CN117987274A - Identification and application of fungus strain for preventing and controlling pine wood nematodes - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P5/00—Nematocides
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention provides a fungus strain Arthrobotrys robusta nefuMH for preventing and treating pine wood nematodes, which has a microorganism preservation number of: CGMCC 40258. Also provides the morphological identification characteristic of fungi and the time sequence change condition in the process of preventing and controlling pine wood nematodes. Comprising the following steps: a method for obtaining a fungus strain Arthrobotrys robusta nefuMH from pine wood nematode epidemic wood; morphology and molecular recognition characteristics of fungal strains; the fungus strain can prevent and treat the time sequence change of the pine wood nematodes. The fungus strain Athrobotrys robusta has higher activity for preventing and controlling pine wood nematodes.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to morphological identification and application of a fungus strain Arthrobotrys robusta nefuMH for preventing and treating pine wood nematodes.
Background
Pine wood nematodes are important quarantine pests in forestry in China, and are mainly harmful to conifers such as Korean pine and Pinus massoniana in China. By the end of 2021, pine wood nematode disease caused by pine wood nematodes has occurred in 731 county administrative regions of 19 provinces nationwide, the occurrence area is 171.65 ten thousand hectares, which results in death of 1407.92 ten thousand pine trees, and a situation of rapid spread from punctiform distribution to flaky development and westernization and northwards is shown in China. The pine wood nematodes have great damage to pine forests, seriously affect the safety of the world forest ecosystem, and prevent the ecological civilization construction process. The existing control method mainly uses chemical pesticides such as emamectin benzoate and the like to carry out dry injection and pesticide application treatment, but the long-term use of the chemical pesticides is easy to cause insect resistance and environmental pollution. Meanwhile, the transpiration, photosynthesis and respiration of pine tree lead to great loss of medicines, thus greatly increasing the prevention and treatment cost.
As a tree species in rural areas in China, the pinus massoniana has the characteristics of short growth cycle, strong stress resistance and wide adaptability, and is an important ecological artificial forestation pioneer tree species in the south. The pinus massoniana is extremely wide in distribution range in China, the north is from Henan and Shandong south, the south is from south to Guangdong and Guangxi Provinces, taiwan, the east is from coastal, the west is from Sichuan middle and Guizhou, and the pinus massoniana is spread over China and south China, and is a main material tree species in the south of China, and the economic value and the ecological value are high. The Korean pine is mainly distributed in Changbai mountain and Xiaoxinganling of northeast China, has strong cold resistance and is favored by slightly acidic soil or neutral soil. The wood is light and soft, fine, has straight texture and strong corrosion resistance, and is an excellent material for buildings, bridges, sleepers and furniture; the bark can extract tannin extract, and the trunk can extract rosin; the seeds can be used for eating or medical use, and can be used for eating or industrial use by pressing oil. Is mainly used for forestation of tree species and ornamental tree species in producing places.
The natural enemy microorganism becomes an important foundation for the development of the green wire killing agent due to the advantages of high safety, good environmental compatibility and the like. At present, the aschersonia parvula (Arthrobotrys oligospora), the protonematophagous variety of the aschersonia parvula (Arthrobotrys cladodes) is a widely studied nematophagous fungus, and can generate a specific adhesion network to capture nematodes, and the hyphae promote the adhesion, fixation, penetration and destruction of the nematodes. Spores show better activity against livestock parasitic nematodes. Is considered to be a potentially effective biological agent for controlling parasitic nematodes.
The obtained tree is used for the tree. The tree endophytic fungi-alternaria alternata capable of predating nematodes are obtained in the trunk, so that the ecological environment of the trunk is not destroyed, and a large number of artificial propagation can be realized. On the other hand, the agaricus bisporus can grow stably on the xylem of the pine tree, and can be used for predating the pine wood nematodes in a host environment so as to achieve the control effect. Saving a great deal of manpower and material resources in terms of prevention and treatment cost and having no pollution to the environment. The development requirement of China on green cycle ecological control and the increasing importance of China on forest ecological systems are raised, and an ecological environment treatment system is innovated. The patent has important targeting application value for developing and applying the biological wire killing agent for targeting the accurate pine wood nematodes. The application of the aschersonia aleyrodis for controlling soil nematodes is the focus of the current biological control research, but the utilization of the aschersonia aleyrodis for controlling pine nematodes is not reported.
