Biological disease-preventing and growth-promoting preparation and application thereof

Abstract

This application discloses a biological disease control and growth-promoting agent and its application, belonging to the technical field of biological disease control and growth-promoting agents. The biological disease control and growth-promoting agent includes the Pantotheca acuminata strain NFD34, which is used not only to control anthracnose in Trichosanthes kirilowii caused by pathogens, but also to promote seed germination, seedling growth, and fruit yield in Trichosanthes kirilowii. According to the biological disease control and growth-promoting agent of this application, the Pantotheca acuminata strain NFD34 is used to control anthracnose caused by pathogens in Trichosanthes kirilowii production, thereby solving the current problems of fungal diseases and slow growth in Trichosanthes kirilowii production, ultimately improving the quality and yield of Trichosanthes kirilowii. The control cost is low, avoiding environmental pollution and pesticide residues, and preventing the development of drug resistance in pathogens.

Description

Technical Field

[0001] This application relates to the field of control agents, and more specifically, to a biological disease prevention and growth promotion agent and its application. Background Technology

[0002] Trichosanthes kirilowii Maxim., belonging to the genus Trichosanthes in the family Cucurbitaceae, is a perennial herbaceous vine. In traditional Chinese medicine, the dried, mature fruit, seeds, pericarp, and roots of Trichosanthes kirilowii are all used medicinally, making it a traditional Chinese medicinal herb. Also known as Gualou, wild gourd, and hanging gourd, it can clear heat and resolve phlegm, moisten the lungs and relieve cough, and soothe the chest and dissipate nodules. Besides its important medicinal uses, the mature seeds of Trichosanthes kirilowii are plump and nutritious, containing abundant protein, unsaturated fatty acids, minerals, and various vitamins, making them a popular nut food. Given its dual medicinal and edible properties, its cultivation has been expanding, but yields are affected by various factors such as nutritional conditions, growing environment, and disease infection, with disease problems becoming an increasingly important concern.

[0003] Currently, existing technologies still primarily rely on chemical pesticides and fertilizers for the control and growth promotion of medicinal plants. However, long-term dependence on chemical pesticides (such as thiamethoxam, abamectin, metalaxyl, etc.) not only leads to increased resistance of pathogens in medicinal plants and reduced control efficacy, but also causes multiple problems such as product safety hazards and environmental pollution.

[0004] Regarding the aforementioned technologies, the inventors believe that the current production of Trichosanthes kirilowii still faces problems such as fungal diseases and slow growth, and these are difficult to control. Summary of the Invention

[0005] To address the aforementioned problems, this application provides a biological disease prevention and growth promotion agent and its application.

[0006] The biological disease prevention and growth-promoting agent provided in this application adopts the following technical solution: A biological disease prevention and growth promotion agent, comprising Pantotheca acuminata strain nfd34, is used to prevent and control anthracnose of Trichosanthes kirilowii caused by pathogens, and can also be used to promote seed germination, seedling growth and fruit yield of Trichosanthes kirilowii.

[0007] Through the above technical solution, the Pan-Mycobacterium strain nfd34 is used to control anthracnose caused by pathogens in the production of Trichosanthes kirilowii, thereby solving the current problems of fungal diseases and slow growth in the production of Trichosanthes kirilowii, ultimately improving the quality and yield of Trichosanthes kirilowii, with low control costs, avoiding environmental pollution and pesticide residues, and preventing the development of drug resistance in pathogens.

[0008] Optionally, the pantothenic strain nfd34 is used to produce indoleacetic acid.

[0009] Optionally, the pantothenic strain nfd34 is used to dissolve inorganic phosphorus.

[0010] Optionally, the pantothenic strain nfd34 is used to produce cellulase.

[0011] Optionally, the pantothenic strain nfd34 is used to produce siderophores.

[0012] Optionally, the pantothenic strain nfd34 has nitrogen-fixing capabilities.

[0013] Optionally, the biological disease prevention and growth promotion agent is the fermentation broth of Pantotheca strain NFD34.

[0014] Optionally, the biological disease prevention and growth promotion agent is used to prevent and control at least one of the genera *Anthracis*, *Mesochrysis*, *Fusarium*, and *Cytomyces*.

