Beauveria bassiana QXNYCJSZX2025-08 and tobacco field application thereof

Abstract

The invention discloses beauveria bassiana QXNYCJSZX2025-08 and application of the beauveria bassiana QXNYCJSZX2025-08 in a tobacco field, and belongs to the technical field of plant protection. The problems that in existing tobacco planting, pesticide residues exceed the standard, the pesticide resistance of pests is enhanced and the ecological environment is damaged due to the fact that chemical pesticides are relied on for preventing and controlling underground pests (such as grubs and cutworms) for a long time, and existing biocontrol bacteria (such as single growth-promoting bacteria or insecticidal bacteria) can only achieve the single function of growth promotion or insect killing are solved. The invention provides an environment-friendly alternative scheme which is characterized in that continuous biological control is realized through endophytic colonization of the beauveria bassiana QXNYCJSZX2025-08 strain, and triple functions of promoting tobacco plant growth (increasing yield and improving quality), enhancing disease resistance and preventing and controlling underground pests are integrated into a whole; efficient utilization of resources and field synergistic interaction are realized. Meanwhile, the invention further develops a matched field application scheme, and empirical support is provided for technical landing.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and in particular to a strain of Beauveria bassiana QXNYCJSZX2025-08 and its application in tobacco fields. Background Technology

[0002] Endophytic microorganisms are ubiquitous in the healthy tissues of vascular plants, collectively forming a complex micro-ecosystem within the plant. They play an irreplaceable role in nutrient absorption, growth and development, and resistance to abiotic stress, with endophytic fungi being a research hotspot. Endophytic fungi have adapted well to the endophytic environment of host plants, forming a mutually beneficial symbiotic relationship with them during co-evolution. Among plant endosymbiotic fungi, a special group of fungi—insect pathogenic fungi—is receiving increasing attention. These fungi can cause disease and death in insects and are an important resource for pest microbial control, commonly used in plant protection. Their pathogenic mechanism involves the secretion of various hydrolytic enzymes such as chitinase, protease, and lipase by fungal spores, which degrade the cuticle of the insect's body and enter the insect's hemocoel. The mycelium inside the body continuously multiplies and grows, destroying the insect's tissues and causing death. These fungal insecticides occupy an increasingly large proportion of the biopesticide industry, possessing advantages such as a wide variety, safety and effectiveness, broad insecticidal spectrum, ease of large-scale production, and low resistance in pests. However, they also have disadvantages such as spore activity being easily affected by the environment and slow knockdown rate.

[0003] Increasing research indicates that, in addition to their role as insecticides, entomopathogenic fungi play multiple ecological roles in nature, including endophytic colonization in plants, antagonism against plant diseases and pests, and promotion of plant growth and rhizosphere colonization. Exploring diverse application methods of entomopathogenic fungi, and avoiding the adverse effects of direct application to the field surface such as solar radiation, high temperatures, and low humidity, is a crucial research direction for addressing the shortcomings of fungal insecticides. Among these, exploring the role of entomopathogenic fungi as plant endophytes in promoting growth and enhancing stress resistance is a feasible direction that could improve the utilization efficiency of these fungi in integrated pest management systems.

[0004] Endophytic colonization by entomopathogenic fungi can promote the growth and development of host plants and help them antagonize various pathogens and feeding insects in the field. Its main mechanisms of action include: promoting the synthesis of secondary metabolites (enzymes, toxins, or antibiotics), initiating plant defenses, competing for nutrients and ecological niches, and the secondary metabolites, in turn, regulating plant-microbe interactions and dynamically influencing the overall composition of the plant-root microbial community. For example, entomopathogenic fungi can synthesize plant hormones such as indole-3-acetic acid, cytokinins, indole-3-acetonitrile, and salicylic acid to promote the growth and adaptability of host plants. After colonization by *Beauveria bassiana* in tobacco, it can induce the upregulation of salicylic acid and other immune pathways, thereby promoting the disease resistance of tobacco. Furthermore, these metabolites also participate in mechanisms such as signal transduction, defense, and genetic regulation of symbiosis between the host and endophytic fungi; they can enhance the adaptability of fungal endophytes and their host plants, and improve tolerance to biotic and abiotic stresses. In addition, entomopathogenic fungi are closely connected with plant roots. They form a complex network of material and information exchange with other symbiotic microorganisms through hyphae in the plant roots. Through this network, nutrients (essential elements such as nitrogen, phosphorus, and potassium) can be transferred from the soil and even insects to the host plant, thereby promoting plant nutrient absorption and thus promoting plant growth. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Beauveria bassiana QXNYCJSZX2025-08 and its application in tobacco fields to solve the problems existing in the prior art. The Beauveria bassiana QXNYCJSZX2025-08 strain provided by this invention can integrate the three functions of "promoting tobacco plant growth (increasing yield and quality), enhancing disease resistance and controlling underground pests", so as to achieve efficient resource utilization and synergistic effect in the field.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a strain of Beauveria bassiana QXNYCJSZX2025-08, which was deposited on September 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42209. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides the application of the aforementioned Beauveria bassiana QXNYCJSZX2025-08 in the preparation of biocontrol agents.

