Beauveria bassiana QSE-F1 and application of Beauveria bassiana QSE-F1 in preparation of biological preparation for preventing and treating botrytis cinerea

Abstract

The invention discloses beauveria bassiana QSE-F1 and application of the beauveria bassiana QSE-F1 in preparation of a biological preparation for preventing and treating botrytis cinerea. The beauveria bassiana QSE-F1 is a plant endophyte separated and screened from tomato stems, the classification name of the beauveria bassiana QSE-F1 is Beauveria bassiana, the beauveria bassiana QSE-F1 is preserved in the China Center for Type Culture Collection, and the preservation number of the beauveria bassiana QSE-F1 is CCTCC NO: M 20241661. A bacterial colony of the beauveria bassiana QSE-F1 is flat, villus-shaped, thick in middle and thin in edge, the surface of the bacterial colony is initially milky white and gradually becomes faint yellow along with the increase of culture time, and the bacterial colony is milky white powder after spores are generated in the later period; the QSE-F1 hyphae are colorless and transparent and are provided with diaphragms, and most conidia are spherical and oval. Experiments prove that the fermentation filtrate of the beauveria bassiana QSE-F1 can remarkably inhibit spore germination and hypha growth of botrytis cinerea, has a remarkable prevention and treatment effect on the occurrence of the botrytis cinerea, can be used for preparing biological agents for preventing and treating the botrytis cinerea, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, and particularly relates to a strain of Beauveria bassiana QSE-F1 and application of the strain in preparing a biological agent for controlling tomato gray mold. Background Art

[0002] Beauveria bassiana is a tricholoma insect pathogenic fungus widely used in biological control of agricultural and forestry pests. It can parasitize more than 700 species of insects and has a wide host range. At the same time, as a dual biological control fungus, Beauveria bassiana can not only effectively control pests by colonizing in plants, but also inhibit the growth of various pathogens such as Gaeumannomyces graminis, Fusarium oxysporum, Rhizoctonia solani, and Exser ohilumturcicum. In recent years, studies have found that the genome of Beauveria bassiana contains abundant secondary metabolic gene clusters, which can synthesize non-ribosomal peptides and benzoquinone antibiotics. These substances are secreted outside the cell to antagonize pathogens. Beauveria bassiana also has the effect of promoting plant growth and improving plant tolerance to abiotic stress. As a model insect pathogenic fungus, the biological characteristics of Beauveria bassiana have a good research foundation, are safe for non-target organisms and the environment, and are in line with the purpose of integrated pest management. Beauveria bassiana is easy to cultivate and mass-produce, and has a competitive control cost, giving it broad application prospects in the biological control of plant diseases and insect pests.

[0003] Tomato gray mold is a common fungal disease caused by Botrytis cinerea infecting tomatoes, which mainly occurs in the stems, leaves, flowers, fruits and other parts of tomatoes. White or gray mold spots will appear on the surface of the infected plants, and the infected parts will become soft and rotten and brown. As an important vegetable and economic crop, tomatoes are widely grown around the world. Botrytis cinerea can infect tomatoes during the planting process, storage, transportation and retail links, causing significant economic losses to tomato production. In recent years, the area of ​​protected tomato cultivation has continued to expand, providing suitable conditions for the occurrence and spread of gray mold. The disease has spread rapidly, and the area and severity of the disease have shown an upward trend. At present, the most effective means of prevention and control of this disease is still chemical control, but because Botrytis cinerea has the characteristics of a wide host range, a short life cycle, and easy genetic variation, it has a high resistance to pesticides, which poses a serious challenge to the prevention and control of this disease. Due to the problem of drug resistance, it has further led to the excessive use of traditional chemical pesticides, causing serious environmental pollution and ecological balance damage. Therefore, in actual production, it is urgent to find green and safe prevention and control measures to ensure and achieve the safe, green and sustainable development of the tomato industry. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention aims to provide a strain of Beauveria bassiana QSE-F1 and its application in the preparation of a biological agent for preventing and treating tomato gray mold. The Beauveria bassiana QSE-F1 can significantly inhibit the growth of Botrytis cinerea and has a significant preventive effect on the occurrence of tomato gray mold.

