A strain of Beauveria bassiana QSE-F1 and its application in the preparation of biological agents for controlling tomato gray mold.

Abstract

This invention discloses a strain of Beauveria bassiana QSE-F1 and its application in the preparation of biological agents for controlling tomato gray mold. The Beauveria bassiana QSE-F1 is a plant endophytic fungus isolated and screened from tomato stems, classified as... Beauveria bassiana The *Beauveria bassiana* QSE-F1 strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number M 20241661. The colonies of *Beauveria bassiana* QSE-F1 are flat, fluffy, thick in the middle and thin at the edges. The colony surface is initially milky white, gradually turning pale yellow with increasing culture time, and later becoming a milky white powder after spore production. The hyphae of *Beauveria bassiana* QSE-F1 are colorless, transparent, and septate, and the conidia are mostly spherical and oval. Experiments have demonstrated that the fermentation filtrate of *Beauveria bassiana* QSE-F1 can significantly inhibit the spore germination and hyphal growth of *Botrytis cinerea*, exhibiting a significant control effect on tomato gray mold. It can be used to prepare biological agents for the control of tomato gray mold and has good market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial control technology, specifically relating to a strain of Beauveria bassiana QSE-F1 and its application in the preparation of biological agents for controlling tomato gray mold. Background Technology

[0002] Beauveria bassiana is a filamentous insect pathogenic fungus widely used in the biological control of agricultural and forestry pests. It can parasitize over 700 insect species, exhibiting a wide host range. Simultaneously, as a dual biocontrol fungus, its colonization within plants not only effectively controls pests but also inhibits the growth of various pathogens such as Gaeumannomyces g raminis (wheat take-all), Fusarium oxysporum, Rhizoctonia solani, and Exser ohilumturcicum. Recent studies have revealed that the Beauveria bassiana genome contains abundant secondary metabolic gene clusters, enabling the synthesis of non-ribosomal polypeptides and benzoquinone antibiotics. These substances are secreted extracellularly to exert antagonistic effects against pathogens. Furthermore, Beauveria bassiana also promotes plant growth and enhances plant tolerance to abiotic stresses. As a model entomopathogenic fungus, Beauveria bassiana has a solid research foundation in biological characteristics, good safety against non-target organisms and the environment, and aligns with the principles of integrated pest management. Furthermore, Beauveria bassiana is easy to cultivate and scale up for production, and its control costs are competitive, making it a promising candidate for widespread application in the biological control of plant diseases and pests.

[0003] Gray mold, a common fungal disease of tomatoes, is caused by *Botrytis cinerea*, primarily affecting the stems, leaves, flowers, and fruits. Infected plants develop white or gray mold spots, and infected areas exhibit soft rot and browning. Tomatoes are widely cultivated globally as an important vegetable and economic crop. *Botrytis cinerea* can infect tomatoes during cultivation, storage, transportation, and retail, causing significant economic losses to tomato production. In recent years, the expanding area of ​​protected tomato cultivation has provided suitable conditions for the occurrence and spread of gray mold, leading to its rapid spread and increasing incidence and severity. Currently, chemical control remains the most effective method for controlling this disease. However, due to *Botrytis cinerea*'s wide host range, short life cycle, and high genetic mutation rate, it exhibits high resistance, posing a serious challenge to disease control. This resistance further leads to the overuse of traditional chemical pesticides, causing severe environmental pollution and ecological imbalance. Therefore, in actual production, there is an urgent need 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] To address the aforementioned problems in the existing technology, the purpose of this invention is to provide a strain of Beauveria bassiana QSE-F1 and its application in the preparation of biological agents for the control of tomato gray mold. The Beauveria bassiana QSE-F1 can significantly inhibit the growth of Botrytis cinerea and has a significant control effect on the occurrence of tomato gray mold.

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

[0006] This invention provides a strain of Beauveria bassiana QSE-F1 for controlling tomato gray mold, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M20241661.

[0007] Furthermore, the colonies of the *Beauveria bassiana* QSE-F1 are flat, fluffy, thick in the middle and thin at the edges. The colony surface is initially milky white, gradually turning pale yellow as the culture time increases, and later becomes milky white powder after spores are produced. The vegetative hyphae of the QSE-F1 fungus are colorless, transparent, and septate, 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 present invention also provides a biocontrol preparation containing Beauveria bassiana QSE-F1.