[0009] The present invention also provides a biocontrol agent comprising the aforementioned Beauveria bassiana QXNYCJSZX2025-08.

[0010] The present invention also provides the application of the aforementioned Beauveria bassiana QXNYCJSZX2025-08 or the aforementioned biocontrol agent in the control of underground pests, the underground pests including small cutworms, large cutworms, yellow cutworms, eight-character cutworms, wireworms, fine-breasted wireworms, black-brown gill beetles, black-velvet gill beetles and mole crickets.

[0011] The present invention also provides the application of the aforementioned Beauveria bassiana QXNYCJSZX2025-08 or the aforementioned biocontrol agent in promoting tobacco growth.

[0012] The present invention also provides the application of the aforementioned Beauveria bassiana QXNYCJSZX2025-08 or the aforementioned biocontrol agent in improving the economic benefits of tobacco leaves.

[0013] The present invention also provides a method for controlling underground pests, including the step of applying the aforementioned Beauveria bassiana QXNYCJSZX2025-08;

[0014] The underground pests include small cutworms, large cutworms, yellow cutworms, eight-character cutworms, wireworms, fine-breasted wireworms, black-brown gill beetles, black-velvet gill beetles, and mole crickets.

[0015] The present invention also provides a method for promoting tobacco leaf growth and / or improving the economic benefits of tobacco leaves, including the step of applying the aforementioned Beauveria bassiana QXNYCJSZX2025-08.

[0016] Optionally, the method includes the step of applying the Beauveria bassiana QXNYCJSZX2025-08 to the field and the surface of tobacco leaves.

[0017] Optionally, the Beauveria bassiana QXNYCJSZX2025-08 applied to the field is a granule formulation with a spore content of 1.5 billion spores / g and an application rate of 5 kg / mu.

[0018] The Beauveria bassiana QXNYCJSZX2025-08 applied to the surface of tobacco leaves is a suspension with a spore content of 1×10⁻⁶. 8 Spores / mL, application rate is 100 mL / plant.

[0019] The present invention discloses the following technical effects:

[0020] To address the problems in existing tobacco cultivation, such as the long-term reliance on chemical pesticides to control underground pests (e.g., grubs and cutworms), leading to excessive pesticide residues, increased pest resistance, and ecological damage, as well as the limitations of existing biocontrol bacteria (such as single growth-promoting or insecticidal bacteria) which only achieve single functions of promoting growth or killing insects and cannot simultaneously improve the overall resilience of crops, this invention provides an environmentally friendly alternative: continuous biological control through the endophytic colonization of Beauveria bassiana strain QXNYCJSZX2025-08. This integrates the three functions of "promoting tobacco plant growth (increasing yield and quality), enhancing disease resistance, and controlling underground pests," achieving efficient resource utilization and synergistic benefits in the field. Furthermore, this invention has developed a corresponding field application plan, providing empirical support for the technology's implementation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 These are some Beauveria bassiana specimens collected in the field; A, L, and E represent 16 specimens respectively.

[0023] Figure 2 The following are sporulation structures, spore morphology, and phylogenetic trees of some Beauveria bassiana strains: A: Sporulation structure and spore morphology of strain XYML-1; B: Sporulation structure and spore morphology of strain ST389-1; C: Sporulation structure and spore morphology of strain XYML-6; D: Sporulation structure and spore morphology of strain XYML-7; E: Phylogenetic tree of Beauveria bassiana strains and closely related species.

