Endophytic chaetomium globosum and application thereof in prevention and treatment of corn diseases and insect pests

Abstract

This invention belongs to the field of biological control technology for plant diseases and pests, and provides a strain of Chaetomium globosum ( Chaetomium globosum EF-14 and its applications. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40618. The *Chaetomium globosum* EF-14 provided by this invention is an endophytic fungus of maize that can simultaneously inhibit *Fusarium graminearum* (…). Fusarium gramineae ) and Corn spores ( Bipolaris maydis It can effectively reduce corn stalk rot and corn leaf spot caused by fall armyworm ( ) and can also effectively reduce the incidence of fall armyworm ( ). Spodoptera frugiperda This invention provides a highly efficient and safe microbial resource for the green control of major corn diseases and pests, and broadens the application scope of Chaetomium globosum in agricultural biocontrol, achieving synergistic control of diseases and pests through "one fungus targeting multiple targets".

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases and pests, and specifically relates to a strain of Chaetomium globosum that can be used for the control of diseases and pests in corn. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Corn, as a vital global food crop, feed source, and industrial raw material, plays an irreplaceable role in ensuring food security and agricultural economic stability. However, throughout its growth cycle, corn is frequently subjected to the synergistic damage of various diseases and pests, severely impacting its yield and quality and hindering the sustainable development of the industry. Among these, corn stalk rot is caused by Fusarium spp. (…). Fusarium spp.) and Pythium ( Pythium Soil-borne diseases caused by complex infections of multiple pathogens such as *Fusarium graminearum* (spp.) are predominantly caused by *Fusarium graminearum* in northern my country. Fusarium graminearum The dominant pathogen is characterized by its long survival period and complex transmission routes, making it difficult to control. Meanwhile, corn leaf spot, a common foliar fungal disease, is particularly severe during the tasseling and grain-filling stages, further exacerbating yield losses. In addition, the fall armyworm (… Spodoptera frugiperda As a major migratory pest, it directly feeds on corn plants, seriously threatening the safe production of corn.

[0004] Currently, the control of corn diseases and pests still relies mainly on chemical pesticides. Although these pesticides are fast-acting, long-term use can lead to increased resistance in pathogens and pests, pesticide residues, and environmental pollution, which does not meet the requirements of green and sustainable agricultural development. Biological control is gaining increasing attention due to its environmental friendliness and specific target characteristics. Current research reports that compound microbial agents, such as Metarhizium anisopliae-Bacillus preparations, can show good control efficacy against major diseases and pests of fresh corn, demonstrating the potential of compound microbial agents in synergistic control. However, existing biocontrol agents mostly rely on the combination of strains, and single biocontrol strains that can simultaneously control multiple diseases and also have pest control functions are still rarely reported. Summary of the Invention

[0005] To address the lack of microbial agents for controlling corn diseases and pests in existing technologies, this invention provides a strain capable of effectively antagonizing Fusarium graminearum (…). Fusarium graminearum ) and corn spores ( Bipolaris maydis ) of Chaetomium coccidioides ( Chaetomium globosum It is used to prevent and control corn stalk rot and corn leaf spot, and can also help prevent lepidopteran pests.

[0006] Another objective of this invention is to provide a crude metabolic extract of the above-mentioned Chaetomium globosum that can be used for the prevention and control of corn diseases and pests.

[0007] Another objective of this invention is to provide the application of the above-mentioned *Chaetoceros* in the control of corn stalk rot, small leaf spot, and fall armyworm.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] A strain of Chaetomium globosum ( Chaetomium globosum EF-14 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40618.

[0010] The present invention also provides a fermentation product produced by the above-mentioned Chaetomium globosum, wherein the fermentation product is: a) EF-14 cell-free fermentation broth, concentrate, or... b) Ethyl acetate extract of a.

[0011] The above-mentioned Chaetomium EF-14 and / or fermentation products can be used to prepare microbial agents and pharmaceuticals.

