Pogonatum pseudoactinatum Chryseobacterium sp. And application thereof

Abstract

The invention relates to the technical field of phytopathogen prevention and control, and discloses a pogonatum insignis strain Chryseobacteria insignis KL01 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.34549. The invention further discloses a preparation method of the pogonatum insignis strain Chryseobacteria insignis strain and application of the pogonatum insignis strain Chryseobacteria insignis strain KL01, and the pogonatum insignis strain Chryseobacteria insignis strain KL01, the pogonatum insignis strain KL01 and the preservation number is CGMCC No.34549. According to the present invention, the mycosphaeria farinoides C.polytrichi KL01 obtained through separation and purification shows the good antagonistic effect on a variety of plant pathogens, such that the mycosphaeria farinoides C.polytrichi KL01 obtained through the present invention can be used for the prevention and control of plant pathogens, and has excellent prevention and control effects; and the mycosphaeria farinoides C.polytrichi KL01 can be used for the prevention and control of the plant pathogens, such that the mycosphaeria farinoides C.polytrichi KL01 can be used for the prevention and control of the plant pathogens.

Description

Technical Field

[0001] The present invention relates to the technical field of plant pathogen prevention and control, and in particular to Chryseobacterium pseudotricholoma and applications thereof. Background Art

[0002] The incidence and severity of plant diseases continue to rise, posing a significant threat to primary productivity, food security, and biodiversity in many regions of the world. Fruits and vegetables play a vital role in people's dietary health and economic development. However, due to their nutrient-rich and high water content, fresh fruits and vegetables are highly susceptible to pathogens during cultivation, transportation, and storage, with 30% of losses caused by fungi. Common symptoms include necrosis (leaf spots and leaf blight), rot (root rot, fruit rot, etc.), and wilting (roots, stem bases, vascular bundles, etc.).

[0003] Currently, agricultural pathogen control still requires the use of large quantities of chemical fungicides. However, the single, long-term use of many classic antimicrobial agents can lead to the development of serious pathogen resistance, resulting in rampant resurgence and increasing persistence. This severely hinders the adjustment of agricultural planting structures and the development of the green food industry. Compared to synthetic pesticides, natural strains offer significant advantages in biocompatibility and functional diversity. Furthermore, they have been naturally selected and optimized through long-term evolution, making them highly likely to become an important alternative to synthetic pesticides in the future.

[0004] Numerous microorganisms exist in the soil with varying antagonistic abilities against various pathogens. Some of these microorganisms can inhibit the growth of plant pathogens through their own actions and the secretion of specialized substances. Therefore, identifying biocontrol bacteria from the soil that have antagonistic effects on plant pathogens is of great significance for increasing crop yields and protecting the environment. Studies have shown that some Chryseobacterium species that live in rhizosphere soil, within plants, and on their surfaces can inhibit plant pathogens and are important beneficial bacteria for protecting plant health. Reports have been published on the disease resistance and antibacterial mechanisms of rhizosphere-derived Chryseobacterium species, demonstrating their biocontrol capabilities through nutrient competition, antagonistic competition, and induction of systemic resistance. Studies have found that the indoltheticum C. indoltheticum in the orchid plants Paphiopedilum calyx and Lithospermum nodosa produces auxin that promotes plant rooting and is of great significance to plant growth; C. balustinum AUR9 in the rhizosphere of Arabidopsis plants also plays an important role in reducing soil biological barriers. It can significantly enhance the host plant's disease resistance by inducing plant systemic resistance, reducing the incidence of the disease by at least 74%; a new species of Chryseobacterium sp. HT1 obtained from the dung of cows fed on grain straw can act as a cellulose-degrading bacterium to promote the decomposition of cellulose and xylan, providing sufficient carbon source for plant growth; the bacterial community structure and interaction network of broad beans inoculated with rhizobia changed under salt stress conditions, among which the number of Chryseobacterium increased, significantly improving the plant's tolerance to salt stress. However, the specific applications of C. polytrichastri are still relatively scarce, with only three reports. Researchers identified 10 cold-tolerant protease genes from the genome assembly of C. polytrichastri YG4-6, isolated from the Donglatong Glacier in the Sikkim Himalayas at an altitude of 4,613 meters, indicating the potential of this bacterium in the discovery of cold-tolerant industrial enzymes. The volatile secondary metabolites of C. polytrichastri DSM 26899, a moss grown on the Galong Glacier in Tibet, China, contain 71 compounds, including nitrogen-containing pyrazines and other aromatic heterocycles. Furthermore, reports indicate that the linalool synthase from C. polytrichastri exhibits diterpene synthase activity. However, no reports have been published on the biocontrol application of C. polytrichastri. Summary of the Invention

