CN106591157B - The preparation and application of the Tabin aspergillus and its metabolite of one plant of disease prevention growth-promoting - Google Patents

Abstract

The invention discloses the preparations and application of the Tabin aspergillus and its metabolite of one plant of disease prevention growth-promoting.The bacterial strain number of the Tabin aspergillus is QF05, is CGMCC No.13563 in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Tabin aspergillus QF05 all has inhibiting effect to the botrytis cinerea on fruit trees and vegetables, Colletotrichum capsici, cucumber fusarium axysporum, target bacterium, cucumber bacterial angular leaf spot bacterium.The sterile ferment filtrate of Tabin aspergillus QF05 is remarkably improved the germination percentage of pepper seed and remarkably promotes the elongation of radicle.Tabin aspergillus QF05 metabolin containing bacterium has effectively facilitated the growth of cucumber true leaf, effectively increases the plant height of cucumber, effectively increases the yield of cucumber.

Description

Preparation and application of a strain of Aspergillus tubingensis and its metabolites for disease prevention and growth promotion

technical field

The invention belongs to the technical field of microbial pesticides, and in particular relates to the preparation and application of a disease-preventing and growth-promoting Aspergillus tubingensis and its metabolites.

Background technique

With the development of horticultural facility cultivation and the expansion of planting area in protected areas, diseases of fruit and vegetable crops are common and cause serious economic losses to people's production and life. At present, domestic and foreign research on disease control of fruit and vegetable crops is mainly based on comprehensive control from the aspects of breeding excellent disease-resistant varieties, seed disinfection treatment, strengthening cultivation management measures, spraying chemical agents, etc., and chemical control is the main method. Long-term use of a single type of fungicide and unreasonable application techniques lead to the development of resistance of pathogenic bacteria, and the control effect decreases year by year. At the same time, it brings a series of problems such as excessive chemical pesticide residues, environmental pollution, and destruction of biodiversity. Biological control has the advantages of good environmental compatibility, safety for humans and animals, and not easy to produce drug resistance. Microbial pesticides are gradually leading the development of scientific research and market due to their advantages of high efficiency, broad spectrum, safety and environmental protection. Therefore, the development of new biopesticide products that can gradually replace chemical pesticides is of great significance for the green prevention and control of fruit and vegetable crop diseases.

Contents of the invention

The technical problem to be solved by the invention is how to inhibit the pathogenic bacteria of the fruit and vegetable crops and promote the growth of the fruit and vegetable crops.

In order to solve the above technical problems, the invention provides a strain of Aspergillus tubingensis.

The Aspergillus tubingensis provided by the present invention is Aspergillus tubingensis, its strain number is QF05, and its registration number in the General Microorganism Center of China Microbiological Culture Collection Management Committee is CGMCCNo.13563. Hereinafter referred to as Aspergillus tubingensis QF05.

Aspergillus tubingensis (Aspergillus tubingensis) QF05 has the ITS sequence shown in sequence 1 in the sequence listing and the calmodulin gene sequence shown in sequence 2 in the sequence listing, and Aspergillus tubingensis QF05 grows mycelia on PDA medium Lush, producing a large number of black spores; on the CYA medium, the front of the colony is black-brown, and the back is light yellow; on the MEA medium, the center of the colony is gray-black, the periphery is off-white, and the back is light yellow. Under a 40 times optical microscope, it can be seen that the top of the conidiophores of the strain QF-05 swells into an apical capsule, which is nearly spherical, with small stalks growing on it; the conidia are spherical, dark brown.

Metabolites of Aspergillus tubingensis QF05 also belong to the protection scope of the present invention. The metabolite of Aspergillus tubingensis QF05 is a substance obtained by culturing Aspergillus tubingensis QF05 in a microbial liquid fermentation medium.

In order to solve the above technical problems, the present invention provides pathogenic bacteria inhibitors.

The pathogen inhibitor provided by the present invention contains Aspergillus tubingensis QF05 and/or metabolites of Aspergillus tubingensis QF05.

The active ingredient of the above-mentioned pathogen inhibitor can be Aspergillus tubingensis QF05 and/or the metabolite of Aspergillus tubingensis QF05, and the active ingredient of the above-mentioned pathogen inhibitor can also contain other biological components or non-biological components, Other active ingredients of the above pathogenic bacteria inhibitors can be determined by those skilled in the art according to their inhibitory effect on pathogenic bacteria.