[0012] The aforementioned Chaetomium globosum and its fermentation products, as well as microbial agents, can be used to control plant diseases and fall armyworm.

[0013] The plant disease is caused by the following pathogen: Fusarium graminearum (… Fusarium graminearum ) or Corn spores ( Bipolaris maydis ).

[0014] The plant in question is corn.

[0015] The above application involves applying one of the following to plant diseases or the host plant of the fall armyworm: *Chaetoceros globosum* and its fermentation products, microbial agents, or pesticides, so that the plant diseases or fall armyworms can come into contact with the *Chaetoceros globosum* and its fermentation products, microbial agents, or pesticides.

[0016] The present invention has the following advantages: The present invention relates to *Chaetomium globosum* EF-14, an endophytic fungus with broad-spectrum biocontrol functions. Studies have found that this strain not only exhibits significant control effects against maize stalk rot and maize leaf spot, but its metabolites also interfere with the feeding behavior of fall armyworm larvae and have a repellent effect on adult oviposition, demonstrating its potential for "multi-effect" biocontrol. Currently, single biocontrol strains possessing both pathogen inhibition and pest control functions are extremely rare. The discovery of EF-14 provides a new microbial resource for the green control of maize diseases and pests, possessing significant research value and application prospects. Therefore, developing biocontrol agents based on *Chaetomium globosum* EF-14 is of great significance for promoting green maize production, reducing the use of chemical pesticides, and ensuring food security.

[0017] Biological Preservation Information Chaetomium coccidioides ( Chaetomium globosum EF-14 was deposited on May 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 40618. Attached Figure Description

[0018] Figure 1 These are the morphological characteristics of strain EF-14; where a is the colony morphology of strain Chaetomium EF-14 on PDA; b is the growth morphology of EF-14 in PDB; c is the hyphae of strain EF-14; and d is the ascospores of strain EF-14. Figure 2 It is a phylogenetic tree of EF-14 constructed based on ITS sequences; Figure 3 This study demonstrates the inhibitory effect of EF-14 on Fusarium graminearum 3H-S1 and other pathogens causing maize stalk rot. A represents the plate confrontation test of Chaetomium globosum EF-14 against 3H-S1, 3H-S4, and FV. Left: EF-14 was inoculated first, followed by the pathogen; Right: the pathogen was inoculated first, followed by EF-14; a PDA plate with EF-14 inoculated in the center and the pathogen only inoculated in the center served as a control. BD represents the inhibition rate of EF-14 against the pathogens. Figure 4 This shows the plate confrontation effect of Chaetomium globosum EF-14 against the corn leaf blight pathogen. In Figure A (left): EF-14 was inoculated before the corn leaf blight pathogen; the corn leaf blight pathogen was inoculated before EF-14 (right): the corn leaf blight pathogen was inoculated before the corn leaf blight pathogen. The center of the plates is dominated by the corn leaf blight pathogen. Figure B shows the inhibition rate of EF-14 against the corn leaf blight pathogen. Figure 5The study focuses on the effect of EF-14 metabolite treatment on the feeding selectivity of fall armyworm; where A represents the feeding behavior of fall armyworm on maize leaves; and B represents the statistical data on leaf feeding behavior of fall armyworm. Figure 6 The effect of EF-14 metabolite treatment on oviposition in fall armyworm; Figure 7 The data represents the field control efficacy of EF-14 against maize leaf spot in two plots, Linqing and Guangrao. A represents Linqing, and B represents Guangrao. Figure 8 The disease index represents the field control efficacy of EF-14 against maize stalk rot in Linqing and Tai'an areas; CK is the control group consisting of 3H-S1 (7.5 g / m), and T is EF-14 (15 g / m) + 3H-S1 (7.5 g / m). Detailed Implementation

