Application of Mortierella isabellina 2K4 in biocontrol of poplar anthracnose

Abstract

The application discloses an application of Mortierella isabellina 2K4 in a poplar anthracnose biocontrol bacterium and belongs to the technical field of microbial pesticides. The plate confrontation experiment proves that the strain has a significant antagonistic effect on the poplar anthracnose pathogen Colletotrichum gloeosporioides, and the inhibition rate reaches 50.9%. The fermentation filtrate also shows a concentration-dependent antibacterial effect, and the inhibition rate of 50% concentration is 45.50%. The ex vitro leaf and pot experiments further prove that the application of the strain can effectively reduce the lesion diameter and disease index of the poplar anthracnose, and the control effect is more than 59.45%. The application provides an efficient, environment-friendly biocontrol bacterium resource and an application method for the green prevention and control of the poplar anthracnose.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticide technology, specifically relating to the application of *Morchella 2K4* as a biocontrol agent for poplar anthracnose. Background Technology

[0002] Poplar trees grow quickly, are highly resilient, and easily reproduce asexually. They play a positive role in forestry production, urban greening, and ecological protection, making them an important economic and landscaping tree species. Currently, my country has 8.5 million hectares of planted poplar forests. 2 Poplar cultivation ranks first in the world, generating significant economic, ecological, and social benefits. However, the continuous increase in poplar planting area, the homogenization of poplar varieties, and inadequate management practices have led to a decline in poplar's resilience. Combined with the biological characteristics of diseases themselves, this has resulted in frequent outbreaks of poplar diseases and severe losses. There are over 300 types of poplar diseases, with anthracnose being one of the most serious, severely hindering the sustainable development of the poplar industry.

[0003] Poplar anthracnose is primarily caused by *Colletotrichum gloeosporioides*, and is widespread globally. When infected, poplar leaves and branches turn black and rot, and in severe cases, the entire tree dies, causing significant forestry economic losses and ecological threats. However, traditional chemical and agricultural control methods have risks such as long control cycles, high costs, increased resistance, reduced soil quality, and damage to natural ecosystems. Numerous studies have shown that biocontrol agents (fungi and bacteria) can control plant diseases by competing with pathogens for nutrients and space, secreting antimicrobial substances, producing secondary metabolites, and inducing systemic resistance in plants. These methods are environmentally friendly and sustainable, making the exploration of effective biological control methods for poplar diseases crucial. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, the technical problem to be solved by this application is to provide the application of *Morchella 2K4* as a biocontrol fungus for poplar anthracnose.

[0005] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0006] A method for controlling anthracnose in poplar trees involves applying *Morchella 2K4*, preserved under accession number CGMCC No. 3.26002, and / or its fermentation broth, and / or its metabolites, to poplar trees or their growing environment.

[0007] In some embodiments, the metabolite of *Morchella 2K4* is a sterile fermentation filtrate obtained by centrifuging and filtering the fermentation broth after PDB liquid fermentation.

[0008] In some embodiments, the volume concentration of the sterile fermentation filtrate in the application medium is 10% to 50%.

[0009] In some embodiments, the application method includes spraying, root irrigation, or wound application.

[0010] Application of *Morchella 2K4* with accession number CGMCC No. 3.26002 in the preparation of microbial agents for the control of anthracnose in poplar trees.

[0011] In some embodiments, the microbial agent comprises at least one of the live cells of *Morchella 2K4*, its fermentation broth, or its metabolites.

[0012] A microbial agent for controlling anthracnose in poplar trees, the active ingredients of which include *Morchella 2K4* with preservation number CGMCCNo.3.26002, and / or its fermentation broth, and / or its metabolites.

[0013] In some embodiments, the microbial agent is a liquid microbial agent, a solid microbial agent, or a wettable powder.