Among the above-mentioned pathogenic bacteria inhibitors, the pathogenic bacteria inhibitors can have an inhibitory effect on at least one of the following pathogenic bacteria:

A. Botrytis cinerea;

B. Capsicum anthracnose bacteria;

C. Fusarium wilt of cucumber;

D. Potato blight bacillus;

E, Cucumber horn spot fungus.

In order to solve the above technical problems, the present invention provides a disease inhibitor.

The disease inhibitor provided by the present invention contains Aspergillus tubingensis QF05 and/or a metabolite of Aspergillus tubingensis QF05.

The active ingredient of the above-mentioned disease inhibitor can be a metabolite of Aspergillus tubingensis QF05 and/or Aspergillus tubingensis QF05, and the active ingredient of the above-mentioned disease inhibitor can also contain other biological components or non-biological components, Other active ingredients of the above-mentioned disease inhibitors can be determined by those skilled in the art according to their inhibitory effects on diseases.

Among the above-mentioned disease inhibitors, the disease is at least one of the following:

a. Botrytis cinerea;

b. Pepper anthracnose;

c. Cucumber wilt;

d. Potato early blight;

e, Cucumber bacterial angular spot.

Any of the following applications of the metabolites of Aspergillus tubingensis QF05 and/or Aspergillus tubingensis QF05 also belong to the protection scope of the present invention:

1) Application in inhibiting pathogenic bacteria;

2) Application in the preparation of pathogen inhibitors;

3) Application in disease suppression;

4) Application in the preparation of disease inhibitors.

In the above application, the pathogenic bacteria can be at least one of the following:

A. Botrytis cinerea;

B. Capsicum anthracnose bacteria;

C. Fusarium wilt of cucumber;

D. Potato blight bacillus;

E, Cucumber horn spot fungus.

The disease can be at least one of the following:

a. Botrytis cinerea;

b. Pepper anthracnose;

c. Cucumber wilt;

d. Potato early blight;

e, Cucumber bacterial angular spot.

In the above, the pathogen inhibitor and disease inhibitor contain a carrier in addition to the active ingredient. The carrier can be commonly used in the field of pesticides and is biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and At least one of diatomaceous earth; the plant material can be at least one of corn flour, soybean flour and starch; the polymer compound can be polyvinyl alcohol and/or polyglycol; the liquid carrier can be Organic solvent, vegetable oil, mineral oil or water; said organic solvent may be decane and/or dodecane.

Among the pathogen inhibitors and disease inhibitors, Aspergillus tubingensis QF05 can exist in the form of conidia, hyphae or cultures containing conidia and/or hyphae.

The dosage form of the pathogenic bacteria inhibitor and disease inhibitor can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the pathogen inhibitors and disease inhibitors as required.

Any of the following applications of the metabolites of Aspergillus tubingensis QF05 and/or Aspergillus tubingensis QF05 also belong to the protection scope of the present invention:

H, application in promoting plant seed germination;

1. Application in promoting plant seed radicle growth;

J, application in promoting the growth of true leaves of plants;

K, application in improving plant strain high school;

L, application in improving plant yield.

In the above application, the plant can be any of the following plants:

P1) cucumber or pepper;

P2) plants of the genus Cucumber or plants of the genus Capsicum;

P3) Cucurbitaceae or Solanaceae;

P4) seed plants;

P5) Dicotyledons.

In the above, the metabolites of Aspergillus tubingensis QF05 can be obtained from the fermentation broth of Aspergillus tubingensis QF05. The metabolite of Aspergillus tubingensis QF05 may be a sterile metabolite of Aspergillus tubingensis QF05 or a bacterium-containing metabolite of Aspergillus tubingensis QF05. Aspergillus tubingensis (Aspergillus tubingensis) QF05 sterile metabolite (sterile fermentation filtrate) can be specifically prepared according to the following method, culture Aspergillus tubingensis (Aspergillus tubingensis) QF05 in a liquid medium, filter to remove the liquid culture (fermentation liquid) Aspergillus tubingensis QF05 obtained from Aspergillus tubingensis QF05 is a sterile metabolite of Aspergillus tubingensis QF05. The bacterium-containing metabolites of Aspergillus tubingensis QF05 can be specifically prepared according to the following method. Cultivate Aspergillus tubingensis QF05 in a liquid fermentation medium to contain Aspergillus tubingensis QF05 and its metabolites. The fermented liquid is sterilized to obtain the bacterium-containing metabolite of Aspergillus tubingensis QF05.

