Application of mortierella 2K4 as biocontrol bacterium for poplar anthracnose

Abstract

The invention discloses application of mortierella 2K4 as a biocontrol bacterium for poplar anthracnose, and belongs to the technical field of microbial pesticides. A plate confrontation experiment proves that the strain has a remarkable antagonistic effect on poplar anthracnose pathogenic bacteria colletotrichum gloeosporioides, and the bacteriostasis rate reaches 50.9%. The fermentation filtrate also shows a concentration-dependent antibacterial effect, and the antibacterial rate of 50% concentration is 45.50%. In-vitro leaf and pot experiments further prove that the scab diameter and disease index of the poplar anthracnose can be effectively reduced by applying the strain, and the prevention and treatment effect exceeds 59.45%. The invention provides an efficient and environment-friendly biocontrol bacterium resource for green prevention and control of poplar anthracnose and an application method of the biocontrol bacterium resource.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial pesticides, and particularly relates to the application of Mortierella 2K4 as a poplar anthracnose biocontrol fungus. BACKGROUND

[0002] Poplar grows fast, has strong stress resistance, and is easy to reproduce asexually, and thus plays a positive role in forestry production, urban greening and ecological protection, and is an important economic tree species and greening tree species. At present, the area of poplar plantations in China reaches 8.5 million hm 2 , ranks first in the world, and creates high economic, ecological and social benefits. However, due to the continuous increase of poplar planting area, the single variety of poplar, and the imperfect management measures, the stress resistance of poplar declines, combined with the biological characteristics of the disease itself, which leads to frequent occurrence of poplar diseases and serious losses. There are more than 300 kinds of poplar diseases, and anthracnose is one of the most serious poplar diseases, which seriously restricts the sustainable development of the poplar industry.

[0003] Poplar anthracnose is mainly caused by Colletotrichum gloeosporioides, and occurs widely in the world. When the disease occurs, the poplar leaves and branches will become black and rotten, and even the whole poplar will die, which causes serious forestry economic losses and ecological threats. However, the traditional chemical and agricultural control methods have the risk of long control period, high cost, increase of drug resistance, reduction of soil quality and destruction of natural ecological system. Numerous studies have shown that biocontrol fungi (fungi and bacteria) can prevent and control plant diseases through methods such as competition for nutrients and space with pathogenic fungi, secretion of antibacterial substances, production of secondary metabolites, and induction of plant systemic resistance, and thus have the characteristics of environmental friendliness and green sustainability. Therefore, it is very important to explore effective biological control methods for poplar diseases. SUMMARY

[0004] To solve the above problems in the prior art, the technical problem to be solved by the present application is to provide the application of Mortierella 2K4 as a poplar anthracnose biocontrol fungus.

[0005] To solve the above technical problems, the technical scheme of the present application is as follows:

[0006] A method for preventing and treating poplar anthracnose, Mortierella 2K4 with a preservation number of CGMCC No.3.26002, and / or a fermentation liquor thereof, and / or a metabolite thereof are applied to poplar or an environment in which poplar grows.

[0007] In some embodiments, the metabolite of Mortierella 2K4 is a sterile fermentation filtrate obtained by centrifugation and filtration sterilization of the fermentation liquor obtained after PDB liquid fermentation.

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

[0009] In some embodiments, the administration mode comprises spraying, root drenching or wound painting.

[0010] Use of Mortierella 2K4 with a preservation number of CGMCC No. 3.26002 in the preparation of a microbial agent for preventing and treating poplar anthracnose.

[0011] In some embodiments, the microbial agent comprises at least one of the live Mortierella 2K4, fermentation broth thereof or metabolite thereof.

[0012] A microbial agent for preventing and treating poplar anthracnose, wherein the active ingredient comprises Mortierella 2K4 with a preservation number of CGMCC No. 3.26002, and / or fermentation broth thereof, and / or metabolite thereof.

