CN114774291A - Trichoderma asperellum 10564 strain and application thereof in biocontrol of tomato blight - Google Patents
Trichoderma asperellum 10564 strain and application thereof in biocontrol of tomato blight Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A01N63/38—Trichoderma
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N3/00—Spore forming or isolating processes
Abstract
The invention provides a trichoderma asperellum 10564 strain and application thereof in biocontrol of tomato wilt, and particularly relates to the technical field of biological control of plant diseases. The trichoderma asperellum 10564 strain with the biocontrol effect shows the inhibition effect of the trichoderma asperellum 10564 strain on fusarium oxysporum; the tomato seedlings treated by the mixed spore liquid of the 10564 strain and the fusarium oxysporum show a more remarkable disease-resistant effect on the fusarium oxysporum, and meanwhile, the related parameters of chlorophyll fluorescence and hormone content in leaves are measured, so that the 10564 strain can protect a photosynthetic system II in the tomato seedlings and can induce the change of the plant hormone content to improve the disease resistance, and the corresponding biocontrol microbial inoculum can be prepared by applying the biocontrol microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a trichoderma asperellum 10564 strain and application thereof in biocontrol of tomato blight.
Background
Trichoderma spp belongs to Deuteromycotina, class Hyphomycetes, order Moniliales, family Moniliaceae. Trichoderma can induce plant systemic resistance, and has competition effect, resistance effect and heavy parasitic effect on pathogenic bacteria, and is a kind of fungi with great biocontrol potential.
The current studies on Trichoderma reesei mainly focus on Trichoderma harzianum (Trichoderma harzianum), Trichoderma atroviride (Trichoderma atroviride), Trichoderma viride (Trichoderma virens), Trichoderma reesei (Trichoderma reesei) and Trichoderma longibrachiatum (Trichoderma longibrachiatum), and there are a few studies on the Trichoderma asperellum species, and it is known that Trichoderma asperellum (Trichoderma asperellum) wood-8 strain has a significant growth promoting effect in chinese patent CN107142213A, but there are few studies on tomato blight.
Disclosure of Invention
In view of this, the present invention aims to provide a trichoderma asperellum 10564 strain and an application thereof in tomato blight biocontrol, wherein the trichoderma asperellum 10564 strain has an inhibitory effect on fusarium oxysporum, can protect photosynthetic system II in tomato seedlings, and can induce plant hormone content change so as to improve disease resistance.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Trichoderma asperellum 10564 strain with biocontrol effect, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 40090.
Preferably, the biocontrol effect comprises inhibition of Fusarium oxysporum (Fusarium oxysporum).
The invention also provides application of the 10564 strain in preparation of a biocontrol microbial inoculum.
Preferably, the types of the biocontrol microbial inoculum include conidium eluate and conidium suspension.
The invention provides a preparation method of a conidium eluent of a trichoderma asperellum 10564 strain, which comprises the following steps: inoculating the trichoderma asperellum 10564 strain on a PDA (personal digital assistant) plate, and culturing for 3d under the conditions of 25 ℃ and illumination to obtain a single colony;
and (3) picking edge hyphae of the single colony, inoculating the edge hyphae to a PDA (personal digital assistant) plate culture for 7d, adding sterile water, and scraping and washing conidia on the surface of the colony to obtain the conidia eluent.
The invention also provides a preparation method of the conidium suspension of the trichoderma asperellum 10564 strain, which comprises the following steps: inoculating the trichoderma asperellum 10564 strain on a PDA (personal digital assistant) flat plate for culturing to obtain a single colony;
selecting hypha blocks at the edge of the single colony, inoculating the hypha blocks into a PDB culture solution, and culturing for 3-7 days to obtain a culture;
and (3) centrifuging the culture, filtering the centrifuged supernatant, and collecting the filtrate to obtain the conidium suspension.
The invention also provides application of the 10564 strain, conidium eluent obtained by the preparation method or conidium suspension obtained by the preparation method in preventing and treating tomato blight.
