CN116622517B - 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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Abstract
Trichoderma harzianum (Trichoderma afroharzianum) LMNS-M9 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.40523 and the preservation address in 2023, 3 and 10 days: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, beijing, chao's area. The method for preparing the solid biological agent by utilizing the Trichoderma harzianum (Trichoderma afroharzianum) LMNS-M9 comprises the following steps: activating the strain, preparing spore-forming strains and preparing solid biological agents. The application method of the prepared solid biological agent 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 growth of root systems and enhancing the activity of the root systems.
Description
Technical Field
The invention belongs to the field of control of quinoa fungal diseases, and particularly relates to a method for controlling quinoa fungal diseases and promoting germination of quinoa seeds, promoting root development and enhancing root activity by using trichoderma harzianum (Trichodermaafroharzianum).
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 harzianum (Trichodermaafroharzianum) has remarkable nutrition competition, re-mailing and other effects, so that germs can stop growing or infecting, and the trichoderma harzianum is used as biocontrol bacteria. However, in practical application, the strains of Trichoderma harzianum of different sources have quite different spore-producing capacity, secondary metabolites, culture conditions and the like, the control effect is uneven, the seedling strengthening effect is not obvious, and the method for promoting the growth and preventing the diseases of different crops cannot be used for a plurality of technical problems.
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 are Trichoderma harzianum (Trichodermaafroharzianum) LMNS-M9 which is grown in quinoa seeds, and the strain is preserved in China general microbiological culture collection center (CGMCC) for 3 months and 10 days in 2023, wherein the preservation number is CGMCC No.40523, and the preservation address is: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, beijing, chao's area.
The trichoderma harzianum (Trichodermaafroharzianum) LMNS-M9 is obtained by collecting mature quinoa ears in a buckeye county buckeye of Xinzhou city in Shanxi province in 2021 and 9 months, separating and screening seeds of the mature quinoa ears.
The trichoderma harzianum LMNS-M9 spore-forming clusters are 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). The phylogenetic tree was constructed with tef1 gene sequence, LMNS-M9 was closest to the relatedness of T.afroharzianum, and 98% of self-expanding support rate gathered as 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.
Firstly, trichoderma harzianum LMNS-M9 has remarkable inhibition effect on pathogens (B.cinerea, A.calulina, F.citri, A.alternata and T.roseum) of 5 quinoa fungus diseases, and the inhibition rate is 33.3% -61.5%; and secondly, the Trichoderma harzianum LMNS-M9 can inhibit diseases such as A.calulina, A.alternata, T.roseum and the like from generating conidium, and the reduction of the sporulation is 70.9% -100%. Thirdly, the Trichoderma harzianum LMNS-M9 has obvious nutrition and space competition advantages, can cover the bacterial colonies of the pathogens such as A.calulina, F.citri, A.alternate and the like, and invades the living space of the bacterial colonies of the pathogens. Fourthly, the trichoderma harzianum LMNS-M9 has obvious re-mailing effect, and can contact and wind hyphae of pathogens such as B.cinerea, A.calulina, F.citri, A.alternata and the like, so that the hyphae of pathogens such as B.cinerea, F.citri, A.alternata and the like are broken or digested. In a word, through the multiple comprehensive effects, the African trichoderma harzianum LMNS-M9 has remarkable control effects on the quinoa fungus diseases, the control effects of the quinoa gray mold, the quinoa leaf spot, the quinoa black stem disease, the quinoa ear rot and the like are 62.6% -72.2%, besides remarkable control effects on the quinoa fungus diseases, after the African trichoderma harzianum LMNS-M9 is prepared into a solid fermentation substrate, the early 2d emergence of quinoa can be promoted, and the growth promoting rate reaches 130.0%; 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) Bacterial strain activation, inoculating Trichoderma harzianum LMNS-M9 to a mycelium culture medium, culturing in a dark environment at 30deg.C for 12h under light/12 h for 3d, and standing until mycelium grows full of the culture medium;
(2) Preparing spore-producing strains, namely taking 25-30 bacterial cakes with the diameter of 5mm from the edge of a bacterial colony of the mycelium culture medium in the step (1), inoculating the bacterial cakes to the spore-producing culture medium, shaking uniformly, culturing for 7d in a dark environment at 30 ℃ for 12h under illumination/12 h, and standing for later use after the strains are produced;
(3) And (3) preparing a solid biological agent, inoculating the spore-producing culture medium of the spore-producing strain prepared in the step (2) into a solid fermentation matrix according to the mass of 9% -15%, uniformly mixing, and finally culturing the solid biological agent in an environment of 30 ℃ for 7d to prepare the solid biological agent.