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

[0014] Compared with the prior art, the present application has the following beneficial effects:

[0015] The present application first discovers that the Mortierella 2K4 strain and its fermentation products exhibit significant and multi-level biological prevention and treatment effects on poplar anthracnose. The plate confrontation test directly proves that the strain has strong antagonistic effect on the pathogenic fungus Colletotrichum gloeosporioides, and the inhibition rate is more than 50%. More importantly, the sterile fermentation filtrate also exhibits clear concentration-dependent antibacterial activity in vitro, and the inhibition rate is 45.50% at a concentration of 50%, which clearly indicates that the metabolites are rich in antibacterial active substances. The in vitro leaf and pot experiments further verify the actual disease prevention and treatment ability of the strain, and the strain treatment can effectively inhibit the expansion of the lesion, significantly reduce the disease index of poplar anthracnose, and the final prevention and treatment effect is as high as 59.45%, fully embodying the high efficiency and reliability of the strain in the near real habitat. In summary, the present application provides a green prevention and control resource for poplar anthracnose using Mortierella 2K4, which has a clear mechanism and excellent effect, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a plate confrontation effect diagram of the Mortierella strain on the plant pathogenic fungi Aspergillus fumigatus, Colletotrichum gloeosporioides and Aspergillus aculeatus, and the lower figure is the growth phenotype of the pathogenic fungi cultured on PDA plates and double-cultured with Mortierella 2K4, wherein G1 (Colletotrichum gloeosporioides), G2 (Aspergillus fumigatus) and G17 (Aspergillus aculeatus) are the growth morphology of the pathogenic fungi cultured alone (control), and 2K4 / G1, 2K4 / G2 and 2K4 / G17 are the confrontation culture results of Mortierella and the pathogenic fungi; the upper figure is the antagonistic rate of Mortierella on the plant pathogenic fungi;

[0017] Figure 2 Figure 2K4 of Mortiella is 2K4 cellulase (a), protease (b), beta-1-3 glucanase (c), chitinase (d) and siderophore (e) detection results chart;

[0018] Figure 3 Figure 2K4 of Mortiella is 2K4 different proportion of fermentation filtrate (10%, 30% and 50%) on the growth of the pathogen of poplar anthracnose Gomphus anthracnose inhibition results; The left graph is the growth of the pathogen on the ordinary PDA and the growth morphology in the 10%, 30% and 50% fermentation filtrate mixed PDA medium, and the right graph is the pathogen growth diameter and the antagonistic rate of Mortiella to the pathogen;

[0019] Figure 4 Figure 2K4 of Mortiella is the antagonistic effect on poplar in vitro leaves, the left graph is the growth condition of leaves inoculated with Mortiella and pathogen, a is the control group inoculated with sterile water, a1, a2, a3 are the leaf pictures of 1, 3, 5 days after inoculation with sterile water respectively; b1, b2, b3 are the leaf pictures of 1, 3, 5 days after inoculation with Mortiella 2K4; c1, c2, c3 are the leaf pictures of 1, 3, 5 days after inoculation with Gomphus anthracnose; d1, d2, d3 are the leaf pictures of 1, 3, 5 days after inoculation with Mortiella 2K4 and Gomphus anthracnose G1; The right graph is the statistical graph of the prevention and control efficiency of Mortiella on poplar anthracnose;

[0020] Figure 5 Figure 2K4 of Mortiella is the growth condition of poplar fine roots after inoculation with Mortiella 2K4, which is the scanning electron microscope graph of the antagonistic effect on poplar in vitro fine roots; CK is the fine root microstructure inoculated with sterile water on the fine root, G1 is the microstructure of the pathogen cultured alone, 2K4 is the fine root microstructure inoculated with Mortiella alone, and 2K4 / G1 is the microstructure of Mortiella and pathogen inoculated together;

[0021] Figure 6 Figure 2K4 of Mortiella is the effect graph of preventing and treating anthracnose in pot experiment; a is inoculated with sterile water (CK), b is inoculated with Mortiella 2K4, c is inoculated with Gomphus anthracnose G1, and d is inoculated with Mortiella 2K4 and Gomphus anthracnose G1. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described below in combination with specific examples. In the following examples, if not specified in detail, the technical means used are all conventional means familiar to those skilled in the art. Or according to the kit and product instructions. The materials, reagents and the like used in the following examples, if not specially specified, can be obtained from commercial channels.

