CN116622517A - Method for preventing and controlling quinoa fungus diseases and promoting quinoa growth by utilizing trichoderma harzianum - Google Patents
Method for preventing and controlling quinoa fungus diseases and promoting quinoa growth by utilizing trichoderma harzianum Download PDFInfo
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- DOQPXTMNIUCOSY-UHFFFAOYSA-N [4-cyano-4-(3,4-dimethoxyphenyl)-5-methylhexyl]-[2-(3,4-dimethoxyphenyl)ethyl]-methylazanium;chloride Chemical compound [H+].[Cl-].C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 DOQPXTMNIUCOSY-UHFFFAOYSA-N 0.000 description 3
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
Trichoderma harzianum strainTrichoderma afroharzianum) The LMNS-M9 strain is preserved in China general microbiological culture Collection center (CGMCC) at the 3 rd month and 10 th year 2023, and the preservation number is CGMCC No.40523, and the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing. Utilizing Trichoderma harzianumTrichoderma afroharzianum) The method for preparing the solid biological agent by LMNS-M9 comprises the following steps: activating the strain, preparing spore-forming strains and preparing solid biological agents. Prepared solid raw materialThe application method of the microbial inoculum comprises a seedling culture application method, a transplanting application method, an open field direct seeding application method and a growing period application method. The Trichoderma harzianum LMNS-M9 strain has the functions of preventing and controlling quinoa fungus diseases, promoting the germination of quinoa seeds, promoting the development of root systems and enhancing the activity of the root systems.
Description
Technical Field
The invention belongs to the field of control of quinoa fungus diseases, and in particular relates to a method for controlling trichoderma harzianum by utilizing trichoderma harzianumTrichoderma afroharzianum) A method for preventing and treating fungal disease of quinoa and promoting germination of quinoa seeds, promoting root development and enhancing root activity.
Background
Quinoa is small grains, the nutrition of seeds is limited, and technical problems of no emergence, uneven emergence, weak seedlings and the like in conventional planting often occur. In addition, quinoa shallow root plants are young and underdeveloped in root system after emergence of seedlings, and weak seedlings are easy to form. Another important factor affecting quinoa quality and yield in production is fungal disease. At present, the quinoa fungal diseases tend to be aggravated year by year, and the main reason is that the number of the pathogenic fungi group is more accumulated, so that the diseases are easy to occur in a large area; the heavier leaf spot disease, ear rot, black stem disease, gray mold and the like, the yield loss is about 10% -30%, and the serious loss is more than 80%. However, quinoa belongs to small crops, available control methods are limited, and the problem of 'no standard can be used and no medicine can be used' is faced in actual production. Therefore, it is urgent to find an environmentally friendly and efficient control method.
Trichoderma harzianumTrichoderma afroharzianum) The microbial inoculum has obvious effects of nutrition competition, re-mailing and the like, and can stop the growth or infection of germs, so that the microbial inoculum is applied as biocontrol bacteria. However, there are differences in practical applicationsThe strain of Trichoderma harzianum from which the strain has diverse spore-producing capability, secondary metabolites, culture conditions and the like has uneven control effect and unobvious seedling strengthening effect, and the method can not be used for solving a plurality of technical problems of different crops such as growth promotion and disease prevention methods.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preventing and controlling quinoa fungus diseases by utilizing trichoderma harzianum, promoting the early germination of quinoa seeds, effectively promoting root system development and enhancing root system activity.
The biocontrol bacteria obtained by screening of the invention are trichoderma harzianum strain which is endophytic to quinoa seedsTrichoderma afroharzianum) LMNS-M9, the strain is preserved in China general microbiological culture collection center (CGMCC) for 3 months and 10 days in 2023, with the preservation number of CGMCC No.40523 and the preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The Trichoderma harzianum isTrichoderma afroharzianum) LMNS-M9 was obtained by harvesting mature quinoa ears from the planting area of Chenopodium aesculum in Xin Jing le county, shanxi province, and separating and screening the seeds thereof in 2021 month 9.
The trichoderma harzianum LMNS-M9 spore-forming cluster is yellow-green, conidiophores are pyramid-shaped, have acute main shaft angles or right-angle branches, and have 3-4 bottle peduncles at the tail end in multiple rounds, and are flask-shaped, and conidiophores are spherical to oval; the average size of the conidiophores was 9.3X2.9. Mu.m, and the average size of the conidiophores was 4.1X2.9. Mu.m (FIGS. 1-4). Construction of phylogenetic Tree with tef1 Gene sequence, LMNS-M9 andT. afroharzianumis the closest, the 98% self-display support rate is clustered into one branch (fig. 5).
