Use of a fungus of the genus emericella F-XTBG8 with broad-spectrum antibacterial activity in the preparation of antifungal active substances

Abstract

The application discloses application of a broad-spectrum antibacterial biocontrol Cytospora fungus F-XTBG8 in preparation of antifungal active substances. Chaetomella raphigera The application of the broad-spectrum antibacterial biocontrol Cytospora fungus (F-XTBG8) is applied in preparation of antifungal active substances. The strain has obvious antagonism to more than 100 environmental fungi and plant pathogenic fungi including Pyricularia oryzae (Monascus oryzae), Mycosphaerella fijiensis (Mycosphaerella fijiensis), Alternaria tenuissima (Alternaria tenuissima), Fusarium graminearum (Fusarium graminearum), Fusarium oxysporum (Fusarium oxysporum) and Fusarium verticillioides (Fusarium verticillioides), and fermentation liquor of the strain can significantly reduce occurrence of corn large spot, tobacco black spot and sclerotinia rot. Magnaporthe oryzae Setosphaeria turcica Alternaria tenuissima Fusarium graminearum Fusarium oxysporum Fusarium concentricum The application provides an excellent biocontrol strain for development and research of microbial source pesticides and provides a new idea for green prevention and control of plant diseases.​​​​​

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to the application of a broad-spectrum antibacterial fungus, F-XTBG 8, in the preparation of antifungal active substances. Background Technology

[0002] Plant infectious diseases have had a serious impact on global agriculture. Food crop diseases caused by fungi have become a global food security problem. With the large-scale flow of people and goods in the era of globalization, plant diseases that were originally limited to a certain place are now more likely to spread, and this trend is showing signs of aggravation.

[0003] Rice blast is the most damaging rice disease worldwide, caused by the fungus *Magnaporthe oryzae*. Magnaporth rice Rice blast fungus can damage leaves, stems, and grains throughout the entire growth cycle of rice, generally reducing yield by 10-20%, and in severe cases by 40-50%, or even causing total crop failure. Infection with rice by rice blast fungus causes various symptom types, the most serious of which is neck blast, which damages the neck of the panicle and can lead to empty or white panicles. Furthermore, due to somatic cell recombination, mutation, and adaptive variation, rice blast fungus is prone to mutation, resulting in numerous physiological races and a wide host range, capable of proliferating in a wide variety of plants.

[0004] Large spotted setae cocci ( Setosphaeria turcica *Cladosporium glomeratum* is a semi-living parasitic ascomycete fungus that can infect plants such as corn, sorghum, and Sudan grass, forming water-soaked, grayish-brown lesions that affect photosynthesis and thus reduce yield. *Cladosporium glomeratum*, causing leaf blight in northern maize, is a serious fungal disease threatening maize production worldwide, with severe outbreaks in cool maize-producing areas of Northeast China, northern North China, Northwest China, and southern mountainous regions of my country.

[0005] Tobacco black spot disease is caused by Alternaria spp. (Alternaria microphylla) Alternaria tenuissima This disease is caused by a large number of lesions on the lower and middle leaves of tobacco plants during their vigorous growth period to maturity, resulting in significant yield losses.

[0006] Fusarium graminearum ( Fusarium gramineae ) can infect wheat ( Wheat ),barley( Barley ), rice ( White rice ),oat( Sand gray It can cause ear rot, stem rot, stem base rot and root rot in cereal crops such as ears, stems, stem base and roots, as well as Fusarium head blight in cereal crops, causing yield and quality loss, and the fungal toxins produced can harm human and animal health.

[0007] Fusarium oxysporum ( Fusarium oxysporumIt can cause vascular bundle wilt in a variety of plants, resulting in serious losses to crops such as melons, passion fruit, tomatoes, cotton, and bananas, and can even infect immunocompromised humans.

[0008] In order to control plant diseases, humans have used large amounts of chemical pesticides in an unreasonable manner, which has led to problems such as the development of pesticide resistance in plant pathogens, excessive pesticide residues in agricultural products, and serious damage to the ecological environment. Biological control of plant diseases has received increasing attention and is gradually being applied in production.