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

[0024] Potato dextrose agar (PDA): Potato infusion powder 20 g, glucose 20 g, agar 15 g, distilled water to 1 L, sterilized at 121 °C for 30 min.

[0025] Potato dextrose broth (PDB): Potato infusion powder 20 g, glucose 20 g, distilled water to 1 L, sterilized at 121 °C for 30 min.

[0026] Sodium carboxymethyl cellulose medium: Proteose peptone 10 g, KH2PO4 1 g, NaCl 5 g, yeast powder 10 g, sodium carboxymethyl cellulose 10 g, deionized water to 1 L, pH = 7, sterilized at 121 °C for 30 min.

[0027] Colloidal chitin medium: Magnesium sulfate heptahydrate 0.2 g, diammonium phosphate 1 g, potassium chloride 0.2 g, colloidal chitin 10 g, agar 18 g, deionized water to 1 L, pH = 7, sterilized at 121 °C for 30 min.

[0028] Skim milk solid medium: Skim milk 10 g, magnesium sulfate heptahydrate 0.5 g, potassium phosphate dibasic 1 g, potassium chloride 5 g, ferrous sulfate heptahydrate 0.1 g, agar 20 g, deionized water 1000 ml, pH = 7, sterilized at 115 °C for 30 min.

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

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

[0031] Mortierella sp 2K4 in the following examples was isolated from the rhizosphere soil of poplar, and was classified and named as Mortierella sp 2K4. It has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 3.26002, the preservation date is December 13, 2022, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing. Colletotrichum gloeosporioides G1, Aspergillus fumigatus G2, and Aspergillus aculeatus G17 are plant pathogenic fungi obtained by the research group in the early stage.

[0032] Example 1

[0033] Determination of the fungal inhibition spectrum of Mortierella sp 2K4

[0034] Plate confrontation method was used to inoculate Mortierella sp 2K4 and pathogenic fungi Colletotrichum gloeosporioides, Aspergillus fumigatus, and Aspergillus aculeatus on PDA plates, and cultured at 28°C for 7d. The specific steps of plate confrontation method are as follows: under sterile conditions, punch samples with a 5mm puncher on the plates covered with Mortierella sp and pathogenic fungi, and carefully transfer the strains into fresh PDA plates with tweezers, and the distance between the two is 2cm from the edge of the culture dish. The control is the plate inoculated with pathogenic fungi alone in the center, and then it is placed in a 28°C constant temperature incubator for dark inverted culture for 7d. Each confrontation test has 3 replicates. The colony radius of the pathogenic fungus control group and the radius of the pathogenic fungus in the confrontation culture are measured, and the inhibition rate is calculated. Inhibition rate (%) = (colony radius of control group - colony radius of pathogenic fungus group) / colony radius of control group x 100%.

[0035] The fungal inhibition spectrum and inhibition rate of Mortierella sp 2K4 are shown in Table 1. Figure 1 As shown in Table 1, Mortierella sp has significant inhibition effect on the three pathogenic fungi, and the inhibition rate on Colletotrichum gloeosporioides is 50.9%, the inhibition rate on Aspergillus fumigatus is 50.63%, and the inhibition rate on Aspergillus aculeatus is 51.23%.

[0036] Example 2

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

[0038] In order to evaluate the antagonistic ability of Mortierella sp 2K4, the present application detects cellulase, protease, β-1-3 glucanase, chitinase, and siderophore of Mortierella sp.