Trichoderma harzianum LMNS-M9 has the functions of bacteriostasis, prevention and treatment of fungal diseases, nutrition and space competition advantages, heavy parasitism, growth promotion and the like.
First, the pathogen of Trichoderma harzianum LMNS-M9 against 5 quinoa fungus diseasesB.cinerea、A.caulina、F.citri、A.alternata、T.roseum) Has remarkable inhibition effect, and the inhibition rate is 33.3% -61.5%; second, trichoderma harzianum LMNS-M9 inhibitsA.caulina、A.alternata、T.roseumThe isopathy produces conidium, and the reduction range of the sporulation quantity is 70.9% -100%. Third, trichoderma harzianum LMNS-M9 has remarkable nutrition and space competition advantages and can coverA.caulina、F.citri、A.alternateColonies of the pathogen invade the living space of the colonies of the pathogen. Fourth, trichoderma harzianum LMNS-M9 has significant re-mailing effect, and can be contacted and woundB. cinerea、A. caulina、F. citri、A. alternataHyphae of the same pathogen causingB. cinerea、F. citri、A. alternataMycelium of the pathogen breaks or is digested. In a word, through the multiple comprehensive effects, the trichoderma harzianum LMNS-M9 has remarkable control effect on the fungal diseases of the quinoa, and the control effects of the gray mold, the leaf spot, the black stem, the ear rot and the like of the quinoa are 62.6-72.2 percent
Besides remarkable effect of preventing and controlling the fungal diseases of the quinoa, after the trichoderma harzianum LMNS-M9 is prepared into the solid fermentation substrate, the quinoa can be further promoted to emerge at 2 d early, and the growth promoting rate reaches 130.0 percent; the root growth of quinoa seedlings is accelerated, and the growth of the quinoa seedlings is improved by 71.9 percent; the root fresh weight, the total fresh weight and the total dry weight of quinoa are increased by 104.7%, 30.8% and 28.6% respectively.
The method for preparing the solid biological agent by the Trichoderma harzianum LMNS-M9 comprises the following steps:
(1) Strain activation
Inoculating Trichoderma harzianum LMNS-M9 to mycelium culture medium, culturing at 30deg.C under 12 h light/12 h dark environment for 3 d, and keeping after mycelium grows to be full of culture medium;
(2) Preparation of spore-producing strains
Taking 25-30 bacterial cakes with the diameter of 5 mm from the colony edge of the mycelium culture medium in the step (1), inoculating the bacterial cakes to the spore-producing culture medium, shaking uniformly, culturing 7 d in an environment with the temperature of 30 ℃ and the illumination of 12 h/the darkness of 12 h, and standing for later use after spawn is produced;
(3) Preparation of solid biological agent
Inoculating 9% -15% of the spore-producing culture medium of the spore-producing strain prepared in the step (2) to a solid fermentation substrate, uniformly mixing, and finally placing the solid fermentation substrate in an environment of 30 ℃ for culturing 7 d to prepare the solid biological microbial inoculum.
The preparation method of the mycelium culture medium comprises the following steps: weighing glucose 20.0 g, potassium dihydrogen phosphate 1.0 g, peptone 2.0 g, magnesium sulfate 0.5 g, agar 20.0 g and corn flour 40.0 g, mixing completely, adding distilled water 1L, stirring to dissolve completely, adjusting pH to 5 with HCl 1 mol/L, and sterilizing with 121 deg.C high pressure steam for 20 min.
The preparation method of the spore-producing culture medium comprises the following steps: weighing soaked 6 h oat grains 900 g, soaked 1 h quinoa grains 100 g, glucose 20.0 g, dipotassium hydrogen phosphate 1.0 g, ammonium nitrate 2.0 g, magnesium sulfate 0.5 g and corn flour 40.0 g, fully and uniformly mixing the above materials, adjusting pH to 5 by using 1 mol/L HCl, subpackaging in 250 mL triangular flasks, bottling to 100 mL, and sterilizing at 121 ℃ under high pressure steam for 20 min.
The preparation method of the solid fermentation substrate comprises the following steps: fully and uniformly mixing turf and vermiculite according to a ratio of 1:1; then the mixture of turf and vermiculite 100 parts by mass, glucose 2.0 parts, dipotassium hydrogen phosphate 0.1 parts, ammonium nitrate 0.2 parts, magnesium sulfate 0.05 parts and corn flour 4.0 parts are uniformly mixed, the water content is adjusted to 25% -28%, the pH is adjusted to 5, and the mixture is subjected to high-pressure steam sterilization at 121 ℃ for 20 min.