In the present application, the tomato cinerea can be Botrytis cinerea, the pepper anthracnose can be Colletotrichum capsici, and the cucumber wilt can be Fusarium oxysporum. type (Fusarium oxysporum f.sp.cucumerinum), the potato early blight bacteria can be Alternaria tenuissima (Alternaria tenuissima), and the cucumber bacterial angular spot bacteria can be Pseudomonas syringae pv. Lachrymans).

The present invention takes the pathogenic bacteria of fruit and vegetable diseases as the target, and obtains a microbial strain-Aspergillus tubingensis (Aspergillus tubingensis) QF05 with the function of disease prevention and growth promotion by screening microbial resources in special habitats, which is a new method for the preparation of microbial pesticides. Products provide new resources.

Aspergillus tubingensis (Aspergillus tubingensis) QF05 has inhibitory effects on tomato cinerea, capsicum anthracnose, cucumber wilt, potato early blight and cucumber horn spot on fruit and vegetable crops; it can promote pepper seed germination and cucumber plant growth effect. Compared with the control, the 10-fold dilution of the sterile fermentation filtrate of Aspergillus tubingensis QF05 reduced the germination rate of pepper seeds, but significantly increased the radicle length of pepper, and the increase reached 26.0%; Aspergillus tubingensis (Aspergillus tubingensis) tubingensis) QF05 aseptic fermentation filtrate 50 times of dilution liquid treatment of the pepper seed germination rate is the same as the control, but significantly increased the radicle length of pepper, an increase of 58.9%; Tabingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate 100 The pepper seeds treated with the double dilution solution not only increased the germination rate by 11.1% compared with the control, but also significantly increased the radicle length of the pepper, and the increase reached 149.3% (Table 2). It shows that Aspergillus tubingensis QF05 can significantly increase the germination rate of pepper seeds and significantly promote the elongation of radicle. Aspergillus tubingensis (Aspergillus tubingensis) QF05-containing metabolites can promote the growth of cucumber true leaves, increase cucumber plant height, and increase cucumber yield. Aspergillus tubingensis QF05 has a fast propagation speed, can be cultured artificially, has simple culture conditions, is easy to preserve, is suitable for industrial production, and has good development and application prospects.

Preservation instructions

Species name: Aspergillus tubingensis

Strain number: QF05

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Depository institution abbreviation: CGMCC

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: January 19, 2017

Registration number of the collection center: CGMCC No.13563

Description of drawings

Fig. 1 is the colony and spore morphology of Aspergillus tubingensis QF05, wherein a, c, e and b, d, f are respectively bacterial strain QF05 adopting three-point method to inoculate Saccharomyces chabai extract agar (CYA) medium and malt Colony morphology, sporangia and conidia on juice agar (MEA) medium.

Figure 2 is a phylogenetic tree based on the calmodulin gene (calmodulin) of Aspergillus tubingensis QF05, in which strain QF05 and Aspergillus tubingensis strains (FN594558, EF661151, KC796385) are clustered on the same branch, with a similarity of 99% . In the figure, the strain in the frame is Aspergillus tubingensis QF05.

Figure 3 shows the space competition effect of Aspergillus tubingensis strain QF05 on common plant pathogens, where a, b, c, and d are the effects of strain QF05 on cucumber wilt, capsicum anthracnose, tomato cinerea, and potato blight respectively competitive inhibition. In a, b, c, d, the upper plate is the control of single inoculation of pathogenic bacteria, and the lower plate is the confrontation culture of pathogenic bacteria and Aspergillus tubingensis strain QF05.

Figure 4 shows the inhibitory effect of Aspergillus tubingensis (Aspergillus tubingensis) QF05 sterile fermentation filtrate on Xanthomonas cucumber (a) and Capsicum anthracnose (b), wherein CK is the control.