Preferably, the method comprises dipping roots by using the conidium eluent before transplanting the tomato seedlings;
and/or irrigating roots by using the conidium suspension after transplanting the tomato seedlings.
The invention also provides a method for preventing and treating tomato leaf blight, which comprises the following steps: before transplanting the tomato seedlings, root dipping is carried out on the root systems of the seedlings by using the conidium eluent obtained by the preparation method.
The invention also provides a method for protecting the photosynthetic system II of the tomato seedlings and improving the content of the resistance-related hormones, which comprises the following steps: and after transplanting the tomato seedlings, irrigating roots by using the conidium suspension obtained by the preparation method.
Has the advantages that: the invention provides a trichoderma asperellum 10564 strain with a biocontrol effect, which is characterized in that the trichoderma asperellum 10564 strain and fusarium oxysporum are utilized to carry out a flat plate confrontation experiment to show the inhibition effect of the trichoderma asperellum 10564 strain on the fusarium oxysporum; the tomato seedlings treated by the mixed spore liquid of the 10564 strain and the fusarium oxysporum show a more remarkable disease-resistant effect on the fusarium oxysporum, and the measurement of related parameters of chlorophyll fluorescence and hormone content in leaves shows that the 10564 strain can protect a photosynthetic system II in the tomato seedlings and can induce the change of plant hormone content so as to improve the disease resistance.
Biological preservation information
Trichoderma asperellum 10564 has been preserved in China general microbiological culture Collection center, the specific preservation address is microorganism institute of China academy of sciences, No. 3, West Lu No. 1 Hopkin, Chaoyang, the preservation time is 2022 years, 03 months, 04 days, and the preservation number is CGMCC No. 40090.
Drawings
FIG. 1 shows the inhibition effect of Trichoderma asperellum 10564 strain and Fusarium oxysporum on the growth of Fusarium oxysporum hyphae by co-culture on PDA culture medium for 7 days;
FIG. 2 shows phenotype of plants after roots of tomato seedlings are soaked by spore eluent of Trichoderma asperellum 10564 strain, wherein control represents clear water treatment, F.oxy represents Fusarium oxysporum treatment, 564& F.oxy represents Fusarium oxysporum and 10564 mixed treatment, 564 represents treatment by using the strain 10564, the same applies below;
FIG. 3 shows the disease index of tomato seedlings after the roots of the tomato seedlings are soaked by the trichoderma asperellum 10564 strain spore eluate;
FIG. 4 shows the plant morbidity of tomato seedlings after the tomato seedlings are root-soaked by trichoderma asperellum 10564 strain spore eluate;
FIG. 5 shows that after the tomato seedlings are soaked in the trichoderma asperellum 10564 strain spore suspension liquid, chlorophyll fluorescence related parameters in tomato leaves are changed, so that the stress resistance of the tomatoes is enhanced;
FIG. 6 shows the jasmonic acid content change in tomato plants after the tomato seedlings are root-watered with the trichoderma asperellum 10564 strain spore suspension.
Detailed Description
The invention provides a Trichoderma asperellum 10564 strain with a biocontrol effect, wherein the 10564 strain is preserved and has a preservation number of CTCCSJ-W-AW 10564. The 10564 strain is separated from a tidal flat wetland, and the result is Trichoderma asperellum by molecular level identification.
The trichoderma asperellum 10564 disclosed by the invention can generate a large amount of aerial hyphae and generate dark green conidiospore clusters after being cultured on a PDA (potato dextrose agar) culture medium for one week, and is beneficial to rapid growth and sporulation after being cultured at the temperature of 25-28 ℃ under illumination.
The biocontrol effect preferably comprises the inhibition of Fusarium oxysporum (Fusarium oxysporum), and the plate confrontation experiment proves that the trichoderma asperellum 10564 strain can inhibit the growth of the Fusarium oxysporum, and the biocontrol effect is specifically represented as follows: the trichoderma asperellum 10564 strain can take up more growth space and nutrition of a flat plate in the same time, can generate resistant substances to obviously inhibit the growth of fusarium oxysporum hyphae, and forms an inhibition zone between the two, so the 10564 strain can be applied to the preparation of biocontrol microbial inoculum.
