A strain of Acremonium divaricata NtSi1 and its application in preparing a microbial agent for controlling root rot

Abstract

The present invention belongs to the technical field of microbial agents and discloses a strain of Acremonium crossbranchii NtSi1 and its use in preparing a root rot prevention and control agent. The Acremonium crossbranchii NtSi1 bacterial solution provided by the present invention has a wide range of inhibitory effects on plant pathogens and can prevent and control the root rot caused by Fusarium oxysporum ( Fusarium oxysporum )、Fusarium solani( Fusarium solani ), Fusarium fusae ( Fusarium sickle-shaped ), Alternaria ( Alternaria sp. ) or Curvularia spp. Curvularia spicifera ) and other plant root rot. Tests have shown that the biocontrol agent provided by the present invention has a 66.21% control effect on mung bean root rot and a 63.46% control effect on tobacco root rot under potted conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agents, and particularly relates to a strain of Acremonium truncatum NtSi1 and an application thereof in preparing a root rot prevention and control agent. Background Art

[0002] Root rot is a plant disease that develops at the root level, resulting in underdeveloped root systems, stunted and weak above-ground growth, pale green leaves, and a significant reduction in branching and fruiting. In its early stages, root rot affects only a few lateral and fibrous roots, gradually spreading to the taproot. Initially, the taproot is diseased, but as root rot progresses, the plant's ability to absorb water and nutrients gradually weakens, and the leaves wilt. In severe cases, the root bark turns brown and separates from the pith, the leaves wilt, and the plant dies suddenly.

[0003] Taking mung bean root rot as an example, the main pathogens of mung bean root rot include fungi (such as Pythium Pythium spp. and Fusarium spp. Fusarium spp. ) and bacteria (such as Xanthomonas spp. Xanthomonas spp. Root rot is caused by a variety of pathogens. In China, mung bean root rot is a common and serious disease, leading to widespread yield and quality reductions, especially in continuous cropping conditions.

[0004] Taking tobacco root rot as an example, root rot is caused by a variety of pathogens. At present, the pathogens of tobacco root rot reported in my country mainly include: Pythium ultimum ( Pythium ultimum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium solani ( Fusarium solani graminearum ( Fusarium graminearum ), Rhizoctonia solani ( Rhizoctonia solani According to statistics, tobacco root rot causes a yield reduction of over 30% each year, and in severe cases, even total crop failure. Tobacco root rot is not only caused by a complex array of pathogens but can also involve multiple infections, making it challenging to control.

[0005] Current methods for controlling root rot include: 1. Using disease-resistant varieties of plants. However, due to the complex pathogenic bacteria population and rapid virulence variation, plants can easily lose resistance. 2. Using chemical agents. While these can control root rot to a certain extent, excessive use can lead to the development of drug resistance in pathogens. Furthermore, excessive residues can pollute the environment and harm human health.

[0006] Many beneficial microorganisms exist in the environment surrounding plants. These microorganisms not only do not cause plant disease, but some can even induce plant resistance, improving their ability to survive biotic and abiotic stresses. Furthermore, these microorganisms originate from natural sources and are environmentally friendly. Using them to control plant diseases helps maintain ecological balance. Currently, a growing number of researchers are dedicated to finding environmental probiotics as a biocontrol method for plant diseases. Therefore, there is an urgent need for a green, highly effective environmental probiotic for preventing plant root rot. Summary of the Invention

[0007] The present invention aims to provide a strain of Acremonium crossbranchii NtSi1 and its use in preparing a root rot control agent. The Acremonium crossbranchii NtSi1 and the root rot control agent of the present invention have excellent biological control effects on various pathogens such as mung bean root rot and tobacco root rot.

[0008] The present invention is achieved through the following technical solutions:

[0009] A strain of Acremonium divaricata ( Sarocladium implicatum ) NtSi1, the Acremonium crossbranchum NtSi1 was deposited in the General Microbiology Center of China Culture Collection Administration on January 20, 2025, with the deposit number CGMCCNO.41760.

[0010] The present invention also provides the use of the above-mentioned Acremonium truncatula NtSi1 in the preparation of a root rot prevention and control bacterial agent.

[0011] Furthermore, the preparation method of the root rot disease controlling bacterial agent includes: inoculating the above-mentioned Acremonium crossbranchii NtSi1 into PDA liquid culture medium, culturing on a shaker, removing the supernatant of the cultured bacterial liquid and resuspending it with sterile water to obtain the root rot disease controlling bacterial agent.