Use method of Trichoderma harzianum LMNS-M9 solid biological agent
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacterial agent into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa is sown until seedlings emerge.
(2) Transplanting application method
And (3) applying solid biological bacteria to the position 8-10 cm away from the quinoa plants, about 10-15 kg of solid biological bacteria per mu, applying a solid fermentation matrix, covering a mulching film, and transplanting quinoa seedlings after 7-10 d.
(3) Live broadcast application method
Ditching according to a row spacing of 35-45 cm, wherein the ditch depth is 5-8 cm, applying solid biological bacteria agent to the ditch, and covering a mulching film after sowing, wherein each mu is about 15-20 kg.
(4) Method of use in growth phase
And (3) applying solid biological bacteria to the positions 10-15 and cm away from quinoa plants in a hole mode, wherein 35-40 kg of solid biological bacteria are applied to each mu of plants.
Identification of trichoderma harzianum LMNS-M9 and test for preventing and treating quinoa fungus diseases and promoting quinoa growth
1. Identification of quinoa seed endophytic biocontrol strain LMNS-M9
(1) Test method
Mature quinoa ears are collected in the area of planting of the quinoa in the Qin county of Xin Zhou, shanxi province, and endophytic fungi in quinoa seeds are obtained by applying an endophyte separation screening method from the seeds. Sequentially soaking quinoa seeds in 75% ethanol for 1 min,1% sodium hypochlorite for 5 min,75% ethanol for 1 min, rinsing with sterile water for 3 times, and taking the rinsing liquid of the last 1 times as blank control. And (3) naturally air-drying the rinsed seeds in a sterile environment, and then inoculating the seeds to a PDA (personal digital assistant) for dark culture at 25 ℃. After the bacterial colony is formed on the surface of the seed, picking the edge hypha on PDA by using an inoculating needle, culturing at 25 ℃, repeatedly purifying for 3-5 generations, and preserving the purified bacterial strain at 4 ℃ for later use. Inoculating the strain to PDA25 ℃ for culturing 7 d, and observing colony morphology; the morphology of conidia, bottle peduncles, etc. was observed under a BX53 microscope using the modified insert method, and the relevant microscopic data were measured.
(2) Test results
The endophytic fungi 9 strain is separated from quinoa seeds, and the pre-experiment shows that LMNS-M9 has better antibacterial effect on quinoa disease pathogen. Morphological characteristics of strain LMNS-M9: on PDA, yellowish green spore-forming clusters were observed (fig. 1); conidiophores are pyramid-shaped, the main shaft is acutely or right-angle branched, 3-4 bottle peduncles are generated at the tail end in multiple rounds, the bottle peduncles are flask-shaped, and conidiophores are spherical to oval (figure 2); the average size of the conidiophores was 9.3X2.9. Mu.m, and the average size of the conidiophores was 4.1X2.9. Mu.m (FIG. 3); compared with the existing Trichoderma harzianum related strain, the LMNS-M9 has larger conidia.
Molecular biology characteristics: the length of the tef1 gene sequence of LMNS-M9 is 1241 bp, and the number of the gene is OQ509672 in GenBank. At tConstruction of phylogenetic tree, LMNS-M9 and Ef1 Gene sequenceT. afroharzianumThe self-exhibition support rate was 98% (fig. 4). Comprehensive morphological and phylogenetic analysis to determine LMNS-M9 as Trichoderma harzianumTrichoderma afroharzianum。
Test of pathogenic bacteria of bacteriostatic quinoa fungus disease
(1) Test method
And (3) antibacterial effect measurement: trichoderma harzianum LMNS-M9 was cultured at 25℃for 5 d, trichoderma harzianum LMNS-M9 was inoculated to 5 mm from the edge of PDA, and 5 kinds of cakes (Alternaria alternata) of quinoa fungal disease pathogens were inoculated at the center symmetry point thereofA. alternataBotrytis cinereaB. cinereaFusarium citriF. citriTrichothecene PinkT. roseumTwo spore of aschersonia aleyrodisA. caulina) Only the pathogenic bacteria were inoculated as a control, and the culture was repeated 3 times at 25 ℃. Colony radius was observed and measured at 7 d and the bacteriostatic rate was calculated. Antibacterial ratio (%) = (colony radius of control-treated colony radius)/colony radius of control x 100.
Competition assay: the Trichoderma antagonistic coefficients were graded, grade I: trichoderma reesei silk coverage rate 100%; stage II: the coverage rate of trichoderma reesei wires is more than 2/3; III grade: 1/3< trichoderma reesei coverage <2/3; grade IV: trichoderma reesei silk coverage <1/3; v level: the coverage rate of pathogenic bacteria hypha is 100%.
