An endophytic fungus in antheroporum chinense seed and application thereof

Abstract

The application discloses an anther smut fungus and application thereof. The strain is preserved in the China General Microbiological Culture Collection Center on June 29, 2026, and the preservation number is CGMCC 3.29441. The endophytic fungus and extracellular metabolites thereof can be used for preventing and treating plant fungal diseases or antagonizing pathogenic fungi. The endophytic fungus isolated in the application has good antagonistic and inhibitory activities on Rhizoctonia solani, Tulasnella prachens, Curvularia lunata, C. intermedia and Brevicaivella ilicis, and can strongly inhibit the mycelium growth of pathogenic fungi. Meanwhile, the extracellular metabolites secreted by the strain also have high inhibitory effects on Rhizoctonia solani, Tulasnella prachens, Curvularia lunata, C. intermedia and Brevicaivella ilicis, and can be used for preparing a biocontrol agent or a pesticide.

Description

Technical Field

[0001] This invention relates to an endophytic fungus and its application, and more particularly to an endophytic fungus found in seeds of *Gnaphalium affine* and its application. Background Technology

[0002] Leaf spot disease in grasses is caused by *Rhizoctonia solani* and *Epicoccum latusicollum*. These pathogens infect not only grasses but also many other economic crops belonging to the legume, solanaceae, and cruciferous families. The disease develops rapidly at temperatures of 21–32°C, especially 25–30°C, and relative humidity exceeding 80%. It is commonly found in lawns during rainy summers or in areas with persistent morning dew. Early symptoms include water-soaked, bluish-gray spots on leaves and leaf sheaths with reddish edges. These spots then enlarge and merge, leading to yellowing and dieback of the leaves and stems, eventually causing the plant to wilt and die from the bottom up.

[0003] Curvularia leaf blight in grasses is caused by Curvularia lunata and Curvularia intermedia. Initially, infected grass leaves develop small, leaf-colored spots that gradually expand into oval lesions with a grayish-white center and darker edges. Later, these lesions merge, leading to leaf death. Curvularia fungi have a wide host range and can cross-infect cool-season grasses such as Kentucky bluegrass, ryegrass, and fine-leaved fescue. When Curvularia leaf blight occurs, leaves, leaf sheaths, and stem bases of grasses can all be affected. This disease typically causes a sharp decline in the population of grasses, with an incidence rate as high as 45–82%, causing devastating damage to the normal growth of grasses.

[0004] Root rot in grasses can be caused by *Bipolaris sorokiniana*, a pathogen that can damage various economic crops and turfgrass species within the grass family, including wheat, barley, ryegrass, and Kentucky bluegrass, severely impacting agricultural production and the quality of green landscapes. This fungus spreads through airflow, irrigation water, agricultural operations, or infected seeds, primarily penetrating and invading through root wounds, natural openings, or young tissues, leading to root and leaf rot, stem base necrosis, and leaf spot development. In severe cases, it can cause widespread death of grasses. Currently, the control of leaf spot, Curvularia leaf blight, and root rot in grasses mainly includes the following methods: 1. Cultivating high-quality disease-resistant seedlings. This method is the most economical and effective way to control diseases in grass production. However, due to the difficulty in large-scale production and the relatively unclear mechanism, it cannot be widely promoted and applied at present. 2. Chemical control. Although this method can control leaf spot, Curvularia leaf blight, and root rot in grasses to a certain extent, the large-scale use of chemical agents poses a great threat to the ecological environment and food safety, and also leads to the continuous enhancement of drug resistance in pathogens.

[0005] With the development of ecological agriculture, biological control has become a key research direction in disease control. Plant endophytic fungi can help plants obtain nutrients, induce resistance, and improve their survival ability under biotic or abiotic stress conditions. Because plant endophytic fungi originate from plants, they are safe for plants, environmentally friendly, and less prone to developing drug resistance. Using these microorganisms to control plant diseases is beneficial for maintaining ecological balance. Therefore, plant endophytic fungi are a novel biological control method for promoting organic agriculture. Currently, no endophytic fungi have been reported that can simultaneously control leaf spot, Curvularia leaf blight, and root rot, as well as their pathogens. Summary of the Invention