Fig. 5 is the promoting effect of aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate to pepper seed germination, wherein CK is a contrast, 10 *, 50 *, 100 * are respectively aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate 10 Times, 50 times, 100 times dilution.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The pathogenic bacteria used in the following examples can be collected from the wild, or can be obtained from Beijing Academy of Agriculture and Forestry Sciences, to repeat the application experiment:

Botrytis cinerea, Fusarium oxysporum f.sp.cucumerinum, Rhizoctonia cereali, Fusarium moniliforme, Monilinia fructicola, Pepper anthracnose (Colletotrichum capsici), eggplant Ralstonia solanacearum (Ralstonia solanacearum), Chinese cabbage black rot (Xanthomonas campestris pv. campsetris) (Wang Junli et al. Identification and biocontrol characteristics analysis of a Bacillus sp. QD-10. Chinese Journal of Biological Control, 2014, 30(4):564-572.);

Alternaria tenuissima (Zheng, et al. Characterization of Alternaria species associated with potato foliar diseases in China. Plantpathology, 2015, 64:425-433.);

Fusarium oxysporum f.sp.conglutinans, Pseudomonas syringae pv.lachrymans (Lu Caige et al. Screening, identification and antibacterial activity of a strain of cabbage wilt antagonistic bacteria. Journal of North China Agricultural Science, 2014,29(1):195-202.).

Embodiment 1, isolation and identification of Aspergillus tubingensis QF05

1.1 Strain isolation

The strain was isolated from soil samples in special habitats of Qinghai-Tibet Plateau, Qinghai Province, China. Bacterial strains were separated by the dilution plate method, and the specific operation method was as follows: weigh 10 g of the soil sample, put it into 90 ml of sterile water and oscillate evenly to obtain a bacterial solution with a concentration of 10 ^-1 , and use a 1 ml sterile pipette to draw 1 ml of a bacterial solution with a concentration of 10 ^-1 . solution in a tube of 9ml sterile water, shake well to obtain a bacterial solution with a concentration of 10 ^-2 , and dilute to 10 ^-4 successively in the same way. Spread the above-mentioned 10 ^-2 , 10 ^-3 and 10 ^-4 soil bacteria liquids on PDA plates respectively, dry them and place them upside down in an incubator at 25°C for 3-5 days, and then transfer the isolated fungal single colonies to Cultivate it on a PDA slope, and store it in a refrigerator after it grows well. The strain numbered QF05 was taken for the following identification.

1.2 Strain identification

1.2.1 Observation of strain morphology

The mycelium of strain QF05 was picked and inoculated on PDA medium, and cultivated at 25°C for 4 days to obtain a plate seed. The strain QF05 was inoculated on the Saccharomyces chabai extract agar (CYA) medium and malt extract agar (MEA) medium by the three-point method, cultured at 25°C for 4 days, the colony morphology was observed, and the sporangia and conidia were observed under an optical microscope. The results showed that the mycelia of strain QF05 grew luxuriantly on the PDA medium and produced a large number of black spores; on the CYA medium, the front of the colony was black and brown, and the back was light yellow; Under a 40-fold optical microscope, it can be seen that the top of the conidiophores of the strain QF05 swells into an apical capsule, which is nearly spherical, with small stalks growing on it; the conidia are spherical and dark brown (Figure 1). In view of the above morphological characteristics, strain QF05 was initially identified as Aspergillus sp.

Among them, potato dextrose agar (PDA) medium: peeled potatoes, cut 200g into small pieces, boiled with water for 30min, filtered through 4 layers of gauze, added 20g of glucose, 17g of agar, distilled water to 1000mL, boiled and mixed, 121°C Sterilize for 20 minutes to obtain PDA medium.

Chabai Yeast Extract Agar (CYA) medium: K ₂ HPO ₄ 1g, Chase concentrated solution 10ml, yeast extract 5g, sucrose 30g, agar 15g, distilled water to 1000mL, sterilized at 121°C for 20min to obtain Chabai extract agar (CYA) medium. Chase concentrate: NaNO ₃ 30g, KCl 5g, MgSO ₄ 7H ₂ O 5g, FeSO ₄ 7H ₂ O 0.1g, ZnSO ₄ 7H ₂ O 0.1g, CuSO ₄ 5H ₂ 0 0.05g, distilled water to volume to 100mL.

Malt extract agar (MEA) medium: malt extract 20g, peptone 1g, glucose 20g, agar 15g, distilled water to 1000mL, sterilized at 121°C for 20min to obtain malt extract agar medium.

1.2.2 Molecular biological identification of strains

The mycelium of strain QF05 was picked and inoculated on PDA medium, and the mycelium was collected after culturing at 25°C for 4 days. Genomic DNA of strain QF05 was extracted by CTAB method, and the gene sequence of its ITS region was amplified by general primer ITS1/ITS4. Primer CMD5/CMD6 amplified its calmodulin (calmodulin) gene sequence. The sequencing results were compared using Blast software in GenBank, Clustal X software for multiple sequence alignment, and Neighbor-Joining method in MEGA5.0 software to construct a phylogenetic tree.