[0012] Furthermore, the culture temperature of the Acremonium truncatum NtSi1 is 28-32° C., and the culture time is 2-7 days.

[0013] Furthermore, the root rot control bacterial agent is used in one or more of the following applications:

[0014] Inhibit the growth of root rot pathogens;

[0015] Promotes plant growth after root rot infection;

[0016] Prevent and control root rot caused by plant root rot pathogens;

[0017] Increases the content of defense enzymes in plants.

[0018] Furthermore, the plants include mung beans and tobacco.

[0019] Furthermore, the root rot pathogen is Fusarium oxysporum ( Fusarium oxysporum )、Fusarium solani( Fusarium solani ), Fusarium fusae ( Fusarium falciforme ), Alternaria ( Alternaria sp ) or Curvularia spp. Curvularia spicifera ).

[0020] Furthermore, the defense enzymes include peroxidase, polyphenol oxidase, and catalase.

[0021] Furthermore, the promoting the growth of plants infected with root rot includes increasing the above-ground plant height, above-ground fresh weight, root length, root fresh weight and chlorophyll content of the plants infected with root rot.

[0022] The present invention also provides a method for preventing and controlling plant diseases, which comprises applying the above-mentioned Acremonium crossbranchii NtSi1 to the growth environment of plant roots, wherein the plant disease is root rot.

[0023] The method for preparing a bacterial liquid of Acremonium crossbranchingensis NtSi1 includes: inoculating Acremonium crossbranchingensis NtSi1 into a PDA liquid culture medium, culturing the culture on a shaker at a rotation speed of 220 rpm, a culture temperature of 30°C, and a culture time of 48 to 72 hours. The cultured bacterial liquid is centrifuged at 8000 rpm / min for 5 minutes, the supernatant is removed, and the liquid is resuspended in sterile water to obtain a bacterial liquid of Acremonium crossbranchingensis NtSi1.

[0024] The present invention also provides a biocontrol bacterial liquid, which comprises the above-mentioned Acremonium truncatum NtSi1.

[0025] Beneficial effects

[0026] The biocontrol agent provided by the present invention has the function of preventing and treating multiple fungal diseases, has antagonistic functions against multiple root rot pathogens, has a prevention effect of 66.21% on mung bean root rot, and a prevention effect of 63.46% on tobacco root rot, and effectively curbs the phenomena of plant shrinkage, leaf yellowing, and root rot caused by root rot infection. The agent has broad-spectrum resistance, is environmentally friendly, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the phylogenetic tree of Acremonium intergenicum NtSi1;

[0028] Figure 2 The control effect of the plate and fermentation filtrate of Acremonium crossbranch NtSi1 root rot fungus; Figure 2 Middle A is the experiment of the confrontation effect of Acremonium dioica NtSi1 on five soil-borne pathogenic fungi; Figure 2 Middle B is the fermentation filtrate test to verify the inhibitory effect of the fermentation broth of Acremonium divaricata NtSi1 on five soil-borne pathogenic fungi;

[0029] Figure 3 This is the phenotype of mung bean plants, leaves and roots after treatment with biocontrol liquid; Figure 3 A in the middle is a mung bean plant treated with biocontrol liquid; Figure 3 Middle B is the mung bean leaves treated with biocontrol solution; Figure 3 Middle C is the phenotype of mung bean roots after treatment with biocontrol solution;

[0030] Figure 4 This is the phenotype of tobacco plants, leaves and roots after being treated with biocontrol liquid; Figure 4 Middle A is a morphological diagram of tobacco plants; Figure 4 Middle B is a picture of tobacco leaves; Figure 4 Middle C is a picture of the tobacco root; Figure 4 D in the middle is a slice of tobacco root;

[0031] Figure 5 These are the physiological indicators of mung bean after biocontrol bacteria treatment, including fresh weight of aboveground parts, fresh weight of underground parts, plant height, root length and chlorophyll content;

[0032] Figure 6 Physiological indicators of tobacco after biocontrol bacteria treatment, including fresh weight of aboveground parts, fresh weight of underground parts, plant height, root length and chlorophyll content;

[0033] Figure 7 It is the antioxidant enzyme activity of tobacco after treatment with biocontrol bacteria. DETAILED DESCRIPTION

[0034] In order to better understand the present invention, the present invention is further described below with reference to the accompanying drawings and examples, but the present invention is not limited thereto. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0035] The Acremonium spp. of the present invention ( Sarocladium implicatum ) NtSi1 was deposited in the General Microbiology Center of the China Culture Collection Administration on January 20, 2025, with the culture collection number CGMCC NO.41760, and the deposit location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; its ITS gene sequence is shown in SEQ ID NO.1.