[0019] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0020] Example 1 Identification of the target strain 1. Isolation of target bacteria Healthy corn stalks were collected in Linqing City, Shandong Province. After rinsing the samples, the stalks were cut into 1 cm segments using a sterile scalpel in a laminar flow hood. These segments were placed in sterile PDA medium and surface-sterilized with 75% alcohol for 60 seconds, followed by 1.05% sodium hypochlorite for 10 minutes, and rinsed three times with sterile water. The corn stalk segments were then dried on sterile filter paper to remove any residual moisture. After thorough drying, the segments were placed on potato dextrose agar (PDA) plates, with five segments per plate. To check the effectiveness of surface sterilization, the stalks were left to stand for 10 minutes before being removed as a control. The samples were incubated upside down in the dark at 25°C for 5–7 days until colonies grew around the stalks. The resulting fungal colonies were then inoculated onto new PDA plates.

[0021] 2. Identification of strains The strain EF-14, which showed good activity after initial screening, was identified as follows: In PDA medium, EF-14 colonies were pale yellowish-white in the early stages of growth, with milky-white aerial hyphae; later, the colony color deepened, and a yellowish-green exudate was produced during cultivation. The hyphae aged and turned olive-green or yellowish-brown. Figure 1 a). After culturing in PDB medium at 25°C and 150 rpm for 5 days, a large number of mycelial balls were produced ( Figure 1b). After 7 days of incubation at 25℃, the colony diameter reached 4.27 cm, and ascocarps began to form. After 10 days of incubation, the colony color darkened significantly, and the culture medium also turned pale yellow. The hyphae were pale yellow, septate, and had a slender, filamentous structure. Figure 1 c), ascospores are brown, lemon-shaped or obovate in shape ( Figure 1 d).

[0022] EF-14 hyphae were picked, and DNA was extracted using a fungal genomic DNA extraction kit (BioFlux). PCR amplification was performed using the DNA as a template with universal primers ITS1 / ITS4. After the reaction, 2 μL of the PCR product was analyzed by agarose gel electrophoresis (1%). The unpurified PCR product was sent to Qingke Biotechnology Co., Ltd. for sequencing. The rDNA-ITS sequence of this strain was compared with NCBI, and a phylogenetic tree was constructed using MEGA 7.0 software and the NJ method. Based on the phylogenetic tree of EF-14 and seven ITS sequences derived from the GenBank genus *Chaetoceros*, it was found that the biocontrol bacteria clustered with *Chaetoceros*, with a similarity of 99%. Figure 2 ).

[0023] Based on morphological observation and ITS sequence analysis, strain EF-14 was identified as Chaetomium globosum ( Chaetomium globosum It was deposited on May 26, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40618.

[0024] Example 2: Antibacterial effect of Chaetomium EF-14 against different pathogens causing corn stalk rot The plate confrontation method was used to determine the susceptibility of EF-14 to Fusarium graminearum, the pathogen of maize stalk rot. Fusarium graminearum ) 3H-S1, 3H-S4 and Fusarium pseudomorphum ( Fusarium verticillioides Inhibition rate of FV. All tested strains were cultured on PDA plates, and mycelial cakes were collected from the edge of the colony using a 5 mm diameter sterile punch. Both preventative and therapeutic inoculation methods were employed. In the prevention group, four EF-14 plates were first symmetrically inoculated on PDA plates. Then, Fusarium graminearum 3H-S1, 3H-S4 and Fusarium verticillatum (FV) were inoculated in the center of the plates at intervals of 0 days, 1 day and 2 days, respectively. In the treatment group, *Fusarium graminearum* 3H-S1, 3H-S4, and *Fusarium verticillatum* (FV) were first inoculated in the center of the plates, followed by EF-14 inoculated symmetrically around the periphery of the plates at 0, 1, and 2 day intervals. PDA plates inoculated only with the pathogens served as a control. The experiment was repeated three times. The inhibitory effect was observed after 5 days of dark incubation at 25°C.