[0005] To achieve the above invention objectives, the present invention is implemented through the following scheme:

[0006] The invention provides a strain of Beauveria bassiana QSE-F1 for preventing and controlling tomato gray mold. The strain has a classification name of Beauveria bassiana and is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M20241661.

[0007] Furthermore, the colony of the Beauveria bassiana QSE-F1 is flat, villi-like, thick in the middle and thin at the edge. The colony surface is milky white initially, gradually turns light yellow with the increase of culture time, and becomes milky white powder after spores are produced in the later stage. The QSE-F1 bacterial body vegetative hyphae is colorless, transparent and has septa, and the conidia are spherical and oval.

[0008] Furthermore, the ITS sequence of the Beauveria bassiana QSE-F1 is shown in SEQ ID No: 1.

[0009] Furthermore, the B-locus sequence of the Beauveria bassiana QSE-F1 is shown in SEQ ID No: 2.

[0010] The invention also provides a biocontrol preparation containing Beauveria bassiana QSE-F1.

[0011] The present invention also provides the use of the Beauveria bassiana QSE-F1 in the preparation of a biological agent for preventing and controlling tomato gray mold.

[0012] Furthermore, the Beauveria bassiana QSE-F1 can inhibit the hyphae growth and spore germination of Botrytis cinerea.

[0013] Furthermore, when used, the Beauveria bassiana QSE-F1 is prepared into Beauveria bassiana QSE-F1 fermentation liquid.

[0014] Furthermore, the Beauveria bassiana QSE-F1 fermentation broth can be used alone or prepared into a biological preparation with other materials.

[0015] Furthermore, the biological preparation contains Beauveria bassiana QSE-F1 fermentation broth.

[0016] Furthermore, the preparation method of the Beauveria bassiana QSE-F1 fermentation broth is as follows: Beauveria bassiana QSE-F1 is inoculated into a YEPD liquid culture medium, shaken and cultured at 25°C and 180rpm for 4 days, the culture broth is centrifuged at 12000rpm, and the supernatant is filtered through a 0.45μm sterile filter to obtain a Beauveria bassiana QSE-F1 fermentation broth free of hyphae and spores.

[0017] Furthermore, the volume ratio of the Beauveria bassiana QSE-F1 fermentation broth added to the biological preparation is 12.5-50%.

[0018] Furthermore, the optimal concentration of Beauveria bassiana QSE-F1 for inhibiting the hyphae growth and spore germination of Botrytis cinerea is 50% (v / v).

[0019] Furthermore, the method for using the biological agent is: after making a solution, spray it evenly on the entire tomato plant.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: the Beauveria bassiana QSE-F1 of the present invention is extracted from greenhouse tomato plants, and has a significant inhibitory effect on Botrytis cinerea, and can not only inhibit the growth of Botrytis cinerea hyphae, but also inhibit the spore germination of Botrytis cinerea, thereby inhibiting the growth of Botrytis cinerea on plants from multiple aspects. Beauveria bassiana QSE-F1 is made into a strain fermentation liquid, which can be used alone or prepared with other materials into a biological preparation for preventing and controlling tomato gray mold, and the biological preparation does not contain bacteria, is simple to use, has good effect, is safe and reliable, and has a long storage period, so it has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the colony morphology of the Beauveria bassiana QSE-F1 on the PDA culture medium.

[0022] Figure 2 The mycelium and mycelium morphology of Beauveria bassiana QSE-F1 are described.

[0023] Figure 3 This is the conidia morphology of the Beauveria bassiana QSE-F1.

[0024] Figure 4 is the phylogenetic tree of Beauveria bassiana QSE-F1.

[0025] Figure 5 The fermentation liquid of Beauveria bassiana QSE-F1 with different concentrations inhibits the growth of Botrytis cinerea hyphae on the PDA plate. The abscissa of the bar graph represents the concentration of the fermentation liquid of the QSE-F1 strain in the PDA plate, and the ordinate represents the inhibition rate of the growth of Botrytis cinerea hyphae.

[0026] Figure 6 The fermentation liquid of Beauveria bassiana QSE-F1 with different concentrations inhibits the germination of Botrytis cinerea spores. The abscissa represents the concentration of the fermentation liquid of QSE-F1 strain in the spore liquid, and the ordinate represents the germination rate of Botrytis cinerea spores.