[0005] In view of this, the present invention proposes a Chryseobacterium pseudoginicola and its application, aiming to solve the technical problem that Chryseobacterium pseudoginicola has not been applied to the prevention and control of plant pathogens in the current technology.

[0006] The invention provides a Chryseobacterium polytrichastri KL01, which is a Chryseobacterium polytrichastri KL01 strain. The single colony is orange-yellow and short rod-shaped, and is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 34549.

[0007] The present invention also provides a bacterial agent, comprising Chryseobacterium polytrichastri KL01 described in the above technical solution.

[0008] The present invention also provides the use of the Chryseobacterium truncatum described in the above technical solution or the bacterial agent described in the above technical solution in the prevention and control of plant pathogens.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention provides a strain of Chryseobacterium polytrichastri KL01, isolated from the rhizosphere soil of Oxytropis yunnanensis on the Baima Snow Mountain in Deqin County, Diqing Tibetan Autonomous Prefecture, Yunnan Province, at an altitude of 4,600 meters. Oxytropis yunnanensis, a plant of the genus Oxytropis in the Leguminosae family, exhibits a pronounced "rhizosphere effect." Driven by the dual unique ecological environments of high altitude cold and the leguminous rhizosphere, the strain C. polytrichastri KL01 exhibits strong antagonistic effects against various plant pathogens.

[0011] The Chryseobacterium polytrichastri KL01 of the present invention is effective against Lasiodiplodia theobromae LY01 (longan scorch rot), Corynesporacassiicola XB02 (rubber rod spore leaf spot), Curvularia geniculate YW03 (corn Curvularia geniculate leaf spot), C. cassiicola HY04 (cucumber-like spore leaf disease), Colletotrichum boninense KT05 (cocoa anthracnose), C. siamense XT06 (rubber anthracnose), Magnaporthe oryzae XW07 (rice blast), Botryosphaeria dothidea MT08 (mango stem rot), Fusarium concentricum MO09 (mango fusarium), and Rhizoctonia solani. solani SK10 (rice sheath blight) and Fusarium oxysporum XK11 (banana wilt), and the C. polytrichastri KL01 of the present invention has a good control effect on plant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0013] Figure 1 This is a schematic diagram of the growth environment and appearance of Oxytropis yunnanensis described in Example 1;

[0014] Figure 2 This is a schematic diagram of the colony morphology of Chryseobacterium polytrichastri KL01 described in Example 1;

[0015] Figure 3 This is a schematic diagram of the microscopic morphology of the Chryseobacterium polytrichastri KL01 colony described in Example 1;

[0016] Figure 4This is a graph showing the antibacterial effect of Chryseobacterium polytrichastri KL01 described in Example 1 on 11 plant pathogens. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0018] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] The invention provides a Chryseobacterium polytrichastri KL01, wherein the Chryseobacterium polytrichastri KL01 is a Chryseobacterium polytrichastri KL01 strain, a single colony of which is orange-yellow and short rod-shaped, and is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 34549.

[0023] The present invention also provides a bacterial agent, comprising Chryseobacterium polytrichastri KL01 described in the above technical solution.

[0024] The present invention also provides the use of the Chryseobacterium truncatum described in the above technical solution or the bacterial agent described in the above technical solution in the prevention and control of plant pathogens.