[0036] SEQ ID NO.1:

[0037] .

[0038] The screening and sequencing process of the biocontrol strains having the effect of preventing and controlling multiple plant pathogenic fungi of the present invention is as follows:

[0039] 1. Sample Collection

[0040] Tobacco rhizosphere soil was collected from diseased tobacco fields in Zhengzhou City, Henan Province, and the soil was labeled and taken back to the laboratory for separation.

[0041] 2. Isolation of Acremonium truncatula NtSi1

[0042] A dilution-spreading and temperature-screening method was used: The collected soil was air-dried and ground finely, and 10 g was weighed into a conical flask. 90 mL of sterile water was added. A gradient of dilutions of varying concentrations was prepared. The dilutions, from low to high, were evenly spread onto air-dried LB and PDA plates and incubated at 28°C for 2 days. Colonies with distinct morphology, color, and other characteristics were selected for further purification on PDA plates. The purified strains were stored in glycerol. Single hyphae were picked and plated onto PDA plates for further culture. Purification was repeated three times to obtain a pure strain. The purified strains were stored on slants for future use.

[0043] 3. Molecular Identification of Acremonium truncatula NtSi1

[0044] After the genomic DNA of the isolated strain was extracted using the CTAB method, the ITS region of the eukaryotic organism was amplified using PCR premix (2× Taq Master Mix enzyme from Vazyme) and primer pair ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO. 2) / ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO. 3). The nucleotide sequence information was obtained by sequencing at Qingke Biotechnology Co., Ltd. The obtained sequence was submitted to the sequence database (GenBank) for analysis and comparison. The sequences with higher homology were selected and the phylogenetic tree was constructed using molecular evolution genetic analysis software (Mega 5.0). Figure 1 ), and confirmed that the strain isolated from the soil this time was Acremonium intergeniculate NtSi1.

[0045] The following examples illustrate the effects of the biocontrol bacterial strain solution of the present invention for controlling various plant pathogenic fungi in controlling tobacco root rot.

[0046] Example 1

[0047] Antagonistic assay of Acremonium truncatum NtSi1 on plates

[0048] The root rot pathogen was used as the target to detect the antagonistic activity of Acremonium crossbranchingensis NtSi1. The specific method is: a root rot pathogen cake with a diameter of 6 mm is placed in the center of a PDA plate with a diameter of φ=90 mm, and Acremonium crossbranchingensis NtSi1 is inoculated around the pathogen that has been cultured for 3 days, with a distance of 30 mm; the pathogens include Fusarium oxysporum (Fo), Fusarium fusae (Ff), Fusarium solani (Fs), Alternaria alternata (As) and Curvularia spp. (Cs). Using the control without Acremonium crossbranchingensis NtSi1, the culture was carried out at 28°C, and the degree of antagonism was determined according to the inhibition area, and repeated 3 times. The plate confrontation test in Example 1 verified that Acremonium crossbranchingensis NtSi1 has a confrontation effect on five soil-borne pathogenic fungi, such as Figure 2 As shown in A. This proves that Acremonium crossbredum NtSi1 has a significant antagonistic effect on root rot pathogens.

[0049] Example 2

[0050] Cultivation of root rot pathogen using fermentation filtrate of Acremonium truncatum NtSi1

[0051] The preserved Acremonium truncatum NtSi1 strain was activated. Plates with good growth and no foreign bacteria were selected and clumps of equal size were harvested from appropriate locations under a clean bench. These clumps were inoculated into 250 mL of PDB medium, with 10 clumps of equal size per bottle. The culture was shaken in an incubator at 30°C and 220 rpm for one week. The culture broth was filtered through three layers of gauze, the filtrate was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected. Sterilized PDA medium and the fermentation filtrate were mixed at a volume ratio of 9:1. Under a clean bench, the mycelial side of the pathogen clumps, including Fusarium oxysporum (Fo), Fusarium fusae (Ff), Fusarium solani (Fs), Alternaria alternata (As), and Curvularia spp. (Cs), were inoculated into the center of the PDA medium containing the fermentation filtrate. The control group was cultured in PDA medium without fermentation filtrate at 30°C in the dark for about one week, and the pathogen growth inhibition rate was calculated. Inhibition rate (%) = (control group pathogen colony radius - treatment group pathogen colony radius) / control group pathogen colony radius × 100. The results are shown in Table 1 and Figure 2 As shown in B, the fermentation broth of Acremonium truncatula NtSi1 has a significant inhibitory effect on five soil-borne pathogenic fungi.

