stress, which shows that the Trichoderma harzianum ZL-811 can obviously promote the growth of the seedlings under the salt stress and increase the biomass of the seedlings.
TABLE 3 influence of Trichoderma harzianum ZL-811 on wheat seedling growth under salt stress
TABLE 4 influence of Trichoderma harzianum ZL-811 on cucumber seedling growth under salt stress
Example 6
Test of prevention and treatment of plant diseases by Trichoderma harzianum ZL-811 microbial inoculum:
the test was set up with 2 treatments, including a fresh water control, trichoderma harzianum ZL-811 spore liquid treatment.
Tobacco black shank field inhibition experiment: each agent was applied 3 times, 1 st time at the time of transplanting, 2 nd time 10d after transplanting, 3 rd time 20d after transplanting. Spraying the whole plant, and spraying the stem base to make the liquid medicine flow along the stem base to the surface soil around the rhizosphere. And (5) after transplanting for 30 days, observing whether phytotoxicity occurs, performing control effect investigation, sampling 5 points in each cell, and selecting 30 plants in each point.
Wheat powdery mildew field inhibition experiment: the powdery mildew is sprayed by a manual sprayer in the initial stage. And (3) performing control investigation 30d after the medicine, sampling 5 points in each cell, and selecting 30 strains in each point.
Tomato wilt field inhibition experiment: and selecting a field with serious tomato wilt and continuous cropping for more than 3 years. Each treatment was repeated 3 times. Transplanting the tomato 6-7 true leaves, and inoculating the tomato fusarium wilt germs by adopting a spore suspension root irrigation method during transplanting. And (3) transplanting for 10 days, performing primary root irrigation treatment, wherein each seedling is 300mL, and root irrigation is performed for 1 time at 15d intervals, and 2 times of total control are performed. And (5) after 30d, investigation of the disease condition of the wilt of the tomato plants, and calculation of the control effect.
Control effect (%) = (control disease She Pingjun severity-treatment disease She Pingjun severity)/control disease She Pingjun severity×100.
The result shows that: after the trichoderma harzianum ZL-811 spore liquid is sprayed, the incidence rate of wheat powdery mildew, tobacco black shank and tomato fusarium wilt is obviously reduced, and the control effect is good (table 5).
TABLE 5 field control Effect of Trichoderma harzianum ZL-811 spore liquid spray on wheat powdery mildew, tobacco black shank and tomato blight
Example 7
Preparation and activity detection of Trichoderma harzianum ZL-811 microbial inoculum:
the trichoderma multifunctional microbial agent is formed by mixing trichoderma harzianum strain ZL-811 chlamydospore liquid and microbial agent carrierThe chlamydospore content of the Trichoderma harzianum strain ZL-811 was 2X (10 6 -10 8 )CFU·g -1 The microbial inoculum carrier comprises diatomite, turf and bran.
Culturing and collecting spores: preparing chlamydospore culture medium (15 g/L KH) 2 PO 4 ,5g/L(NH 4 ) 2 SO 4 ,0.6g/L MgSO 4 ,0.6g/L CaCl 2 ,0.005g/L FeSO 4 ·7H 2 O,0.0016g/L MnSO 4 ·H 2 O,0.0014g/L ZnSO 4 ·7H 2 O,0.0037g/L CoCl 2 ·6H 2 O,20g/L glucose, 1% wheat straw, natural pH value), inoculating 5mL of activated Trichoderma harzianum ZL-811 fermentation broth into 200mL of culture medium, culturing for 10d at 28 ℃ in a constant-temperature shaking box at 180rpm, performing sterile centrifugation, concentrating to collect spore liquid, and calculating chlamydospore yield of Trichoderma harzianum ZL-811 by using a viable bacteria counting method.
Preparation of a microbial inoculum carrier: respectively weighing 3000g of diatomite, 1500g of turf and 750g of bran, carrying out jet milling, sieving with a 300-mesh sieve, and collecting to obtain a culture medium carrier.
Preparation of a composite microbial inoculum: trichoderma harzianum ZL-811 chlamydospore liquid (10) 9 CFU·mL -1 ) Mixing with 200g of microbial inoculum carrier, standing at 35deg.C, oven drying, pulverizing, sieving with 200 mesh sieve, and collecting Trichoderma harzianum ZL-811 microbial inoculum.
Detection of effective active spores in the composite microbial inoculum: taking 1g of Trichoderma harzianum ZL-811 microbial inoculum, taking a long test tube containing 9mL of deionized water, preparing spore suspension, and respectively setting 7 dilution concentrations (10 -1 -10 -7 ) Then, 100. Mu.L of each was added to a plate culture dish, and the PDA medium containing Triton X-100 was poured into the plate containing the bacterial liquid and shaken well. After cooling and solidifying, the culture dish is placed in an incubator upside down, and is cultured for 3 days at 28 ℃ to count colonies, thus completing viable bacteria count.
The results show that: the prepared Trichoderma harzianum ZL-811 microbial inoculum contains active chlamydospore with the spore content of 1.8x10 8 cfu/g. After 6 months and 12 months of storage at normal temperature, the content of active chlamydospores is 1.6X10% 8 cfu/g and 1.3X10 8 cfu/g, maintained high chlamydosporesVitality as shown in figure 7.