[0006] Objectives of this invention: The first objective is to provide an endophytic fungus for *Gnaphalium affine* seeds, addressing the problems of controlling leaf spot, *Curvularia spp.* leaf blight, and root rot, and their pathogens. The second objective is to propose the application of this endophytic fungus in controlling or antagonizing plant fungal diseases, addressing the problem of how to control or antagonize plant fungal diseases. The third objective is to provide a biological control agent, addressing the problem of how to obtain such an agent. The fourth objective is to propose the application of this biological control agent in controlling or antagonizing plant fungal diseases, addressing the problem of how to control or antagonize plant fungal diseases.

[0007] Technical solution: This invention provides an endophytic fungus in the seeds of *Epichloë bromicola*, the strain of which is deposited under the preservation name *Epichloë bromicola* EGCans002, with the accession number CGMCC No. 3.29441. This strain was deposited on June 29, 2026, at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The second aspect of this invention discloses the application of the above-mentioned endophytic fungi in the seeds of *Gnaphalium affine* in the prevention and control of fungal diseases in plants or in the antagonism of pathogenic fungi.

[0009] Preferably, the plant includes at least one of the following: grasses, legumes, solanaceae, and cruciferous plants.

[0010] Preferably, the fungal disease includes at least one of leaf spot, Curvularia leaf blight, and root rot.

[0011] Preferably, the pathogenic fungus includes at least one of Rhizoctonia solani, Acromegalys thunbergii, Curvularia crescentis, Curvularia intercalans, and Helicobacter spp.

[0012] A third aspect of the present invention provides a biological control agent comprising at least one of the following: hyphae, conidia, and extracellular metabolites of the endophytic fungi in the seeds of *Gnaphalium affine*.

[0013] The fourth aspect of this invention provides the application of the above-mentioned biological control agent in the control of plant fungal diseases or in the antagonism of pathogenic fungi.

[0014] Preferably, the plant is a grass, and the fungal disease includes at least one of leaf spot, Curvularia leaf blight, and root rot.

[0015] Furthermore, the pathogens of the leaf spot disease include Rhizoctonia solani and / or Phytococcus thunbergii, the pathogens of Curvularia leaf blight include Curvularia crescentis and / or Phytococcus thunbergii, and the pathogens of root rot include Helicobacter spp.

[0016] Preferably, the pathogenic fungus includes at least one of Rhizoctonia solani, Acromegalys thunbergii, Curvularia crescentis, Curvularia intercalans, and Helicobacter spp.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The endophytic fungus *Epichloë bromicola* EGCans002 of this invention exhibits good antagonistic and inhibitory activity against *R. solani*, *E. latusicollum*, *C. lunata*, *C. intermedia*, and *B. sorokinina*, strongly inhibiting the mycelial growth of these pathogenic fungi with inhibition rates of 28.68%, 18.64%, 41.18%, 50.43%, and 59.48%, respectively. Simultaneously, the extracellular metabolites secreted by this strain also show high inhibitory effects on *R. solani*, *E. latusicollum*, *C. lunata*, *C. intermedia*, and *B. sorokinina*, and can be used to prepare biocontrol agents or pesticides. Attached Figure Description

[0018] Figure 1The diagram shows the antagonistic effects of Epichloë bromicola EGCans002 against different pathogens. Figure 2 for Figure 1 Statistical results of pathogen diameter in plate confrontation experiment; Figure 3 The antagonistic effect of the fermentation broth of Epichloë bromicola EGCans002 on different pathogens is shown in the figure. Figure 4 Statistical results on the inhibition of pathogenic bacteria growth by the fermentation broth of Epichloë bromicola EGCans002; Figure 5 Phylogenetic tree of Epichloë bromicola EGCans002 based on tef and tub (NJ method); Figure 6 Disease index of wheat after treatment with fermentation broth of Epichloë bromicola EGCans002; Figure 7 This is a graph showing the disease index of E+ and E- plants in a potted plant experiment of *Gnaphalium affine* under the infection of five different pathogens. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0020] Example 1: An endophytic fungus Epichloë bromicola EGCans002 was screened using the following method: 1. Isolation of potential biocontrol bacteria 1.1 Obtaining test materials: Select healthy, vigorous *Gnaphalium affine* plants from the field and collect the seeds from the spike-like inflorescence at the top of the plant.