[0015] This application also provides a biological disease prevention and growth-promoting agent, which employs the following technical solution: The application of the above-mentioned biological disease prevention and growth promotion agent includes its use in the prevention and control of at least one of the genera *Anthracis*, *Mesochrysis*, *Fusarium*, and *Cytomyces*.

[0016] The above-mentioned technical solutions offer low prevention and control costs, avoid environmental pollution and pesticide residues, and prevent pathogens from developing drug resistance. Biological Preservation

[0017] The nfd34 strain (Pantoea eucrina) described in this invention is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on October 22, 2024, with accession number CCTCC No: M 20242294. Attached Figure Description

[0018] Figure 1 The results of species identification for strain nfd34 are as follows: (A) Colony morphology observation; (B) Gram staining; (C) Hemolysis test results; Phylogenetic tree of the strain based on the 16S rRNA gene (D) and a single copy of the orthologous gene (E); Figure 2 A circulated diagram of the Pantoea eucrina nfd34 genome: (A) chromosome; (B) plasmid; Figure 3 AntiSMASH prediction results for the Pantoea eucrina nfd34 genome: (A) chromosome; (B) Pnx1 plasmid; Figure 4Metabolic pathways related to IAA biosynthesis in the genome of Pantoea eucrina nfd34: enzymes marked in red in the figure can be produced by the nfd34 strain; Figure 5 Qualitative and quantitative analysis of metabolites of strain nfd34; Figure 6 Image showing the effect of strain nfd34 on promoting the germination of Trichosanthes kirilowii seeds; Figure 7 The effect of strain nfd34 on the growth of Trichosanthes kirilowii tissue culture seedlings: (A) Representative images of Trichosanthes kirilowii tissue culture seedlings at 0 dpi (0 days after inoculation) and 10 dpi (10 days after inoculation); (B) Difference in plant height of tissue culture seedlings in different treatment groups; (C) Difference in weight of tissue culture seedlings in different treatment groups; Different letters in the same bar chart represent significant differences (P<0.05). Figure 8 The effect of strain NFD34 on the growth of potted Trichosanthes kirilowii seedlings: (A) Overview of potted Trichosanthes kirilowii seedlings; (B) Growth of potted seedlings under different treatment groups (B1-B4: water control group, 1×10⁻⁶). 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 (C) Growth index plots (C1-C5: plant height, whole plant fresh weight, leaf area in the middle of the plant, root length, chlorophyll content); different lowercase letters in the same bar chart represent significant differences (P<0.05). Figure 9 Figure showing the growth of Trichosanthes kirilowii plants under field conditions using strain nfd34; Figure 10 The plate inhibition effect of strain nfd34 on the pathogenic fungus of Trichosanthes kirilowii (A) and other pathogenic fungi of traditional Chinese medicine (B); Figure 11 The in vitro biocontrol efficacy of strain nfd34 against anthracnose (A) on leaves and anthracnose (B) on fruit of Trichosanthes kirilowii is shown in the figure. ** indicates a highly significant difference (P<0.01); * indicates a significant difference (P<0.05). Detailed Implementation

[0019] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0020] The present application will be further described in detail below with reference to the accompanying drawings.

[0021] This application discloses a biological disease prevention and growth promotion agent, which includes the pantothecin strain nfd34. The biological disease prevention and growth promotion agent is used to prevent and control anthracnose caused by pathogens, and can also be used to promote the germination of Trichosanthes kirilowii seeds, seedling growth and fruit yield.

[0022] According to the biological disease prevention and growth promotion agent of this application, the Pan-Mycobacterium strain nfd34 is used to control anthracnose caused by pathogens in the production of Trichosanthes kirilowii, thereby solving the problems of fungal diseases and slow growth encountered in the current production of Trichosanthes kirilowii, ultimately improving the quality and yield of Trichosanthes kirilowii, with low control costs, avoiding environmental pollution and pesticide residues, and preventing the development of drug resistance in pathogens.

[0023] This application also provides an application of a biological disease prevention and growth-promoting agent. The biological disease prevention and growth-promoting agent is based on the above-described biological disease prevention and growth-promoting agent, and its application includes controlling at least one of *Anthracis*, *Mesochrysis*, *Fusarium*, and *Cyperus*. Through the above technical solution, the control cost is low, environmental pollution and pesticide residues can be avoided, and the development of drug resistance in pathogens can be prevented.

