A strain of PNP-1 truncated anthracnose fungus and its application in inhibiting the growth of Phytophthora in Panax notoginseng.

Abstract

This invention discloses a truncated anthracnose bacterium, PNP-1, and its application in inhibiting the growth of *Phytophthora cactorum*, a pathogen of *Phytophthora insica*, belonging to the field of biotechnology. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42013. Its cell-free filtrate showed an inhibition rate of 61.76% against *Phytophthora cactorum*, and a potted plant control effect of 51.03%. It also exhibited broad-spectrum inhibitory activity against eight pathogens, including *Fusarium culmorum*. PNP-1 can be used to prepare inoculants from fermentation broth or filtrate, achieving environmentally friendly disease control and providing a new resource for the biological control of *Phytophthora insica*. This invention utilizes an environmentally friendly strategy to control *Phytophthora insica*.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of truncated anthracnose fungus PNP-1 and its application in inhibiting the growth of Phytophthora infestans. Background Technology

[0002] Currently, wild populations of Panax notoginseng are extinct worldwide, and resource supply relies entirely on artificial cultivation. However, this species is extremely sensitive to habitat conditions. During production, Panax notoginseng is susceptible to infection by pathogenic microorganisms, affecting its safety and efficacy. Phytophthora blight has become a devastating disease of Panax notoginseng, causing huge losses to the industry and seriously affecting its yield and quality. Therefore, the prevention and control of Phytophthora blight has become a major problem that urgently needs to be solved in this industry.

[0003] Plant disease control primarily relies on chemical pesticides and disease-resistant breeding. However, no Panax notoginseng variety is currently highly resistant to Phytophthora blight and suitable for large-scale cultivation. Therefore, chemical control remains the main method for controlling Phytophthora blight in Panax notoginseng. Given the long growth period of Panax notoginseng, using chemical agents to control its diseases results in pesticide residues and reduces the quality of the plant. Therefore, there is an urgent need to develop biocontrol agents for biological control, providing support for achieving an ecologically safe and environmentally friendly strategy for controlling Phytophthora blight in Panax notoginseng. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Pythium spp.* PNP-1 and its application in inhibiting the growth of *Phytophthora insectii*, thereby addressing the problems existing in the prior art. This invention isolates *Pythium spp.* PNP-1 from healthy *Panax notoginseng* plants, filling a gap in the application of *Pythium spp.* in this field and providing an efficient and green alternative for the prevention and control of *Phytophthora insectii*.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of truncated anthracnose bacterium (Annulohypoxylon annulatum) PNP-1, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.42013.

[0007] The present invention also provides a microbial inoculum containing the above-mentioned truncated carbonaceous bacteria PNP-1 or its fermentation broth.

[0008] Furthermore, the fermentation broth is obtained by inoculating the truncated carbonaceous bacteria PNP-1 into PDA medium and culturing it.

[0009] The present invention also provides a cell-free filtrate, which is obtained by filtering the fermentation broth through a 0.22 μm filter membrane.

[0010] The present invention also provides the application of the above-mentioned truncated charcoal bacterium PNP-1, the above-mentioned microbial inoculum, or the above-mentioned cell-free filtrate in inhibiting the growth of plant pathogens.

[0011] Furthermore, the plant pathogens include Phytophthora cactorum, Fusarium culmorum, Curvularia lunata, Boeremia exigua, Fusarium oxysporum, or Colletotrichum sublineola.

[0012] The present invention also provides a method for reducing the incidence of Phytophthora blight in Panax notoginseng, comprising the step of applying the above-mentioned microbial agent or the above-mentioned cell-free filtrate to Panax notoginseng plants.

[0013] The present invention also provides a broad-spectrum plant pathogen inhibitor, the active ingredient of which includes the above-mentioned truncated anthracnose fungus PNP-1.