[0025] Plate confrontation tests showed that *Chaetomium globosum* EF-14 had a good inhibitory effect on all three pathogens. Figure 3 A). Specifically, after EF-14 administration followed by 3H-S1 administration at 0, 1, and 2 days, the inhibition rates were 80.48%, 86.87%, and 100%, respectively; after 3H-S1 administration followed by EF-14 administration at 0, 1, and 2 days, the inhibition rates were 80.26%, 65.93%, and 39.82%, respectively. Figure 3 B). After EF-14 was administered first, followed by 3H-S4 at 0, 1, and 2 days intervals, the inhibition rates were 60.10%, 67.93%, and 80.43%, respectively. After 3H-S4 was administered first, followed by EF-14 at 0, 1, and 2 days intervals, the inhibition rates were 58.16%, 41.51%, and 20.88%, respectively. Figure 3 C). After EF-14 inoculation, followed by FV inoculation at 0, 1, and 2 days, the inhibition rates were 65.61%, 80.67%, and 89.68%, respectively; after FV inoculation, followed by EF-14 inoculation at 0, 1, and 2 days, the inhibition rates were 61.04%, 43.55%, and 26.29%, respectively. Figure 3 D).

[0026] The results of the confrontation experiment showed that EF-14 had a significant inhibitory effect on Fusarium graminearum 3H-S1, 3H-S4 and Fusarium verticillatum FV. The inhibition rate was above 80% 2 days after inoculation with EF-14, and the inhibitory effect of inoculating with EF-14 first was higher than that of inoculating with pathogens first.

[0027] Example 3: Antibacterial effect of Chaetomium EF-14 on the pathogen of maize leaf spot. The inhibitory effect of biocontrol bacterium EF-14 on the pathogen of maize leaf spot disease was determined using the plate confrontation method. All strains were activated on PDA plates, and mycelial cakes were collected from the edge of the colony using a 5 mm diameter sterile punch. Two inoculation methods were employed: preventative and therapeutic. In the preventative group, four EF-14 plates were symmetrically inoculated on PDA plates, followed by inoculation of the pathogen of maize leaf spot disease in the center of the plate at 0, 1, and 2 day intervals. In the therapeutic group, the pathogen of maize leaf spot disease was first inoculated in the center of the plate, followed by symmetrical inoculation of the biocontrol bacterium EF-14 around the perimeter of the plate at 0, 1, and 2 day intervals. PDA plates inoculated only with the pathogen of maize leaf spot disease served as a control group. The experiment was repeated three times. The inhibitory effect was observed after dark incubation at 25℃ for 3-5 days. The colony diameter was measured using the cross-cross method, and the inhibition rate was calculated.

[0028] Plate confrontation experiments showed that, compared with the control group, the co-culture group significantly inhibited the growth of mycelium of the pathogen of maize leaf spot (Spotted Leaf Spot). Figure 4 A). Specifically, the inhibition rates of inoculating with EF-14 first, followed by inoculation with the corn leaf blight pathogen at intervals of 0 days, 1 day, and 2 days were 87.01%, 89.13%, and 100%, respectively; the inhibition rates of inoculating with the corn leaf blight pathogen first, followed by inoculation with EF-14 at intervals of 0 days, 1 day, and 2 days were 83.58%, 59.09%, and 34.98%, respectively. Figure 4 B). The results of the confrontation experiment showed that the inhibition rate reached 100% when EF-14 was inoculated first for 2 days, and the inhibition effect was significantly higher than that of the group inoculated first with pathogens.

[0029] Example 4: Effects of fermentation products from strain EF-14 on fall armyworm 1. Preparation of crude EF-14 metabolites EF-14 was inoculated into 20 Erlenmeyer flasks (1000 mL each) containing 400 mL of PDB. The mixture was shaken at 25°C and 150 rpm for 7 days. The mycelia were filtered through a single layer of gauze, and an equal volume of ethyl acetate was added to the filtrate. After thorough mixing, the mixture was allowed to stand and separate into layers. The crude extract of the fermentation broth was obtained by rotary evaporation at 37°C, dissolved in methanol, and sterilized using a 0.22 μm microporous membrane. The crude extract of EF-14 was diluted to 10 mg / mL and 20 mg / mL.