The rDNA-ITS sequence (sequence 1 in the sequence listing) and calmodulin gene sequence (sequence 2 in the sequence listing) of the bacterial strain QF05 were obtained by sequencing. According to the ITS sequence analysis results, the bacterial strain QF05 was similar to Aspergillus tubingensis, Aspergillus niger, Aspergillus vadensis, etc. All reached 99%, so it was classified as Aspergillus (Aspergillus sp.). According to the results of calmodulin gene sequencing and phylogenetic analysis, the similarity between strain QF05 and Aspergillus tubingensis was 99%, and it was clustered in the same system branch with Aspergillus tubingensis strains (FN594558, EF661151, KC796385). Based on the above results, the strain QF05 was identified as Aspergillus tubingensis.

Aspergillus tubingensis QF05 was deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms on January 19, 2017, with the preservation number CGMCC No.13563. Hereinafter referred to as Aspergillus tubingensis (Aspergillus tubingensis) QF05 or strain QF05.

Embodiment 2, Aspergillus tubingensis (Aspergillus tubingensis) QF05 is to the competitive inhibitory effect of testing pathogenic bacteria

Pick the Aspergillus tubingensis (Aspergillus tubingensis) QF05 bacterium cake with a diameter of 7mm grown on the flat plate and the bacteria cake of the pathogenic bacteria for testing, and inoculate them symmetrically on the PDA medium respectively at a position 2cm away from the center. The control does not inoculate Aspergillus tubingensis (Aspergillus tubingensis) tubingensis) QF05 bacterium cake, only inoculate the test pathogen bacterium cake, each treatment was repeated three times, and cultured at 25°C for 4 days to observe the growth of the pathogen. The tested pathogens include: Botrytis cinerea, Fusarium moniliforme, Colletotrichum capsici, Fusarium oxysporum f.sp.cucumerinum, Rhizoctonia cereali), Alternaria tenuissima, Fusarium oxysporum f. sp. conglutinans, Monilinia fructicola.

The results showed that Aspergillus tubingensis QF05 was effective against Botrytiscinerea, Colletotrichum capsici, Fusarium oxysporum f.sp.cucumerinum and Alternaria tenuissima. Competitive inhibition has no inhibitory effect on the other four tested pathogens (Fig. 3 and Table 1).

Table 1, Aspergillus tubingensis (Aspergillus tubingensis) QF05 is to the competitive inhibitory effect of testing pathogenic fungus

Note: "+" means inhibitory effect, "-" means no inhibitory effect.

Embodiment 3, Aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate is to the inhibitory effect of testing pathogenic bacteria

1. Preparation of Aspergillus tubingensis QF05 sterile fermentation filtrate

Pick up the Aspergillus tubingensis QF05 grown on the plate with the inoculation loop and inoculate it into a 500mL Erlenmeyer flask containing 100mL of liquid fermentation medium, culture it on a shaker at 150rpm at 25°C for 4 days, collect the fermentation broth, and use a 0.22μm micropore Filtering the fermentation liquid with a filter membrane to remove Aspergillus tubingensis QF05 cells in the culture to obtain Aspergillus tubingensis QF05 sterile fermentation filtrate.

Among them, the liquid fermentation medium is maltose 20g, glycerin 20ml, beef extract 30g, peptone 30g, distilled water to 1000mL, sterilized at 121°C for 20min to obtain the liquid fermentation medium.

2. Antibacterial effect

The pathogens tested included Pseudomonas syringae pv. lachrymans, Ralstonia solanacearum, Xanthomonas campestrispv. campsetris, and Colletotrichum capsici. Pseudomonas syringae pv. lachrymans, Ralstonia solanacearum, and Xanthomonas campestris pv. campsetris were evenly spread on LB solid medium respectively, and pepper anthracnose ( Colletotrichum capsici) were spread evenly on the PDA solid medium.

The LB solid medium is: tryptone 10g, yeast extract 5g, NaCl 10g, agar 17g, distilled water to 1000mL, sterilized at 121°C for 20min. The PDA solid medium is: peeled potatoes, cut 200g into small pieces, add water to boil for 30min, filter through 4 layers of gauze, add 20g of glucose, 17g of agar, distilled water to 1000mL, boil and mix well, and sterilize at 121°C for 20min.