[0014] The present invention discloses the following technical effects:

[0015] This invention screened and isolated a strain of *Annulohypoxylon annulatum*, PNP-1. This strain significantly inhibits *Phytophthora insectii* through a dual mechanism (secretion of antibacterial substances and teratogenic hyphae), achieving a control efficacy of 51.03% in potted plants. It also exhibits broad-spectrum activity against beet root rot and corn leaf spot. The fermentation broth is easy to prepare, overcoming the problem of chemical residues and providing key technical support for the sustainable development of the *Panax notoginseng* industry. The core of this invention lies in the first discovery of the biocontrol potential of *Annulohypoxylon annulatum* PNP-1 against *Phytophthora insectii*. This strain was isolated from the petioles of healthy Panax notoginseng in the disease-affected area and identified by molecular identification as Annulohypoxylon annulatum. Its unique features are: (1) Highly effective antagonism: Through plate confrontation and double-layer plate tests, it was confirmed that PNP-1 cell-free filtrate can inhibit Phytophthora teratogens hyphae with an inhibition rate of 61.76%, which is significantly higher than that of conventional biocontrol bacteria; (2) Broad spectrum: It is effective against 8 kinds of crop pathogens, especially Fusarium culmorum, with an inhibition rate of over 60%, which expands the application range of anthracnose fungi; (3) Convenient application: The fermentation liquid can be directly used for potted plant control. Inoculation with the fungicide first can increase the control efficacy to 51.03%. The operation process is compatible with existing agricultural practices; (4) Environmentally friendly: It replaces chemical pesticides, reduces drug residues in Panax notoginseng, and meets the needs of ecological planting. This invention uses healthy plants surviving in areas affected by Phytophthora in Panax notoginseng as materials to discover functional endophytic fungi with biocontrol effects against Phytophthora in Panax notoginseng, and analyzes their diversity. This provides effective strains for the development of biological control agents for Phytophthora in Panax notoginseng, plays a positive role in promoting the green control of Phytophthora in Panax notoginseng, and provides new ideas for the development of biological pesticides. Attached Figure Description

[0016] 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.

[0017] Figure 1 The antagonistic effect of cell-free filtrate of strain PNP-1 on Phytophthora cactorum was investigated; where A: control group; B: treatment group; C: colony diameter statistics of the control and treatment groups.

[0018] Figure 2 To observe the hyphal changes of Phytophthora cactorum using an optical microscope; where A: control group; B: treatment group;

[0019] Figure 3 To determine the pot control effect of strain PNP-1; where A: negative control group; B: positive control group; C: treatment group 1; D: treatment group 2;

[0020] Figure 4 The antibacterial spectrum of strain PNP-1 was determined; where A: Fusarium culmorum; B: Boeremia exigua var. Exigua; C: Curvularia lunata; D: Alternaria tenuissima; E: Fusarium oxysporum; H: Boeremia exigua; I: Colletotrichum sublineola; J: Phona multirostrata; -1: Control culture of pathogenic fungi; -2: Dual culture of strain PNP-1 and pathogenic fungi. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] 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.

[0026] Example 1

[0027] 1. Materials and Methods

[0028] 1.1 Test Materials

[0029] 1.1.1 Test Culture Medium

[0030] PDA medium was used for both fungal isolation and screening of antagonistic strains.

[0031] 1.1.2 Test strains

[0032] The pathogens of Phytophthora cactorum, Boeremia exigua, Boeremia exigua var. exigua, Alternaria tenuissima, Phoma multirostrata, Fusarium oxysporum, Fusarium culmorum, Curvularia lunata, and Colletotrichum sublineola were provided by Wenshan Panax notoginseng Research Institute.

[0033] 1.2 Test Methods

[0034] 1.2.1 Sample Collection and Surface Treatment

[0035] Samples were collected in September 2024 from the Huaxin Forest Panax notoginseng planting base in Wenshan Zhuang and Miao Autonomous Prefecture. This region has an average annual temperature of 19.1℃, average annual precipitation of 1087mm, and average annual sunshine duration of 2028.2h. Its latitude and longitude are 104°18'E, 23°39'N, classifying it as a subtropical monsoon climate. Healthy plant samples were collected from the diseased area. The samples were thoroughly rinsed with running water for 30 minutes to remove surface dirt, then rinsed with sterile water for 5 minutes, air-dried, and transferred to a clean bench for surface disinfection. The samples were then sequentially immersed in 75% ethanol for 1 minute, 0.1% mercuric chloride for 1 minute, and rinsed with sterile water for 30 seconds, for a total of 5 rinses. The surface disinfection effect of the obtained plant tissues was tested using the rinsing solution test and tissue culture method. For the rinsing solution test, under sterile conditions, 0.1 mL of the final rinsing solution was spread onto a prepared PDA plate. After culturing the plate for 5 days, the presence of bacterial growth in the culture dish was observed. The tissue block culture method involves placing the tissue block into PDA culture medium, and the remaining procedures are the same as those for the rinsing solution method.