[0011] The present invention also provides the application of the aforementioned Beauveria bassiana QSE-F1 in the preparation of biological agents for the control of tomato gray mold.

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

[0013] Furthermore, in application, the Beauveria bassiana QSE-F1 is prepared into Beauveria bassiana QSE-F1 fermentation broth.

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

[0015] Furthermore, the biological agent 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 YEPD liquid medium and cultured with shaking at 25°C and 180 rpm for 4 days. The culture broth is centrifuged at 12000 rpm, and the supernatant is filtered through a 0.45 μm sterile filter to obtain Beauveria bassiana QSE-F1 fermentation broth free of mycelia and spores.

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

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

[0019] Furthermore, the biological agent is used as follows: after being prepared into a solution, it is sprayed evenly onto the entire tomato plant.

[0020] Compared with existing technologies, this invention has the following advantages and beneficial effects: The *Beauveria bassiana* QSE-F1 strain described in this invention is extracted from greenhouse tomato plants and has a significant inhibitory effect on *Botrytis cinerea*. It not only inhibits the mycelial growth of *Botrytis cinerea* but also inhibits the spore germination, thus inhibiting the growth of *Botrytis cinerea* on plants from multiple aspects. The fermentation broth of *Beauveria bassiana* QSE-F1 can be used alone or with other materials to prepare a biological agent for controlling tomato gray mold. Furthermore, the biological agent does not contain mycelial cells, is simple to use, effective, safe, reliable, and has a long shelf life, thus possessing good market application prospects. Attached Figure Description

[0021] Figure 1 The colony morphology of Beauveria bassiana QSE-F1 on PDA medium is shown.

[0022] Figure 2 This refers to the mycelium and mycelial morphology of the *Beauveria bassiana* QSE-F1 strain.

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

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

[0025] Figure 5 The different concentrations of fermentation broth of Beauveria bassiana QSE-F1 inhibited the growth of Botrytis cinerea mycelia on PDA plates. The horizontal axis of the bar chart represents the concentration of fermentation broth containing the QSE-F1 strain in the PDA plate, and the vertical axis represents the inhibition rate of Botrytis cinerea mycelia growth.

[0026] Figure 6 The different concentrations of fermentation broth of Beauveria bassiana QSE-F1 inhibited the germination of Botrytis cinerea spores. The horizontal axis represents the concentration of fermentation broth containing the QSE-F1 strain in the spore solution, and the vertical axis represents the germination rate of Botrytis cinerea spores.

[0027] Figure 7 This study describes the in vitro experimental effects of different concentrations of Beauveria bassiana QSE-F1 fermentation broth on the control of tomato gray mold. QSE-F1 fermentation broth was mixed with Botrytis cinerea spores, and the diameter of lesions was measured 2 days after inoculation. CK indicates the treatment of leaves with H2O instead of QSE-F1 strain fermentation broth.

[0028] Figure 8 The study describes the in vivo experimental effect of the QSE-F1 fermentation broth of Beauveria bassiana in controlling tomato gray mold. After treating the entire plant with the QSE-F1 fermentation broth, the plant was sprayed with Botrytis cinerea spore solution, and the disease status of the tomato plants was observed 4-6 days later. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the following embodiments are all commercially available products. The quantitative statistics in the following embodiments are all based on three repeated experiments, and the average value is taken.

[0030] Example 1

[0031] I. Isolation and Screening of QSE-F1 Strains

[0032] Five tomato samples were collected from a greenhouse in Qingdao on October 2, 2022. Stems approximately 1 cm long were cut from the main stem of each plant, disinfected with 0.1% sodium hypochlorite for 3 minutes, then with 70% ethanol for 1 minute, and rinsed three times with sterile water. The samples were then ground in a sterile mortar to extract juice, which was serially diluted with sterile water. 100 μl of each of the 100-fold and 1000-fold diluted tissue suspensions were evenly spread onto PDA plates (200g of potatoes were cut into pieces, boiled in water for 15 minutes, filtered through three layers of gauze, and the supernatant was mixed with 20g glucose, 15g agar powder, and water to a final volume of 1 L, then autoclaved at 121℃ for 20 minutes). The plates were then incubated at 28℃ for 24-48 hours. Single colonies grown on the PDA plates were picked and inoculated onto new PDA plates to obtain single colonies.