The preparation method of the mycelium culture medium comprises the following steps: weighing 20.0g of glucose, 1.0g of potassium dihydrogen phosphate, 2.0g of peptone, 0.5g of magnesium sulfate, 20.0g of agar and 40.0g of corn flour, fully and uniformly mixing the above materials, adding 1L of distilled water, stirring to fully dissolve, adjusting the pH to 5 by using 1mol/L of HCl, and sterilizing for 20min by high-pressure steam at 121 ℃.
The preparation method of the spore-producing culture medium comprises the following steps: weighing 900g of oat grains soaked for 6h, 100g of quinoa grains soaked for 1h, 20.0g of glucose, 1.0g of dipotassium hydrogen phosphate, 2.0g of ammonium nitrate, 0.5g of magnesium sulfate and 40.0g of corn flour, fully and uniformly mixing the above materials, adjusting the pH to 5 by using 1mol/L HCl, subpackaging in 250mL triangular bottles, and sterilizing for 20min at 121 ℃ by high-pressure steam.
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 20min.
The method for using the Trichoderma harzianum LMNS-M9 solid biological agent (1) for seedling cultivation comprises the steps of subpackaging the Trichoderma harzianum LMNS-M9 solid biological 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 plant, 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) The open field direct seeding application method comprises the steps of ditching according to a row spacing of 35-45 cm, ditching with a depth of 5-8 cm, applying solid biological bacteria agent to ditches, and covering a mulching film after seeding, wherein each mu of ditches is about 15-20 kg.
(4) The application method in the growing period is that the solid biological microbial inoculum is applied in holes at the position 10-15 cm away from the quinoa plants, and 35-40 kg of solid biological microbial inoculum is applied per mu.
The method for identifying and controlling the quinoa fungus disease and promoting the quinoa growth test 1 by trichoderma harzianum LMNS-M9 and identifying (1) the quinoa seed endophytic fungi LMNS-M9 comprises the steps of collecting mature quinoa ears in a Xinzhou Jingle county buckeye area of Shanxi province, aesculus, and applying an endophyte separation screening method to the seeds of the mature quinoa ears. Quinoa seeds were sequentially soaked in 75% ethanol for 1min,1% sodium hypochlorite for 5min,75% ethanol for 1min, rinsed 3 times with sterile water, and the final 1 rinse was used as a blank. 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 7d culture, 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 LMNS-M9 has better antibacterial effect on quinoa disease pathogen in preliminary experiments. 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, LMNS-M9 has larger conidia.
Molecular biology characteristics: the length of the tef1 gene sequence of LMNS-M9 is 1241bp, and the number of the gene is OQ509672 in GenBank. The phylogenetic tree was constructed with tef1 gene sequence, LMNS-M9 was closest to the genetic relationship of T.afroharzianum, with a self-display support rate of 98% (FIG. 4). Combining morphological and phylogenetic analyses, LMNS-M9 was determined to be Trichoderma harzianum Trichodermaafroharzianum.
Antibacterial quinoa fungus disease pathogen test
(1) Test method
And (3) antibacterial effect measurement: trichoderma harzianum LMNS-M9 was cultured at 25deg.C for 5d, trichoderma harzianum LMNS-M9 was inoculated to the 5mm distance from the edge of PDA, 5 kinds of quinoa fungal disease pathogens were inoculated to the center symmetry point of the culture, and the culture was repeated 3 times at 25deg.C. At 7d, the colony radius was observed and measured, and the bacteriostasis 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, LMNS-M9 and the pathogen to be tested at 25deg.C for 3d, collecting mycelium, eluting with sterile water to obtain mycelium suspension. The PDA to be sterilized was cooled to about 50℃and 1mL of the solution was uniformly applied to a sterile slide glass having a size of about 4.0 cm. Times.2.5 cm. Times.0.1 cm. After the bacterial suspension is solidified, taking LMNS-M9 and 20L of mycelium suspension of the pathogen to be tested respectively, inoculating the mycelium suspension to two ends of the smear (with a spacing of 2 cm), placing the smear in a culture dish paved with sterilized wet filter paper, culturing for 1-2d at 25 ℃ and repeating for 3 times, and observing the re-mailing phenomenon under a microscope.