The invention also provides application of the 10564 strain in preparation of a biocontrol microbial inoculum.
The types of the biocontrol microbial inoculum according to the present invention preferably include conidium eluents and conidium suspensions.
The invention provides a preparation method of a conidium eluent of a trichoderma asperellum 10564 strain, which comprises the following steps: inoculating the trichoderma asperellum 10564 strain on a PDA (personal digital assistant) plate, and culturing for 3d under the conditions of 25 ℃ and illumination to obtain a single colony;
and (3) picking edge hyphae of the single colony, inoculating the edge hyphae to a PDA (personal digital assistant) plate culture for 7d, adding sterile water, and scraping and washing conidia on the surface of the colony to obtain the conidia eluent.
The trichoderma asperellum 10564 strain is inoculated on a PDA (personal digital assistant) plate to be cultured to obtain a single colony, wherein the culturing temperature is preferably 25 ℃, and the culturing time is preferably 3 d. The composition of the PDA plate is not particularly limited in the present invention, and a plate medium which is conventional in the art may be preferably used. The formulation of the PDA plate used in the examples preferably includes: 200g of potato, 20g of glucose and 10g of agar powder, and distilled water is supplemented to 1000 ml. In the preparation of the PDA plate, the invention preferably slices the cleaned peeled potatoes, adds water to boil for about half an hour, filters the potatoes by gauze, then adds glucose, adds water to agar powder to stir evenly, adds the agar powder into the solution, adds water to a constant volume, stirs to dissolve, subpackages and sterilizes.
After obtaining single colonies, picking edge hyphae of the single colonies, inoculating the hyphae to a PDA (personal digital assistant) plate, culturing the hyphae for 7 days at 25 ℃ under the illumination condition, adding sterile water, and scraping and washing conidia on the surfaces of the colonies to obtain conidia eluent. According to the invention, preferably, the marginal hyphae of the single colony is picked and inoculated to a new PDA plate for culture, the culture temperature is preferably 25 ℃, the culture time is preferably 7d, then water is added into a culture dish containing the PDA plate, conidia on the surface of the colony are scraped and washed, and the conidia eluent is obtained after uniform mixing. After the conidium eluate is obtained, the invention preferably further comprises counting, such as counting under a microscope by using a blood counting plate, and adjusting the concentration of the conidium eluate to be 1x107Each ml was used as a working solution.
The invention also provides a preparation method of the conidium suspension of the trichoderma asperellum 10564 strain, which comprises the following steps: inoculating the trichoderma asperellum 10564 strain on a PDA (personal digital assistant) flat plate for culturing to obtain a single colony;
selecting hypha blocks at the edge of the single colony, inoculating the hypha blocks into a PDB culture solution, and culturing for 3-7 days to obtain a culture;
and (4) centrifuging the culture, filtering the centrifuged supernatant, and collecting the filtrate to obtain the conidium suspension.
The trichoderma asperellum 10564 strain is inoculated on a PDA flat plate to be cultured to obtain a single colony. The culturing according to the present invention preferably comprises culturing at 25 ℃ for 3 days under light conditions to produce single colonies.
After single colony is obtained, selecting hypha blocks at the edge of the single colony, inoculating the hypha blocks into PDB culture solution, and culturing for 3-7 days to obtain a culture. The formula of the PDB culture solution is completely different from that of a PDA flat plate except that agar is not contained. The culture according to the present invention preferably comprises shaking culture at a temperature of 25 ℃ and 150r/min for 7 days.