[0030] 2. The effect of EF-14 on the feeding of fall armyworm Maize seedlings were grown under controlled conditions (25±1℃, 70% relative humidity, 16 / 8 h light / dark cycle) in plastic pots (15 cm in diameter) containing sterile vermiculite. Fourteen days after emergence, uniform leaves (of equal area) were cut from fully expanded leaves. A methanol solution of EF-14 crude extract (20 mg / mL) was evenly applied to the maize leaves. For the control group, methanol was used instead of the EF-14 crude extract. After the maize leaves dried, they were placed in sterile petri dishes (9 cm in diameter), with control leaves on the left half and treated leaves on the right half. Five third-instar fall armyworms were placed in each petri dish (20 petri dishes per replicate, 3 replicates). Feeding activity was monitored for 15 h under controlled conditions (25℃, 60% relative humidity), and the moths were divided into three groups (a, b, and c) based on leaf area consumption. Group A: Leaf loss area in the control group > Leaf loss area in the treatment group; Group b: Leaf area consumed in the control group < Leaf area consumed in the treatment group; Group C: Leaf area consumed in the control group = Leaf area consumed in the treatment group.

[0031] After applying EF-14 crude extract to corn leaves, the leaves were fed to fall armyworms, and the feeding area was compared. Results were as follows: Figure 5 As shown, the fall armyworm fed on corn leaves. Group a had 19 petri dishes, group b had 0.33 petri dishes, and group c had 0.67 petri dishes. The number of petri dishes in group a was significantly higher than that in groups b and c. After EF-14 treatment, the fall armyworm's feeding selectivity on corn was significantly reduced.

[0032] 3. Field trial effect of EF-14 on oviposition of fall armyworm The field experiment was conducted outdoors in cages. Corn kernels were planted in plastic pots containing soil. Twelve pots (30 cm in diameter) of uniformly growing corn plants, with three plants per pot, were placed on each side of the cage. The cage measured 3 m × 2 m × 1 m (length × width × height), with a 10 cm spacing between plants in each pot. The control and treatment groups were spaced 1 m apart. Each pot in the treatment group was sprayed with 1 mL of EF-14 crude extract (10 mg / mL) in methanol, while the control group received methanol instead. After air-drying, 20 pairs of male and female adults (3 days after emergence) were randomly placed at any location within the cage. The experiment was conducted in triplicate. Oviposition by the females on the corn plants on both sides was assessed 3 days later.

[0033] The results are as follows Figure 6 As shown, the number of eggs laid by the fall armyworm in the treatment group was reduced by 60.65% compared with the control group, and the number of eggs laid in the treatment group was significantly lower than that in the control group. This indicates that the EF-14 fermentation product can repel the fall armyworm from laying eggs on corn.

[0034] Application Example 1: Field control effect of Chaetomium EF-14 on maize leaf spot. The field trial was conducted simultaneously in 2024 on two plots: Linqing (fertile soil) and Guangrao (saline-alkali land). Each plot was 30 m². 2 The rows were 12 m long and 3 m wide, with 3 plots per treatment and protective rows between plots. The planting density was 4500 plants / mu. Five EF-14 atomizers (5 mm in diameter) were punched from the edge of the colony using a sterile punch and inoculated into 1000 mL bottles of corn sand culture medium (mass ratio = 1:1) and incubated at 25℃ for 7 days. The culture (including the medium and mycelium) was then air-dried at low temperature to obtain the EF-14 inoculum. Four treatments were set up in both Linqing and Guangrao trials: EF-14 (7.5 g / m²), EF-14 (15 g / m²), EF-14 (30 g / m²), and a control group. Field management for each treatment plot followed standard field practices, with sufficient water supply throughout the growing season. When the corn reached the grain-filling stage, 100 corn plants were randomly selected from each treatment using a 5-point sampling method to investigate the incidence of corn leaf spot and calculate the disease index.