Determination of the redirection effect: culturing by smear inoculation method, culturing LMNS-M9 and the pathogen under test at 25deg.C for 3 d, collecting mycelium, eluting with sterile water to obtain mycelium suspension. PDA to be sterilized was cooled to about 50 c and 1 mL was uniformly applied to a sterile slide of about 4.0 cm x 2.5 cm x 0.1 cm. After the bacterial suspension is solidified, LMNS-M9 and mycelium suspension 20L of the pathogen to be tested are respectively taken and inoculated to the two ends of the smear (interval 2 cm), placed in a culture dish paved with sterilized wet filter paper, cultured for 1-2 d times at 25 ℃ and repeated for 3 times, and the re-mailing phenomenon is observed under a microscope.
(2) Test results
Trichoderma harzianum LMNS-M9 was cultivated against 5 pathogenic bacteria, and a zone of inhibition was produced at the junction with 5 pathogenic bacteria (FIGS. 5-9), indicating that strain LMNS-M9 inhibitedB.cinerea、A.caulina、F.citri、A.alternataAndT.roseumis grown on colony wires. Meanwhile, the strain LMNS-M9 has remarkable competitive advantage; when the strain is cultivated in the opposite direction of 12 d, the strain LMNS-M9 can coverA.caulina(FIG. 6),F.citri(FIG. 7),A.alternate(FIG. 8) colonies of 3 pathogens. Trichoderma harzianum LMNS-M9 pairB.cinerea、A.caulina、F.citri、A.alternata、T.roseumThe antibacterial ratio of (a) was 35.9%, 61.5%, 33.3%, 41.9% and 59.1% in this order (see Table 1 for details).
TABLE 1 inhibition of the pathogen of quinoa fungus by Trichoderma harzianum LMNS-M9
As shown in Table 1, the production of Trichoderma harzianum LMNS-M9 inhibits the growth of pathogenic hyphae of 5 quinoa fungal diseases. At the same time, can significantly inhibitA.caulina、A.alternata、T.roseumThe pathogenic fungi generate conidium, so that the population quantity of the pathogenic fungi is controlled. The strain LMNS-M9 can be obviously reducedA.caulina、A.alternata、T.roseumThe generation and reduction of conidium of isopathogenic species were 70.9% -100% (Table 2).
TABLE 2 inhibition of Trichoderma harzianum LMNS-M9 on the pathogenic spore production of quinoa mycosis
Trichoderma harzianum LMNS-M9 mycelium can be contacted and woundB. cinerea(FIG. 10),A. caulina(FIG. 11),F. citri(FIG. 12),A. alternataThe hyphae (indicated by black arrows) of FIG. 13 can be used to makeB. cinerea、A. caulina、F. citri、A. alternataMycelium rupture and digestion of the strain LMNS-M9 shows that the strain LMNS-M9 has a re-mailing effectFig. 10 to 13).
Trichoderma harzianum LMNS-M9 spore production optimization test
(1) Test method
The spore-producing culture medium is configured: weighing soaked 6 h oat grains 900 g, soaked 1 h quinoa grains 100 g, glucose 20.0 g, dipotassium hydrogen phosphate 1.0 g, ammonium nitrate 2.0 g, magnesium sulfate 0.5 g and corn flour 40.0 g, and mixing. Adjusting pH to 5 with 1 mol/L HCl, packaging in 250 mL triangular flask with bottling volume of 100 mL, and sterilizing with 121 deg.C high pressure steam for 20 min. Inoculating the activated strain LMNS-M9 to a spore-forming culture medium, shaking uniformly, and finally culturing in an environment of 30 ℃ and 12 h light/12 h darkness for 7 d; the test shows that the spore yield of the Trichoderma harzianum LMNS-M9 on the spore-producing culture medium is about 2.5X10 9 CFU/mL, significantly higher than other media (table 3).
TABLE 3 production of Trichoderma harzianum LMNS-M9 on different media
Preparing a solid biological agent: fully and uniformly mixing turf and vermiculite according to a ratio of 1:1; then the mixture of turf and vermiculite 100 parts by mass, glucose 2.0 parts, dipotassium hydrogen phosphate 0.1 parts, ammonium nitrate 0.2 parts, magnesium sulfate 0.05 parts and corn flour 4.0 parts are uniformly mixed, the water content is adjusted to 25% -28%, the pH is adjusted to 5, and the mixture is subjected to high-pressure steam sterilization at 121 ℃ for 20 min. After the strain is cooled, inoculating the spore-forming strain to a sterile solid fermentation substrate according to the mass percentage of 10%, uniformly mixing, and culturing in an environment of 30 ℃ for 7 d to prepare the solid biological microbial inoculum.