[0024] The pantothecin strain nfd34 of this application is a bacterium with disease prevention and growth promotion potential isolated and screened from the fruit of *Trichosanthes kirilowii*. This strain is deposited at the China Center for Type Culture Collection. Test plants: *Trichosanthes kirilowii* leaves, fruits, seeds, and tissue culture seedlings were all obtained from the *Trichosanthes kirilowii* production base of Chuanwen Melon Seed Co., Ltd. in Qianshan City, Anhui Province (30°33'59″N, 116°32'19″E).

[0025] I. Preparation of nfd34 strain suspension and fermentation broth Fresh nfd34 colonies were inoculated into LB liquid medium (Luria-Bertani medium, 5 g yeast extract, 5 g tryptone, 10 g NaCl, 1000 mL water) and cultured on a shaker for 24 h (37℃, 180 r / min) to obtain the seed culture, i.e., the bacterial suspension. The seed culture was then inoculated into LB liquid medium at a 1% inoculation rate and cultured on a shaker at 37℃, 200 r / min for 3 days. The supernatant was collected by centrifugation at 12000 r / min for 30 min and then filtered three times through a 0.22 μm microporous membrane to obtain the sterile fermentation supernatant.

[0026] II. Morphological observation, hemolysis test, and physiological and biochemical tests of strain nfd34 to determine the strain type. Reference Figure 1A. Fresh strain nfd34 was inoculated onto LB agar plates using the five-step streak method and incubated in a 28℃ biochemical incubator for 1 day. The growth and morphological characteristics of the nfd34 strain, including colony size, texture, color, and edge morphology, were observed. The results showed that the colonies were yellow, round, and had a smooth, opaque surface.

[0027] Reference Figure 1 B. Gram staining of strain nfd34 and observation using an optical microscope showed that it was a Gram-negative bacterium.

[0028] Reference Figure 1 A hemolysis test was performed on strain C, nfd34, and the results showed that nfd34 had no hemolytic properties.

[0029] As shown in Table 1, physiological and biochemical tests were conducted on strain nfd34, including sugar or alcohol fermentation tests, MR tests (methyl red test), VP tests (acetylmethane test), and hydrogen sulfide tests. The results showed that strain nfd34 can utilize four carbon sources during its growth: glucose, maltose, mannitol, and sucrose. Reactions with urea and hydrogen sulfide were negative, while tests for methyl red, acetylmethane, and Simon's citrate were positive.

[0030] Table 1

[0031] Reference Figure 1 D. Phylogenetic analysis based on the 16S rRNA gene of strain nfd34 (also known as "bacterial fossil," belonging to conserved genes in bacterial structure and function) showed that strain nfd34 clustered with Pantoea eucrina KT03, Pantoeaeucrina Cf7, and Pantoea eucrina IHB B10086, while it was on a more distant branch with Pantoea ananatis and Pantoeaacypripedii. The whole genome sequences of 12 closely related strains to nfd34 were downloaded from the NCBI (National Center for Biotechnology Information) database. ANI (Average Nucleotide Identification) analysis (Table 2) showed that the ANI value between strain nfd34 and Pantoea eucrina OB49 was 99.20%, higher than the 95% interspecies identification standard. Furthermore, referring to… Figure 1E. Phylogenetic analysis of single-copy orthologous genes using the above whole genome sequence showed that strain nfd34 was located on the same minimal branch of the phylogenetic tree as *Pantoea eucrina* XL123, and was significantly separated from *P. agglomerans* and *P. vagans*. Therefore, based on the combined results of morphological observation, physiological and biochemical tests, phylogenetic tree analysis, and ANI value, strain nfd34 was identified as *Pantoea eucrina*.

[0032] Table 2

[0033] III. Genome-wide profile and bioinformatics analysis of Pantoea eucrina nfd34 Total DNA was extracted from pantothenic acid nfd34 according to the instructions in the bacterial genome kit (Applied Biosystems, USA). After the purity and concentration of the extracted sample were qualified by agarose gel electrophoresis, whole genome sequencing was performed.