Add the aspergillus tubingensis (Aspergillustubingensis) QF05 sterile fermentation filtrate of step 1 in the hole of 7mm in the diameter that the hole puncher gets, each process repeats three times, cultivates 2d at 25 ℃ and measures the diameter of the inhibition zone to the test pathogenic bacteria , cultured at 25°C for 4 days to measure the diameter of the inhibition zone to the tested pathogenic fungus, and the control was the liquid fermentation medium in step 1 that was not inoculated with Aspergillus tubingensis QF05. The results showed that the aseptic fermentation filtrate of Aspergillus tubingensis QF05 had inhibitory effects on Pseudomonas syringaepv. ±0.58mm (Figure 4), no inhibitory effect on Ralstonia solanacearum and Chinese cabbage black rot (Xanthomonas campestris pv. campsetris).

Embodiment 4, Aspergillus tubingensis (Aspergillus tubingensis) QF05 sterile fermentation filtrate (sterile metabolite) promotes seed germination

1, Aspergillus tubingensis (Aspergillus tubingensis) QF05 sterile fermentation filtrate dilution preparation

Aspergillus tubingensis QF05 grown on the plate was picked with an inoculation loop and inoculated into a 500 mL Erlenmeyer flask containing 100 mL of liquid fermentation medium, cultured on a shaker at 150 rpm at 25°C for 4 days, and the fermentation broth was collected. The fermentation broth was centrifuged at 12000 rpm for 10 min to take the supernatant, and the supernatant was filtered with a 0.22 μm microporous membrane to sterilize to obtain Aspergillus tubingensis QF05 sterile fermentation filtrate. Aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate is diluted 10 times, 50 times, 100 times respectively with sterile water, obtains aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate 10 times of dilutions, aspergillus tubingensis (Aspergillus tubingensis) tubingensis) QF05 sterile fermentation filtrate 50-fold dilution and Aspergillus tubingensis (Aspergillus tubingensis) QF05 sterile fermentation filtrate 100-fold dilution.

Among them, the liquid fermentation medium is maltose 20g, glycerin 20ml, beef extract 30g, peptone 30g, distilled water to 1000mL, sterilized at 121°C for 20min to obtain the liquid fermentation medium.

2. Seed germination test

Select pepper (Jingyan Guoxi 113) seeds with full particles and uniform size, soak the seeds with 10-fold dilution, 50-fold dilution and 100-fold dilution of Aspergillus tubingensis QF05 sterile fermentation filtrate, and soak the seeds with an equal volume of The sterile liquid fermentation medium of step 1 was used as a control. Cultivate at 28°C for 24 hours, collect the seeds, rinse the seeds with sterile water, put them on a petri dish covered with two layers of sterile water-soaked filter paper, and culture them at 28°C for 5 days, count the germination rate, measure the hypocotyl, embryo root length. Three replicates were set for each treatment, and 10 seeds were set for each replicate.

The experimental results show that compared with the control, the 10-fold dilution of Aspergillus tubingensis QF05 sterile fermentation filtrate reduces the germination rate of pepper seeds, but significantly increases the radicle length of pepper, and the increase reaches 26.0%; Tabingensis Aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate 50-fold dilution of pepper seeds germination rate is the same as the control, but significantly increased the radicle length of pepper, an increase of 58.9%; The 100-fold dilution of the fermentation filtrate not only increased the germination rate of the pepper seeds compared with the control by 11.1%, but also significantly increased the radicle length of the pepper by 149.3% (Fig. 5 and Table 2). It shows that Aspergillus tubingensis QF05 can increase the germination rate of pepper seeds and significantly promote the elongation of pepper radicle.

Table 2, the effect of aspergillus tubingensis (Aspergillus tubingensis) QF05 aseptic fermentation filtrate on the germination of pepper seeds

deal with Germination rate% Root length (mm) Growth rate of radicle length (%) control 90 10.56 - 10 80 13.31 26.0 50 90 16.78 58.9 100 100 26.33 149.3

Note: 10, 50 and 100 represent 10-fold dilution of Aspergillus tubingensis QF05 sterile fermentation filtrate, 50-fold dilution of Aspergillus tubingensis QF05 sterile fermentation filtrate and Aspergillus tubingensis (Aspergillus tubingensis) respectively 100-fold dilution of QF05 sterile fermentation filtrate.