Disclosure of Invention
The invention aims to provide a strong alternaria Athrobotrys robusta nefuMH for preventing and controlling pine wood nematodes and application thereof.
In one aspect, the invention claims a fungal strain Arthrobotrys robusta nefuMH.
The fungus strain Arthrobotrys robusta nefuMH claimed in the invention is preserved in China general microbiological culture collection center (CHINA GENERAL Microbiological Culture Collection Center, CGMCC) in 8 months of 2022, and the preservation number of microorganisms is: CGMCC 40258.
In another aspect, the claimed fungal strain Arthrobotrys robusta nefuMH is used in any of the following applications:
(1) Preventing and controlling pine wood nematodes;
(2) And preparing a product for preventing and controlling pine wood nematodes.
The invention collects pine wood nematode disease wood from masson pine in Linan area of Hangzhou, separates to obtain nematophagous fungus strain, screens out a biocontrol strain for effectively controlling pine wood nematodes by measuring the toxicity of the separated strain, and identifies the biocontrol strain as the strongylus robusta (Arthrobotrys robusta) through morphological and molecular biological analysis, wherein the strain nefuMH has remarkable control effect on pine wood nematodes. The effect of the trichlella tenuifolia nefuMH on controlling pine wood nematodes is obviously enhanced along with the increase of time, and the increment of 24-30 h is maximum. Up to 54.52% at 42 h. At 42h, the insect body starts to be seriously digested and the body wall is broken. Simultaneously, hyphae grow in large quantity near the insect bodies to generate conidium. And basically digesting the insect bodies at 48 hours. The biocontrol strain provided by the invention can be used for efficiently preventing and controlling pine wood nematodes, has good biocontrol potential, provides new resources for biological prevention and control of forest quarantine pest pine wood nematodes, and simultaneously provides assistance for protection of masson pine and Korean pine forest.
Drawings
FIG. 1 is a strain morphology diagram of the present invention: wherein A, B and C are conidiophores; d is a predation ring; e and F are conidia; g is chlamydospores.
FIG. 2 shows the binding of fungus strain Arthrobotrys robusta nefuMH of the present invention at various times during predation by pine wood nematodes.
FIG. 3 shows the morphological changes of nematodes during interaction of fungal strain Arthrobotrys robusta nefuMH of the invention with pine wood nematodes: a is nematode morphology after 18h inoculation; b is nematode morphology 24h after inoculation; c is the nematode morphology after 30h inoculation; d is nematode morphology 36h after inoculation; e is the nematode morphology after 42h inoculation; f is the nematode morphology 48h after inoculation.
Detailed Description
Example 1
1. Collection of nematophagous fungi
Branches, trunks and roots of pine wood nematode epidemic are collected in the masson pine forest land in Linan area of Hangzhou, zhejiang province.
2. Isolation and purification of nematophagous fungi
Cutting the specimen into tissue blocks with the size of 1cm multiplied by 1cm, placing the tissue blocks on an LCMA culture medium, culturing at the constant temperature of 26 ℃, and purifying grown fungi to obtain pure strains.
3. Strain virulence assay
Pine wood nematodes were collected from the trunks of Pinus massoniana by the Bellman funnel method. The collected pine wood nematodes were sterilized with 15% H 2O2 solution for 10min, washed 3 times with phosphate buffer containing 0.1% Tween 20, centrifuged at 5000rpm for 1min and the supernatant discarded. 6mL of pine wood nematode suspension was collected for testing.
The purified fungus was inoculated with 5mm diameter pieces of LCMA medium of 9cm diameter at 26℃for 7 days in pure culture, and 10mL of LCMA medium without pieces was used as a control. 200 mu L of nematode suspension is dripped on each flat plate, after 24 hours of inoculation, 200 pine wood nematodes are recorded for binding the number of insects and the number of insect rings, experiments are carried out for 3 times in parallel, observation and detection are carried out on the insect bodies, the strains are determined to be consistent with the original strains, and the insect body binding rate is determined.
The results show that a plurality of separated and purified nematophagous fungi can kill pine wood nematodes, wherein one strain of nematophagous fungi has the highest toxicity of the number of the glabrous greenbrier nefuMH.