[0034] Sequencing results refer to Figure 2 The genome of strain *P. eucrina* nfd34 contains a chromosome of 3,353,234 bp and two circular plasmids, Pnx1 (418,326 bp) and Pnx2 (250,356 bp). The GC content of the chromosome is 56.30%, and the GC contents of the plasmids are 55.20% and 52.94%, respectively. Non-coding RNA prediction results show that strain nfd34 contains 182 ncRNAs, including 80 tRNAs (0.16% of the total gene sequence) and 22 rRNAs (7 16S, 7 23S, and 8 5S), accounting for 0.80% of the total gene sequence. In addition, the genome sequence is predicted to contain 41 gene islands and 1 prophage (Table 3).

[0035] Table 3

[0036] Anti-SMASH prediction results show that the nfd34 genome includes six secondary metabolite synthesis gene clusters (Table 4), referring to... Figure 3 A, three of which are located on chromosomes: thiopeptides, hesrlactones, and redox cofactors. (See reference...) Figure 3B, three genes located on the Pnx1 plasmid, are acyl amino acids, RiPP-like compounds, NI-siderophores, and terpenes. Further investigation revealed that thiopeptides, redox cofactors, NI-siderophores, and terpenes are disease-preventing and growth-promoting substances, especially NI-siderophores, and key genes for substance synthesis on the gene cluster were identified. Further gene cluster comparison with other bacteria revealed a 100% similarity to the thiopeptide synthesis gene cluster of *Pantoea eucrina* Russ, and a 95% similarity to the synthesis gene clusters of the other three substances of *Pantoea eucrina* XL123 and *Pantoea sp. M9*. Simultaneously, genes related to the biosynthesis of disease-preventing and growth-promoting substances in the genome of strain nfd34 were mined, revealing 11 genes related to IAA (indoleacetic acid) biosynthesis (Table 5). Based on this, an IAA biosynthetic metabolic pathway diagram was extracted from the KEGG website (see reference). Figure 4 In addition, 9 genes related to siderophore biosynthesis, 5 genes related to cytokinin biosynthesis (Table 6), 8 genes related to nitrogen fixation, 5 genes related to colonization (Table 7), 15 genes related to phosphate solubility, and 12 genes related to phosphate metabolism were identified (Table 8).

[0037] Table 4

[0038] Table 5

[0039] Table 6

[0040] Table 7

[0041] Table 8 Statistics on genes related to phosphate solubility and sulfate metabolism in the Pantoea eucrina nfd34 genome

[0042] IV. Metabolite Analysis of Pantoea eucrina NFD34 Reference Figure 5 This experiment detected and analyzed the metabolites of Pantoea eucrina nfd34.

[0043] Qualitative tests showed that strain nfd34 could grow and produce a clear zone on inorganic phosphorus medium, cellulase identification medium, and nitrogen-free aspartate medium. It could also grow on siderophore detection medium, exhibiting an orange halo around its growth. These phenomena indicate that strain nfd34 can dissolve inorganic phosphorus, possesses nitrogen-fixing ability, and can produce cellulase and siderophores. Strain nfd34 was also observed to grow on tryptophan-containing (Trp... + ) and lack of tryptophan (Trp) - After shaking culture in LB liquid medium for 4 days, the fermentation supernatant was collected and centrifuged. After adding Salkawski colorimetric solution, the supernatant turned red and light red, respectively, indicating that nfd34 can produce indoleacetic acid (IAA).

[0044] Quantitative analysis showed that nfd34 exhibited varying abilities in four areas—IAA production, cellulase production, siderophore production, and phosphate solubilization—at different cultivation days. IAA production reached its maximum on day 5, with a yield of 18.12 mg / mL without tryptophan and 44.96 mg / mL with tryptophan. The other three growth-promoting properties—cellulase production, siderophore production, and phosphate solubilization—reached their maximum values ​​on days 3, 2, and 4, respectively, at 131.20 U / mL, 0.40 SU (siderophore activity units), and 1.21 mg / mL.

[0045] V. Effects of strain nfd34 on the germination of Trichosanthes kirilowii seeds A bacterial suspension and sterile fermentation supernatant of strain NFD34 were prepared. This embodiment included 6 experimental groups and 2 control groups, with each experimental group containing 1×10⁻⁶ cells. 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 The treatment groups consisted of CFU / mL nfd34 bacterial suspension and sterile fermentation supernatant diluted 10, 100, and 1000 times. The control groups were sterile water and LB medium.