[0027] Figure 7 The results are as follows: the in vitro experimental effects of different concentrations of fermentation liquid of Beauveria bassiana QSE-F1 on preventing and treating tomato gray mold; the QSE-F1 fermentation liquid was mixed with Botrytis cinerea spores, and the diameter of the lesions was measured 2 days after inoculation; CK indicates that H2O was used instead of the fermentation liquid of QSE-F1 to treat the leaves.

[0028] Figure 8 The present invention is a live experimental effect of the Beauveria bassiana QSE-F1 fermentation liquid in preventing and treating tomato gray mold. After the whole plant is treated with the QSE-F1 fermentation liquid, the plant is sprayed with and inoculated with Botrytis cinerea spore liquid, and the disease condition of the tomato plant is observed after 4-6 days. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials, drugs, instruments, etc. used in the following embodiments are all commercially available products unless otherwise specified. The quantitative statistics in the following embodiments are all repeated experiments three times and the average value is taken.

[0030] Example 1

[0031] 1. Isolation and screening of QSE-F1 strain

[0032] On October 2, 2022, 5 tomato samples were collected in a greenhouse in Qingdao. A stem of about 1 cm was cut from the main stem of the plant, disinfected with 0.1% sodium hypochlorite for 3 minutes, 70% ethanol for 1 minute, and washed three times with sterile water; then placed in a sterile mortar to grind out the juice, and the juice was diluted with sterile water; 100 μl of the 100-fold and 1000-fold diluted tissue suspensions were taken respectively, and evenly spread on the PDA plate with a spreader (200 g of potato was cut into pieces and boiled in boiling water for 15 minutes, filtered through three layers of gauze, the supernatant was taken, 20 g of glucose, 15 g of agar powder, water was added to 1 L, and autoclaved at 121°C for 20 minutes), and placed in a 28°C incubator for 24-48 hours. Pick a single colony grown on the PDA plate and inoculate it on a new PDA plate to obtain a single colony.

[0033] The purified strain was inoculated into YEPD liquid medium (10g yeast powder, 20g peptone, 20g glucose, add water to 1L, and sterilize at 121℃ for 20min), and cultured at 25℃, 180rpm for 4 days. The culture solution was centrifuged at 12000rpm, and the supernatant was filtered through a 0.45μm sterile filter to obtain a fermentation solution without bacteria. The fermentation solution was mixed with PDA medium and poured onto a plate, and then inoculated with Botrytis cinerea cake, and continued to be cultured in a 25℃ incubator for 2-3 days. The inhibition of Botrytis cinerea hyphae growth was observed, and a strain with a strong inhibitory effect of the fermentation solution on the growth of Botrytis cinerea hyphae was obtained, named QSE-F1.

[0034] 2. Classification and Identification of QSE-F1 Strain

[0035] 1. Morphological identification of strains: The QSE-F1 strain was inoculated on PDA medium and cultured at 28°C. The colonies formed were flat and fluffy, thick in the middle and thin at the edges. The surface of the colonies was milky white at first, and gradually turned into light yellow with the increase of culture time. Spores began to form after 5-7 days, and the surface of the colonies was milky white powder ( Figure 1 ); QSE-F1 aerial hyphae were selected and observed under a microscope. The hyphae were colorless, transparent, and had septa, with a diameter of about 1.73±0.27 μm ( Figure 2 Conidia are mostly spherical and oval, with a size of about 1.52±0.17μm - 3.52±0.23μm ( Figure 3 ).

[0036] 2. Molecular identification: The genomic DNA of QSE-F1 strain was extracted and used as a template to perform PCR amplification using specific primers; the amplified specific genes and primers are shown in Table 1;

[0037] Table 1 Amplified genes and primers used

[0038]

[0039] The PCR reaction system was as follows: 5×TransStart R FastPfu Buffer 10 μl; 10 mM dNTP mix1 μl; template 2 μl; primer-F (10 μM) 1 μl; primer-R (10 μM) 1 μl; TransStart R FastPfuDNA polymerase 1 μl; ddH2O 34 μl; Total 50 μl.

[0040] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 40 s; extension at 72°C for 5 min; 34 cycles; and hold at 4°C.