[0014] Compared with the prior art, the beneficial effects of this application are as follows:

[0015] This invention marks the first discovery that *Morchella 2K4* strain and its fermentation products exhibit significant and multi-layered biocontrol effects against poplar anthracnose. Plate confrontation experiments directly demonstrated that this strain possesses a strong antagonistic effect against the pathogen *Colletotrichum gloeosporioides*, with an inhibition rate exceeding 50%. More importantly, its sterile fermentation filtrate also showed clear concentration-dependent antibacterial activity in vitro, achieving an inhibition rate of 45.50% at a 50% concentration, clearly indicating that its metabolites are rich in antibacterial active substances. In vitro leaf and pot experiments further verified its actual disease control ability; treatment with this strain effectively inhibited lesion expansion, significantly reduced the disease index of poplar anthracnose, and achieved a final control effect of up to 59.45%, fully demonstrating its high efficiency and reliability in near-realistic habitat conditions. In conclusion, this invention utilizes *Morchella 2K4* to provide a biocontrol resource with a clear mechanism and excellent efficacy for the green control of poplar anthracnose, showing promising application prospects. Attached Figure Description

[0016] Figure 1 The images show the plate antagonistic effects of *Morchella* strains against plant pathogens *Aspergillus fumigatus*, *Colletotrichum gloeosporioides*, and *Aspergillus echinosporioides*. The bottom image shows the growth phenotypes of the pathogens in normal culture on PDA plates and in double culture with *Morchella* in 2K4. G1 (*Colletotrichum gloeosporioides*), G2 (*Aspergillus fumigatus*), and G17 (*Aspergillus echinosporioides*) represent the growth morphology of the pathogens cultured alone (control). 2K4 / G1, 2K4 / G2, and 2K4 / G17 represent the results of confrontation culture between *Morchella* and the pathogens. The top image shows the antagonistic rate of *Morchella* against the plant pathogens.

[0017] Figure 2 The graph shows the detection results of cellulase (a), protease (b), β-1-3-glucanase (c), chitinase (d) and siderophore (e) in *Morchella 2K4*.

[0018] Figure 3 The results show the inhibitory effect of different proportions of fermentation filtrate of *Morchella 2K4* (10%, 30%, and 50%) on the growth of *Colletotrichum gloeosporioides*, the pathogen of poplar anthracnose. The left figure shows the growth morphology of the pathogen on ordinary PDA and in PDA medium mixed with 10%, 30%, and 50% fermentation filtrate. The right figure is a statistical graph of the growth diameter of the pathogen and the antagonistic rate of *Morchella gloeosporioides* against the pathogen.

[0019] Figure 4 To illustrate the antagonistic effect of *Morchella 2K4* on detached poplar leaves, the left figure shows the growth of leaves inoculated with *Morchella* and the pathogen, with a representing the control group inoculated with sterile water, where a1, a2, and a3 are leaf images at 1, 3, and 5 days after inoculation with sterile water, respectively; b1, b2, and b3 are leaf images at 1, 3, and 5 days after inoculation with *Morchella 2K4*; c1, c2, and c3 are leaf images at 1, 3, and 5 days after inoculation with *Colletotrichum gloeosporioides*; d1, d2, and d3 are leaf images of leaves simultaneously inoculated with *Morchella 2K4* and *Colletotrichum gloeosporioides* G1; the right figure is a statistical chart showing the control efficiency of *Morchella 2K4* against poplar anthracnose.

[0020] Figure 5 Scanning electron micrographs showing the antagonistic effect of the growth of poplar fine roots after inoculation with *Morchella esculenta* 2K4 on detached poplar fine roots; where CK represents the microscopic morphology of fine roots inoculated with sterile water, G1 represents the microscopic growth morphology of the pathogen cultured alone, 2K4 represents the microscopic morphology of fine roots inoculated with *Morchella esculenta* alone, and 2K4 / G1 represents the microscopic morphology of fine roots co-inoculated with *Morchella esculenta* and the pathogen.