[0046] Seeds were soaked for 24 hours according to the above groups. After soaking, 20 seeds were placed in glass petri dishes lined with moist sterile cotton, with 5 replicates per group. The petri dishes were statically cultured in an artificial climate incubator (25℃, 12-hour light-dark cycle, 90% humidity). The germination potential of the Trichosanthes kirilowii seeds was investigated on day 8, and the germination rate was calculated on day 16 (formula shown below). The fresh weight of the germinated seeds was also measured, as well as the length of the plumule and radicle.

[0047] Seed germination potential calculation method: Germination rate = Number of germinated seeds on day 8 after treatment / Number of tested seeds × 100%; Seed germination rate calculation method: Germination rate = Number of germinated seeds on day 16 after treatment / Number of tested seeds × 100%.

[0048] Reference Figure 6 During seed germination and seedling growth, it was found that both the bacterial suspension and sterile fermentation supernatant of strain nfd34 promoted the germination of Trichosanthes kirilowii seeds and the growth of seedlings to varying degrees, especially at a concentration of 1×10⁻⁶. 8 The CFU / mL bacterial suspension showed the most significant effect, increasing germination potential and germination rate by 23.00% and 26.00%, respectively, compared to the sterile water control. The next most effective was 1×10⁻⁶ CFU / mL suspension. 9 The CFU / mL treatment of Trichosanthes kirilowii seeds also showed a significant growth-promoting effect, with radicle, plumule, and fresh weight reaching 10.45 cm, 2.33 cm, and 0.77 g, respectively. Furthermore, compared to the LB medium control, all seeds treated with the aseptic fermentation supernatant of the nfd34 strain exhibited higher germination potential, germination rate, fresh weight, and longer radicle and plumule lengths. The 100-fold diluted aseptic fermentation supernatant treatment group showed the most significant growth-promoting effect, with germination potential and germination rate increasing by 6.00% and 5.00%, respectively, and radicle, plumule, and seed fresh weight reaching 10.60 cm, 1.08 cm, and 0.78 g, respectively (Table 9).

[0049] Table 9

[0050] VI. Effects of strain nfd34 on the growth of Trichosanthes kirilowii tissue culture seedlings Prepare a bacterial suspension of strain NFD34 and a sterile fermentation supernatant of PDB. Dilute the bacterial suspension to a concentration of 1×10⁻⁶. 8 CFU / mL was prepared for use, and the aseptic fermentation supernatant was diluted 100 times for use. Healthy and uniformly growing Trichosanthes kirilowii tissue culture seedlings were selected, and 1×10⁻⁶ of (1) nfd34 strain was injected around the roots of the seedlings. 8 (2) CFU / mL bacterial suspension; (3) 100-fold dilution of nfd34 strain aseptic fermentation supernatant. 50 μL was injected into each Trichosanthes kirilowii seedling. Aseptic water treatment was used as a control. Five bottles of Trichosanthes kirilowii tissue culture seedlings were set up in each treatment group and cultured statically in an intelligent artificial climate incubator (25℃, 12 h light and dark alternation, 90% humidity). The plant height and weight were measured 10 days (10 dpi) after inoculation.

[0051] Reference Figure 7 Throughout the entire observation period of *Trichosanthes kirilowii* tissue culture seedling growth, the aseptic water control group showed relatively slow growth in seedling height and fresh weight, increasing by only 2.55 cm and 0.45 g, respectively. In contrast, the seedlings inoculated with 10... 8The growth changes of tissue culture seedlings treated with CFU / mL nfd34 strain bacterial suspension and 100-fold diluted sterile fermentation supernatant were more pronounced. The 100-fold diluted sterile fermentation supernatant treatment group showed a particularly significant growth-promoting effect, with seedling height and fresh weight increasing significantly by 1.23 cm and 0.86 g, respectively. Furthermore, the nfd34 strain significantly promoted root development in the tissue culture seedlings; compared to the sterile water control group, the seedlings had longer taproots, more lateral roots, and more vigorous growth.