[0021] 1.2 Isolation and culture of endophytic fungi from seeds of *Gnaphalium affine*: Take healthy *Gnaphalium affine* seeds and disinfect their surface. Soak in 2.5% (v / v) sodium hypochlorite solution for 2 minutes, then soak in 70% (v / v) ethanol for 30 seconds, and finally rinse with sterile water 3 times. Four times, blot dry the surface with sterile absorbent paper. Place the seeds on a PDA agar plate under sterile conditions. Seal the petri dish and incubate at 25°C in the dark for 1 day. 2d. When the mycelium emerges from the seed surface, carefully transfer the small piece of culture medium growing at the edge of the medium, along with the mycelium, onto a new PDA plate.

[0022] 2. Screening of antagonistic bacteria using the plate confrontation method Rhizoctonia solani, C. lunata, and B. sorokinina were purchased from the China Agricultural Microbiological Culture Collection Center (strain numbers ACCC37444, ACCC38966, and ACCC36341, respectively). E. latusicollum and C. intermedia were isolated and identified by our laboratory from diseased grass leaves (NCBI numbers PX411273.1 and PX436246.1, respectively).

[0023] The PDA plate confrontation method was used. Mycelial discs (5 mm in diameter) of the tested endophytic fungi, cultured for 7 days, were symmetrically inoculated 2 cm from the edge of 15 mL of PDA medium on 9 cm diameter plates. Then, mycelial discs (5 mm in diameter) of *Rhizoctonia solani*, *Pseudomonas aeruginosa*, *Curvaria crescentis*, *Curvaria intercalata*, and *Helicobacter rotundus* strains cultured for 3-5 days were inoculated in the center of the medium. Plates inoculated only with mycelial discs of *Rhizoctonia solani*, *Pseudomonas aeruginosa*, *Curvaria crescentis*, *Curvaria intercalata*, and *Helicobacter rotundus* strains served as controls. The plates were incubated at 25 ℃ in the dark. After 5-7 days, the colony diameters of each pathogen were measured, and the inhibition rate was calculated. The inhibition rate was calculated using the formula: Inhibition rate = (Coronary diameter of control group colonies) / (Coronary diameter of control group colonies) Treatment group colony diameter / control group colony diameter × 100%.

[0024] Finally, one strain was selected that showed good antagonistic activity against Rhizoctonia solani, Acromegalys thunbergii, Curvularia crescentis, Curvularia intercalans, and Helicobacter spp. on agar plates, specifically exhibiting strong inhibition of pathogen mycelial growth. This strain was designated EGCans002.

[0025] like Figure 1 As shown, compared with the control, the colonies of pathogenic bacteria in the experimental group were significantly smaller, indicating that EGCans002 inhibited *Rhizoctonia solani*, *E. latusicollum*, *C. lunata*, *C. intermedia*, and *B. sorokinina* by 28.68%, 18.64%, 41.18%, 50.43%, and 59.48%, respectively. Figure 2As shown, the average growth diameters of the control groups of Rhizoctonia solani, Acromegalys blight, Curvularia crescentis, Curvularia intercalans, and Helicobacter spp. were 8.23±0.15cm, 3.97±0.55cm, 5.27±0.61cm, 4.70±0.26cm, and 5.43±0.15cm, respectively. The average growth diameters of the EGCans002 treatment groups were 5.87±0.55cm, 3.23±0.21cm, 3.10±0.17cm, 2.33±0.15cm, and 2.20±0.2cm, respectively. The comparison shows that the mycelial growth of the pathogens causing leaf spot, Curvularia leaf blight, and root rot was inhibited by EGCans002.