After the culture is obtained, the invention centrifuges the culture, filters the supernatant fluid of the centrifugation and collects the filtrate to obtain the conidium suspension. The centrifugation is 8000 rpm. The supernatant after centrifugation is filtered, preferably the filtration comprises two-stage filtration, filter paper filtration is firstly carried out, the obtained primary filtrate is filtered again by a bacterial filter, and conidium suspension is obtained.
The invention also provides application of the 10564 strain, conidium eluent obtained by the preparation method or conidium suspension obtained by the preparation method in preventing and treating tomato blight.
The application preferably comprises the steps of dipping roots by using the conidium eluent before transplanting the tomato seedlings; and/or irrigating roots by using the conidium suspension after transplanting the tomato seedlings.
The invention also provides a method for preventing and treating tomato leaf blight, which comprises the following steps: before transplanting tomato seedlings, root dipping is carried out on seedling root systems by using the conidium eluent obtained by the preparation method.
In the invention, during root dipping, root dipping is preferably carried out by using a working solution of a conidium eluent, and the root dipping time is preferably 15-20 min. Before root dipping, the method preferably further comprises the steps of taking tomato seedlings growing for 3 weeks out of the plug tray, washing the roots with clear water, and then carrying out the root dipping. The root dipping agent can inhibit fusarium oxysporum parasitized in tomato root and tomato rhizosphere soil, and effectively prevent and treat tomato leaf blight.
The invention also provides a method for protecting the photosynthetic system II of the tomato seedlings and improving the content of resistance-related hormones, which comprises the following steps: after transplanting tomato seedlings, the conidium suspension obtained by the preparation method is used for adjusting the concentration to 1x107And (4) irrigating roots per ml. The root irrigation is preferably carried out for 3 times in 3 weeks of transplantation, wherein the interval is 3d each time, and the root irrigation volume is 20ml each time.
The invention utilizes the root irrigation to protect the photosynthetic system II of the tomato seedlings from being damaged, thereby improving the withering resistance of tomato plants, which is specifically represented by: chlorophyll fluorescence related parameters in leaves of tomato seedlings treated by the trichoderma asperellum 10564 strain spore liquid show a trend of promoting photosynthesis of tomatoes; and the root irrigation can also improve the content of tomato resistance related hormone, which is particularly shown in that the jasmonic acid content of tomato seedlings is obviously improved.
The Trichoderma asperellum 10564 strain provided by the present invention and the use thereof in the biocontrol of tomato wilt will be described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Separation and purification of trichoderma asperellum 10564
Taking back the soil containing trichoderma from the field, naturally drying the collected soil sample, grinding the soil sample, and sieving the ground soil sample. Weighing 10g of soil sample, placing the soil sample in a flask containing 90mL of sterile water, fully oscillating the soil sample by using a vortex instrument, sucking 10mL of suspension liquid to dilute the suspension liquid in 90mL of sterile water, sucking 10mL of suspension liquid from the diluted suspension liquid to dilute the suspension liquid in 90mL of sterile water, and so on until the volume fraction is 10-3、10-4、10-5. 0.1mL of suspension liquid is sucked from the soil suspension liquid with each content by a micro-pipetting gun and is dispersed and dripped on a Martin-Bengal red plate, a sterilized triangular glass rod is used for coating evenly, the mixture is placed still for 20min and then is placed upside down in a constant temperature incubator at 25 ℃, and the mixture is immediately purified after bacterial colony is formed.
Identification of Trichoderma species
Culturing Trichoderma in PDA plate for 3 days, punching the bacterial cake with a punch with diameter of 0.5cm, placing on PDA plate, culturing at 25 deg.C under illumination for 5 days, and observing colony shape and color. And (3) observing the branching condition of conidiophores of the trichoderma harzianum under a microscope and the morphology of the conidiophores, and performing species identification by combining culture traits and referring to a trichoderma harzianum population classification system of Rifai and Bissett.