[0024] Figure 3 Colony morphology of highly virulent strains ST389-1(A) and XYML-1(B);

[0025] Figure 4 To screen the virulence of different Beauveria bassiana strains; A: Survival curves of each strain infecting the wax moth; B: Median lethal time of different Beauveria bassiana strains on 3-year-old wax moths.

[0026] Figure 5 To screen the endophytic colonization ability of two highly virulent Beauveria bassiana strains in tobacco seedlings.

[0027] Figure 6 The three-stage propagation of Beauveria bassiana strain QXNYCJSZX2025-08: A: Activated strain; B: Liquid seed culture; C: Solid fermentation culture; D: Mature spore powder; E: Solid granular inoculant product;

[0028] Figure 7 Evaluation and analysis of the overall insect control effect of different treatment groups; A: replanting rate; B: number of live insects per 100 plants; C: number of dead insects per 100 plants; D: incidence rate of root and stem diseases; E: incidence rate of viral diseases;

[0029] Figure 8 The growth of tobacco leaves in different treatment groups during the rosette stage, vigorous growth stage, and maturity stage;

[0030] Figure 9 Analysis of the main agronomic traits of tobacco leaves in different treatment groups; A: plant height; B: number of effective leaves; C: length of the middle leaf; D: width of the middle leaf;

[0031] Figure 10 Analysis of yield and quality data of flue-cured tobacco leaves for different treatment groups; A: Yield per mu; B: Output value per mu; C: Proportion of top-grade tobacco and proportion of top-grade tobacco in the middle section;

[0032] Figure 11 Sampling methods for soil (A) and tobacco leaf tissue (B);

[0033] Figure 12 Nested PCR was used to detect the presence of strain QXNYCJSZX2025-08 in different tissues of tobacco leaves and rhizosphere soil; M is the marker, - is the negative control ddH2O, and + is the positive control QXNYCJSZX2025-08 DNA amplification.

[0034] Figure 13 The isolation of Beauveria bassiana from tobacco soil (A), roots (B), stems (C), and leaves (D) is shown; the red arrows indicate Beauveria bassiana colonies.

[0035] Figure 14 The isolation rate of Beauveria bassiana in different tissues of tobacco plants. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Example 1: Isolation, identification, and preservation of Beauveria bassiana QXNYCJSZX2025-08

[0042] From 2023 to 2024, a total of 18 Beauveria bassiana specimens were collected from Xingyi City and other areas in Qianxinan Prefecture, Guizhou Province. Figure 1 (Sixteen specimens are shown). Fungal strains were isolated from the specimens by disinfecting the washroom tables and the surrounding environment with 75% alcohol. After steam sterilization for 30 minutes, the entomopathogenic fungi collected in the field were isolated into pre-prepared PDA agar plates around an alcohol lamp flame. A total of 18 Beauveria bassiana strains were isolated from these specimens.

[0043] Observation of Beauveria bassiana spore structure using an optical microscope ( Figure 2 China A- Figure 2 The ITS sequence of the strain was extracted using the CTAB (hexadecyltrimethylammonium bromide) method and sent to Sangon Biotech Co., Ltd. (Shanghai) for DNA sequencing. The sequenced sequences were spliced ​​using Bioedit software to remove chimeric, overlapping, and other abnormal sequences, and then compared in the NCBI (National Center for Biotechnology Information) database. Species information was classified based on a 97% sequence similarity threshold, and a phylogenetic tree was constructed for species identification. Figure 2 (E).

[0044] The isolated strains had milky-white colonies. The colonies were powdery, thin, wrinkled in the middle, and grew radially. The base of the colonies was colorless or pale yellow. Under a light microscope, the conidiophores were whorled or solitary, repeatedly producing conidia apically, with geniculate conidiophores bearing small teeth. The conidiophores were smooth and spike-like, and the conidia were transparent, spherical, 2.1 μm × 2.7 μm in size, with a smooth surface, consistent with the characteristics of *Beauveria bassiana*. Phylogenetic analysis showed that, except for XYML-7, all isolated strains clustered with *Beauveria bassiana* (TNAU ENT BB1), indicating a close phylogenetic relationship. Combined morphological and molecular identification results showed that, except for XYML-7, the similarity of the 17 isolated strains to the type species was greater than 98%. Figure 2 These strains are all Beauveria bassiana.