[0009] Biocontrol bacteria have advantages such as strong targeting, thorough control effect, no soil pollution, and no adverse impact on product quality. This invention aims to provide a *Rhizopus* fungus with broad-spectrum antibacterial activity. Summary of the Invention

[0010] The purpose of this invention is to provide a fungus of the genus *Nyctaginis* with broad-spectrum antibacterial activity (…). Chaetomel quicksilver Application of F-XTBG8 in the preparation of antifungal active substances.

[0011] The objective of this invention is achieved by using *Rhizopus* fungi with broad-spectrum antibacterial activity. Chaetomel quicksilver Application of F-XTBG8 in the preparation of antifungal active substances. This strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 40274, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0012] The beneficial effects of this invention are as follows: The *F. XTBG8* fungus of the genus *F.* exhibits high efficacy in controlling various pathogenic fungi. The control rates against *Bacillus oryzae*, *Cladosporium macrocarpa*, *Alternaria spp.*, *Fusarium graminearum*, *Fusarium oxysporum*, and *Fusarium graminearum* are 84.17%, 45.36%, 65.74%, 60.28%, 71.67%, and 52.90%, respectively. In semi-in vitro inoculation experiments on plant leaves, it shows significant inhibitory effects on lesions caused by *Bacillus oryzae*, *Cladosporium macrocarpa*, *Alternaria spp.*, *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium graminearum*, demonstrating high application value in the development of biological agents for the control of plant or crop diseases. Its fermented bacterial broth can significantly reduce the occurrence of corn leaf blight, tobacco black spot, and Fusarium head blight. This invention provides an excellent biocontrol strain for the research and development of microbial pesticides and offers a new approach to the green control of plant diseases. Attached Figure Description

[0013] Figure 1A, B, C, D, E, and F in the figure represent the comparative effects of the fungus F-XTBG8 of the genus *Nyctaginus* on *Magnaporum oryzae*, *Cladosporium macrocarpa*, *Alternaria spp.*, *Fusarium graminearum*, *Fusarium oxysporum*, and *Fusarium graminearum*, respectively.

[0014] Figure 2 A, B, C, D, E, and F in the figure represent the comparative effects of 4-isopropylbenzoic acid (50 μg / mL), a metabolite of the fungus F-XTBG8, on *Strombus haematobium*, *Cladosporium macrocarpa*, *Alternaria spp.*, *Fusarium graminearum*, *Fusarium oxysporum*, and *Fusarium graminearum*.

[0015] Figure 3 A, B, C, and D in the figure are comparison diagrams of the semi-in vitro antibacterial effects of the fermentation broth of the fungus F-XTBG8 against *Cladosporium macrocarpa*, *Alternaria spp.*, *Fusarium graminearum*, and *Fusarium oxysporum* on corn, tobacco, corn, and tomato leaves, respectively.

[0016] Figure 4 Phylogenetic classification tree for strain F-XTBG8 constructed using maximum likelihood analysis of ITS sequence. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0018] This invention provides fungi of the genus *Nyctaginis* with broad-spectrum antibacterial activity (… Chaetomella raphigera Application of F-XTBG8 in the preparation of antifungal active substances. This strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 40274, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on August 17, 2022.

[0019] The antagonistic fungi belong to the genus *Polyporus* (also known as velutipes). Abundispore Alternaria ( ) Alternaria Aspergillus ( ) Aspergillus ), Top of the Trichophyton genus ( Bartolinia ), genus Colletotrichum ( Colletotrichum ), Corynebacterium ( Corynespora ), genus *Rhizoctonia* ( Emma Fusarium ( ) Fusarium ), Pyreosporium ( Firefly Mucor ( ) I am sick. ), Neo-red crustacean ( Neocosmospore ), Neodiscochaeta ( Neopestalotiopsis ), *Pyrtomyces* genus ( Neosetophoma ), Penicillium ( Penicillium ), Perennial genus of *Porphyra* ( Perennial ), Phaeophlebiopsis genus *Schizophyllum* ( Schizophyllum ), genus Helicobacter ( Exerohyl ), Basilella Talaromyces ), spp. Trametes Trichoderma ( ) Trichoderma ) and the genus *Yamada* ( Yamadazyma).