[0025] Example 1

[0026] 1. Prepare samples and culture medium

[0027] 1. Sample Source

[0028] Oxytropis yunnanensis plant samples and their rhizosphere soil were collected. The specific sampling site was a scree beach at an altitude of 4600 meters (99°05′E, 28°30′N) on Baima Snow Mountain in Deqin County, Diqing Tibetan Autonomous Prefecture, Yunnan Province. It was surrounded by rivers and many rocks, and the soil was moist. The whole plant of Oxytropis yunnanensis sample was dug out, and then the soil 5 cm around the whole plant and the root system was shaken off. The samples were placed in large sterile self-sealing bags, frozen with liquid nitrogen, and brought to the laboratory, and the sample coating process was completed within 48 hours.

[0029] Among them, the growth environment and appearance of Yunnan Oxytropis yunnanensis are as follows Figure 1 As shown, Figure 1 Figure A is a schematic diagram of the sampling environment of Oxytropis yunnanensis, and Figure B is a schematic diagram of the appearance of Oxytropis yunnanensis.

[0030] 2. Preparation of Culture Medium

[0031] Potato dextrose agar (PDA): 200 g peeled potatoes, 20 g glucose, 17 g agar, distilled water to 1 L;

[0032] R2A medium: yeast extract 0.5 g, peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, K2HPO4 0.3 g, MgSO4 (anhydrous) 0.024 g, sodium pyruvate 0.3 g, agar 16 g, and distilled to 1 L.

[0033] LB liquid medium: 5 g sodium chloride, 10 g peptone, 1 g glucose, 5 g yeast powder, add distilled water to 1 L;

[0034] Potato dextrose broth (PDB): 200 g peeled potatoes, 20 g glucose, distilled water to 1 L.

[0035] 2. Bacterial Isolation, Purification, and Species Identification

[0036] Take 1g of fresh soil attached to the roots of Oxytropis yunnanensis and dilute it to 10% by adding sterile water. -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 100 μl of soil solution of different concentrations was aspirated and added to R2A culture dishes for coating, with three parallels for each concentration, and cultured in a culture room at 28°C for 2-5 days. When colonies grew on the plate that were visible to the naked eye, single colonies were obtained by the plate streak method.

[0037] The bacterial species was then identified. The sequencing results were cut and compared with BLAST in the NCBI database. The strain name with the highest similarity was determined as the strain species information. The obtained strain was identified as Chryseobacterium polytrichastri KL01.

[0038] The pure cultured strain was streaked on a culture medium and cultured at 28°C for 2-3 days. The bacteria were yellow, with moist, sticky colonies that were easily picked up by an inoculation needle. Single colonies appeared orange-yellow. Carbolic acid fuchsin stain was used for staining, and irregular colonies were observed under a biological microscope at 10×40 magnification.

[0039] Among them, the colony morphology of Chryseobacterium polytrichastri KL01 is as follows Figure 2 As shown; the microscopic morphology of the colony of strain C. polytrichastri KL01 is as shown Figure 3 shown.

[0040] 3. Determination of the antagonistic activity of Chryseobacterium polytrichastri KL01 against plant pathogens

[0041] 1. Select plant pathogenic bacteria

[0042] Eleven plant pathogenic bacteria were selected, including Lasiodiplodia theobromae LY01, Corynespora cassiicola XB02, Curvularia geniculate YW03, Corynespora cassiicola HY04, Colletotrichum boninense KT05, Colletotrichum siamense XT06, Magnaporthe oryzae XW07, Botryosphaeria dothidea MT08, Fusarium concentricum MO09, Rhizoctonia solani SK10, and Fusarium oxysporum XK11, the above strains were provided by the Key Laboratory of Tropical Medicinal Resources of Hainan Normal University, and all strains were identified and preserved in the Key Laboratory of Screening and Research on Pathogen-Resistant Plant Resources in Western Yunnan Province.

[0043] 2. Determination of the Antagonistic Activity of Chryseobacterium polytrichastri KL01 against Plant Pathogens

[0044] The purified Chryseobacterium polytrichastri KL01 was inoculated into LB liquid culture medium and cultured at 37°C and 180 rpm for 12-18 hours to activate the bacteria.