[0021] Figure 6 The image shows the effect of *Morchella 2K4* in controlling anthracnose in a pot experiment; where a is the inoculation with sterile water (CK), b is the inoculation with *Morchella 2K4* and c is the inoculation with *Colletotrichum gloeosporioides* G1, and d is the co-inoculation with *Morchella 2K4* and *Colletotrichum gloeosporioides* G1. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0023] The culture medium preparation method used in the following examples is as follows:

[0024] Potato glucose broth (PDA): 20g potato extract powder, 20g glucose, 15g agar, distilled water to a final volume of 1L, sterilize at 121℃ for 30min.

[0025] Potato glucose liquid culture medium (PDB): 20g potato extract powder, 20g glucose, distilled water to a final volume of 1L, sterilize at 121℃ for 30min.

[0026] Sodium carboxymethyl cellulose medium: 10g peptone, 1g KH2PO4, 5g NaCl, 10g yeast extract, 10g sodium carboxymethyl cellulose, deionized water to a final volume of 1L, pH=7, sterilize at 121℃ for 30min.

[0027] Colloidal chitin medium: 0.2g magnesium sulfate heptahydrate, 1g diammonium hydrogen phosphate, 0.2g potassium chloride, 10g colloidal chitin, 18g agar, and deionized water to a final volume of 1L. pH=7. Sterilize at 121℃ for 30min.

[0028] Skim milk solid culture medium: 10g skim milk, 0.5g magnesium sulfate heptahydrate, 1g dipotassium hydrogen phosphate, 5g potassium chloride, 0.1g ferrous sulfate heptahydrate, 20g agar, 1000ml deionized water, pH=7, sterilize at 115℃ for 30min.

[0029] β-1-3 glucanase detection medium: β-1-3 glucan 5g, ammonium sulfate 3.5g, dipotassium hydrogen phosphate 1g, potassium sulfate 0.5g, magnesium sulfate heptahydrate 0.5g, agar 20g, distilled water 1000ml, sterilized at 115℃ for 30min.

[0030] CAS detection medium: CAS 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, 1 mmol / L FeCl3·6H2O (prepared with 10 mmol / L HCl) 10 mL, 0.1 mol / L phosphate buffer 50 mL, agar 9.0 g, deionized water 940 mL, pH=7, sterilized at 115℃ for 30 min.

[0031] The *Mortierella* 2K4 in the following examples was isolated from the rhizosphere soil of poplar trees and classified as *Mortierellasp.* 2K4. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo. 3.26002, deposit date: December 13, 2022, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. *Colletotrichum gloeosporioides* G1, *Aspergillus fumigatus* G2, and *Aspergillus aculeatus* G17 were all plant pathogens previously obtained by our research group.

[0032] Example 1

[0033] Determination of the antifungal spectrum of *Morchella 2K4* fungi

[0034] The plate confrontation method was used, inoculating *Aspergillus 2K4* and pathogens *Colletotrichum gloeosporioides*, *Aspergillus fumigatus*, and *Aspergillus echinosporum* onto PDA plates and incubating them at 28°C for 7 days. The specific steps of the plate confrontation method were as follows: Under aseptic conditions, 5mm diameter holes were punched in plates contaminated with *Aspergillus 2K4* and pathogens, respectively, to obtain samples. The inoculum was carefully transferred to fresh PDA plates using forceps, with each plate 2cm from the edge of the plate. The control was a plate inoculated solely with pathogens in the center. The plates were then incubated in the dark and upside down for 7 days. Each confrontation experiment was performed in triplicate. The colony radius of the pathogens in the control group and the radius of the pathogens in the confrontation culture were measured, and the inhibition rate was calculated. Inhibition rate (%) = (Coronary radius of control group - Colony radius of pathogen group) / Colony radius of control group × 100%.