[0020] The antifungal active substance is 4-isopropylbenzoic acid.

[0021] This invention also provides the application of the genus *Rhizopus* fungus F-XTBG8, which has broad-spectrum antibacterial activity, in the prevention and control of plant diseases.

[0022] The plant diseases mentioned include rice blast, maize leaf spot, tobacco black spot, Fusarium head blight, or Fusarium wilt.

[0023] The present invention further provides a microbial preparation based on the fungus F-XTBG8 of the genus *Rhizopus*, wherein the effective component of the microbial preparation is the fungus F-XTBG8 of the genus *Rhizopus* and its fermentation products or fermentation broth.

[0024] The fermentation medium for the fungus F-XTBG8 of the genus *Rhizopus* is 1L, containing 12g of potato extract powder, 20g of glucose, and a pH of 5.4-5.8. The fermentation conditions are: a culture temperature of 28-30℃, a shaking speed of 180-200 rpm, and a culture time of 120-170h.

[0025] This invention further provides the application of the aforementioned microbial preparation in the prevention and control of plant diseases, wherein the microbial preparation should...

[0026] It can be applied by foliar spraying or root irrigation.

[0027] Example 1: Isolation, purification, and screening of strain F-XTBG8

[0028] 1. Isolation and purification of strain F-XTBG8

[0029] Aerial root mucilage of *Melastoma candida* was collected from the Xishuangbanna Tropical Botanical Garden (Mengla, Yunnan) in May 2020 and stored in centrifuge tubes in the dark at low temperature before being brought back to the laboratory for processing. 200 μL of the mucilage was aspirated using a sterile pipette tip in a laminar flow hood and placed on potato dextrose agar (PDA) plates. The plates were then spread using a spreader, sealed, and incubated at 30°C for 72 hours. Single colonies were then picked and re-agared on PDA medium for propagation.

[0030] 2. Screening of strain F-XTBG8

[0031] 1. Initial screening of the biocontrol function of the strain

[0032] The initial screening of the biocontrol function of the strain was carried out using a plate confrontation experiment. The specific steps are as follows: *Magnaporum oryzae* (a type of orchid) was inoculated into PDA medium. Magnaporthe rice ), Large Spotted Cladosporium ( Setosphaeria turcica ), Alternaria spp. ( Alternaria tenuissima Fusarium graminearum ( ), Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum ) and Fusarium moniliforme ( Fusarium concentricum Common pathogens such as *Melastoma candida* (prepared into 5mm d-cell fungi using a sterile pipette tip and inoculated in the center of the culture medium) were inoculated. Different isolated and purified fungi were inoculated into 5mm d-cell fungi at a 2cm perimeter around the pathogenic fungi inoculated into the fungi, with four strains arranged in a cross shape. Plates without fungal inoculation served as controls. The plates were incubated at 30℃ for 7-10 days. When the blank plates were fully colonized, the colony morphology of plates inoculated with *Melastoma candida* derived from its aerial root slime fungi was observed, recorded, and fungi exhibiting antibacterial effects were selected for further use.

[0033] 2. Rescreening of strains for biocontrol function

[0034] The screening method is the same as above, but after each plate is inoculated with the pathogen, the same candidate strain is inoculated four weeks later. The control is not inoculated and the plate is fully colonized. Then, the colony diameter is counted using the cross-hatching method, repeated three times, and the strain diameter is recorded (e.g., ...). Figure 1 The growth rate of the test bacteria, specifically the control growth (colony diameter) and the treatment growth (growing diameter after inoculation with antagonistic bacteria), is expressed as the inhibition rate.