[0036] 1.2.2 Isolation and Identification of Endophytic Fungi

[0037] Each Panax notoginseng plant's main root, lateral roots, stems, leaves, petioles, and fruits were cut into 0.5cm × 0.5cm × 0.5cm pieces using a sterile knife. 10g pieces were weighed, placed in a sterile mortar, and ground into a homogenate. 100mL of sterile water was added to dilute the homogenate, and the mixture was poured into a sterile Erlenmeyer flask and incubated at 180rpm and 25℃ for 30min with shaking. Endophytic fungi were isolated using the tissue block culture method. After inoculating single colonies onto plates, the plates were inverted and incubated at 25℃ for 3-5 days. Pure single colonies were obtained and stored at -20℃ using slant agar. The isolated endophytic fungi were inoculated onto PDA solid medium and incubated at 25℃ for 7 days, and their single colony morphology was observed. The Fungal DNA Kit was used to extract DNA from the purified endophytic fungi isolated from different Panax notoginseng tissues, and the DNA was stored at -20℃.

[0038] PCR amplification was performed using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGCGG-3'(SEQ ID NO.1)) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'(SEQ ID NO.2)). The PCR reaction mixture (30 μL) consisted of 15 μL PCR Mix, 2 μL DNA, 0.9 μL each of primers ITS1 and ITS4, and ddH2O to make up the volume. PCR conditions were: 95℃ for 3 min; 95℃ for 30 s; 56℃ for 30 s; 72℃ for 30 s; 30 cycles; 72℃ for 10 min. The PCR products were sent to Shanghai Sangon Biotech for sequencing.

[0039] 1.2.3 Evaluation of the antagonistic function of endophytic fungi of Panax notoginseng against Phytophthora pathogens

[0040] The plate confrontation method was used. A 0.6 cm diameter mycelial disc was collected from the edge of a Phytophthora colony. An endophytic fungal strain was inoculated at a distance of 2.5 cm from the pathogenic mycelial disc, with four inoculation points per petri dish. A PDA medium inoculated only with the pathogen served as a control group. Each treatment was repeated three times. The dishes were incubated upside down at 28°C. Colony size was measured using the cross-sectional method, and the inhibition rate was calculated. Inhibition rate (%) = (Control pathogen colony diameter - Treatment colony diameter) / Control group colony diameter × 100%.

[0041] A double-layer plate method was used to conduct a preliminary antibacterial experiment on cell-free filtrate of strains with good antagonistic effects. The strain with the best antibacterial effect was screened and named PNP-1. Then, the fermentation broth of PNP-1 (4 days) was filtered through a 0.22 μm sterile membrane. The cell-free filtrate of this strain was prepared into double plates at a 1:1 ratio. Pathogen mycelial cakes were inoculated on both double-layer plates and PDA plates. After 5 days of confrontation, the diameter of the pathogen was measured. The antibacterial effect was observed using the slide insertion method. The results showed that strain PNP-1 had a teratogenic effect on the hyphae of Phytophthora insicae.

[0042] The antagonistic function of endophytic fungi was determined using a pot experiment. Phytophthora infestans pathogenic fungal cakes were placed in PDA liquid culture medium and cultured at 25°C with shaking at 180 rpm for 7 days to prepare 1×10⁻⁶ fungal cakes. 5 A spore suspension of CFU / mL was prepared. Then, single colonies of the Panax notoginseng endophytic fungus PNP-1, which exhibits the best antagonistic effect, were picked and cultured in PDA liquid medium at 25°C with shaking at 180 rpm for 4 days. The bacterial suspension was then diluted to 1×10⁻⁶. 7 CFU / mL. Then, select healthy, disease-free, and uniformly aged potted seedlings, with 15 seedlings per experimental treatment, divided into 4 groups: Treatment 1 was inoculated with fungal inoculum solution first, followed by pathogen inoculation 12 hours later; Treatment 2 was inoculated with pathogen first, followed by fungal inoculum solution 12 hours later; Treatment 3 was a negative control, sprayed only with sterile water; Treatment 4 was a positive control, inoculated only with pathogen. Observe and record the disease incidence, disease index, and control effect.