[0035] The antifungal spectrum and inhibition rate of the fungus *Morchella 2K4* are as follows: Figure 1 As shown, *Morchella* exhibited significant inhibitory effects against all three pathogens, with an inhibition rate of 50.9% against *Colletotrichum gloeosporioides*, 50.63% against *Aspergillus fumigatus*, and 51.23% against *Aspergillus echinosporium*.

[0036] Example 2

[0037] Detection of physiological and biochemical characteristics of strain Mucor 2K4

[0038] To evaluate the antagonistic ability of *Morchella 2K4*, this application conducted cellulase, protease, β-1-3-glucanase, chitinase, and siderophore detection on *Morchella 2K4*.

[0039] Determination of cellulase production capacity of the strain: Inoculate *Epipremnum aureum* 2K4 into PDA medium and culture for 72 h. Then, use a punch to take 5 mm diameter mycelial cakes and inoculate them onto sodium carboxymethyl cellulose medium. After placing them in an incubator at 28 ℃ for 3 days, pour an appropriate amount of Congo red solution (1 mg / mL) onto the plate and let it stand for 15 min to stain. Then, wash three times with sodium chloride solution (1 mol / L) and observe whether a yellow halo appears around the colony. If so, it indicates that the strain can produce cellulase.

[0040] Determination of the strain's ability to produce protease: The strain was inoculated into PDA medium and cultured for 72 hours. Then, a 5 mm diameter mycelial cake was taken with a punch and inoculated into skim milk solid medium and cultured for 3 days. The presence of a clear zone was observed. The presence of a clear zone indicated that the strain had the ability to produce protease.

[0041] Determination of the ability of the strain to produce β-1-3 glucanase: The fungus was inoculated into PDA medium and cultured for 72 h. Then, 5 mm diameter mycelial cakes were taken with a punch and inoculated into β-1-3 glucanase detection medium and cultured for 3 days. An appropriate amount of Congo red solution (1 mg / mL) was poured into the plate and allowed to stand for staining for 15 min. After that, the plate was rinsed several times with sodium chloride solution (1 mol / L). The presence of a white halo around the colony was observed. If a white halo appeared, it indicated that the strain had the ability to produce β-1-3 glucanase.

[0042] Chitinase production capacity assay: The strain was inoculated on a colloidal chitin medium plate and cultured at 28°C for 72 h. Samples were taken by punching holes with a 5 mm diameter punch. The strain was carefully transferred to a chitin test medium with tweezers and cultured for 3 days. The presence of a clear zone indicates that the strain has the ability to produce chitinase.

[0043] Test for the ability of the strain to produce siderophores: The purified strain was inoculated in the center of the CAS test medium and incubated upside down at 30°C for 3 to 5 days. If an orange-yellow halo was formed around the colony, it was determined that the strain had the ability to produce siderophores.

[0044] The test results are shown in Table 1, indicating that *Morchella 2K4* possesses the ability to produce cellulase, protease, β-1-3-glucanase, chitinase, and siderophores. Figure 2 The image shows the growth morphology of *Morchella 2K4* in the test medium.

[0045] Table 1. Physiological and biochemical test results of strain 2K4

[0046]

[0047] Note: + indicates positive, - indicates negative.

[0048] The ability of strain 2K4 to produce multiple functional factors such as cellulase, β-1-3-glucanase, chitinase, protease, and siderophores indicates that it has the potential to act as a biocontrol bacterium with multiple antagonistic mechanisms, such as degrading the cell wall of pathogens (chitinase, glucanase) and competing for iron nutrition (siderophores).

[0049] Example 3

[0050] Inhibitory effect of *Morchella 2K4* fermentation filtrate on *Colletotrichum gloeosporioides*, the pathogen of poplar anthracnose.

[0051] Fermentation filtrate preparation method: Select strain 2K4 and incubate it in 1 mL of PDB liquid medium at 30℃ and 180 rpm for 24 h as seed culture. Take 1% of the seed culture and incubate it in new PDB medium for 24 h to obtain the fermentation broth. Centrifuge the fermentation broth at 10000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter to remove bacteria. The filtrate is the fermentation filtrate.