(2) Test results
Experiments show that the spore production capacity and the spore production quantity of the Trichoderma harzianum LMNS-M9 can be obviously improved by about 6.0 multiplied by 10 on the solid biological microbial inoculum 9 CFU/mL, significantly higher than other media (see table 3).
Test for promoting germination of quinoa seeds and test for promoting growth of quinoa seedlings by trichoderma harzianum LMNS-M9
(1) Test method
The treatment is that the Trichoderma harzianum LMNS-M9 solid biological agent is subpackaged into nutrition bowls (caliber 11 cm, bottom diameter 8 cm, basin height 10 cm), and 10 seeds of quinoa seeds to be tested are sown in each basin; the control is unfermented sterile matrix, and the surface of the matrix is covered with plastic film after the quinoa is sown until seedlings emerge, and the process is repeated for 5 times. And counting the emergence rate every day after sowing by 2-4 d.
(2) Test results
The test result shows that compared with the control, the quinoa on the solid biological microbial agent containing the trichoderma harzianum LMNS-M9 can be advanced by 2 d to achieve 130.0 percent of seedling promotion rate, and the seedling rate can be up to 69.0 percent when the quinoa is 2 d on the solid fermentation substrate containing the trichoderma harzianum LMNS-M9 (table 4).
TABLE 4 promotion of germination and root development of quinoa seeds by Trichoderma harzianum LMNS-M9
The root length of the seedlings of the treatment group is obviously higher than that of the control group, which can reach 9.32 and cm, and is improved by 71.9 percent (table 4); meanwhile, the fresh weight, the total fresh weight and the total dry weight of the treated group root are respectively 0.06 g, 1.02 g and 0.09 g, which are improved by 104.7%, 30.8% and 28.6% compared with a control group (Table 4), which shows that the germination of the quinoa seeds is improved, the root development is effectively promoted and the root activity is enhanced on the solid biological inoculant containing the trichoderma harzianum LMNS-M9.
Field control effect test of strain LMNS-M9
(1) Test method
The open field direct seeding method is adopted, ditches are carried out according to the line spacing of 35-45 cm (about 5 cm-8 cm), the solid biological inoculant of trichoderma harzianum LMNS-M9 in ditches Shi Feizhou is about 15-20 kg/mu, and mulching films are covered and moisture is kept after seeding. At the position 8-10 cm away from quinoa plants in the seedling stage, 35-40 kg of solid biological inoculant of trichoderma harzianum LMNS-M9 is applied per mu, and no inoculant is applied to make blank group control. In the disease occurrence period of quinoa, the morbidity of gray mold, leaf spot, black stem, spike rot and the like of quinoa are counted by adopting a random investigation method.
(2) Test results
The test result shows that the solid biological inoculant of trichoderma harzianum LMNS-M9 disclosed by the invention has remarkable control effect on quinoa fungus diseases, compared with a control group, the morbidity of quinoa gray mold, quinoa leaf spot, quinoa black stem disease, quinoa ear rot and other diseases is remarkably reduced, the control effect on the 4 quinoa fungus diseases is remarkable, and the control effect is 62.6% -72.2% (Table 5).
TABLE 5 control Effect of Trichoderma harzianum LMNS-M9 on quinoa fungal diseases
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention relates to a method for treating pathogeny of 5 quinoa fungus diseases by Trichoderma harzianum LMNS-M9B.cinerea、A.caulina、F.citri、A.alternata、T.roseum) Has remarkable inhibition effect, and the inhibition rate is 33.3% -61.5%. Meanwhile, the strain LMNS-M9 has remarkable nutrition and space competition advantages and can coverA.caulina、F.citri、A.alternateColonies of the pathogen invade the living space of the colonies of the pathogen. In addition, LMNS-M9 can inhibit pathogenic fungi from generating conidium, and can reduce the population quantity of pathogenic fungi controlled by the conidium; will beA.caulina、A.alternata、T.roseumThe content of the conidium of the isopathogenic agent is greatly reduced by 70.9% -100%. The strain LMNS-M9 has obvious re-mailing effect and can be contacted and woundB. cinerea、A. caulina、F. citri、A. alternataHyphae of the same pathogen causingB. cinerea、F. citri、A. alternataMycelium of the pathogen breaks or is digested.