[0026] 3. Molecular biological identification of the screened strains 3.1 The fungus was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results are as follows: The tefA gene sequence of strain EGCans002 is shown in SEQ ID NO.1: GACCTGTATCATTCGATGTATTGGATAACAGTCGCTAACTTGTCTGCTAACAGGGGTACGTACTGCGAAATATCACTCGCCGTCGCCGAAATTCACGTACTGACTGAAGCGTAGCCACGTCGACTCTGGCAAGTCTACCACCACCGGTCACTTGATTTACCAGTGCGGTGGAATTGACAAGCGTACCATCGAGAAGTTCGAGAAGGTAAGACATTCTTCTTTCAATTCACGCATAATATGTGATGTTCACTCGTTGCAATGCGAGCCTGCCTTGTGTGTCGCTTTGCAACCTTGGTGGGCGAGCAAGCATCTGCCCCTCTTTTCGCCCGCGATACGAATTTTTTTTTTTCGGTCGCGGGGCTCAGTCTGACTTTTGGTGGGGCTCCTCTCAACCCGTCACTGGTCTAAGCTAGAGACGCAAACGAGAGAGGCATGACATGACATTCGCGTGCCCTCCCAAAAAAAAATTGTGATGACAAATCACTGACTTGCCTTCGCTCTATAGGAAGCCGCCGAACTCGGAAAGGGTTCTTTCAAATATGCGTGGGTTCTTGACAAGCTCAAGGCCGAGCGTGAGCGTGGTATCACCATCGACATTGCCCTCTGGAAGTTCGAGACTCCCAAGTACTATGTCACCGTCATTGGTAAGCCTTGGTCGACACAGTAGACTCTATCAACCTGATCTGTATCACTAACGTGCATCTACTAGACGCTCCCGGTCACCGTGATTCATCAAGAA The tubB gene sequence of strain EGCans002 is shown in SEQ ID NO.2: TCCTCGACGCGTTCCAGTGTTGAGCCCCTGATTTCGTACCCCGCCGAGCCCGGCCACGACGTGCACGCCCAATGGACAGTCGTGATGAGAGGCGGACCGAGACAAAAAAAATTAATGATTGCGGTATTCGAGAACTGTCGCTGACATTTTACTTCCCCTCTAGGTTCATCTTCAAACCGGTCAGTGCGTAAGTGTGACAAATCCGCCGACCTCGAACGACAGGCACAAATAACATGAAAAACTCACATTTCATTGGGCAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGTCTCGACAGCAATGGTGTGTACAACGGTACCTCCGAGCTCCAGCTGGAGCGTATGAGTGTCTACTTCAACGAGGTAAGTCTTCATAATTTAAAGTCTCCATTGAGCTACATACCGCCCTGGAGATGAGACGGAAAGAGAACGATGAAAAGTGTTAACATGCTAATCTATGTGACAGGCTTCTGGCAACAAGTATGTTCCTCGCGCTGTCCTCGTCGATCTCGAGCCTGGTACCATGGATGCAGTCCGTGCCGGTCCCTTCGGTCAGCTTTTCCGTCCCGACAACTTCGTCTTCGGTCAGTCTGGTGCTGGCAACAACTGGGCCAAGGGTCACTACACTGAGGGTGCTGAGCTGGTTGACCAAGTCCTCGACGTTGTGCGTCGCGAGGCCGAAGGCTGTGACTGTCTCCAGGGTTTCCAGATCACCCACTCGCTTGGTGGTGGTACCGGTGCCGGTATGGGTACATTGTTGATCTCCAAGATCCGTGAGGAGTTCCCCGACCGGATGATGGCCACTTTCTCCGTCGTCCCCTCTCCCAAGGTCTCTGATACCGTTGTCGA 3.2. Align the two sequences of SEQ ID NO. 1 and SEQ ID NO. 2 on the NCBI database website. Download the reference sequence most similar to the above two morphotype sequences, using the Neighbor Phylogenetic analysis was performed using joining (NJ) to determine the phylogenetic position of the strain to be identified, such as Figure 5 As shown. The endophytic fungal strain of this invention shares 99.59% sequence homology with three reference strains of Epichloë bromicola: Epichloë bromicola (MW961387.1) and Epichloë bromicola (PP801123.1). It belongs to the order Spheroidales, family Clavicipitaceae, genus Epichloë, and is classified as Epichloë bromicola EGCans002. It was deposited at the China General Microbiological Culture Collection Center on June 29, 2026, with the accession number CGMCC No. 3.29441.