Wherein:
1. preparation of trichoderma asperellum 10564 spore eluate:
1) inoculating the trichoderma asperellum 10564 strain on a PDA culture medium for culturing to obtain a trichoderma asperellum 10564 strain colony;
2) inoculating marginal hyphae of trichoderma asperellum 10564 bacterial colony obtained in the step 1) to a PDA culture dish for culture;
3) adding sterile water into the culture dish in the step 2), scraping and washing conidia on the surfaces of the bacterial colonies, and uniformly mixing to obtain the trichoderma asperellum 10564 strain conidia eluent.
4) Counting the conidium eluate of step 3) under microscope with blood counting plate, and adjusting the concentration of conidium eluate to 1 × 107One per ml.
2. Preparation of conidium suspension of trichoderma asperellum 10564 strain:
1) inoculating the trichoderma asperellum 10564 strain on a PDA culture medium for culturing to obtain a trichoderma asperellum 10564 strain colony;
2) inoculating the hypha blocks on the edges of the trichoderma asperellum 10564 bacterial colonies obtained in the step 1) into a PDB culture solution for culture to obtain a culture;
3) centrifuging the culture obtained in the step 2), filtering the centrifuged supernatant through filter paper, and filtering the obtained primary filtrate again through a bacterial filter to obtain filtrate.
Example 2
Inhibition effect of trichoderma asperellum 10564 strain on fusarium oxysporum
Respectively activating the stored 10564 strain and fusarium oxysporum on a PDA (personal digital assistant) plate, punching a hole on a PDA culture medium plate by using a puncher in a clean bench to obtain a 5 x 5mm fungus cake, transferring the two kinds of fungi to a new PDA plate, and culturing for 7 days at room temperature.
As shown in figure 1, the Trichoderma asperellum 10564 strain can take up more growth space and nutrition of the plate in the same time, and can generate resistant substances to obviously inhibit the growth of Fusarium oxysporum hyphae, so that an inhibition zone is formed between the two.
Example 3
Prevention and treatment effect of trichoderma asperellum 10564 strain on tomato fusarium wilt
Tomato seedlings growing for 3 weeks are taken out of the plug tray, roots are washed clean by clear water, and the tomato seedlings are respectively soaked in clear water and the trichoderma asperellum 10564 strain spore eluent (1x 10) prepared in example 17Seed/ml), Fusarium oxysporum spore eluate (1X 10)7Piece/ml), mixed eluent of trichoderma asperellum 10564 strain and fusarium oxysporum spore (1x 10)7Pieces/ml), and planting in a plug for 15-20 min, wherein 24 pieces are treated each. After the treatment, the tomatoes have phenotype difference in the seedling stage, and as shown in fig. 2, the tomato seedlings of the mixed spore solution treatment group and the trichoderma-independent treatment group grow strongly and the disease resistance of the plants is obviously enhanced.
The preparation method of the fusarium oxysporum spore eluent comprises the following steps:
inoculating fusarium oxysporum on a PDA culture medium and culturing for 3d at 25 ℃ to obtain fusarium oxysporum colonies;
inoculating the marginal hyphae of the fusarium oxysporum to a PDA culture dish for culture for 7 d;
adding sterile water into the culture dish, scraping and washing conidia on the surfaces of the bacterial colonies, and uniformly mixing to obtain fusarium oxysporum conidia eluent.
Counting the obtained conidium eluate with a blood counting plate under microscope, adjusting the concentration of conidium eluate to 1 × 107Each/ml.
The preparation steps of the mixed eluent of the trichoderma asperellum 10564 and the fusarium oxysporum are the same, so that the concentrations of the two strains are both 1x107Each/ml.
The tomato plants are classified into 4 grades according to the disease states:
stage 0: the plants grow strongly, the leaves are naturally unfolded, the green color is bright, and the stems are thick;
level 1: the plant stands upright, the leaf yellowing wilting area is less than 50 percent of the total area of the leaves, and the stem base is browned;
and 2, stage: the yellowing wilting area of the plant leaves is 50 percent larger than the total area of the leaves, the top leaves are yellow and shriveled, and the stem is withered yellow and shriveled;
and 3, stage: the plant is short and withered, the leaves are withered, yellow, curled and shriveled, only the growing point survives, and the stem is wilted;
and 4, stage 4: the plants wither to death.