[0043] 1.2.4 Evaluation of the broad spectrum of antagonistic strains

[0044] To determine the broad-spectrum inhibitory effect of the antagonistic strain PNP-1, plate confrontation experiments were conducted against eight pathogens: *Boeremia exigua*, *Boeremia exigua* var. *exigua*, *Alternaria tenuissima*, *Phoma multirostrata*, *Fusarium oxysporum*, *Fusarium culmorum*, *Curvularia lunata*, and *Colletotrichum sublineola*. The specific procedures were as follows: After inoculating the pathogens in the center of the plate, PNP-1 strain was inoculated at equal intervals around the perimeter as the treatment group, with plates inoculated only with the pathogens serving as the control group. Three replicates were set up. After 5 days of confrontation culture, the diameter of the pathogen colonies was measured using the cross-cross method, and the inhibition rate was calculated.

[0045] 1.2.5 Data Statistics and Analysis

[0046] SPSS 22.0 and Origin 2021 software were used for statistical analysis of the data.

[0047] 2. Results and Analysis

[0048] 2.1 Isolation and Identification of Endophytic Fungi in Panax notoginseng

[0049] This invention isolated the strain PNP-1, which exhibited the best antibacterial effect, from the petioles of healthy plants collected from areas affected by Phytophthora infestation in Panax notoginseng. After 7 days of culture, the colony morphology was observed to be circular, black (with grayish-grayish threads), and exhibiting a rough (granular) texture. The colony edges were neat and orderly, and the texture was leathery. The colonies grew rapidly, and the color distribution was very uniform. The hyphae had a low-gloss appearance and appeared grayish-black, while the reverse side was black. Further sequencing analysis of its rDNA ITS region was performed, and BLAST alignment was conducted in the GenBank database. Based on sequence similarity, the species was identified as being most closely related to *Annulohypoxylon annulatum* (GenBank accession number PP860316.1), belonging to the Ascomycota phylum.

[0050] The preservation information for strain PNP-1 is as follows: it was deposited on May 22, 2025 at the China General Microbiological Culture Collection Center (CGMCC); the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit telephone number is 010-64807288; and the accession number is CGMCC No. 42013.

[0051] 2.2 Evaluation of the antagonistic function of endophytic fungi of Panax notoginseng against Phytophthora pathogens

[0052] To verify whether the resistance of healthy plants in diseased fields to pathogens is related to the activity of endophytic fungi, this invention conducted a confrontation experiment between the isolated endophytic fungus PNP-1 and the pathogen *Phytophthora cactorum*. The results showed that the isolated endophytic fungus PNP-1 had an antagonistic effect on *Phytophthora cactorum*. The inhibition diameter (mm) was 19.50 ± 0.20; the inhibition rate (%) was 61.76 ± 0.40.

[0053] Double-layer plate culture was used for secondary screening of cell-free filtrates of antagonistic strains. The antibacterial effect of cell-free filtrate of strain PNP-1 was as follows: Figure 1 As shown, both the treatment group and the control group (the treatment group had the pathogen inoculated in the center of the plate, and PNP-1 strain was inoculated at equal intervals around it; the control group had plates inoculated only with the pathogen) were cultured for 5 days. The diameter of the pathogen on the PNP-1 cell-free filtrate double-layer plate (1.73±0.07) was significantly smaller than that of the pathogen in the control group (4.88±0.30). Furthermore, according to... Figure 1 As can be seen from A, strain PNP-1 contains extracellular antibacterial substances.

[0054] The mycelial morphology of strain PNP-1 against the pathogen *Phytophthora cactorum* was analyzed using the slide method. Results are as follows: Figure 2 As shown, the control group without cell-free filtrate of strain PNP-1 exhibited better hyphal morphology and more branching; the treated group samples treated with cell-free filtrate of strain PNP-1 showed that the hyphae were bent, less extensible, and had significantly fewer branches, indicating that strain PNP-1 has a significant inhibitory effect on the hyphae of Phytophthora cactorum, the pathogen of Phytophthora cactorum.

[0055] To further clarify the antagonistic effect of strain PNP-1 against *Phytophthora cactorum*, the pathogen of *Phytophthora* in Panax notoginseng, this invention also conducted a pot experiment on the antibacterial activity of the fermentation broth of strain PNP-1. The results of the pot experiment on the control of *Phytophthora* disease by the fermentation broth of strain PNP-1 are as follows: Figure 3 As shown in Table 1.

[0056] Table 1. Effects of strain PNP-1 on potted plant control of Phytophthora in Panax notoginseng.