(2) The trichoderma harzianum LMNS-M9 disclosed by the invention has good application effects on the emergence and growth promotion of quinoa in the field, and compared with the control, the quinoa on the solid fermentation substrate containing the trichoderma harzianum LMNS-M9 can be advanced by 2 d, and the growth promotion rate is up to 130.0%. The root length of quinoa seedlings on the solid fermentation substrate containing trichoderma harzianum LMNS-M9 is obviously higher than that of a control group, and the improvement is 71.9%; meanwhile, the root fresh weight, the total fresh weight and the total dry weight of the quinoa are obviously higher than those of a control group, and are respectively improved by 104.7%, 30.8% and 28.6%.
(3) The invention optimizes the spore production condition of the Trichoderma harzianum LMNS-M9 and obviously improves the spore production capacity of the Trichoderma harzianum LMNS-M9 on the solid biological microbial agent. The results of the examples show that: the spore yield is 2.5 multiplied by 10 9 CFU/mL was increased to 6.0X10 9 CFU/mL。
(4) The trichoderma harzianum LMNS-M9 disclosed by the invention can reduce the incidence rate of quinoa fungus diseases and has a remarkable control effect. The results of the examples show that: the control effect on diseases such as gray mold, leaf spot, black stem, spike rot and the like of quinoa is 62.6% -72.2%.
Brief description of the drawings and the accompanying tables
FIG. 1 shows the spore former of Trichoderma harzianum LMNS-M9 of the present invention on PDA.
FIG. 2 shows the conidiophore and phialide of Trichoderma harzianum LMNS-M9 of the present invention.
FIG. 3 shows conidia of Trichoderma harzianum LMNS-M9 of the present invention.
FIG. 4 is a phylogenetic tree of Trichoderma harzianum LMNS-M9 of the present invention.
FIG. 5 shows Trichoderma harzianum LMNS-M9 and Trichoderma harzianum of the present inventionB. cinereaIs a counter culture of the strain.
FIG. 6 shows Trichoderma harzianum LMNS-M9 and Trichoderma harzianum of the present inventionA. caulinaIs a counter culture of the strain.
FIG. 7 shows Trichoderma harzianum LMNS-M9 and Trichoderma harzianum of the present inventionF. citriIs a counter culture of the strain.
FIG. 8 shows Trichoderma harzianum LMNS-M9 and Trichoderma harzianum of the present inventionA. alternateIs a counter culture of the strain.
FIG. 9 shows Trichoderma harzianum LMNS-M9 and Trichoderma harzianum of the present inventionT.roseumIs a counter culture of the strain.
FIG. 10 shows a Trichoderma harzianum LMNS-M9 pair of the present inventionB. cinereaIs a re-parasitic effect of (a) on the host.
FIG. 11 shows a Trichoderma harzianum LMNS-M9 pair of the present inventionA. caulinaIs to be redirected toIs used.
FIG. 12 shows a Trichoderma harzianum LMNS-M9 pair of the present inventionF. citriIs a re-parasitic effect of (a) on the host.
FIG. 13 shows a Trichoderma harzianum LMNS-M9 pair of the present inventionA. alternataIs a re-parasitic effect of (a) on the host.
Detailed Description
Example 1
The invention promotes the germination test of quinoa seeds and the growth of quinoa seedlingsTrichoderma afroharzianum) LMNS-M9 has been preserved in China general microbiological culture collection center (CGMCC) for 3 months and 10 days 2023, with a preservation address: beijing, chaoyang area, north Chenxi Lu No. 1, 3; the preservation number is CGMCC No.40523.
Example 2
The method for preparing the solid biological agent by the Trichoderma harzianum LMNS-M9 comprises the following steps:
(1) Strain activation
Inoculating Trichoderma harzianum LMNS-M9 to mycelium culture medium, culturing at 30deg.C under 12 h light/12 h dark environment for 3 d, and keeping after mycelium grows to be full of culture medium;
(2) Preparation of spore-producing strains
Taking 25-30 bacterial cakes with the diameter of 5 mm from the colony edge of the mycelium culture medium in the step (1), inoculating the bacterial cakes to the spore-producing culture medium, shaking uniformly, culturing 7 d in an environment with the temperature of 30 ℃ and the illumination of 12 h/the darkness of 12 h, and standing for later use after spawn is produced;
(3) Preparation of solid biological agent
Inoculating 9% -15% of the spore-producing culture medium of the spore-producing strain prepared in the step (2) to a solid fermentation substrate, uniformly mixing, and finally placing the solid fermentation substrate in an environment of 30 ℃ for culturing 7 d to prepare the solid biological microbial inoculum.