[0035] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%;

[0036] Results: After two plate screenings, strain F-XTBG8, a potential biocontrol bacterium, was obtained through the second plate screening step, exhibiting inhibitory effects against all six pathogens. The plate confrontation experiment, as shown in Table 1, demonstrated that strain F-XTBG8 inhibited the growth of rice blast fungus (…). Magnaporthe rice ), Large Spotted Cladosporium ( Setosphaeria turcica ), Alternaria spp. ( Alternaria tenuissima Fusarium graminearum ( ), Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum ) and Fusarium moniliforme ( Fusarium concentricum The strain showed good inhibitory effects, with inhibition rates of 84.17%, 45.36%, 65.74%, 60.28%, 71.67%, and 52.90%, respectively, and was therefore selected as the target strain for screening.

[0037] Table 1. Inhibitory effect of strain F-XTBG 8 against six pathogens.

[0038]

[0039] Example 2: Determination of the antibacterial spectrum of strain F-XTBG8

[0040] The antimicrobial spectrum of strain F-XTBG8 was determined using a plate confrontation experiment. The specific steps were as follows: Activated strain F-XTBG8 was prepared into 5mm (d) mycelial discs using a sterile pipette tip and inoculated into the center of PDA medium. Then, approximately 100 activated common pathogenic fungi and other fungal contaminants were prepared into 5mm mycelial discs and inoculated around the perimeter of the F-XTBG8 mycelial discs, forming a cross shape. The culture was incubated at 30℃ for 10 days. The colony morphology of the common pathogenic fungi and other fungal contaminants was observed and recorded. Strains whose colony edges became straightened were identified as those antagonized by strain F-XTBG8.

[0041] Results: Through plate confrontation experiments, as shown in Table 2, we found that 77 strains, representing 60 species and belonging to 22 genera, were significantly inhibited by F-XTBG8. Clearly, F-XTBG8 possesses broad-spectrum antifungal activity.

[0042]

[0043] Table 2. Antibacterial spectrum of strain F-XTBG 8 (partial)

[0044] Example 3 Identification of strain F-XTBG8

[0045] 1. Select a single spore of strain F-XTBG8 and inoculate it into PDA medium for culture. Then scrape off the mycelium from the edge of the colony and add it to PDB liquid medium for 5 days. After precipitation and centrifugation, extract genomic DNA from the bacterial cells. Use the Solarbio FungiGenomic DNA Extraction Kit D2300-100T (containing RNase A) extraction kit to extract sample DNA according to the instructions.

[0046] 2. Using the extracted DNA as a template, PCR sequences were amplified from the internal transcribed spacer (ITS) rRNA gene sequence and the ribosomal large subunit (LSU) rRNA gene sequence. The primer sequences ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS4R (5′-TCCTCCGCTTATTGATATGC-3′), LSUF (5′-ACCCGCTGAACTTAAGC-3′) and LSUR (5′-TCCTGAGGGAAACTTCG-3′) were synthesized by Beijing Qingke Biotechnology Co., Ltd. For specific operating procedures, please refer to the kit instructions.

[0047] 3. The bacterial PCR products were detected using an electrophoresis instrument. The DNA gel recovery kit from Beijing TransGen Biotech Co., Ltd. was used to cut and recover the bands. The recovered DNA samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to determine the ITS rRNA and LSU rRNA gene sequences, as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0048] 4. The DNA sequences generated from sequencing were compared with the fungal ITS rDNA in the public database NCBI using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Homology detection, phylogenetic classification tree construction, and morphological identification were then performed. Figure 4 As shown, strain F-XTBG8 was finally identified as... Chaetomelaraphigera .

[0049] Example 4: Preparation of fermentation broth of strain F-XTBG8

[0050] Single spores of fungal strains such as F-XTBG8 of the genus *Rhizopus* were incubated in PDA medium (potato extract powder: 12 g / L, glucose: 20 g / L, agar: 20 g / L, pH 5.6, sterilized at 121°C for 15 minutes) at 30°C for 5 days. After colony growth, fresh mycelia at the edge were picked and transferred to Erlenmeyer flasks containing 50 mL of PDB liquid medium. These flasks were then incubated in a shaker at 30°C and 180 rpm for 10 days to obtain the fermentation broth. The fermentation broth was found to contain the antibacterial substance 4-isopropylbenzoic acid.