[0033] The purified strain was inoculated into YEPD liquid medium (10g yeast extract, 20g peptone, 20g glucose, water to 1L, autoclaved at 121℃ for 20min), and cultured with shaking at 25℃ and 180rpm for 4 days. The culture broth was centrifuged at 12000rpm, and the supernatant was filtered through a 0.45μm sterile filter to obtain a fermentation broth free of bacterial cells. The fermentation broth was mixed with PDA medium, poured onto a plate, and then inoculated with Botrytis cinerea mycelial cakes. The mixture was incubated at 25℃ for 2-3 days, and the inhibition of Botrytis cinerea mycelial growth was observed. A strain with a strong inhibitory effect on Botrytis cinerea mycelial growth was obtained and named QSE-F1.

[0034] II. Classification and Identification of QSE-F1 Strains

[0035] 1. Strain morphology identification: QSE-F1 strain was inoculated onto PDA medium and cultured at 28℃. The resulting colonies were flat, fluffy, thick in the middle and thin at the edges; the colony surface was initially milky white, gradually turning pale yellow with increasing culture time. Spores began to be produced after 5-7 days, and the colony surface became milky white powdery. Figure 1 ); Aerial hyphae of QSE-F1 were selected and observed under a microscope. The hyphae were colorless, transparent, and septate, with a diameter of approximately 1.73 ± 0.27 μm. Figure 2 Conidia are mostly spherical and oval, with a size of approximately 1.52±0.17μm - 3.52±0.23μm. Figure 3 ).

[0036] 2. Molecular identification: Genomic DNA was extracted from the QSE-F1 strain and used as a template for PCR amplification with specific primers; the specific genes amplified and the primers are shown in Table 1.

[0037] Table 1. Genes amplified and primers used.

[0038]

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

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

[0041] After PCR product gel running, the gel was excised and recovered, and the recovered product was sent to a sequencing company for sequencing. Sequencing revealed the ITS and B-locus gene fragment sequences of strain QSE-F1 as shown in SEQ ID No: 1 and SEQ ID No: 2. The sequenced sequences were compared with sequences in the NCBI database. The ITS and B-locus fragment sequences of the tested strain QSE-F1 showed the highest homology with those of *Beauveria bassiana*. Based on the gene fragment sequence alignment, a phylogenetic tree was constructed. Figure 4 Through comprehensive analysis, strain QSE-F1 was identified as Beauveria bassiana.

[0042] The Beauveria bassiana QSE-F1 strain screened in this invention was deposited at the China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China; deposit date: July 22, 2024; the accession number for Beauveria bassiana QSE-F1 is CCTCC NO: M 20241661.

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

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

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

[0046] QSE-F1 fermentation broth was added to PDA medium at volume ratios of 12.5%, 25%, and 50% to prepare PDA plates containing different concentrations of QSE-F1 fermentation broth. A PDA plate without added QSE-F1 fermentation broth served as a control (CK). Four plates were prepared for each of the CK and the three different QSE-F1 fermentation broth concentrations. *Botrytis cinerea* mycelial cakes were inoculated at the center of each plate, and the plates were incubated at 25°C for 3 days. The colony diameters (a and b) of *Botrytis cinerea* on the plates were measured using the cross-sectional method. The colony area was calculated using the ellipse area formula (S = πab / 4), and the inhibition rate of different concentrations of QSE-F1 fermentation broth on *Botrytis cinerea* mycelial growth was calculated based on the area using Formula 1.

[0047] (1)

[0048] Where, S CK S represents the average colony area of ​​Botrytis cinerea in the control group. F1 This represents the average colony area of ​​Botrytis cinerea in the treatment group where QSE-F1 fermentation broth was added.