The preparation method of the mycelium culture medium comprises the following steps: weighing glucose 20.0 g, potassium dihydrogen phosphate 1.0 g, peptone 2.0 g, magnesium sulfate 0.5 g, agar 20.0 g and corn flour 40.0 g, mixing completely, adding distilled water 1L, stirring to dissolve completely, adjusting pH to 5 with HCl 1 mol/L, and sterilizing with 121 deg.C high pressure steam for 20 min.
The preparation method of the spore-producing culture medium comprises the following steps: weighing soaked 6 h oat grains 900 g, soaked 1 h quinoa grains 100 g, glucose 20.0 g, dipotassium hydrogen phosphate 1.0 g, ammonium nitrate 2.0 g, magnesium sulfate 0.5 g and corn flour 40.0 g, fully and uniformly mixing the above materials, adjusting pH to 5 by using 1 mol/L HCl, subpackaging in 250 mL triangular flasks, bottling to 100 mL, and sterilizing at 121 ℃ under high pressure steam for 20 min.
The preparation method of the solid fermentation substrate comprises the following steps: fully and uniformly mixing turf and vermiculite according to a ratio of 1:1; then the mixture of turf and vermiculite 100 parts by mass, glucose 2.0 parts, dipotassium hydrogen phosphate 0.1 parts, ammonium nitrate 0.2 parts, magnesium sulfate 0.05 parts and corn flour 4.0 parts are uniformly mixed, the water content is adjusted to 25% -28%, the pH is adjusted to 5, and the mixture is subjected to high-pressure steam sterilization at 121 ℃ for 20 min.
Example 3
The application method of the Trichoderma harzianum LMNS-M9 solid biological agent comprises the following steps:
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacterial agent into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa is sown until seedlings emerge.
(2) Transplanting application method
And (3) applying solid biological bacteria to the holes of the quinoa plants at a distance of 8-cm, applying solid fermentation matrix to about 10 kg/mu, covering the holes with mulching film, and transplanting quinoa seedlings after 7-d.
(3) Live broadcast application method
Ditching according to the row spacing of 35 cm, ditching depth of 5 cm, applying solid biological bacteria to ditches, about 15 kg per mu, and covering mulching film after sowing.
(4) Method of use in growth phase
At a distance of 10 to cm from quinoa plants, 35 kg of solid biological microbial inoculum is applied in holes per mu.
Example 4
The application method of the Trichoderma harzianum LMNS-M9 solid biological agent comprises the following steps:
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacterial agent into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa is sown until seedlings emerge.
(2) Transplanting application method
And (3) applying solid biological bacteria to the holes of the quinoa plants at a distance of 10 to cm, applying solid fermentation matrix at a ratio of about 15 kg/mu, covering a mulching film, and transplanting quinoa seedlings after 10 d.
(3) Live broadcast application method
Ditching according to the row spacing of 45 cm, wherein the ditch depth is 8 cm, applying solid biological bacteria agent to the ditch, and covering the ditch with a mulching film after sowing, wherein each mu is about 20 kg.
(4) Method of use in growth phase
At a distance of 15 to cm from quinoa plants, the solid biological inoculant is applied in holes, and 40 kg of the solid biological inoculant is applied per mu.
Example 5
The invention relates to a method for using Trichoderma harzianum LMNS-M9 solid biological agent
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacterial agent into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa is sown until seedlings emerge.
(2) Transplanting application method
And (3) applying solid biological bacteria to the holes of the quinoa plants at a distance of 8 cm, applying solid fermentation matrix at a ratio of about 15 kg/mu, covering a mulching film, and transplanting quinoa seedlings after 7 d.
(3) Live broadcast application method
Ditching according to the row spacing of 45 cm, ditching depth of 5 cm, applying solid biological bacteria to ditches, and covering mulch film after sowing, wherein each mu is about 20 kg.
(4) Method of use in growth phase
At a distance of 10 to cm from quinoa plants, the solid biological inoculant is applied in holes, and 40 kg of the solid biological inoculant is applied per mu.
Example 6
The invention relates to a method for using Trichoderma harzianum LMNS-M9 solid biological agent
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacterial agent into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa is sown until seedlings emerge.
(2) Transplanting application method
And (3) applying solid biological bacteria to the holes of the quinoa plants at a distance of 10 to cm, applying solid fermentation matrix to about 10 kg per mu, covering a mulching film, and transplanting quinoa seedlings after 10 d.
(3) Live broadcast application method
Ditching according to the row spacing of 35 cm, wherein the ditch depth is 8 cm, applying solid biological bacteria to the ditch, and covering the ditch with a mulching film after sowing, wherein each mu is about 15 kg.