[0051] Example 5: Preparation of fermentation broth of strain F-XTBG8

[0052] Single spores of fungal strains such as F-XTBG8 of the genus *Rhizopus* were incubated in PDA medium (potato extract powder: 12 g / L, glucose: 20 g / L, agar: 20 g / L, pH 5.8, sterilized at 121°C for 15 minutes) at 28°C for 7 days. After colony growth, fresh mycelia at the edge were picked and transferred to Erlenmeyer flasks containing 50 mL of PDB liquid medium. These flasks were then incubated in a shaker at 30°C and 200 rpm for 10 days to obtain the fermentation broth. The fermentation broth was found to contain the antibacterial substance 4-isopropylbenzoic acid.

[0053] Example 6 Preparation of fermentation broth of strain F-XTBG8

[0054] Single spores of fungal strains such as F-XTBG8 of the genus *Rhizopus* were incubated in PDA medium (potato extract powder: 12 g / L, glucose: 20 g / L, agar: 20 g / L, pH 5.4, sterilized at 121°C for 15 minutes) at 29°C for 6 days. After the colonies emerged, fresh mycelia at the edge were picked and transferred to a conical flask containing 50 mL of PDB liquid medium. The flask was then placed in a constant temperature shaker at 30°C and 190 rpm for 10 days to obtain the fermentation broth.

[0055] Experimental Example 1: Identification of the antibacterial effect of compound 4-isopropylbenzoic acid

[0056] 1. Weigh 1g of 4-isopropylbenzoic acid (purchased from Maclean, purity 98%) and dissolve it in 10mL of methanol solution to prepare a working stock solution with a concentration of 100mg / mL.

[0057] 2. Add 250 μL of working stock solution to 499.75 mL of LPDA medium (55℃, mix well), pour 20 mL into each plate to prepare a 50 μg / mL drug-containing plate.

[0058] 3. Using a sterilization nozzle, sterilize F-XTBG8 and rice blast fungus (…). Magnaporthe rice ), Large Spotted Cladosporium ( Setosphaeria turcica Alternaria spp. ( ) Alternaria tenuissima Fusarium graminearum ( ), Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum ) and Fusarium moniliforme ( Fusarium concentric Fresh colonies at the edge were prepared into mycelial discs with a diameter of 5 mm (d=5 mm) and inoculated onto a 50 μg / mL drug-containing plate. A blank control was prepared by inoculating a plate containing the same volume of methanol. Figure 2 The culture conditions were: constant temperature incubator at 30℃ for 10 days.

[0059] 4. Colony diameter was determined using the cross-cross method, repeated three times, and the strain diameter was recorded. The control growth (colon diameter) and treatment growth (growth diameter inoculated onto drug-containing plates) of the test bacteria were expressed as inhibition rate:

[0060] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%;

[0061] The results are shown in Table 3. 50 μg / mL of 4-isopropylbenzoic acid was effective against *Magnapordica oryzae* (…). Magnaporthe rice ), Large Spotted Cladosporium ( Setosphaeria turcica Alternaria spp. ( ) Alternaria tenuissima Fusarium graminearum ( ), Fusarium gramineae Fusarium oxysporum ( Fusarium oxysporum ) and Fusarium moniliforme ( Fusarium concentricum The inhibition rates of the α, β, and α were 9.75%, 38.60%, 28.08%, 16.94%, 39.83%, and 29.98%, respectively.

[0062] Table 3. Inhibitory effects of 4-isopropylbenzoic acid on six pathogens.

[0063]

[0064] Experiment Example 2: Semi-in vitro control efficacy of fermentation broth of strain F-XTBG8 against four pathogens.