[0041] The PCR products were cut and recovered after running on the gel, and the recovered products were sent to a sequencing company for sequencing. After sequencing, the fragment sequences of the ITS and B-locus genes of the strain QSE-F1 are shown as SEQ ID No: 1 and SEQ ID No: 2. The sequences obtained by sequencing were compared with the sequences in the NCBI database, and the ITS and B-locus fragment sequences of the strain QSE-F1 to be tested had the highest homology with the sequence of Beauveria bassiana. Based on the sequence alignment of the gene fragments, a phylogenetic evolutionary tree was constructed ( Figure 4 ), through comprehensive analysis, strain QSE-F1 was identified as Beauveria bassiana.

[0042] The Beauveria bassiana QSE-F1 screened by the present invention is deposited in China Center for Type Culture Collection; address: Wuhan University, Wuhan, China; deposit date: July 22, 2024; the deposit number of Beauveria bassiana QSE-F1 is CCTCC NO: M 20241661.

[0043] Example 2: Inhibitory effect of QSE-F1 fermentation broth on the growth of Botrytis cinerea

[0044] The Beauveria bassiana QSE-F1 was inoculated into a YEPD liquid culture medium and cultured at 25°C and 180 rpm for 4 days. The culture solution was centrifuged at 12,000 rpm and the supernatant was filtered through a 0.45 μm sterile filter to obtain a QSE-F1 fermentation solution free of QSE-F1 hyphae and spores.

[0045] 1. Inhibitory effect of QSE-F1 fermentation broth on the growth of Botrytis cinerea hyphae

[0046] QSE-F1 fermentation liquid was added to PDA medium at a volume ratio of 12.5%, 25%, and 50%, and PDA plates containing different concentrations of QSE-F1 fermentation liquid were prepared. The PDA plate without QSE-F1 fermentation liquid was used as the control (CK), and 4 plates were prepared for CK and 3 different concentrations of QSE-F1 fermentation liquid. Botrytis cinerea cake was inoculated in the center of the plate and cultured in an incubator at 25℃ for 3 days. The colony diameter (a and b) of Botrytis cinerea on the plate was measured by the cross method, and the colony area was calculated according to the ellipse area formula (S=πab / 4). According to the area, the inhibition rate of different concentrations of QSE-F1 fermentation liquid on the growth of Botrytis cinerea hyphae was calculated according to Formula 1.

[0047] (1)

[0048] Among them, S CK represents the average colony area of ​​Botrytis cinerea in the control group, S F1 It represents the average colony area of ​​Botrytis cinerea in the treatment group added with QSE-F1 fermentation liquid.

[0049] The results are as follows Figure 5 As shown in the figure, the three concentrations of QSE-F1 fermentation broth had an inhibitory effect on the growth of Botrytis cinerea hyphae, and with the increase of concentration, the inhibition rate on hyphae growth increased significantly. When the concentration of QSE-F1 fermentation broth was 50%, the inhibition rate was 36.48%.

[0050] 2. Inhibitory effect of QSE-F1 fermentation broth on spore germination of Botrytis cinerea

[0051] Use 1 / 10 concentration of YEPD medium to collect Botrytis cinerea conidia and adjust the concentration to 10 4 cfu. Add QSE-F1 fermentation liquid with a final concentration of 12.5%, 25% and 50% to the spore suspension as the treatment group, and add an equal volume of YEPD medium as the CK group. Pipette 20μl of spore liquid and drop it on the slide, place it in a dark box to keep it moist, and culture it in a 25℃ incubator. When the spores in the CK group germinate more than 90%, observe the spore germination in the treatment group and the CK group.

[0052] The spore germination results are as follows Figure 6 As shown in the figure, when the spore suspension was cultured for about 6 hours, the spore suspension in the CK group germinated completely and the germ tube was long, while the spores containing 25% QSE-F1 fermentation liquid germinated completely but the growth rate of the germ tube was significantly reduced, and the spores containing 50% QSE-F1 fermentation liquid only partially germinated and the length of the germ tube was extremely short, indicating that when the fermentation liquid concentration reached 50%, it had a strong inhibitory effect on the germination of Botrytis cinerea spores, and the inhibition rate exceeded 50% ( Figure 6 ).