(2) Test results
Trichoderma harzianum LMNS-M9 was cultivated against 5 pathogens and a zone of inhibition was produced at the junction with 5 pathogens (FIGS. 5-9), indicating that strain LMNS-M9 inhibited colony wire growth of B.cinerea, A.calulina, F.citri, A.alternata and T.roseum. Meanwhile, the strain LMNS-M9 has remarkable competitive advantage; when cultivated in the opposite direction for 12d, strain LMNS-M9 was able to cover colonies of 3 pathogens, A.calulina (FIG. 6), F.citri (FIG. 7), A.alternate (FIG. 8). The antibacterial rate of Trichoderma harzianum LMNS-M9 against B.cinerea, A.calulina, F.citri, A.alternata, T.roseum 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, trichoderma harzianum LMNS-M9 produced was able to inhibit the growth of pathogenic hyphae of 5 quinoa fungal diseases. Meanwhile, pathogenic fungi such as A.calulina, A.alternata, T.roseum and the like can be obviously inhibited from generating conidium, and the population quantity of the pathogenic fungi is further controlled. The strain LMNS-M9 can obviously reduce the generation of conidium of pathogens such as A.calulina, A.alternata, T.roseum and the like, and the reduction range is 70.9% -100% (Table 2).
TABLE 2 inhibition of Trichoderma harzianum LMNS-M9 on the pathogenic spore production of quinoa mycosis
The mycelia of Trichoderma harzianum LMNS-M9 can be contacted with and wound around mycelia of B.cinerea (FIG. 10), A.calulina (FIG. 11), F.cintri (FIG. 12) and A.alternata (FIG. 13) (indicated by black arrows), and the mycelia of B.cinerea, A.calulina, F.cintri and A.alternata can be broken and digested, which shows that the strain LMNS-M9 has a re-hosting effect (FIGS. 10-13).
Trichoderma harzianum LMNS-M9 spore production optimization test
(1) Test method
The spore-producing culture medium is configured: 900g of oat grains soaked for 6 hours, 100g of quinoa grains soaked for 1 hour, 20.0g of glucose, 1.0g of dipotassium hydrogen phosphate, 2.0g of ammonium nitrate, 0.5g of magnesium sulfate and 40.0g of corn flour are weighed and mixed. Then, the pH was adjusted to 5 with 1mol/L HCl, the mixture was packed in 250mL Erlenmeyer flasks, the bottling amount was 100mL, and the mixture was autoclaved at 121℃for 20min. Inoculating the activated strain LMNS-M9 to a spore-forming culture medium, shaking uniformly, and finally culturing in a dark environment at 30 ℃ for 12h under illumination/12 h for 7d; experiments showed that Trichoderma harzianum LMNS-M9 produced about 2.5X109 CFU/mL of spores on the spore-forming medium, significantly higher than the other medium (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 20min. 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 7d to prepare the solid biological microbial inoculum.
(2) Test results
Experiments show that the spore production capacity of Trichoderma harzianum LMNS-M9 on a solid biological microbial inoculum can be obviously improved, the spore production amount is about 6.0X109 CFU/mL, and the spore production amount is obviously higher than that of other culture mediums (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 solid biological inoculant of Trichoderma harzianum LMNS-M9 is subpackaged into nutrition bowls (the caliber is 11cm, the bottom diameter is 8cm, the pot height is 10 cm), and 10 seeds of quinoa seeds to be tested are sown in each pot; 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 after sowing for 2-4 d, counting the emergence rate every day.
(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 emerge in advance by 2d, the growth promoting rate reaches 130.0 percent, and the emergence rate reaches 69.0 percent when the quinoa is 2d 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 9.32cm, 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 0.06g, 1.02g and 0.09g, which are improved by 104.7%, 30.8% and 28.6% and are obviously higher than those of a control group (Table 4), which shows that the germination of quinoa seeds is improved, the root development is effectively promoted and the root activity is enhanced on the solid biological inoculant containing Trichoderma harzianum LMNS-M9.