[0045] Subsequently, using the "classic" body wall inoculation method, the virulence of the isolated *Beauveria bassiana* strains was determined using third-instar *Malus baccata* larvae. The concentration of the *Beauveria bassiana* spore suspension used for inoculation was 2 × 10⁻⁶. 7 The *Beauveria bassiana* strain was soaked in a spore suspension for 15 seconds (conidia / mL), filtered, and then placed in a 26°C incubator with cotton balls to maintain humidity. Survival rates were recorded starting 48 hours after infection and observed every 12 hours until the survival rate of infected *Beauveria bassiana* reached zero. *Beauveria bassiana* strain GZUIFR-JR1, previously isolated, identified, and preserved by the Institute of Fungal Resources, Guizhou University, served as the control group. A negative control group was prepared using the same treatment with 0.05% (v / v) Tween-80. Three replicates were set up for each strain, with 30 *Beauveria bassiana* organisms selected for each replicate. The experiment was repeated three times in parallel.

[0046] Bioassay results showed that the most virulent strains were ST389-1 and XYML-1, and their median lethal time (LT) against the larvae of the large wax moth was [not specified]. 50 The LT values ​​were 4.123 d and 4.422 d, respectively, which were lower than those of the control strain. 50 (5.117 d) decreased by 19.43% and 13.58% ( Figure 4 Therefore, through virulence screening, highly virulent Beauveria bassiana strains ST389-1 and XYML-1 were obtained, with colony morphologies as shown in the figure. Figure 3 China A- Figure 3 As shown in B.

[0047] The two strains were then screened for their ability to colonize tobacco. They were cultured on PDA medium (20 g glucose, 200 g potato spores, 20 g agar, and 1000 mL water) at 25°C and 75% RH for 14 days until abundant conidia were produced. The obtained Beauveria bassiana spore powder was suspended in water (spore germination rate ≥95% was checked before use), and the concentration was adjusted using a hemocytometer to obtain a concentration of 1×10⁻⁶. 8 Conidia / mL Beauveria bassiana spore suspension. The colonization treatment of Beauveria bassiana was carried out using tobacco seedlings grown in flower pots at a planting density of 1 seedling per pot (pot height 13.0 cm, width 12.0 cm) under outdoor conditions.

[0048] Foliar spraying was used for propagation, and the treatment time was after no direct sunlight in the evening; the spraying amount per seedling in the treatment group was 4 mL and 1×10⁶. 8 Conidia / mL spore suspension; control group was sprayed with an equal volume of distilled water; before each spraying, the roots of the tobacco seedlings were watered with 100 mL of water to ensure the seedlings had the necessary moisture for growth. The management measures for the tobacco seedlings in the control group and the treatment group were the same.

[0049] The colonization results showed that, three weeks after foliar spraying, the colonization detection rate of strain ST389-1 in the root, stem, and leaf tissues of tobacco seedlings was higher than that of strain XYML-1; especially, the colonization detection rate in the stem tissue reached 0.601±0.086, which was significantly different from that of strain XYML-1 (0.486±0.037). Figure 5 ).

[0050] After comprehensive evaluation, strain ST389-1 showed good colonization ability in tobacco seedlings, therefore it was selected for further field application. To ensure standardized management, strain ST389-1 was deposited at the Institute of Fungal Resources, Guizhou University, and renumbered as QXNYCJSZX2025-08.

[0051] The aforementioned Beauveria bassiana (QXNYCJSZX2025-08) was deposited on September 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42209. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0052] Example 2: Application in Tobacco Fields

[0053] The strain QXNYCJSZX2025-08 provided by this invention has high toxicity against major underground pests in tobacco fields. The main underground pests to be controlled are common tobacco underground pests in Guizhou Province. These pests include: Lepidoptera (Noctuidae) cutworms such as *Agrotis ypsilon (Rottemberg)*, *Agrotis tokionis Butler*, *Agrotis segetum (Denis et Schiffermüller)*, and *Agrotis cnigrum (Linnaeus)*; Coleoptera (Elateridae) wireworms such as *Pleonomus canaliculatus aldermann* and *Agriotes subrittatus Motschulsky*; and Coleoptera (Melolonthidae) wireworms such as *Apogonia cupreoviridis Kolbe* and *Serica orientalis*. Motschulsky]; mole crickets [Gryllotalpa spps] of the order Orthoptera and the family Gryllotalpidae.