Embodiment 5, the bacterium-containing metabolite of Aspergillus tubingensis QF05 promotes plant growth

1. Preparation of bacterium-containing metabolites of Aspergillus tubingensis QF05

Aspergillus tubingensis (Aspergillus tubingensis) QF05 grown on the plate was picked with an inoculation loop and inoculated into a 500mL Erlenmeyer flask containing 100mL liquid fermentation medium, cultured on a shaking table at 25°C for 4 days, and the fermentation broth (containing Aspergillus tubingensis ( Aspergillus tubingensis) QF05 and its metabolites). The fermentation broth was sterilized at 115° C. for 20 minutes to obtain the bacterium-containing metabolite of Aspergillus tubingensis QF05.

Among them, the liquid fermentation medium is maltose 20g, glycerin 20ml, beef extract 30g, peptone 30g, distilled water to 1000mL, sterilized at 121°C for 20min to obtain the liquid fermentation medium.

2. Cucumber plant cultivation

Cucumbers were routinely sown and raised in greenhouse seedling trays, and the cucumber variety was Jingyan Xiamei.

When the cucumber plant grows to the stage of two leaves and one heart, use the bacteria-containing metabolites of Aspergillus tubingensis QF05 in step 1 to dip the roots, and place the cucumber plants in 200ml of Aspergillus tubingensis QF05 containing 200ml of step 1. in a beaker containing bacterial metabolites for 20min, and then transplanted to a pot. Thereafter, each cucumber plant was treated with 100 ml of the bacterial metabolite of Aspergillus tubingensis QF05 in step 1 until 30 days after transplanting. The whole test process is compared with the aseptic liquid fermentation medium of step 1. Investigate the area of the top two true leaves and plant height at the 30th day of the cucumber plant growth, and count the cucumber yield until the plant grows for 90 days. Each treatment was replicated three times, with 10 seedlings per replicate.

The results showed that the bacterial metabolites of Aspergillus tubingensis QF05 promoted the growth of true leaves of cucumber, increased the plant height of cucumber, and increased the yield of cucumber (Table 3).

Table 3 Effects of bacterial metabolites of Aspergillus tubingensis QF05 on biological traits and yield of cucumber plants

Note: The stock solution is the bacterial metabolite of Aspergillus tubingensis QF05.

<110> Beijing Academy of Agriculture and Forestry Sciences

<120> Preparation and application of a strain of Aspergillus tubingensis and its metabolites for disease prevention and growth promotion

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aacgatcgac ttccccggta tgtgatagtc tacgcctgta aggcgggaat gccgtatgga 420

ttgtgattga cttttgccgc cagaattcct taccatgatg gctcgtaaga tgaaggacac 480

cgactccgag gaggaaatcc gcgaggcttt caaggtcttc gaccgcgaca acaatggttt 540

catctccgcc gcggagttgc gccacgttat gacctctatc gg 582

Claims (10)

1. Tabin aspergillus (Aspergillus tubingensis), bacterial strain number is QF05, in Chinese microorganism strain preservation The number of registering on the books of administration committee's common micro-organisms center is CGMCC No.13563.

2. the fermentation liquid of Tabin aspergillus described in claim 1 is inhibiting the application in pathogen, the pathogen is capsicum charcoal Subcutaneous ulcer germ and/or cucumber fusarium axysporum.

3. application of the fermentation liquid of Tabin aspergillus described in claim 1 in preparation cause of disease bacteria inhibitor, the pathogen are Colletotrichum capsici and/or cucumber fusarium axysporum.

4. the fermentation liquid of Tabin aspergillus described in claim 1 is inhibiting the application in disease, the disease is pepper anthracnose And/or cucumber fusarium axysporum.

5. the fermentation liquid of Tabin aspergillus described in claim 1 is capsicum preparing the application in disease suppression agent, the disease Anthracnose and/or cucumber fusarium axysporum.

6. the fermentation liquid of Tabin aspergillus described in claim 1 is promoting the application in Germination of Capsicum Seed.

7. the fermentation liquid of Tabin aspergillus described in claim 1 is promoting the application in pepper seed radicle growth.

8. the fermentation liquid of Tabin aspergillus described in claim 1 is promoting the application in the growth of cucumber true leaf.

9. the fermentation liquid of Tabin aspergillus described in claim 1 is improving the application in cucumber plant height.

10. the fermentation liquid of Tabin aspergillus described in claim 1 is improving the application in cucumber yield.