Calculating disease index according to disease grade: the disease index ∑ (number of diseased plants at each stage × corresponding disease stage)/(total number of plants × highest disease stage) × 100. The disease index is used as the inhibition effect of the trichoderma asperellum 10564 strain conidium eluent on fusarium oxysporum spore eluent. As shown in figure 3 and table 1.
TABLE 1 inhibition effect of different treatment groups on Fusarium oxysporum spore eluate
Calculating the morbidity (judging to be a blight tomato plant when the disease is at grade 2-4): the disease incidence rate is the number of the treated plants/the total number of the treated plants multiplied by 100. The disease incidence is used as the eluent of the conidium of the trichoderma asperellum 10564 strain to prevent and control the tomato fusarium wilt caused by fusarium oxysporum. As shown in fig. 4 and table 2.
TABLE 2 prevention and treatment effects of different treatment groups on tomato wilt
Example 4
Trichoderma asperellum 10564 strain for improving chlorophyll fluorescence content of tomato leaves so as to improve stress resistance of tomato plants
Tomato seedlings growing for 3 weeks are planted in a field, and clear water, the trichoderma asperellum 10564 spore suspension, the fusarium oxysporum suspension, the mixed suspension of the trichoderma asperellum 10564 and the fusarium oxysporum prepared in the embodiment 1 are respectively irrigated to the roots of each tomato seedling. Irrigating the roots once every 3 days for 3 times continuously. Sampling after phenotype difference of the four treatments, picking the leaves of the same part of the tomato, dark treating for 15min, and measuring chlorophyll fluorescence related parameters Fv/Fm, ETR ═ Fm ']xPFD, qP ═ Fm' -Fs)/Fv 'and NPQ ═ Fm')/Fm '═ Fm/Fm' -1 in the samples by using a fluorometer PAM series.
Fv/Fm reflects the potential maximum photosynthetic capacity (i.e., photosynthetic efficiency) of a plant;
ETR is the apparent photosynthetic electron transfer rate;
qP is fluorescence quenching caused by photosynthesis, and reflects the level of photosynthetic activity;
NPQ reflects the ability of a plant to dissipate excess light energy as heat, i.e., photoprotective ability.
The preparation method of the fusarium oxysporum spore suspension comprises the following steps:
inoculating fusarium oxysporum in a PDA culture medium, and culturing for 3d under a dark condition at 25 ℃ to obtain fusarium oxysporum colonies;
inoculating the mycelia on the edge of the fusarium oxysporum into a PDB culture solution for culture to obtain a culture, wherein the culture condition is that the culture is subjected to shaking culture for 5d at the temperature of 25 ℃ and at the speed of 150 r/min;
the resulting culture was centrifuged, the supernatant was filtered through filter paper to obtain a spore suspension, and the concentration was adjusted to 1 × 10 using a hemocytometer7One per ml.
A method of mixing a spore suspension comprising the steps of:
inoculating the trichoderma asperellum 10564 strain into a PDA culture medium to be cultured for 3 days under illumination at the temperature of 25 ℃ to obtain a trichoderma asperellum 10564 strain bacterial colony;
inoculating the hypha blocks at the edges of the trichoderma asperellum 10564 colonies into a PDB culture solution for culture to obtain a culture, wherein the culture condition is that the culture is performed for 5 days under the conditions of 25 ℃ and 150 r/min;
centrifuging the obtained culture, collecting the supernatant, and filtering with filter paper to obtainSpore suspension, and adjusting concentration to 1 × 10 using a hemocytometer7Each/ml.
2x107Fusarium oxysporum spore suspension per ml and 2x107And (3) uniformly mixing the trichoderma asperellum 10564 spore suspension per ml in equal volume to obtain a mixed spore suspension of the trichoderma asperellum 10564 spore suspension and the trichoderma asperellum.