[0054] Primary culture: QXNYCJSZX2025-08 strain was inoculated into PDA medium and cultured for 12–15 days at 25±1℃, relative humidity (RH) 75±5%, and light intensity (L / D) = 12 h: 12 h. Figure 6 (A)

[0055] Secondary culture: Liquid shake flask culture medium (4% glucose, 1% peptone, the remainder water) was inoculated with the primary culture and cultured at 25℃ and 150 r / min for 5 days. Figure 6 (B)

[0056] Tertiary culture: Solid fermentation medium (pure wheat) was soaked in a heat-resistant polyethylene plastic bag for 4 hours, then sterilized by moist heat for 40 minutes. After cooling overnight in a clean bench, 200 mL of secondary inoculum was inoculated and spread out in a ceramic dish, covered with spores, and cultured at 25℃ and 75% RH. After 4 days, the dish was turned over, and after 6 days, the spores were fragmented into 4-9 cm pieces. 3 Small pieces, at this time the humidity increases to above RH 85% ( Figure 6 C- Figure 6 (D). Culture for 12-15 days, then naturally dry and collect spore powder through a 36-mesh sieve for later use. Figure 6 (E).

[0057] 1. Application method of strain QXNYCJSZX2025-08 in tobacco fields

[0058] Select plots of land with a history of severe infestation of underground pests (cutworms, grubs, etc.) in tobacco cultivation, and apply the treatment in three stages:

[0059] When tilling the tobacco field, take 5 kg of granular biocontrol agent per acre (spore powder prepared through the above three-stage culture, with a spore content of 1.5 billion spores / g) and mix it with 20 kg of fine soil (organic fertilizer, wood ash, etc. can be used as substitutes). After thorough mixing, spread it evenly in the field and use a rotary tiller to turn the agent into the soil in time.

[0060] When ridging tobacco fields, take 5 kg of granular biocontrol agent per acre and mix it with 20 kg of fine soil (organic fertilizer, wood ash, etc. can be used as substitutes). After thorough mixing, apply the mixture evenly in strips within the ridges, about 10 cm away from the ridge surface. Do not spread it directly on the ridge surface, and avoid direct sunlight, which can cause the biocontrol agent to lose its activity. After ridging, the granular biocontrol agent should be evenly distributed within the ridges to a depth of 5-10 cm.

[0061] During the vigorous growth period of the *Clerodendrum trichotomum* plant, take an additional 500 g of biocontrol fungal high-spore powder per acre (using a cyclone separator to extract conidia from the mycelial blocks prepared through tertiary culture; the high-spore powder contains over 5 billion spores / g), dilute with 200 kg of water, add 2 g of detergent (spore dispersant), and stir thoroughly to form a suspension (spore content 1×10⁻⁶). 8 The concentration of spores per mL was prepared and sprayed evenly on both sides of the tobacco leaves using an electric sprayer (or drone if available) within 2 hours, with 100 mL sprayed per plant. The blank control group (no chemical pesticides used throughout the process) and the conventional control group (same as large-scale conventional production management, using high-efficiency cyhalothrin microemulsion, 15-20 g / mu) were both sprayed with water (with 2 g of detergent added). The replanting rate, number of live insects per 100 plants, number of dead insects per 100 plants, and the number and incidence of root and stem diseases and viral diseases in tobacco seedlings were recorded for each group.

[0062] 2. Insect-repellent and disease-resistant effects of QXNYCJSZX2025-08

[0063] Seedling replenishment rate results are as follows Figure 7As shown in Figure A: Compared with the blank control and the conventional control, the replanting rate in the treatment group supplemented with QXNYCJSZX2025-08 showed a decreasing trend. The average replanting rate of the blank control group was 9.44%, the conventional control group was 6.01%, while the treatment group was 2.25%, which was 76.2% and 62.6% lower than the blank control and the conventional control, respectively. Overall, the differences in replanting rate between the treatment group and the blank control group and the conventional control group were statistically significant (P < 0.05), indicating that the application of QXNYCJSZX2025-08 has a good effect on the control of underground pests during the transplanting period.