[0045] Adjust the bacterial solution concentration: Take the activated bacterial solution and vortex it for about 1 minute to obtain the supernatant. Measure the bacterial solution concentration under UV conditions at 600nm to ensure that the OD is within the range of 0.6-0.8. If multiple experimental groups and control groups are to be performed simultaneously, prepare the corresponding number of plates according to the experimental design.

[0046] Plant pathogenic fungi grow well in the PDA culture medium, and the fungus blocks are removed with a hole puncher. Punch a hole in the center of the solid culture medium and place the removed indicator fungus block. Use sterile tweezers to pick up the sterilized filter paper and place it about 2.5 cm away from the center point of the solid culture medium to avoid mutual influence. Take 15-20μl of the antagonistic bacteria supernatant and add the bacterial liquid to the filter paper at a slow and even speed. Treatment of the control group filter paper: For the control group, the treatment method is the same as that of the experimental group to eliminate the interference of other factors on the experimental results. Place 3-4 filter paper pieces on each plate, and perform three replicates for each antagonistic bacteria and indicator bacteria. At the same time, mark them to distinguish the experimental group from the control group.

[0047] Allow the plate with the filter paper to rest for 3-5 minutes. Once the bacterial solution has been absorbed by the filter paper, invert the plate and place it in a constant temperature incubator. Cultivate the plate according to the growth temperature requirements of the indicator bacteria. Bacteria are generally cultured for 18-24 hours, and fungi for 2-3 days. Inverting the plate prevents condensation from dripping onto the surface of the culture medium and affecting the experimental results.

[0048] After the culture was completed, the diameter of the pathogenic fungal colony was measured using the cross method, and the inhibition rate of mycelial growth was calculated according to the following formula.

[0049]

[0050] The antibacterial rates of Chryseobacterium polytrichastri KL01 against 11 plant pathogens are shown in Table 1. The antibacterial effects of strain C. polytrichastri KL01 against 11 plant pathogens are shown in Table 1. Figure 4 As shown;

[0051] Table 1 Inhibition rate of Chryseobacterium polytrichastri KL01 against 11 plant pathogens

[0052]

[0053] Based on Table 1 and Figure 4 It can be seen that Chryseobacterium pseudoginicola KL01 has moderate to strong antagonism against 11 plant pathogens;

[0054] Among them, the control effect of the four pathogens, including Lasiodiplodia theobromae LY01 (longan scorch rot), Corynespora cassiicola XB02 (rubber stick spore leaf spot), Curvularia geniculata YW03 (corn Curvularia leaf spot), and Corynespora cassiicola HY04 (cucumber stick spore leaf spot), was good, with the inhibition rates of 67.05%, 65.48%, 64.70%, and 60.00%, respectively.

[0055] The inhibition rates against five plant pathogens, including Colletotrichum boninense KT05 (cocoa anthracnose), Colletotrichum siamense XT06 (rubber anthracnose), Magnaporthe oryzae SW07 (rice blast), Botryosphaeria dothidea MT08 (mango stem rot) and Fusarium concentricum ML09 (mango fusarium), were all between 50% and 60%, namely 58.82%, 56.47%, 55.29%, 54.11% and 50.61%, respectively; the inhibition rates against Rhizoctonia solaniSK10 (rice sheath blight) and Fusarium oxysporum XK11 (banana wilt) were 48.23% and 46.10%, respectively.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A Chryseobacterium pseudotricholoma, characterized in that The Chryseobacterium polytrichastri KL01 is a Chryseobacterium polytrichastri KL01 strain, the single colony is orange-yellow and short rod-shaped, and is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 34549.

2. A bacterial agent, characterized in that The method comprises the Chryseobacterium polytrichastri KL01 described in claim 1.

3. Use of the Chryseobacterium truncatum described in claim 1 or the bacterial agent described in claim 2 in the prevention and treatment of plant pathogens.

Images