Test of field control Effect of Strain LMNS-M9
(1) Test method
The open field direct seeding method is adopted, ditches are carried out according to a row spacing of 35-45 cm (about 5-8 cm), the solid biological inoculant of the trichoderma harzianum LMNS-M9 in the ditches Shi Feizhou is about 15-20 kg/mu, and the mulching film is covered after seeding, and moisture is kept. The solid biological inoculant of the trichoderma harzianum LMNS-M9 is applied to the hole Shi Feizhou at the position 8-10 cm away from the quinoa plant in the seedling stage, 35-40 kg of the solid biological inoculant is applied to each mu, and no inoculant is applied to the solid biological inoculant for blank group comparison. 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 has remarkable control effect on quinoa fungus diseases, compared with a control group, the solid biological inoculant has remarkable control effect on 4 quinoa fungus diseases, wherein the incidence rate of the diseases such as quinoa gray mold, quinoa leaf spot, quinoa black stem disease and quinoa ear rot is remarkably reduced, and the control effect on the 4 quinoa fungus diseases is remarkable and 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 Trichoderma harzianum LMNS-M9 has remarkable inhibition effect on 5 pathogens (B.cinerea, A.calulina, F.citri, A.alternata and T.roseum) of quinoa fungus diseases, and the inhibition rate is 33.3% -61.5%. Meanwhile, the strain LMNS-M9 has obvious nutrition and space competition advantages, can cover pathogenic bacterial colonies such as A.calulina, F.citri, A.alternate and the like, and invades the living space of the pathogenic bacterial colonies. In addition, LMNS-M9 can inhibit the pathogenic fungi from generating conidium, so that the number of the pathogenic fungi colony controlled by the conidium can be reduced; the content of conidium of pathogens such as A.calulina, A.alternata, T.roseum and the like is greatly reduced by 70.9% -100%. The strain LMNS-M9 has obvious re-parasitic effect, and can contact and wind the hyphae of pathogens such as B.cinerea, A.calulina, F.cintri, A.alternata and the like, so that the hyphae of pathogens such as B.cinerea, F.cintri, A.alternata and the like are broken or 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 trichoderma harzianum LMNS-M9 can be early developed for 2d, 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 increased from 2.5X109 CFU/mL to 6.0X109 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-forming cluster of Trichoderma harzianum LMNS-M9 on PDA according to the present invention.
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 the culture of Trichoderma harzianum LMNS-M9 and B.cinerea according to the present invention.
FIG. 6 shows the culture of Trichoderma harzianum LMNS-M9 of the present invention against A.calulina.
FIG. 7 shows the culture of Trichoderma harzianum LMNS-M9 in opposition to F.citri in accordance with the present invention.
FIG. 8 shows the culture of Trichoderma harzianum LMNS-M9 of the present invention in opposition to A.alternate.
FIG. 9 shows the culture of Trichoderma harzianum LMNS-M9 in opposition to T.roseum according to the present invention.
FIG. 10 shows the re-mailing effect of Trichoderma harzianum LMNS-M9 on B.cinerea according to the present invention.
FIG. 11 shows the re-mailing effect of Trichoderma harzianum LMNS-M9 on A.calulina according to the present invention.
FIG. 12 shows the re-mailing effect of Trichoderma harzianum LMNS-M9 on F.citri according to the present invention.
FIG. 13 shows the re-mailing effect of Trichoderma harzianum LMNS-M9 on A.alternata according to the present invention.
Detailed Description
Example 1
The invention promotes the germination test of quinoa seeds and promotes the growth of quinoa seedlings, trichoderma harzianum (Trichodermaafroharzianum) LMNS-M9 is preserved in China general microbiological culture collection center (CGMCC) for 3-10 days in 2023, and the preservation address is: the dynasty district North Star, department 1, hospital 3 in Beijing; 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 of:
(1) Strain activation
Inoculating Trichoderma harzianum LMNS-M9 to mycelium culture medium, culturing at 30deg.C in 12 hr light/12 hr dark environment for 3d, 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 5mm 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 for 7d in a dark environment at 30 ℃ for 12h under illumination/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 culturing the solid fermentation substrate in an environment of 30 ℃ for 7d to prepare the solid biological microbial inoculum.
The preparation method of the mycelium culture medium comprises the following steps: weighing 20.0g of glucose, 1.0g of potassium dihydrogen phosphate, 2.0g of peptone, 0.5g of magnesium sulfate, 20.0g of agar and 40.0g of corn flour, fully and uniformly mixing the above materials, adding 1L of distilled water, stirring to fully dissolve, adjusting the pH to 5 by using 1mol/L of HCl, and sterilizing for 20min by high-pressure steam at 121 ℃.
The preparation method of the spore-producing culture medium comprises the following steps: weighing 900g of oat grains soaked for 6h, 100g of quinoa grains soaked for 1h, 20.0g of glucose, 1.0g of dipotassium hydrogen phosphate, 2.0g of ammonium nitrate, 0.5g of magnesium sulfate and 40.0g of corn flour, fully and uniformly mixing the above materials, adjusting the pH to 5 by using 1mol/L HCl, subpackaging in 250mL triangular bottles, and sterilizing for 20min at 121 ℃ by high-pressure steam.
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 20min.
Example 3
The application method of the Trichoderma harzianum LMNS-M9 solid biological agent comprises the following steps: (1) The method for using the seedlings comprises the steps of subpackaging Trichoderma harzianum LMNS-M9 solid biological microbial inoculum into a nutrition pot, and covering the surface of a matrix with a plastic film after the quinoa is sown until the seedlings emerge.