[0064] Meanwhile, to comprehensively evaluate the control effect of QXNYCJSZX2025-08 on foliage pests, after topping, the number of live and dead foliage pests (tobacco budworm, beet armyworm, and cotton bollworm) was investigated. Five sites were selected for each treatment, with 20 plants selected at each site, for a total of 100 plants. Figure 7 China B- Figure 7 As shown in Figure C, the treatment group had the fewest live tobacco pests (3.39±0.696) and the most dead insects (9.88±1.137), with significant differences compared to both control groups (P<0.05). In conclusion, QXNYCJSZX2025-08 is more effective in controlling underground pests than the blank control and the conventional control group.

[0065] In addition, from transplanting to maturity, the number of seedlings infected with root and stem diseases and viral diseases, as well as the incidence rate, were statistically analyzed every 15 days in the blank control group, the conventional control group, and the treatment group. This was to comprehensively evaluate the control effect of the biocontrol agent Beauveria bassiana on various diseases in tobacco seedlings. Five sites were selected for each group, with 20 seedlings selected at each site, for a total of 100 seedlings. Figure 7 China D and Figure 7 The results from the study show that the incidence rates of viral diseases and root and stem diseases in both control groups were higher than those in the Beauveria bassiana treatment group. The incidence rate of root and stem diseases in the blank control group was 10.3±1.08%, in the conventional control group it was 6.8±0.67%, and in the treatment group it was 3.5±0.97%. The incidence rates of viral diseases were higher in the blank and conventional control groups (17.1±0.624% and 12.3±0.90%, respectively) than in the treatment group (6.2±0.98%). The incidence rates of both types of diseases in the treatment group were significantly lower than those in the two control groups (P<0.05). This indicates that Beauveria bassiana has a significant effect on the prevention and control of tobacco seedling diseases.

[0066] 3. The effect of QXNYCJSZX2025-08 on the growth promotion of tobacco leaves

[0067] On the 10th day after topping, the plant height, number of effective leaves, and number of middle leaves (10th, 11th, and 12th leaves) of different treatment groups were investigated and analyzed. Figures 8-9 ).Depend on Figure 9 The plant heights of the blank control group, the conventional control group, and the treatment group were 112.2±3.27 cm, 115.0±4.03 cm, and 124.4±2.18 cm, respectively. The plant heights of the treatment group were all greater than those of the two control groups (P<0.05). The mean number of effective leaves in all three groups was 18-19. Among the effective leaves, the lengths of the 10th, 11th, and 12th leaves in the blank control, conventional control, and treatment groups were 68.43±2.411 cm, 71.41±1.731 cm, and 75.91±1.151 cm, respectively. The differences between the treatment group and the two control groups were statistically significant (P<0.05). The leaf widths of the blank control, conventional control, and treatment group were 26.71±1.009 cm, 27.81±0.856 cm, and 30.40±1.020 cm, respectively. The leaf width of the treatment group was significantly greater than that of the blank control and conventional control groups (P<0.05). Considering all agronomic traits, the plant height, leaf length, and leaf width of the QXNYCJSZX2025-08 treatment were significantly better than the blank control; the plant height and leaf width were significantly higher than those of the conventional control group. QXNYCJSZX2025-08 has a growth-promoting effect on tobacco leaves.

[0068] 4. The impact of QXNYCJSZX2025-08 on the economic benefits of tobacco leaves