[0039] Strain cellulase production ability determination: Mortierella 2K4 was inoculated into PDA medium and cultured for 72 h, then a 5 mm diameter fungus cake was taken with a puncher and inoculated into sodium carboxymethyl cellulose medium, placed in a 28°C incubator for 3 d, then a proper amount of congo red solution (1 mg / mL) was poured onto the plate and left to dye for 15 min, then washed with sodium chloride solution (1 mol / L) for 3 times, and whether a yellow halo appeared around the colony was observed, if yes, it indicated that the strain could produce cellulase.

[0040] Strain protease production ability determination: Mortierella was inoculated into PDA medium and cultured for 72 h, then a 5 mm diameter fungus cake was taken with a puncher and inoculated into skim milk solid medium and cultured for 3 d, and whether a transparent circle appeared was observed, if yes, it indicated that the strain had protease production ability.

[0041] Strain β-1-3 glucanase production ability determination: Mortierella was inoculated into PDA medium and cultured for 72 h, then a 5 mm diameter fungus cake was taken with a puncher and inoculated into β-1-3 glucanase detection medium and cultured for 3 d, a proper amount of congo red solution (1 mg / mL) was poured onto the plate and left to dye for 15 min, then washed with sodium chloride solution (1 mol / L) for several times, and whether a white halo appeared around the colony was observed, if yes, it indicated that the strain had β-1-3 glucanase production ability.

[0042] Strain chitinase production ability determination: Mortierella was inoculated into colloidal chitin medium plate and cultured at 28°C for 72 h, a 5 mm diameter puncher was used to punch and sample, and the fungus was carefully transferred into chitin detection medium with tweezers and cultured for 3 d, and whether a transparent circle appeared indicated that the strain had chitinase production ability.

[0043] Strain siderophore production ability determination: the purified strain was inoculated into the center of CAS detection medium and cultured at 30°C for 3-5 d, if an orange-yellow halo appeared around the colony, it was judged that the strain had siderophore production ability.

[0044] The detection results are shown in Table 1, which indicates that Mortierella 2K4 has cellulase, protease, β-1-3 glucanase, chitinase, and siderophore production abilities, Figure 2 Fig. 2 shows the growth morphology of Mortierella 2K4 in the detection medium.

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

[0046]

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

[0048] Strain 2K4 produces cellulase, β-1-3 glucanase, chitinase, protease and siderophore, etc. The ability of the strain to produce multiple functional factors indicates that it has the potential of multiple antagonistic mechanisms as a biocontrol agent, such as degrading pathogenic bacterial cell wall (chitinase, glucanase), competing for iron nutrition (siderophore), etc.

[0049] Example 3

[0050] Inhibition of the pathogenic fungus G. cingulate by fermentation filtrate of Mortierella 2K4

[0051] Fermentation filtrate preparation method: pick 2K4 strain in 1 mL of PDB liquid medium at 30°C, 180 rpm shaking culture for 24 h as seed liquid, take seed liquid to new PDB medium at 1% proportion and shake culture for 24 h, which is fermentation liquid; centrifuge the fermentation liquid at 10000 rpm for 10 min, take the supernatant and filter it with 0.22 μm filter to remove bacteria, and the filtrate is the fermentation filtrate.

[0052] Inoculate the pathogenic fungus G. cingulate in PDA medium and culture for 72 h, take the fungus cake with a puncher and inoculate it in PDA medium containing 10%, 30% and 50% fermentation filtrate respectively, culture for 5 days and observe the growth of the strain, and record the diameter of the growth circle. The results are shown in Table 1. Figure 3 The colony diameters of the pathogenic fungus G1 on PDA plates containing 0%, 10%, 30% and 50% fermentation filtrate are 8.95 cm ± 0.02 cm, 6.97 cm ± 0.03 cm, 5.92 cm ± 0.03 cm and 4.88 cm ± 0.08 cm respectively. The inhibition rate of 10% fermentation filtrate on G. cingulate is 22.19%, the inhibition rate of 30% fermentation filtrate is 33.83%, and the inhibition rate of 50% fermentation filtrate on G. cingulate is 45.50%. The inhibition rate increases significantly with the increase of the concentration of fermentation filtrate.