[0027] Example 2: The effects of Epichloë bromicola EGCans002 fermentation broth on the growth of pathogens causing leaf spot, Curvularia leaf blight, and root rot were investigated. The method is as follows: (1) Rhizoctonia solani, Curvularia lunata, and B. sorokinina were purchased from the China Agricultural Microbiological Culture Collection Center (strain numbers ACCC37444, ACCC38966, and ACCC36341, respectively). E. latusicollum and C. intermedia were isolated and identified by our laboratory from diseased grass leaves (NCBI numbers PX411273.1 and PX436246.1, respectively). These five pathogens and Epichloë bromicola EGCans002 were inoculated into 9 cm diameter petri dishes containing 15 mL of sterile PDA medium (formulation: 200 g potato, 20 g glucose, 1000 mL distilled water, 15 g agar, pH natural) and incubated at 25 °C in the dark for 6 days.

[0028] Using a 5mm diameter punch, take 2-3 pieces of bacterial cake containing the EGCans002 strain from the edge of the colony and inoculate them into 300mL of sterilized PDB medium. Incubate at 25℃ and 180rpm for 7 days with shaking. Filter the filtrate to obtain the fermentation broth of Epichloë bromicola EGCans002.

[0029] (2) Take 50 mL of Epichloë bromicola EGCans002 fermentation broth and add it to 200 mL of PDA medium and mix well to obtain a medium containing 20% ​​EGCans002 fermentation broth, which is used as the experimental group medium. The control group is a PDA medium plate without fermentation broth.

[0030] (3) In a clean bench, use a punch (5 mm in diameter) to make a mycelial cake at the edge of the colony and inoculate it into the plate prepared above. Make sure the mycelial side is facing down, and set up 3 replicates for each.

[0031] (4) Place the inoculated plates in a 25℃ incubator and incubate in the dark for 6 days. Measure the colony size using the cross-hatching method. Calculate the inhibition rate using the formula: Inhibition rate = (Coronary diameter of control group) Treatment group colony diameter / control group colony diameter × 100%; (5) Results are as follows Figure 3 As shown, compared with the control, the colonies of each pathogenic bacteria strain in the experimental group were significantly smaller. The extracellular metabolites (fermentation broth) of strain Epichloë bromicola EGCans002 showed inhibition rates of 10.00%, 18.83%, 13.03%, 15.87%, and 10.06% against Rhizoctonia solani, Curvularia lunata, Curvularia intercalata, and Helicobacter spp., respectively. Figure 4 As shown, the average growth diameters of *Rhizoctonia solani*, *Agrococcus brasiliensis*, *Curvularia crescentis*, *Curvularia intercalatum*, and *Helicobacter rotans* in the control groups were 9.00±0.00 cm, 6.00±1.21 cm, 5.83±0.91 cm, 5.67±1.15 cm, and 4.67±1.17 cm, respectively. The average diameters in the EGCans002-treated groups were 8.10±0.69 cm, 4.87±0.21 cm, 5.07±0.64 cm, 4.77±0.81 cm, and 4.20±0.56 cm, respectively. This indicates that the extracellular metabolites secreted by EGCans002 have a certain inhibitory effect on the hyphal growth of the above pathogens.

[0032] Example 3: Field efficacy test of biocontrol strains The pathogens used in the pot experiment were the five pathogens described in Example 2. Under aseptic conditions, 10 ml of sterile water was added to each of the five cultured pathogen petri dishes. After gently scraping the surface of the colonies with a sterile spreader, the samples were filtered through four layers of sterile gauze to obtain suspensions of different pathogen spores.

[0033] Preparation of endophytic fungi and fermentation broth: The isolated and purified endophytic fungus Epichloë bromicola EGCans002 was inoculated into liquid culture medium with a diameter of about 0.5 cm. The culture medium was placed in an Erlenmeyer flask and cultured in a shaker at a temperature of 25℃ and a rotation speed of 120 rpm for 7-14 days to obtain the fermentation broth of Epichloë bromicola EGCans002. Wheat plants at the three-leaf stage were divided into 10 groups, with 3 replicates per group. A single application of Epichloë bromicola EGCans002 fermentation solution was used to treat the wheat plants. Before application, one uniformly growing wheat plant was retained in each pot. The experimental groups received 10 mL of the bacterial suspension per pot, while the control group received 10 mL of sterile water per pot.