[0052] Table 1 Inhibition rate of fermentation filtrate on the growth of pathogenic fungi (%)

[0053]

[0054] Example 3

[0055] Experiment on the control of root rot with the bacterial solution of Acremonium truncatum NtSi1

[0056] 1. Preparation of Pathogenic Fungal Spore Suspension

[0057] Five root rot pathogens, including Fusarium oxysporum (Fo), Fusarium fusae (Ff), Fusarium solani (Fs), Alternaria alternata (As), and Curvularia spp. (Cs), were cultured on PDA plates (200 g potato, 20 g sucrose, 20 g agar, natural pH, 1000 mL distilled water) for 7 days. The conidia were gently scraped off the PDA plates with a pipette tip, rinsed with 10 mL sterile water, centrifuged at 8000 rpm / min for 5 min, and the supernatant was removed. The conidia were resuspended in sterile water, counted using a hemocytometer, and adjusted to a concentration of 5 × 10 conidia with sterile water. 7 The conidia suspensions of five root rot pathogenic fungi were mixed.

[0058] 2. Preparation of biocontrol bacteria solution

[0059] Inoculate the culture medium in PDA (200 g potatoes, 20 g sucrose, natural pH, 1000 mL distilled water) and culture on a shaker at 220 rpm and 30°C for 48–72 hours. Centrifuge the resulting culture at 8000 rpm / min for 5 minutes, remove the supernatant, and resuspend in sterile water to obtain the Acremonium truncatula NtSi1 bacterial suspension.

[0060] 3. Inoculation of mung bean seedlings with pathogenic fungus suspension and biocontrol solution

[0061] Seven days after mung bean seeds emerged, a vermiculite:nutrient soil ratio of 3:1 was added to the seedlings. Four treatments were set up: a. Control (CK) with neither biocontrol agent nor pathogen suspension; b. 20 mL of biocontrol agent (NtSi1); c. 20 mL of pathogen suspension (P) prepared from Fusarium oxysporum (Fo), Fusarium fusae (Ff), Fusarium solani (Fs), Alternaria alternata (As), and Curvularia spp. (Cs), with the same dosage of each pathogen; and d. 20 mL of pathogen suspension plus 20 mL of biocontrol agent (NtSi1+P). Five independent pots were used for each treatment. Disease development was observed 45 days after inoculation, and the disease control efficacy was analyzed.

[0062] 4. Inoculation of tobacco seedlings with pathogenic fungal suspension and biocontrol solution

[0063] Twenty days after tobacco seedling emergence, a vermiculite:nutrient soil ratio of 3:1 was added to the seedlings. Four treatments were set up: a. Control (CK) with neither biocontrol agent nor pathogen suspension; b. 20 mL of biocontrol agent (NtSi1); c. 20 mL of pathogen suspension (P) prepared with Fusarium oxysporum (Fo), Fusarium fusae (Ff), Fusarium solani (Fs), Alternaria alternata (As), and Curvularia spp. (Cs), with the same dosage of each pathogen; and d. 20 mL of pathogen suspension plus 20 mL of biocontrol agent (NtSi1+P). Five independent pots were used for each treatment. Disease development was observed 15, 30, and 45 days after inoculation, and the efficacy of the control was analyzed.

[0064] 5. The results of the potted mung bean control test showed that adding only NtSi1 bacterial solution can promote the growth of mung beans ( Figure 3 The roots of mung beans inoculated with pathogens but not with biocontrol bacteria turned black and rotted ( Figure 3 The efficacy of NtSi1 inoculation was 66.21%, and the incidence rate and disease index also decreased accordingly (Table 2). The phenomena of plant shrinkage, leaf yellowing and root rot were effectively curbed ( Figure 3 ). Take plant samples to determine relevant indicators. Figure 5 The aboveground plant height, aboveground fresh weight, root length, root fresh weight and chlorophyll content showed that the 45-day biocontrol solution (NtSi1) treatment had a significant biocontrol effect.