[0052] VII. Effects of strain nfd34 on the growth of potted Trichosanthes kirilowii seedlings A bacterial suspension of strain nfd34 and a PDB sterile fermentation supernatant were prepared. (1) a water control (CK) and (2) a 1×10⁻⁶ mol / L supernatant were prepared. 9 CFU / mL bacterial suspension treatment, (3) 1×10 8 CFU / mL bacterial suspension treatment, (4) 1×10 7 Four treatments were administered using CFU / mL bacterial suspension. Each treatment was replicated in eight pots, with one seedling per pot. Water was applied as needed based on soil moisture after transplanting. Six days after transplanting, the seedlings were irrigated with 100 mL of the bacterial suspension per group. At 55 days of cultivation, the leaf area, plant height, whole plant fresh weight, and root length of the central part of the plants in each treatment group were measured, and the chlorophyll content of the leaves in each treatment group was also determined.

[0053] Results reference Figure 8 Pot experiments showed that the NFD34 bacterial suspension had a good growth-promoting effect on Trichosanthes kirilowii seedlings. Compared with the water control, 1×10 9 The CFU / mL bacterial suspension showed the most significant growth-promoting effect, with leaf area, plant height, whole plant fresh weight, and root length reaching 195.38 cm. 2 43.55cm, 41.01g, 22.66cm. Additionally, 1×10 8 The CFU / mL bacterial suspension treatment of Trichosanthes kirilowii plants also showed a significant growth-promoting effect, with the leaf chlorophyll content reaching 0.433 mg / g, which was significantly different from the water control group.

[0054] VIII. Effects of strain nfd34 on the growth and development of Trichosanthes kirilowii plants under field conditions In 2023 and 2024, the following experiments were conducted at the Medicinal Botanical Garden of Anhui University of Traditional Chinese Medicine (31°54'36″N, 117°15'00″E): Trichosanthes seedlings brought back from the Trichosanthes planting base in Qianshan, Anhui Province, were planted in the medicinal garden of Anhui University of Traditional Chinese Medicine. After one week of natural growth, the Trichosanthes plants were divided into two groups: a water control group and an NFD34 bacterial suspension treatment group. Subsequently, the roots were irrigated with water and diluted NFD34 bacterial suspension (bacterial suspension: water = 1:8), with an interval of 14 days between each irrigation. The growth of the Trichosanthes seedlings was observed from the 7th day after irrigation, and the results of each treatment were photographed and recorded.

[0055] Reference Figure 9 This study evaluated the growth-promoting effect of strain nfd34 on field-grown Trichosanthes kirilowii plants in 2023 and 2024. Results showed that compared to the water control group, plants treated with nfd34 bacterial suspension exhibited robust vines, vigorous growth, and a faster growth rate. The plants treated with nfd34 bacterial suspension flowered earlier and had more leaves than the water control group. During the fruiting period, the Trichosanthes kirilowii plants treated with nfd34 bacterial suspension had a higher fruit yield than the water control group, with an average single fruit weight of 594.15 g, significantly higher than the 519.79 g of the water control group. At the end of the observation period, the roots of the Trichosanthes kirilowii plants treated with nfd34 bacterial suspension were found to be thicker, with a more robust taproot and denser fibrous roots compared to the water-treated plants.

[0056] IX. Detection of the plate inhibitory effect of strain nfd34 against pathogenic fungi The antagonistic activity of nfd34 bacterial suspension was detected using the plate confrontation method: Fresh mycelial discs of 13 pathogenic fungi were placed in the center of PDA medium (Potato Dextrose Agar). Nfd34 bacterial suspension was inoculated at 2 cm intervals on both sides of the mycelial discs. A control group without mycelial suspension was used. The experiment was repeated three times, and the mixture was incubated upside down at 28℃ for 6 days. The growth inhibition rate of nfd34 against the pathogenic fungi was calculated as follows: Inhibition rate = [(Control group colony diameter – Treatment group colony diameter) / (Control group colony diameter – Mycelial disc diameter)] × 100%, where the mycelial disc diameter was 5 mm.

[0057] The bacterial suspension of strain NFD34 showed varying degrees of antibacterial activity against different pathogenic fungi of Trichosanthes kirilowii. Figure 10A), among which the highest inhibition rate (62.11%) was observed against *Stagonosporopsis cucurbitacearum* NFD-31, the causal agent of *Trichosanthes kirilowii*. The inhibition rates against *Colletotrichum gloeosporioides* NFD-5 and *Colletotrichum aenigma* YM were 41.48% and 34.98%, respectively. Figure 10 A). Meanwhile, the nfd34 bacterial suspension exhibited varying degrees of antagonistic activity against other pathogenic bacteria found in traditional Chinese medicine, with the highest inhibition rate against pathogen QSRD4, at 44.62% (A). Figure 10 B. Table 10).