[0065] I. Experimental Methods:

[0066] 1. Experimental Design: Control and Treatment Groups. Healthy, uniformly growing corn, tobacco, and tomato leaves were selected. After rinsing with sterile water and patting dry with absorbent paper, the leaves were cut into 3cm × 3cm pieces using sterile surgical scissors in a laminar flow hood. A puncture was made in the center of each leaf piece using a sterile needle. Control Group: Leaf pieces were immersed in blank PDB (with 0.1% Tween 20 added) for 30 seconds, and a uniformly grown pathogenic fungal cake (d=5mm) was inoculated into the puncture site. Treatment Group: Leaf pieces were then immersed in the F-XTBG8 fermentation broth prepared in Example 4 (with 0.1% Tween 20 added) for 30 seconds, and a uniformly grown pathogenic fungal cake (d=5mm) was inoculated into the puncture site. The leaf pieces were then placed in sterile glass petri dishes (14cm in diameter) lined with a single layer of filter paper and containing 5mL of sterile water. Ten leaf pieces were placed in each culture.

[0067] 2. After placing the dish, cover it with the lid and place it in a 30℃ constant temperature incubator. Observe the leaf disease situation every other day.

[0068] 3. After the growth period is 3 days, the disease situation will be counted.

[0069] Level 1: No symptoms, no pathogenic mycelium growth; Level 3: Slight yellowing of a small portion of the leaf, slight necrosis at the wound site, almost no pathogenic mycelium growth; Level 5: Half of the leaf is yellow, less necrosis at the wound site, a small amount of pathogenic mycelium growth; Level 7: More than 3 / 4 of the leaf is yellow, some necrosis at the wound site, a lot of pathogenic mycelium growth; Level 9: The entire leaf is yellow and loses its green color, with a large amount of water-soaked necrosis at the wound site, and vigorous mycelial growth.

[0070] Disease index = [Σ(number of diseased leaf patches at each level × relative level value) / (total number of leaf patches × highest level value)] × 100.

[0071] In this experimental example, each treatment has 10 leaf blocks, and the highest level number represents a value of 9.

[0072] II. Results Analysis:

[0073] Depend on Figure 3 It was found that for *Cladosporium macrocarpa*, *Alternaria spp.*, and *Fusarium graminearum*, the symptoms of the leaf blocks in the treatment group were significantly alleviated after treatment with the F-XTBG8 fermentation broth prepared in Example 3. Compared with the control group, the leaf blocks showed no obvious yellowing or necrosis, and the mycelial growth of the pathogens was inhibited. However, for *Fusarium oxysporum* on tomato leaves, there was no difference between the treatment group and the control group.

[0074] Table 4. Inhibition of disease development by strain F-XTBG8 against four pathogens.

[0075]

[0076] As shown in Table 4, the disease indices of leaf lesions caused by *Cladosporium macrocarpa*, *Alternaria spp.*, and *Fusarium graminearum* in the control group were 56, 58, and 62, respectively, while the disease indices of leaf lesions in the treatment group treated with F-XTBG8 fermentation liquid were 16, 26, and 13, respectively, which were significantly lower than those in the control group.

[0077] In summary, the strain F-XTBG8 of this invention can effectively prevent *Cladosporium macrosporum* (…). Setosphere Turkish Alternaria spp. ( ) Alternaria tenuissima ) and Fusarium graminearum ( Fusarium grasses This strain is also effective against crop diseases caused by blast fungus (such as rice blast fungus). Magnaporthe rice ), broad-spectrum pathogen Fusarium oxysporum ( Fusarium oxysporum ) and Fusarium moniliforme ( Fusarium concentricum It also has metabolite and plate inhibition effects, and can be used for the biological control of a variety of important crop diseases.

Claims

1. Fungi of the genus *Rhizopus* with broad-spectrum antibacterial activity ( Chaetomella raphigera The application of F-XTBG8 in the preparation of antifungal active substances: The strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 40274, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the antifungal active substance is 4-isopropylbenzoic acid.

Images