[0069] After the tobacco harvest, the yield and quality data of the cured tobacco leaves for each treatment group were collected, including yield per mu (unit of land area), output value per mu, and the proportion of medium-grade to high-quality tobacco. Figure 10 As shown in Figure A, in terms of yield per mu (a Chinese unit of area, approximately 0.067 hectares), the average yield per mu in the treatment group was 124.97 kg, which was 8.9% higher than that in the blank control group (114.79 kg) and 7.6% higher than that in the conventional control group (116.17 kg). Regarding the percentage of high-quality tobacco, the conventional control group had the highest percentage (58.56%), followed by the group treated with granular biocontrol bacteria (57.10%), which were 9.1% and 6.4% higher than those in the blank control group, respectively. Figure 10 (C) Regarding the proportion of high-quality tobacco in the middle section, the treatment group had the highest proportion (52.25%), which was 3.6% and 3.1% higher than the blank control group and the conventional control group, respectively. Because the treatment group had a higher proportion of high-quality tobacco and high-quality tobacco in the middle section, the yield per mu was also relatively the highest (4253.03 yuan), which was 8.0% and 4.9% higher than the blank control and the conventional control group, respectively. Figure 10 (B) The economic benefits among different treatments were mainly as follows: treatment group > conventional control group > blank control group, indicating that QXNYCJSZX2025-08 has a certain effect on increasing tobacco yield and income.

[0070] Example 3: Detection of the persistence of QXNYCJSZX2025-08 in different tissues of tobacco plants

[0071] 1. Constant Analysis

[0072] Sampling method: The plots without QXNYCJSZX2025-08 were used as the control group, and the areas where QXNYCJSZX2025-08 was applied were selected as the treatment group. Each group had an area of ​​at least 50 m². 2 Five standard sample plots were selected from each plot. Sampling of the rhizosphere soil and tobacco tissues was conducted at the following stages: the initial growth stage, the vigorous growth stage, and 10 days after topping. Figure 11 (A) One tobacco plant was selected from each of the five sample plots. Fine roots collected from four different locations on the same plant were mixed and gently shaken to remove loose soil bound to the surface of the fine roots, as well as stones and clods between the roots. The soil on the surface of the fine roots was brushed off with a soft brush. Finally, the soil samples from these five samples were mixed for persistence analysis. Figure 11 (B). At the same time, root, stem and leaf tissues of these 5 tobacco plants were sampled, and the tissue samples from each part of the 5 tobacco plants were mixed and used for persistence analysis of QXNYCJSZX2025-08.

[0073] Nested PCR was used to confirm the positive colonization of strain QXNYCJSZX2025-08. PCR primers were designed based on strain specificity. First-round primers: ITS1-F / ITS-4 (ITS1-F: 5′-CTTGTTCGCTATCGGTCTC-3′, SEQ ID NO.1; ITS-4: 5′-TCCGTAGGTGAACCTGCGG-3′, SEQ ID NO.2); Second-round primers: Bb_SF / Bb_SR (5′-CAACTCCCCAACCCTTCTGT-3′, SEQ ID NO.3; 5′-AAGTTGGGTGTTTTACGGC-3′, SEQ ID NO.4), with a product size of 466 bp.

[0074] Three tissue samples (root, stem, and leaf) from tobacco plants under different treatment groups were collected at the plant's rosette stage, vigorous growth stage, and 10 days after topping. These samples were placed in 1.5 mL sterile, enzyme-free centrifuge tubes, and a small amount of quartz sand was added for thorough grinding to extract DNA for PCR detection. The PCR reaction system consisted of 0.5 μL each of 10 mM primers, 2 μL of DNA template, and 22 μL of T3 Super PCRMix. The reaction program was: 98℃ for 2 min, 98℃ for 10 s, 61℃ for 10 s, 72℃ for 10 s, for 35 cycles; the first round was incubated at 72℃ for 2 minutes; the second round was annealed at 55℃ for 10 s, with the remaining program consistent with the first round. Finally, the PCR products were detected by agarose gel electrophoresis at a gel concentration of 2.5%.

[0075] Electrophoresis results showed that the target gene of 466 bp could be detected in different tissues of tobacco treated with QXNYCJSZX2025-08. Figure 12 ).

[0076] 2. Detection of colonization sites and colonization density

[0077] Based on the tissue isolation method, QXNYCJSZX2025-08 was re-isolated from tobacco tissue using potato agar medium (1 / 2 PDA: 10 g glucose, 100 g potato, 20 g agar, and 1000 mL water, with chloramphenicol 200 mg / L added as a selectant). The tissue isolation method was used to detect the colonization site and density of QXNYCJSZX2025-08 in tobacco plants at different tissues during the rosette stage, vigorous growth stage, and 10 days after topping.