(2) Transplanting application method
And (3) applying solid biological bacteria to the holes at a position 8cm away from the quinoa plants, applying a solid fermentation matrix at 10 kg/mu, covering a mulching film, and transplanting quinoa seedlings after 7 d.
(3) Live broadcast application method
Ditching according to a row spacing of 35cm, ditching with a depth of 5cm, applying solid biological bacteria agent to ditches, 15 kg per mu, and covering mulching film after sowing.
(4) The application method in the growing period is that the solid biological microbial inoculum is applied in holes at the position 10cm away from the quinoa plants, and 35 kg of solid biological microbial inoculum is applied 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
Packaging Trichoderma harzianum LMNS-M9 solid biological bacteria 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 at a position 10cm away from the quinoa plants, applying 15 kg of solid fermentation matrix per mu, covering a mulching film, and transplanting quinoa seedlings after 10 d.
(3) Live broadcast application method
Ditching according to a row spacing of 45cm, applying solid biological bacteria to the ditches with a ditch depth of 8cm, 20 kg of solid biological bacteria per mu, and covering a mulching film after sowing.
(4) The application method in the growing period is that the solid biological microbial inoculum is applied in holes at a position 15cm away from quinoa plants, and 40 kg of solid biological microbial inoculum 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
Packaging Trichoderma harzianum LMNS-M9 solid biological bacteria into a nutrition pot, and covering the surface of a matrix with a plastic film after quinoa is sown until seedlings emerge.
(2) The transplanting method comprises the steps of applying solid biological bacteria to the position 8cm away from quinoa plants, applying solid fermentation matrix 15 kg per mu, covering mulching film, and transplanting quinoa seedlings after 7 d.
(3) Live broadcast application method
Ditching according to a row spacing of 45cm, ditching with a depth of 5cm, applying solid biological bacteria to ditches, and covering a mulching film after sowing, wherein each mu of ditches is about 20 kg.
(4) The application method in the growing period is that the solid biological microbial inoculum is applied in holes at the position 10cm away from the quinoa plants, and 40 kg of solid biological microbial inoculum 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
Packaging Trichoderma harzianum LMNS-M9 solid biological bacteria 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 10cm away from the quinoa plants, applying a solid fermentation matrix 10 kg per mu, covering a mulching film, and transplanting quinoa seedlings after 10 d.
(3) Live broadcast application method
Ditching according to a row spacing of 35cm, applying solid biological bacteria to the ditches with a depth of 8cm, 15 kg/mu, and covering the ditches with a mulching film after sowing.
(4) Method of use in growth phase
And applying solid biological bacteria to the plant 15cm away from quinoa, wherein 35 kg of the solid biological bacteria are applied to each mu.
Claims (3)
1. Trichoderma harzianum (Trichoderma afroharzianum) LMNS-M9 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.40523 and the preservation address in 2023, 3 and 10 days: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, beijing, chao's area.
2. A method for preparing a solid biological agent using the trichoderma harzianum (Trichoderma afroharzianum) LMNS-M9 strain of claim 1, comprising the steps of:
(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 3d, and keeping after mycelium grows to be full of culture medium;
(2) Preparation of spore-producing strains
Taking 25-30 bacterial cakes with diameters of 5mm from the edge of a bacterial colony of the mycelium culture medium in the step (1), inoculating the bacterial cakes to the spore-producing culture medium, shaking uniformly, culturing in an environment with the temperature of 30 ℃ and the illumination of 12 h and the darkness of 12 h for 7 d, and waiting for the strain to produce spores for later use;
(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 2.0g, magnesium sulfate 0.5 g, agar 20.0 g and corn flour 40.0 g, fully and uniformly mixing the above materials, adding distilled water 1L, stirring to fully dissolve, adjusting pH to 5 with HCl 1mol/L, and sterilizing with 121 ℃ high-pressure steam 20min;
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 20min parts.
3. The method for using the solid biological agent prepared by the method of claim 2, which comprises the following 4 steps;
(1) Seedling raising using method
Subpackaging Trichoderma harzianum LMNS-M9 solid biological bacteria 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
Applying solid biological bacteria to the position 8-10 cm away from the quinoa plant in a hole, applying 10-15 kg of solid fermentation matrix per mu, 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 ditches, 15-20 kg of solid biological bacteria agent per mu, and covering a mulching film after sowing;
(4) Method of use in growth phase
And (3) applying solid biological bacteria to the positions 10-15 cm away from quinoa plants in holes, wherein 35-40 kg of solid biological bacteria are applied to each mu.
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