[0035] Disease index = 100 × ∑ (number of diseased leaves at each level × representative value of that disease level) / (total number of leaves surveyed × representative value of the highest disease level) The survey results showed that the disease index of the control group during the corn grain-filling stage in the Linqing experimental field was 15.27, while the disease indices of the EF-14 treatment group at T30, T15, and T7.5 were 4.27, 4.87, and 7.00, respectively. The disease indices of the different EF-14 treatment groups during the grain-filling stage in Linqing were significantly different from those of the control group. Figure 7 A). The disease index of the control group during the grouting period in Guangrao was 10.20, while the disease indices of the EF-14 treatment group at T30, T15, and T7.5 were 3.80, 4.43, and 6.97, respectively. The disease index of the EF-14 treatment group was significantly different from that of the control group. Figure 7 B). The results showed that in the field trials conducted at the Linqing and Guangrao experimental sites in 2024, treatment with different concentrations of EF-14 inoculant significantly reduced the incidence of maize leaf spot, demonstrating good control effects. Therefore, EF-14 can effectively control maize leaf spot under field conditions.

[0036] Application Example 2: Field control effect of Chaetomium EF-14 on maize stalk rot The field trial will be conducted simultaneously in Linqing and Tai'an cities, Shandong Province, in 2025. Each plot will have an area of ​​36 m². 2The plots were 12 m long and 3 m wide. Protective rows were set between plots, with a planting density of 4500 plants / mu. The preparation method of EF-14 inoculant was the same as in Application Example 1. Two treatments were set up in Linqing and Tai'an respectively: the treatment group EF-14 (15 g / m) and the control group CK. The experiment was repeated 3 times, and field management was carried out for each treatment according to field standards. When the maize reached the grain-filling stage, a five-point sampling method was used to randomly select 100 maize plants from each treatment to investigate the occurrence of maize stem rot and calculate the disease index.

[0037] The results are as follows Figure 8 As shown, the field survey results in Linqing indicated that the disease index in the treatment group was 6.27, while the disease index in the control group was 13.33, showing a significant decrease in the treatment group ( p <0.05). Similarly, in Tai'an, the disease index during the grouting period in the EF-14 treatment group (13.27) was significantly lower than that in the control group (28.37). p <0.05). The control efficacy of EF-14 inoculant at these two locations was 53.00% and 53.23%, respectively. These results indicate that EF-14 can effectively control maize stalk rot under field conditions.

[0038] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A strain of Chaetomium coccidioides ( Chaetomium globosum EF-14, characterized in that, The accession number is CGMCC No.40618.

2. A fermentation product produced by *Chaetomium globosum* as described in claim 1, characterized in that, The fermentation product is: a) EF-14 cell-free fermentation broth, concentrate, or... b) Ethyl acetate extract of a.

3. The use of Chaetomium globosum as described in claim 1 and / or the fermentation product as described in claim 2 in the preparation of microbial agents and pharmaceuticals.

4. A microbial agent or pharmaceutical preparation using *Chaetoceros globosum* as described in claim 1 and / or the fermentation product as described in claim 2.

5. The application of the *Chaetoceros* strain as described in claim 1, the fermentation product as described in claim 2, and the microbial agent or pesticide as described in claim 3 in the prevention and control of plant diseases and fall armyworm.

6. The application according to claim 5, characterized in that, The plant disease is caused by the following pathogen: Fusarium graminearum (… Fusarium graminearum ) or Corn spores ( Bipolaris maydis ).

7. The application according to claim 5, characterized in that, The plant in question is corn.

8. The application according to claim 5, characterized in that, The *Chaetoceros*, fermentation products, microbial agents, or pesticides are applied to plant diseases or the host plant of the fall armyworm, allowing the plant disease or fall armyworm to come into contact with the *Chaetoceros*, fermentation products, microbial agents, or pesticides.

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