[0057] deal with Incidence rate (%) Disease index Prevention and control efficacy (%) negative control — — — Positive control 100.00 68.06 — Process 1 40.00 33.33 51.03 Process 2 47.37 43.86 35.52

[0058] according to Figure 3 As shown in Table 1, on the 5th day after inoculation with *Phytophthora cactorum*, the negative control group showed no disease, while the positive control group had a disease index of 68.06 and an incidence rate of 100%. Treatment 1 showed a disease index of 33.33 and an incidence rate of 40.00%, with a control effect of 51.03%. Treatment 2 showed a disease index of 43.86 and an incidence rate of 47.37%, with a control effect of 35.52%. Both treatments 1 and 2 reduced the damage of *Phytophthora cactorum* to *Panax notoginseng*. Treatment 1, which involved spraying the fermentation broth of strain PNP-1 followed by the pathogen, showed the best control effect.

[0059] The results of the inhibition experiments of strain PNP-1 against eight crop pathogens are shown in the figure. Figure 4 And Table 2.

[0060] Table 2. Inhibition rate of strain PNP-1 against crop pathogens.

[0061]

[0062]

[0063] Data are expressed as mean ± standard error. According to Duncan's multiple range test (P<0.05 and P<0.01), different letters indicate significant differences.

[0064] according to Figure 4As shown in Table 2, strain PNP-1 exhibited the most significant inhibitory effects against *Fusarium culmorum* (beet root rot) and *Curvularia lunata* (maize leaf spot), with inhibition rates exceeding 50.00% (P<0.01). It also showed good inhibitory effects against *Boeremia exigua* (Panax notoginseng leaf spot), *Fusarium oxysporum* (Panax notoginseng root rot), and *Colletotrichum sublineola* (sorghum anthracnose). Strain PNP-1 possesses good broad-spectrum antibacterial activity and shows promising potential in biocontrol applications.

[0065] The PNP-1 strain isolated in this invention is *Annulohypoxylon annulatum*, a novel antagonistic strain that is currently absent from reports on the biocontrol of plant diseases, laying the foundation for the development of subsequent biocontrol agents. The *Annulohypoxylon annulatum* PNP-1 isolated from healthy plants and its cell-free filtrate exhibit high antagonistic activity against *Phytophthora indicum*, the causal agent of *Phytophthora indicum*. Pot experiments showed a control efficacy of 51.03%, and it also demonstrated good inhibitory effects on *Boeremiaexigua* leaf spot, *Fusarium oxysporum* root rot, *Fusarium culmorum* root rot of beets, *Curvularia lunata* leaf spot of maize, and *Colletotrichum sublineola* anthracnose of sorghum. Therefore, *Annulohypoxylon annulatum* PNP-1 holds promise for development as a biocontrol agent against *Phytophthora indicum*.

[0066] 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 truncated anthracnose fungus ( Annulohypoxylon annulatum PNP-1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42013.

2. A microbial inoculant, characterized in that, Contains the truncated charcoal bacterium PNP-1 as described in claim 1.

3. The application of the truncated charcoal bacterium PNP-1 according to claim 1, the microbial agent according to claim 2, or the fermentation broth or cell-free filtrate of the truncated charcoal bacterium PNP-1 according to claim 1 in inhibiting the growth of plant pathogens, characterized in that, The plant pathogen is Phytophthora cactorum , Fusarium culmorum, Curvularia lunata, Boeremia exigua, Fusarium oxysporum or Colletotrichum sublineola .

4. The application as described in claim 3, characterized in that, The fermentation broth was obtained by inoculating the truncated carbonaceous bacteria PNP-1 into PDA medium and culturing it.

5. The application as described in claim 3, characterized in that, The cell-free filtrate was obtained by filtering the fermentation broth through a 0.22 μm filter membrane.

6. A method for reducing the incidence of Phytophthora blight in Panax notoginseng, characterized in that, The method includes the step of applying the microbial agent of claim 2 or the cell-free filtrate of claim 3 to the Panax notoginseng plants.

7. A broad-spectrum inhibitor of plant pathogens, characterized in that, The active ingredient comprises the truncated charcoal fungus PNP-1 as described in claim 1; the plant pathogen is... Phytophthora cactorum , Fusarium culmorum, Curvularia lunata, Boeremia exigua, Fusarium oxysporum or Colletotrichum sublineola .

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