[0053] Example 4

[0054] Preventive effect of Mortierella 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] The poplar cuttage method is used to cultivate seedlings. After poplar cultivation for 60 days, about 15 leaves grow. At this time, poplar pots are selected with the same size and growth. A 5mm fungus block is inoculated on each leaf. Each poplar is inoculated with about 8 leaves, and is divided into four groups with five repeats in each group. The four groups are: CK, inoculated with sterile water; 2K4, inoculated with only 2K4; G1, inoculated with only G1; and 2K4 / G1, inoculated with both 2K4 and G1. After 15 days of inoculation, the poplar disease index and incidence rate are counted.

[0063] The poplar anthracnose is divided into six grades according to the ratio of the lesion area on the poplar leaf to the total leaf area. The grading standards are as follows:

[0064] 0 grade: no lesion occurs;

[0065] 1 grade: lesion area ≤ 5%;

[0066] 2 grade: lesion area is between 5% and 25%;

[0067] 3 grade: lesion area is between 25% and 45%;

[0068] 4 grade: lesion area is between 45% and 65%;

[0069] 5 grade: lesion area > 65%.

[0070] Incidence rate (%) = number of diseased plants / total number of treated plants x 100%;

[0071] Disease index = ∑(number of plants at each grade x corresponding grade) / (total number of plants surveyed x highest grade) x 100;

[0072] Control effect (%) = (disease index of the control group - disease index of the treated group) / disease index of the control group x 100%.

[0073] The results are shown in Table 2 and Figure 6 The incidence rate and disease index of poplar leaves treated with strain 2K4 significantly decreased, and the control effect on poplar anthracnose was 59.45%. The above results show that strain 2K4 has good disease control ability for poplar anthracnose and is a good microbial agent.

[0074] The fungus block preparation method of Colletotrichum gloeosporioides and G1 is as follows: Colletotrichum gloeosporioides 2K4 and G1 are inoculated into PDA medium and cultured for 72h, and then a 5mm fungus cake is taken with a puncher.

[0075] Table 2: Biocontrol effect of applying Colletotrichum gloeosporioides 2K4 agent on poplar anthracnose

[0076]

[0077] The above description is only illustrative and is not restrictive; many modifications and variations of the application can be possible in light of this teaching without departing from the spirit or scope of the forthcoming claims.

Claims

1. A method of controlling anthracnose on poplar trees, characterized in that, The Mortierella 2K4 with the preservation number of CGMCC No.3.26002, and / or its fermentation liquor, and / or its metabolite is applied to a poplar or an environment where the poplar grows.

2. The method of claim 1, wherein, The metabolite of the Mortierella 2K4 is a sterile fermentation filtrate obtained by centrifuging and filtering bacteria from a fermentation liquor obtained after PDB liquid fermentation.

3. The method of claim 2, wherein, The volume concentration of the sterile fermentation filtrate in the application medium is 10% to 50%.

4. The method of claim 1, wherein, The application mode includes spraying, root irrigation or wound smearing.

5. The Mortierella 2K4 with the preservation number of CGMCC No.3.26002 is applied to the preparation of a microbial agent for preventing and treating poplar anthracnose.

6. Use according to claim 5, characterized in that, The microbial agent comprises at least one of the active components of the Mortierella 2K4, its fermentation liquor or its metabolite.

7. A microbial inoculant for controlling poplar anthracnose, characterized by, The active components thereof comprise the Mortierella 2K4 with the preservation number of CGMCC No.3.26002, and / or its fermentation liquor, and / or its metabolite.

8. The microbial inoculant of claim 7, wherein, The agent is a liquid agent, a solid agent or a wettable powder.

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