[0052] The pathogenic bacterium *Colletotrichum gloeosporioides* was inoculated into PDA medium and cultured for 72 hours. Mycelial cakes were then collected using a punch and inoculated into fermentation filtrate containing 10%, 30%, and 50% of the bacteria, respectively. After 5 days of cultivation, the growth of the strain was observed, and the diameter of the growth zone was recorded. Results are as follows: Figure 3 The colony diameters of pathogen G1 grown on PDA plates with 0%, 10%, 30%, and 50% fermentation filtrate were 8.95cm±0.02cm, 6.97cm±0.03cm, 5.92±0.03cm, and 4.88cm±0.08cm, respectively. The inhibition rate of 10% fermentation filtrate against *Colletotrichum gloeosporioides* was 22.19%, that of 30% fermentation filtrate was 33.83%, and that of 50% fermentation filtrate was 45.50%. The inhibition rate increased significantly with increasing fermentation filtrate concentration.

[0053] Example 4

[0054] Control effects of Motharella 2K4 on detached poplar leaves and fine roots

[0055] Healthy poplar leaves of similar size and growth were collected, rinsed with sterile water, soaked in 75% ethanol for 15 seconds, then soaked in 1% sodium hypochlorite solution for 2 minutes, and rinsed three times with sterile water. The leaves were then air-dried on sterile filter paper and inoculated with *Morchella esculenta* 2K4 and the pathogen *Colletotrichum gloeosporioides* G1 in PDA medium and incubated at 30°C for 5 days. Four treatments were administered, with three leaves per group. The four treatments included: inoculation with sterile water alone, *Morchella esculenta* 2K4, *Colletotrichum gloeosporioides* G1, and simultaneous inoculation with *Morchella esculenta* 2K4 and *Colletotrichum gloeosporioides* G1 (2K4 / G1). Inoculation was performed by cross-scratching the detached leaves with an inoculation loop coated with mycelium. Leaf condition was observed on days 1, 3, and 5 after inoculation, and the leaf disease index and incidence rate were recorded. The control effect of *Morchella esculenta* inoculation on detached diseased poplar leaves was calculated.

[0056] Control efficacy % = (average lesion diameter of blank control - average lesion diameter of treatment) / average lesion diameter of blank control × 100%.

[0057] The results are as follows Figure 4 As shown, treatment of detached poplar leaves with strain 2K4 significantly reduced the diameter of poplar anthracnose lesions and the disease index, with a treatment effect of over 53.33% on poplar anthracnose.

[0058] The treatment of poplar fine roots was the same as that for poplar leaves. However, because the fine root tissue is small and difficult to observe with the naked eye, a scanning electron microscope was used to observe the morphology of the root tissue 15 days after infection. The results are as follows: Figure 5 As shown, after applying *Morchella esculenta* 2K4 and the pathogen *Colletotrichum gloeosporioides* G1, the pathogen successfully colonized the surface of fine roots. The surface tissue of fine roots treated with 2K4 showed little change compared to the control group. In the fine roots treated with G1, the epidermal cells completely lost their normal plump shape, and the surface showed an eroded appearance with a large number of holes and torn structures, accompanied by obvious tissue degradation and fragmentation. The fine root tissue treated with both *Morchella esculenta* and *Colletotrichum gloeosporioides* suffered less damage.

[0059] Example 5

[0060] Biological control effect of *Morchella esculenta* on poplar anthracnose in a pot experiment.