4. Identification of strains
(1) Morphological observation
Inoculating the screened fungus strain Arthrobotrys robusta nefuMH on a low-nutrition corn meal agar culture medium plate, culturing at constant temperature of 26 ℃ for 7-9 days, and fully growing a plate with the diameter of 9cm, wherein hyphae are sparse, colorless and separated. Conidiophores are single-born, unbranched, undivided, base-contracted towards the end, top-unexpanded, forming a plurality of peduncles, one conidiophore per peduncle (fig. 1a, b, c). 2-7 conidia per pedicel. Conidiophores are colorless, oval, distally broad-rounded, proximally contracted, truncated, no septum or with a septum in the middle, no constriction in the septum, 18.3-21.3x7.5-9.8 μm (x=19.3x8.2 μm) (n=50), no septum 13.2-18.6x5.7-8.8 μm (x=15.2x6.7 μm) (n=50) (fig. 1e, f). The food catcher is a three-dimensional bacterial net (figure 1G).
(2) Molecular biological identification
The CTAB method of milling with frozen liquid nitrogen was used to extract the genomic DNA of the fungal strain. The primers used were ribosomal ITS segment universal sequences ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCGCTTATTGATATGC-3'), and were subjected to agarose gel electrophoresis detection after 50. Mu.L of systematic amplification. The PCR product was sent to Shanghai Biotechnology for sequencing.
Sequencing and comparison: and (3) performing sequence comparison analysis on the DNA sequences obtained by sequencing and NCBI database, performing fungus homology analysis, and determining the taxonomic position of the fungus strain by combining morphological identification. The strain is determined to be the Phanerochaete chrysosporium Arthrobotrys robusta through the sequence comparison result and the morphological identification result.
Fungus strain Arthrobotrys robusta nefuMH was deposited at the university of northeast forestry plant pathology laboratory, 6.20.2020 under accession number nefuMH.
Example 2
Indoor test for preventing and controlling pine wood nematodes
Fungus strain Arthrobotrys robusta nefuMH and 5-mm are inoculated onto 9cm diameter culture dish with 10mL of low-nutrition corn flour agar medium poured one day in advance, and pure culture is carried out at 26 ℃ in dark for 7 days. Taking 10mL of LCMA culture medium without bacterial blocks as a control, dripping 200 mu L of nematode suspension on each flat plate, inoculating for 24 hours, recording the number of the binded worms and the number of worm rings of 200 pine wood nematodes, performing experiments in parallel for 3 times, observing and detecting worm bodies, determining that pathogenic bacterial strains are consistent with original bacterial strains, and measuring the binding rate of worm bodies.
Preparation of pine wood nematodes
Pine wood nematodes were collected from the withered pine trunks by the bellman funnel method. Collected pine wood nematodes were sterilized with 15% H 2O2 solution for 10min, washed 3 times with phosphate buffer containing 0.1% Tween 20, centrifuged at 8000rpm for 1min, and the supernatant discarded. 2mL of pine wood nematode suspension was collected for testing.
Investigation of results
After 18h inoculation, 200 pine wood nematodes are used for binding the insect number and the insect body ring number every 6 h.
TABLE 1 grading Standard for nematophagous fungi predating pine wood nematodes
X represents the number of bundled fungus rings on the insect body
The results show that: the effect of the trichlella tenuifolia nefuMH on controlling pine wood nematodes is obviously enhanced along with the increase of time, and the growth of 24-30 h is maximum. 54.52% (FIG. 2) was reached at 42 h. At 42h, the insect body starts to be severely digested and the body wall is broken. Simultaneously, hyphae grow in large quantity near the insect bodies to generate conidium. At 48h, the worms were substantially completely digested (FIG. 3).
Claims (5)
1. An application of a masson pine endophytic fungus strain in preventing and controlling pine wood nematodes, which is characterized in that the fungus strain is Arthrobotrys robustanefuMH and has a microorganism preservation number of: CGMCC 40258.
2. Use of a strain of masson pine endophytic fungi according to claim 1 for controlling or preparing products for controlling pine wood nematodes.
3. Use according to claim 2, wherein the viable amount of fungal product of the pine wood nematode population is inhibited.
4. The use according to claim 2 or 3, wherein the pine wood nematode is a pathogen of pine wood nematode disease, an important disease in forestry.
5. The fungus product for preventing and controlling the survival quantity of the pine wood nematode population, wherein the active ingredient is Arthrobotrys robustanefuMH fungus strain and the product thereof as described in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211374315.3A CN117987274A (en) | 2022-11-04 | 2022-11-04 | Identification and application of fungus strain for preventing and controlling pine wood nematodes |
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