[0049] The results are as follows Figure 5 As shown, the three concentrations of QSE-F1 fermentation broth inhibited the growth of Botrytis cinerea mycelium, and the inhibition rate of mycelial growth increased significantly with increasing concentration. 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 Botrytis cinerea spore germination

[0051] Botrytis cinerea conidia were collected using 1 / 10 concentration YEPD medium and adjusted to a concentration of 10. 4 CFU was added to the spore suspension at final concentrations of 12.5%, 25%, and 50% QSE-F1 fermentation broth as treatment groups, and an equal volume of YEPD medium was added as the control group. 20 μl of spore suspension was added dropwise to a glass slide, placed in a dark box for humidification, and incubated at 25°C. Once spore germination exceeded 90% in the control group, the spore germination status in both treatment and control groups was observed.

[0052] Spore germination results as follows Figure 6 As shown, after about 6 hours of spore culture, the spore suspension in the CK group fully germinated with relatively long germ tubes. In contrast, the spores containing 25% QSE-F1 fermentation broth fully germinated, but the germ tube growth rate was significantly reduced. The spores containing 50% QSE-F1 fermentation broth only partially germinated with extremely short germ tubes. This indicates that a fermentation broth concentration of 50% has a strong inhibitory effect on the germination of *Botrytis cinerea* spores, with an inhibition rate exceeding 50%. Figure 6 ).

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

[0054] 1. In vitro experiment on the control of tomato gray mold by QSE-F1 fermentation broth

[0055] Preparation of Botrytis cinerea conidia suspension (10 6 Using CFU (co-distilled spores), a treatment group was prepared by adding QSE-F1 fermentation broth, and a control group (CK) was prepared by adding an equal proportion of YEPD. Four-week-old tomato seedlings of uniform growth were selected, and ten leaves from each group were placed on moist filter paper. 10 μL of the treated spore suspension was inoculated onto the tomato leaves. After 48 hours of moistening, the infection status of *Botrytis cinerea* was observed; the diameter of the lesion edge was measured using the cross-sectional method, and the area of ​​the ellipse was calculated. The lesion areas of the treatment and control groups were statistically analyzed.

[0056] The results are as follows Figure 7 As shown, the lesion area on leaves in the QSE-F1 fermentation broth treatment group was significantly smaller than that in the control group. The control effect significantly improved with increasing QSE-F1 fermentation broth concentration. At a QSE-F1 fermentation broth concentration of 50%, the inhibition rate against Botrytis cinerea infection exceeded 35%. This experiment was performed in three biological replicates, and the results were largely consistent. 2. Pot experiment on the control of tomato gray mold with QSE-F1 fermentation broth

[0057] Four-week-old potted tomato seedlings with uniform growth were selected. The entire plant was evenly sprayed with 50% QSE-F1 fermentation broth as the treatment group, and water treatment containing 50% YEPD medium was used as the control group (CK). After air-drying for 12 hours, a suspension of Botrytis cinerea conidia (10... 6 CFU was evenly sprayed onto the plant leaves, and the plants were placed in a light incubator with humidity above 85% for further cultivation. After 4 days of cultivation, the disease incidence in 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 broth was significantly less than that in the control group, and the overall growth status of the plants 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 control effect on the occurrence of tomato gray mold. It can be developed into a biological agent for the effective control of tomato gray mold.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The application of the fermentation broth of Beauveria bassiana QSE-F1 in the preparation of biological agents for controlling tomato gray mold, characterized in that, The classification name of the *Beauveria bassiana* QSE-F1 strain is *Beauveria bassiana* (…). Beauveria bassiana The fermentation broth is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241661. The fermentation broth is used by spraying it evenly over the entire plant. The plant is a tomato. The preparation method of the Beauveria bassiana QSE-F1 fermentation broth is as follows: Beauveria bassiana QSE-F1 is inoculated into YEPD liquid medium and cultured with shaking. The culture broth is centrifuged, and the supernatant is filtered through a sterile filter to obtain the Beauveria bassiana QSE-F1 fermentation broth that does not contain hyphae and spores.

2. The application according to claim 1, characterized in that, The volume ratio of the Beauveria bassiana QSE-F1 fermentation broth added to the biological agent is 12.5%-50%.

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