[0061] Mucor 2K4 potted plant disease resistance experiment:

[0062] Poplar seedlings were propagated using cuttings. After 60 days of cultivation, the poplars had grown approximately 15 leaves. At this stage, potted poplars of similar size and growth were selected. Using a syringe, identical cross-shaped wounds were made on the leaves. One 5mm mycelial block was inoculated onto each leaf, with approximately 8 leaves inoculated per poplar tree. The trees were divided into four groups, with five replicates per group. The four group protocols were as follows: CK (inoculation with sterile water); 2K4 (inoculation with only *Morchella esculenta* 2K4); G1 (inoculation with only *Colletotrichum gloeosporioides*); and 2K4 / G1 (co-inoculation with *Morchella esculenta* 2K4 and *Colletotrichum gloeosporioides* G1). Disease index and morbidity were recorded 15 days after inoculation.

[0063] Poplar anthracnose is classified into 6 disease grades based on the ratio of lesion area on poplar leaves to the total leaf area. The grading criteria are as follows:

[0064] Grade 0: No lesions appear;

[0065] Grade 1: Lesion area ≤ 5%;

[0066] Grade 2: Lesions cover an area of ​​5%-25%;

[0067] Grade 3: Lesions cover an area of ​​25%-45%;

[0068] Grade 4: Lesions cover an area of ​​45%-65%;

[0069] Grade 5: Lesion area > 65%.

[0070] Incidence rate (%) = Number of diseased plants / Total number of treated plants × 100%;

[0071] Disease index = ∑(number of diseased plants at each level × corresponding disease level) / (total number of plants surveyed × highest disease level) × 100;

[0072] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0073] The results are shown in Table 2 and Figure 6 As shown, treatment of poplar leaves with strain 2K4 significantly reduced the incidence and disease index, achieving a control efficacy of 59.45% against poplar anthracnose. These results indicate that *Morchella esculenta* 2K4 possesses excellent disease control capabilities against poplar anthracnose and is a highly effective microbial agent.

[0074] Method for preparing mycelial blocks of *Morchella esculenta* and *Colletotrichum gloeosporioides*: Inoculate *Morchella esculenta* 2K4 and *Colletotrichum gloeosporioides* G1 into PDA medium and culture for 72 hours. Then, use a punch to take mycelial blocks with a diameter of 5 mm.

[0075] Table 2: Biocontrol effect of applying Mothorax 2K4 inoculant against poplar anthracnose

[0076]

[0077] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for controlling anthracnose in poplar trees, characterized in that, The *Morchella* species with accession number CGMCC No. 3.26002 was... Mortierella sp 2K4, and / or its fermentation broth, and / or its metabolites are applied to poplar trees or their growing environment. The metabolites of *Morchella 2K4* are the sterile fermentation filtrate obtained after centrifugation and filtration of the fermentation broth obtained through PDB liquid fermentation. The pathogen of poplar anthracnose is *Colletotrichum gloeosporioides*. Colletotrichum gloeosporioides ).

2. The method according to claim 1, characterized in that, The volume concentration of the sterile fermentation filtrate in the application medium is 10% to 50%.

3. The method according to claim 2, characterized in that, The application methods include spraying, root irrigation, or applying to wounds.

4. The application of *Morchella 2K4* with accession number CGMCC No. 3.26002 in the preparation of a microbial agent for controlling poplar anthracnose, wherein the pathogen of poplar anthracnose is *Colletotrichum gloeosporioides*, and the microbial agent contains at least one of live cells of *Morchella 2K4*, its fermentation broth, or its metabolites, wherein the metabolites of *Morchella 2K4* are sterile fermentation filtrate obtained by centrifugation and filtration of the fermentation broth obtained after PDB liquid fermentation.

5. A microbial inoculant for controlling anthracnose in poplar trees, characterized in that, Its active ingredients include *Morchella 2K4* with preservation number CGMCC No. 3.26002, and / or its fermentation broth, and / or its metabolites. The metabolites of *Morchella 2K4* are sterile fermentation filtrate obtained by centrifuging and filtering the fermentation broth after PDB liquid fermentation. The pathogen of poplar anthracnose is *Colletotrichum gloeosporioides*.

6. The microbial agent according to claim 5, characterized in that, The microbial agent can be a liquid microbial agent, a solid microbial agent, or a wettable powder.

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