[0078] Separation method: Rinse the plants with running tap water to remove debris. After rinsing, soak the plants in 75% alcohol for 2 minutes, then in 5% sodium hypochlorite solution for 2 minutes, and rinse three times with sterile water. To test the disinfection effect on the plant surface, spread 100 μL of the sterile water used in the final rinse onto a PDA plate. In a sterile operating table, cut different parts (roots, stems, and leaves) of each tobacco plant into pieces, with roots and stems 1 cm in length and leaves 1 cm² in area. 2 Five segments each of root, stem, and leaf were randomly selected and spread onto selective culture medium. The culture dishes were incubated in the dark at 25°C for 2-3 weeks, and fungal growth was observed and recorded daily. When fungal hyphae emerged from the cut edges of the roots, stems, or leaves, the number of colonies consistent with *Beauveria bassiana* was counted, and the colonization rate of this fungus within the plant was calculated. The colonization rate (%) was calculated using the formula: (Number of plant slices showing fungal growth / Number of plant slices coated) × 100. The identity of the fungi isolated from the tissues to the colonizing strain was confirmed by fungal morphological and molecular biological identification.

[0079] The results are as follows Figure 13 As shown, *Beauveria bassiana* spores could be isolated from the rhizosphere soil, roots, stems, and leaves of tobacco plants, and their colony morphology was consistent with the starting strain. The soil concentration of *Beauveria bassiana* spores was particularly high. The soil suspension was diluted to 10... -3 After coating the plates, five days later the surface of the plates was covered with dense white Beauveria bassiana colonies.

[0080] The detection rate of Beauveria bassiana re-isolated by tissue isolation method showed that the isolation rate in stems was 0.58±0.078; followed by the isolation rate in tobacco roots at 0.32±0.023; and the lowest isolation rate was in leaves at 0.27±0.075. Figure 14This indicates that Beauveria bassiana tends to colonize the stems of tobacco plants, and the longer diffusion path from the roots to the leaf tissue results in a lower detection rate of Beauveria bassiana in the leaf tissue.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Beauveria bassiana QXNYCJSZX2025-08, characterized in that, The Beauveria bassiana strain QXNYCJSZX2025-08 was deposited on September 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42209. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of Beauveria bassiana QXNYCJSZX2025-08 as described in claim 1 in the preparation of biocontrol agents.

3. A biocontrol agent, characterized in that, It contains Beauveria bassiana QXNYCJSZX2025-08 as described in claim 1.

4. The use of Beauveria bassiana QXNYCJSZX2025-08 of claim 1 or the biocontrol agent of claim 3 in the prevention and control of soil pests, characterized in that, The underground pests include small cutworms, large cutworms, yellow cutworms, eight-character cutworms, wireworms, fine-breasted wireworms, black-brown gill beetles, black-velvet gill beetles, and mole crickets.

5. The application of Beauveria bassiana QXNYCJSZX2025-08 as described in claim 1 or the biocontrol agent as described in claim 3 in promoting tobacco leaf growth.

6. The application of Beauveria bassiana QXNYCJSZX2025-08 as described in claim 1 or the biocontrol agent as described in claim 3 in improving the economic benefits of tobacco leaves.

7. A method for controlling underground pests, characterized in that, Includes the step of applying Beauveria bassiana QXNYCJSZX2025-08 as described in claim 1; The underground pests include small cutworms, large cutworms, yellow cutworms, eight-character cutworms, wireworms, fine-breasted wireworms, black-brown gill beetles, black-velvet gill beetles, and mole crickets.

8. A method for promoting tobacco leaf growth and / or improving the economic benefits of tobacco leaves, characterized in that, The procedure includes the application of Beauveria bassiana QXNYCJSZX2025-08 as described in claim 1.

9. The method of claim 8, wherein, The method includes the step of applying the Beauveria bassiana QXNYCJSZX2025-08 of claim 1 to fields and tobacco leaf surfaces.

10. The method of claim 9, wherein, The Beauveria bassiana QXNYCJSZX2025-08 applied to the field is a granule formulation with a spore content of 1.5 billion spores / g and an application rate of 5 kg / mu. The Beauveria bassiana QXNYCJSZX2025-08 applied to the surface of the tobacco leaves is a suspension, and the spore content is 1×10 8 spores / mL, and the application amount is 100 mL / plant.

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