[0065] 6. The results of the tobacco pot plant control test showed that adding only NtSi1 bacterial solution can promote tobacco growth ( Figure 4 ). Roots inoculated with pathogens but not NtSi1 showed root shedding, blackening, and root rot. From the slice diagram ( Figure 4 (D) shows that the root cells were damaged. The efficacy of NtSi1 inoculation was 63.46%, and the incidence rate and disease index also decreased accordingly (Table 3). The phenomenon of plant shrinkage, leaf yellowing and root rot was effectively curbed ( Figure 4 ). Take plant samples to determine relevant indicators. Figure 6 There were no significant differences among the treatment groups in terms of aboveground plant height, aboveground fresh weight, root length, root fresh weight and chlorophyll at 15 days. The NtSi1 bacterial solution treatment had a significant biocontrol effect at 45 days.

[0066] The incidence rate, disease index and biocontrol effect were calculated according to the following formula:

[0067] Incidence rate (%) = number of diseased plants / total number of plants × 100

[0068] Disease index (%) = ∑ (disease level × number of diseased plants at that level) / (highest disease level × total number of plants) × 100.

[0069] Biocontrol effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0070] The tobacco disease classification standards are as follows:

[0071] Level 0: The affected area is less than or equal to 1%;

[0072] Level 1: affected area 1%-25%;

[0073] Level 2: affected area 26%-50%;

[0074] Level 3: affected area 51%-75%;

[0075] Level 4: affected area 76%-89%;

[0076] Level 5: The affected area is greater than or equal to 90%.

[0077] Table 2 Biological control effects of biocontrol bacteria on mung bean root rot

[0078]

[0079] (p<0.05)

[0080] Table 3 Biological control effects of biocontrol bacteria on tobacco root rot

[0081]

[0082] (p<0.05)

[0083] The biocontrol effect of the biocontrol bacterium NtSi1 includes inducing the production of various defense enzymes in plants, including antioxidant enzymes such as peroxidase (POD), polyphenol oxidase (POO), and catalase (CAT). A preliminary study was conducted on the induction of POD, PPO, and CAT enzyme activities by NtSi1 treatment. NtSi1 can significantly increase the activities of POD, PPO, and CAT enzymes ( Figure 7 ). This indicates that the biocontrol fungus NtSi1 improves tobacco disease resistance by inducing the activation of tobacco antioxidant enzymes.

Claims

1. Acremonium spp. Sarocladium implicatum ) Application of NtSi1 in preparing a root rot control agent for promoting the growth of mung beans, characterized in that: The Acremonium crossbranchii NtSi1 was deposited in the General Microbiology Center of China Culture Collection Administration on January 20, 2025, with a deposit number of CGMCC NO.41760; the method of promoting the growth of mung beans is to promote the growth of mung beans after infection with root rot.

2. The use according to claim 1, characterized in that The method for promoting the growth of mung beans includes increasing the above-ground plant height, above-ground fresh weight, root length, root fresh weight and chlorophyll content of plants infected with root rot; the root rot pathogen is Fusarium oxysporum, Fusarium solani, Fusarium fusae, Alternaria alternata or Curvularia spp.

3. The use according to claim 1, characterized in that The application comprises: inoculating the Acremonium truncatula NtSi1 described in claim 1 into a PDA liquid culture medium, culturing on a shaking table, removing the supernatant of the cultured bacterial solution, and resuspending the culture solution with sterile water to obtain a root rot prevention and control bacterial agent.

4. The use according to claim 3, characterized in that The culture temperature of the Acremonium truncatum NtSi1 is 28-32° C., and the culture time is 2-7 days.

5. Acremonium crossbranchii Sarocladium implicatum ) Application of NtSi1 in increasing the content of defense enzymes after tobacco is infected with root rot pathogens, characterized in that: The Acremonium crossbranchingensis NtSi1 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on January 20, 2025, with a deposit number of CGMCC NO.41760; the root rot pathogen is Fusarium oxysporum, Fusarium solani, Fusarium fusae, Alternaria alternata or Curvularia spp.; the defense enzymes are peroxidase, polyphenol oxidase, and catalase.

6. A method for preventing and controlling plant diseases, characterized in that: Acremonium crossbranching NtSi1 is applied to the plant root growth environment, and the plant disease is root rot; the Acremonium crossbranching NtSi1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 20, 2025, with a preservation number of CGMCC NO.41760; the root rot pathogen is Fusarium oxysporum, Fusarium solani, Fusarium fusae, Alternaria alternata or Curvularia spp.; the plant is mung bean or tobacco.

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