The results are shown in fig. 5 and table 3, the trichoderma inhibits the activity of fusarium oxysporum, prevents tomato seedling from being infected with diseases, protects the plant photosynthetic system II from being damaged, and improves the photosynthetic efficiency of the leaves.
TABLE 3 Effect of different treatment groups on photosynthesis
Example 5
Trichoderma asperellum 10564 strain induces the content of jasmonic acid which is related to the resistance of tomato plants to change, and improves the disease resistance of tomatoes
Tomato seedlings growing for 3 weeks are planted in the field, and clear water, the trichoderma asperellum 10564 spore suspension, the fusarium oxysporum suspension, and the mixed suspension of the trichoderma asperellum 10564 and the fusarium oxysporum spores prepared in example 1 are respectively irrigated to the roots of each seedling. Irrigating the root once every 3 days for 3 times. Sampling after phenotype difference occurs in the four treatments, and picking leaves at the same part of the tomato to determine the jasmonic acid content.
As shown in fig. 6 and table 4, trichoderma regulates the change of resistant hormones in plants, induces the defense response of tomato seedlings, and improves disease resistance.
TABLE 4 Effect of different treatment groups on disease resistance of tomato
| CK | F. | 564&F.oxy | 564 | |
| 14.94 | 14.96 | 31.34 | 34.64 | |
| 19.44 | 12.34 | 32.43 | 33.60 | |
| 14.03 | 12.49 | 31.05 | 30.41 | |
| 16.28 | 11.62 | 32.12 | 29.75 |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A Trichoderma asperellum 10564 strain with biocontrol effect, wherein the 10564 strain is preserved and preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 40090.
2. The Trichoderma asperellum 10564 strain of claim 1, wherein said biocontrol effect comprises inhibition of Fusarium oxysporum (Fusarium oxysporum).
3. Use of the trichoderma asperellum 10564 strain of claim 1 or 2 for the preparation of a biocontrol microbial agent.
4. The use according to claim 3, wherein the type of biocontrol agent comprises a conidia eluate and/or a conidia suspension.
5. A preparation method of a conidium eluent of a trichoderma asperellum 10564 strain is characterized by comprising the following steps: inoculating the trichoderma asperellum 10564 strain on a PDA (personal digital assistant) plate, and culturing for 3d under the conditions of 25 ℃ and illumination to obtain a single colony;
picking edge hyphae of the single colony, inoculating the edge hyphae to a PDA plate culture for 7d, adding sterile water, and scraping and washing conidia on the surface of the colony to obtain the conidia eluent.
6. A method for preparing a conidium suspension of Trichoderma asperellum 10564 strain, comprising the steps of: inoculating the trichoderma asperellum 10564 strain on a PDA (personal digital assistant) flat plate for culturing to obtain a single colony;
selecting hypha blocks at the edge of the single colony, inoculating the hypha blocks into a PDB culture solution, and culturing for 3-7 days to obtain a culture;
and (4) centrifuging the culture, filtering the centrifuged supernatant, and collecting the filtrate to obtain the conidium suspension.
7. Use of the trichoderma asperellum 10564 strain of claim 1 or 2 or the conidium eluate obtained by the preparation method of claim 5 or the conidium suspension obtained by the preparation method of claim 6 for preventing and treating tomato wilt.
8. The use of claim 7, which comprises dipping roots with the conidium eluent before transplanting tomato seedlings;
and/or irrigating roots by using the conidium suspension after transplanting the tomato seedlings.
9. A method for preventing and treating tomato leaf blight is characterized by comprising the following steps: before transplanting tomato seedlings, root dipping is carried out on seedling roots by using the conidium eluent obtained by the preparation method of claim 5.
10. A method for protecting the photosynthetic system II of tomato seedlings and increasing the content of resistance-related hormones, which is characterized by comprising the following steps: after transplanting tomato seedlings, irrigating roots by using the conidium suspension obtained by the preparation method of claim 6.
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