Example 8
Improvement test of trichoderma harzianum ZL-811 microbial inoculum on petroleum-contaminated saline-alkali soil:
the collection site of the petroleum polluted saline-alkali soil is near a certain oil extraction well in the solitary east oil extraction area of the victory oil field. The property index of the saline-alkali soil and the petroleum hydrocarbon content are measured before the experiment. 10kg of soil sample is taken, and the prepared Trichoderma harzianum ZL-811 microbial inoculum is added into petroleum polluted saline-alkali soil according to the application amount of 1%, and the mixture is uniformly mixed. After 20d of treatment, soil samples were taken to determine soil property index and petroleum hydrocarbon content again.
Table 6 improvement effect of Trichoderma harzianum ZL-811 microbial inoculum on saline-alkali soil
TABLE 7 degradation effect of Trichoderma harzianum ZL-811 microbial inoculum on petroleum hydrocarbons in petroleum contaminated soil
The results show that: after 20d treatment with Trichoderma harzianum ZL-811 inoculant, the salt content of the petroleum-contaminated saline-alkali soil was significantly reduced, while the organic matter content, compactness, water-stable aggregates, water content and colony number of the soil were significantly increased (Table 6). At the same time, the normal paraffin content, isoparaffin content and aromatic hydrocarbon content in the petroleum-contaminated soil were all significantly reduced (table 7).
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Trichoderma harzianum (Trichoderma harzianum) ZL-811, characterized in that: the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23238 at the 9 th and 18 th of 2021.
2. A microbial agent is characterized in that: spores of Trichoderma harzianum ZL-811 of claim 1 and a carrier, wherein the spores of Trichoderma harzianum ZL-811 are carried on the carrier.
3. The microbial agent of claim 2, wherein: in the microbial agent, the spore loading is 2 multiplied by 10 6 -2×10 8 CFU·g -1 。
4. The microbial agent of claim 2, wherein: the carrier is a mixture of diatomite, turf and bran.
5. The microbial agent of claim 4, wherein: the mass ratio of the diatomite to the turf to the bran is 3-5:1.5-2.5:1.
6. The method for preparing the microbial agent according to any one of claims 2 to 5, which is characterized in that: the method comprises the following steps: and uniformly mixing trichoderma harzianum ZL-811 spore liquid with a carrier, drying, crushing and sieving to obtain the microbial inoculum.
7. The method for preparing the microbial agent according to claim 6, wherein the method comprises the following steps: the drying temperature is 35-40 ℃.
8. The method for preparing the microbial agent according to claim 6, wherein the method comprises the following steps: pulverizing, and sieving with 200 mesh sieve.
9. Use of the trichoderma harzianum (Trichoderma harzianum) ZL-811 of claim 1 or the microbial agent of any one of claims 2-5 in saline-alkali soil remediation or/and petroleum contaminated soil remediation;
or, the application of the trichoderma harzianum (Trichoderma harzianum) ZL-811 of claim 1 or the microbial agent of any one of claims 2-5 in promoting plant seed germination or promoting plant seedling growth under salt stress conditions.
10. Use of the trichoderma harzianum (Trichoderma harzianum) ZL-811 of claim 1 or the microbial agent of any one of claims 2-5 for controlling field diseases.
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| CN102453678A (en) * | 2010-10-26 | 2012-05-16 | 中国科学院生态环境研究中心 | Microorganism composite bacterial agent for restoration of saline alkali soil polluted by petroleum |
| WO2012140304A2 (en) * | 2011-04-15 | 2012-10-18 | Universidad De Jaén | Trichoderma strains that can be used to treat and/or to prevent infections caused by phytopathogenic microorganisms |
| MX2014003305A (en) * | 2014-03-19 | 2015-09-21 | Inst Potosino De Investigación Científica Y Tecnológica A C | Strain of trichoderma harzianum with an antagonistic activity against plant pathogens, compositions containing the same and use thereof. |
| CN111172040A (en) * | 2019-12-09 | 2020-05-19 | 中国农业科学院植物保护研究所 | Trichoderma harzianum, microbial agent and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102453678A (en) * | 2010-10-26 | 2012-05-16 | 中国科学院生态环境研究中心 | Microorganism composite bacterial agent for restoration of saline alkali soil polluted by petroleum |
| WO2012140304A2 (en) * | 2011-04-15 | 2012-10-18 | Universidad De Jaén | Trichoderma strains that can be used to treat and/or to prevent infections caused by phytopathogenic microorganisms |
| MX2014003305A (en) * | 2014-03-19 | 2015-09-21 | Inst Potosino De Investigación Científica Y Tecnológica A C | Strain of trichoderma harzianum with an antagonistic activity against plant pathogens, compositions containing the same and use thereof. |
| CN111172040A (en) * | 2019-12-09 | 2020-05-19 | 中国农业科学院植物保护研究所 | Trichoderma harzianum, microbial agent and application thereof |
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