[0058] Table 10

[0059] 10. Detection of the in vitro biocontrol effect of strain nfd34 against anthrax of Trichosanthes kirilowii The 7-day cultured Colletotrichum gloeosporioides plates were rinsed with sterile water, and the spores were collected by filtration through gauze. The spores were then diluted with sterile water to a concentration of 1×10⁻⁶. 6 A spore suspension of 1 spore / mL was prepared for later use. A bacterial suspension of nfd34 was prepared according to the method described above.

[0060] This experiment used detached leaves and fruits of *Trichosanthes kirilowii* for biocontrol testing. Three control groups were set up: a sterile water control group, a diseased control group treated only with the pathogen, and a treatment group treated with the pathogen plus NFD34 strain. The treated leaves were placed in WA (containing only 1.5% agar) medium and cultured continuously for 7 days in a 25℃ intelligent artificial climate incubator. The treated fruits were wrapped in plastic wrap and cultured continuously for 9 days in a 25℃ intelligent artificial climate incubator. Disease incidence on the leaves and fruits was observed, and the control effect was calculated using the following formula.

[0061] Lesion area = 1 / 4 × π × [(lesion length + lesion width) / 2] 2 The control effect = [(area of ​​lesions in the diseased control group of Trichosanthes kirilowii - area of ​​lesions in the group treated with nfd34 bacteria suspension) / area of ​​lesions in the diseased control group of Trichosanthes kirilowii] × 100%.

[0062] Reference Figure 11Throughout the in vitro biocontrol experiment, the leaves and fruits of the *Trichosanthes kirilowii* in the sterile water control group showed no significant changes, remaining lush and healthy. In contrast, the leaves and fruits inoculated only with the pathogen exhibited large areas of lesions. Inoculation with strain nfd34 effectively inhibited the occurrence of anthracnose in *Trichosanthes kirilowii*, significantly reducing the lesion area. The control efficacy of nfd34 against anthracnose in *Trichosanthes kirilowii* leaves and fruits reached 75.07% and 68.99%, respectively, indicating that strain nfd34 has a good biocontrol effect against anthracnose in *Trichosanthes kirilowii*.

[0063] In summary, the biological disease prevention and growth promotion agent according to this application includes the pan-mycelium strain nfd34, which can be used to control anthracnose caused by pathogens, and can also be used to promote the germination of Trichosanthes kirilowii seeds, seedling growth and fruit yield.

[0064] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A biological disease prevention and growth-promoting agent, characterized in that: The biological disease prevention and growth promotion agent includes the pan-mycelium strain nfd34. This agent is used to prevent and control anthracnose of Trichosanthes kirilowii caused by pathogens, and can also be used to promote seed germination, seedling growth and fruit yield of Trichosanthes kirilowii.

2. The biological disease prevention and growth-promoting agent according to claim 1, characterized in that: The pantothenic strain nfd34 is used to produce indoleacetic acid.

3. The biological disease prevention and growth-promoting agent according to claim 1, characterized in that: The pantothenic strain nfd34 is used to dissolve inorganic phosphorus.

4. The biological disease prevention and growth-promoting agent according to claim 1, characterized in that: The pantothenic strain nfd34 is used to produce cellulase.

5. The biological disease prevention and growth-promoting agent according to claim 1, characterized in that: The pantothecin strain nfd34 is used to produce siderophores.

6. The biological disease prevention and growth-promoting agent according to claim 1, characterized in that: The pantothenic strain nfd34 has nitrogen-fixing ability.

7. The biological disease prevention and growth-promoting agent according to claim 1, characterized in that: The biological disease prevention and growth promotion agent is the fermentation broth of Pantotheca strain nfd34.

8. A biological disease prevention and growth-promoting agent according to any one of claims 1 to 7, characterized in that: The biological disease prevention and growth promotion agent is used to prevent and control at least one of the following genera: Anthrax, Mesospora, Fusarium, and Leptospira.

9. The application of a biological disease prevention and growth-promoting agent according to any one of claims 1 to 8, characterized in that: Including at least one of the genera *Bacillus*, *Melastoma*, *Fusarium*, and *Cytomyces* used for the prevention and control of anthrax.

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