[0034] Seven days after inoculation with the bacterial solution, different pathogenic spore suspensions were inoculated into the experimental and control groups, respectively. After inoculation, the plants were kept moist at a relative humidity of ≥90% for 48 hours, and then transferred to conventional culture conditions. Twenty-one days after inoculation, the disease index of wheat plants in each treatment was investigated according to the corresponding disease grading standards.

[0035] The results are as follows Figure 6 As shown, the results indicated that the wheat disease index was significantly reduced after treatment with the fermentation liquid of the endophytic fungus Epichloë bromicola EGCans002 compared with the sterilized water control group; the wheat disease indices after spraying with the fermentation liquid of Epichloë bromicola EGCans002 were 33.82%, 34.89%, 32.92%, 38.44%, and 38.76%, respectively, which were significantly lower than the disease indices of the control group (49.36%, 67.15%, 77.44%, 63.03%, and 47.67%).

[0036] E+ and E- plants of uniform growth (E+: plants containing endophytic fungi; E-: plants without endophytic fungi) were selected as live inoculation subjects. Before the experiment, the number of green leaves in each pot was counted and recorded. Then, using a sterilized sprayer, a suspension of spores of the five pathogens from Example 2 was evenly sprayed onto each leaf of the E+ and E- plants. Three pots were inoculated for each pathogen, one plant per pot. The E+ and E- plants sprayed with sterilized water served as controls. Immediately after inoculation, each pot was covered with a plastic bag and placed in a greenhouse for 24 hours of moist cultivation. Normal watering was carried out every other day.

[0037] The number of diseased leaves and lesions on the plants were counted 7 days after inoculation. The disease index was calculated using the formula: Disease Index (DI) = ∑(number of diseased plants at each level × corresponding level value) / (total number of leaves surveyed × representative level of the final disease level) × 100%.

[0038] The results are as follows Figure 7 As shown in the pot experiment of *Gnaphalium affine*, after E+ and E- plants were inoculated with five pathogens, the disease index of E+ and E- plants differed significantly within the inoculation time (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

Claims

1. An endophytic fungus on the seeds of *Gnaphalium affine*, characterized in that, The strain is deposited under the name Epichloëbromicola EGCans002, with accession number CGMCC No. 3.29441, and was deposited at the China General Microbiological Culture Collection Center on June 29, 2026.

2. The application of the endophytic fungi in the seeds of *Gnaphalium affine* according to claim 1 in the prevention and control of fungal diseases in plants or in the antagonism of pathogenic fungi.

3. The application according to claim 2, characterized in that, The plants include at least one of the following: grasses, legumes, solanaceae, and cruciferous plants.

4. The application according to claim 2, characterized in that, The fungal diseases include at least one of leaf spot, Curvularia leaf blight, and root rot.

5. The application according to claim 2, characterized in that, The pathogenic fungi include at least one of Rhizoctonia solani, Acromegalys thunbergii, Curvularia crescentis, Curvularia intercalans, and Helicobacter spp.

6. A biological control agent, characterized in that, It contains at least one of the hyphae, conidia, and extracellular metabolites of the endophytic fungus in the seeds of *Gnaphalium affine* as described in claim 1.

7. The application of the biological control agent according to claim 6 in the control of plant fungal diseases or in antagonizing pathogenic fungi.

8. The application according to claim 7, characterized in that, The plant includes at least one of the following: grasses, legumes, solanaceae, and cruciferous plants. The fungal disease includes at least one of the following: leaf spot, Curvularia leaf blight, and root rot.

9. The application according to claim 8, characterized in that, The pathogens of the leaf spot disease include Rhizoctonia solani and / or Phyllostachys nigra; the pathogens of Curvularia leaf blight include Curvularia crescentis and / or Phyllostachys nigra; and the pathogens of root rot include Helicobacter spp.

10. The application according to claim 7, characterized in that, The pathogenic fungi include at least one of Rhizoctonia solani, Acromegalys thunbergii, Curvularia crescentis, Curvularia intercalans, and Helicobacter spp.

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