(4) Method of use in growth phase
At a distance of 15 to cm from quinoa plants, 35 kg of solid biological microbial inoculum is applied in holes per mu.
Claims (3)
1. Trichoderma harzianum strainTrichoderma afroharzianum) The LMNS-M9 strain is preserved in China general microbiological culture Collection center (CGMCC) at the 3 rd month and 10 th year 2023, and the preservation number is CGMCC No.40523, and the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
2. Utilizing Trichoderma harzianumTrichoderma afroharzianum) The method for preparing the solid biological agent by LMNS-M9 comprises the following steps:
(1) Strain activation
Inoculating Trichoderma harzianum LMNS-M9 to mycelium culture medium, culturing at 30deg.C under 12 h light/12 h dark environment for 3 d, and keeping after mycelium grows to be full of culture medium;
(2) Preparation of spore-producing strains
Taking 25-30 bacterial cakes with the diameter of 5 mm from the colony edge of the mycelium culture medium in the step (1), inoculating the bacterial cakes to the spore-producing culture medium, shaking uniformly, culturing 7 d in an environment with the temperature of 30 ℃ and the illumination of 12 h/the darkness of 12 h, and standing for later use after spawn is produced;
(3) Preparation of solid biological agent
Inoculating 9% -15% of the spore-producing culture medium of the spore-producing strain prepared in the step (2) to a solid fermentation substrate, uniformly mixing, and finally placing the solid fermentation substrate in an environment of 30 ℃ for culturing 7 d to prepare a solid biological microbial inoculum;
the preparation method of the mycelium culture medium comprises the following steps: weighing glucose 20.0 g, potassium dihydrogen phosphate 1.0 g, peptone 2.0 g, magnesium sulfate 0.5 g, agar 20.0 g and corn flour 40.0 g, mixing completely, adding distilled water 1L, stirring to dissolve completely, adjusting pH to 5 with HCl 1 mol/L, and sterilizing with 121 deg.C high pressure steam for 20 min;
the preparation method of the spore-producing culture medium comprises the following steps: weighing soaked 6 h oat grains 900 g, soaked 1 h quinoa grains 100 g, glucose 20.0 g, dipotassium hydrogen phosphate 1.0 g, ammonium nitrate 2.0 g, magnesium sulfate 0.5 g and corn flour 40.0 g, fully and uniformly mixing the above materials, adjusting pH to 5 by using 1 mol/L HCl, subpackaging in 250 mL triangular flasks, bottling to 100 mL, and sterilizing at 121 ℃ under high pressure for 20 min;
the preparation method of the solid fermentation substrate comprises the following steps: fully and uniformly mixing turf and vermiculite according to a ratio of 1:1; then the mixture of turf and vermiculite 100 parts by mass, glucose 2.0 parts, dipotassium hydrogen phosphate 0.1 parts, ammonium nitrate 0.2 parts, magnesium sulfate 0.05 parts and corn flour 4.0 parts are uniformly mixed, the water content is adjusted to 25% -28%, the pH is adjusted to 5, and the mixture is subjected to high-pressure steam sterilization at 121 ℃ for 20 min.
3. The method for using the solid biological agent prepared in the claim 2 comprises the following 4 steps:
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacterial agent into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa sowing until seedlings emerge;
(2) Transplanting application method
Applying solid biological bacteria to the position 8-10 cm away from the quinoa plant in a hole mode, applying about 10-15 kg of solid biological bacteria per mu, covering a mulching film after applying a solid fermentation matrix, and transplanting quinoa seedlings after 7-10 d;
(3) Live broadcast application method
Ditching according to a row spacing of 35-45 cm, wherein the ditch depth is 5-8 cm, applying solid biological bacteria to the ditch, and covering a mulching film after sowing, wherein each mu is about 15-20 kg;
(4) Method of use in growth phase
And (3) applying solid biological bacteria to the positions 10-15 and cm away from quinoa plants in a hole mode, wherein 35-40 kg of solid biological bacteria are applied to each mu of plants.
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Non-Patent Citations (2)
| Title |
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| OSCAR M. ROLLANO-PEÑALOZA 等: "Transcriptomic Analysis of Quinoa Reveals a Group of Germin-Like Proteins Induced by Trichoderma", 《FRONTIERS IN FUNGAL BIOLOGY》, 1 December 2021 (2021-12-01), pages 1 - 14 * |
| 田淼 等: "非洲哈茨木霉LMNS-M9的鉴定、生物学特性及其对藜麦的促生作用", 《微生物学通报》, 20 September 2023 (2023-09-20), pages 3848 - 3865 * |
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