[0053] Example 3: Application of QSE-F1 fermentation broth in the prevention and treatment of tomato gray mold

[0054] 1. In vitro experiment on the prevention and treatment of tomato gray mold with QSE-F1 fermentation liquid

[0055] Preparation of Botrytis cinerea conidia suspension (10 6 cfu), with QSE-F1 fermentation liquid added as the treatment group, and YEPD added in equal proportion as the control group (CK). Four-week-old tomato seedlings with consistent growth were selected, and 10 leaves were taken from each of the control group and the treatment group and placed on moist filter paper. 10ul of the treated spore suspension was taken and inoculated on the tomato leaves. Moisturizing was carried out for 48h, and the infection of Botrytis cinerea was observed; the diameter of the edge of the lesion was measured by the cross method, and the elliptical area was calculated. The lesion area of ​​the treatment group and the control group was statistically analyzed.

[0056] The results are as follows Figure 7 As shown, the leaf spot area in the QSE-F1 fermentation liquid treatment group was significantly smaller than that in the control group. As the concentration of QSE-F1 fermentation liquid increased, the control effect was significantly improved. When the QSE-F1 fermentation liquid was added at a concentration of 50%, the inhibition rate of Botrytis cinerea infection exceeded 35%. The experiment was repeated three times and the results were basically consistent. 2. Potted experiment of QSE-F1 fermentation liquid to prevent and control tomato gray mold

[0057] Four-week-old potted tomato seedlings with consistent growth were selected and sprayed evenly with 50% QSE-F1 fermentation liquid as the treatment group, and the control group (CK) was treated with water containing 50% YEPD medium. After drying for 12 hours, the conidia suspension of Botrytis cinerea (10 6 cfu) were evenly sprayed on the plant leaves and placed in a light incubator with a humidity of more than 85% for further cultivation. After 4 days of cultivation, the disease incidence of the control group and the treatment group was observed.

[0058] The results are as follows Figure 8 As shown, the number of diseased leaves in tomato plants treated with QSE-F1 fermentation liquid was significantly less than that in the control group, and the growth state of the whole plant was significantly better than that in the control group.

[0059] Based on all the above results, the fermentation broth of Beauveria bassiana QSE-F1 can directly inhibit the mycelial growth and spore germination of Botrytis cinerea, and has a significant preventive and control effect on the occurrence of tomato gray mold. It can be developed into a biological agent for the effective prevention and control of tomato gray mold.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A strain of Beauveria bassiana QSE-F1, characterized in that: Beauveria bassiana Beauveria bassiana , deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20241661.

2. The Beauveria bassiana QSE-F1 according to claim 1, characterized in that: The colony of the Beauveria bassiana QSE-F1 is flat and fluffy. The surface of the colony is milky white at first, gradually turns to light yellow with the increase of culture time, and becomes milky white powder after spores are produced in the later stage. The QSE-F1 hyphae is colorless, transparent and has septa, and the conidia are spherical and oval.

3. A biocontrol agent containing the Beauveria bassiana QSE-F1 according to claim 1.

4. Use of the Beauveria bassiana QSE-F1 according to claim 1 or the biocontrol agent according to claim 3 in the preparation of a biological agent for preventing and controlling tomato gray mold.

5. The use according to claim 4, characterized in that: The Beauveria bassiana QSE-F1 can significantly inhibit the hyphae growth and spore germination of Botrytis cinerea.

6. The use according to claim 4, characterized in that: The biological preparation contains Beauveria bassiana QSE-F1 fermentation broth.

7. The use according to claim 6, characterized in that: The preparation method of the Beauveria bassiana QSE-F1 fermentation liquid is as follows: inoculating Beauveria bassiana QSE-F1 into a YEPD liquid culture medium for shaking culture, centrifuging the culture liquid, filtering the supernatant through a sterile filter, and obtaining the Beauveria bassiana QSE-F1 fermentation liquid free of hyphae and spores.

8. The use according to claim 6, characterized in that: The volume ratio of the Beauveria bassiana QSE-F1 fermentation broth added to the biological preparation is 12.5%-50%.

9. The use according to claim 4, characterized in that: The method for using the biological agent is to spray it evenly on the entire plant.

10. The use according to claim 4, characterized in that: The plant is tomato.

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