CN116083309A - Pseudomonas L40-14 and application thereof in preventing and treating mulberry diseases - Google Patents
Pseudomonas L40-14 and application thereof in preventing and treating mulberry diseases Download PDFInfo
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Images
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/27—Pseudomonas
-
- 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
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
Abstract
The invention relates to pseudomonas L40-14 and application thereof in preventing and controlling mulberry diseases, belonging to the technical field of plant disease prevention and control. The invention separates a probiotic strain with different degrees of antagonism/prevention and control effects on pathogenic bacteria of bacterial wilt of mulberry from root tissues of mulberry, combines morphological characteristics, physiological and biochemical characteristics and 16s rRNA sequence analysis with whole genome sequence analysis, identifies the probiotic strain as a Pseudomonas sp, and sends the Pseudomonas sp to the Guangdong province microorganism strain collection center for patent collection of new strains, wherein the collection number is GDMCC No:62950. the strain has good antagonism/prevention and control effects on a plurality of pathogenic bacteria of the mulberry wilt and/or the mulberry bacterial wilt, can be used for developing preparations for preventing and treating the mulberry diseases, and lays a foundation for the biological prevention and treatment of the mulberry diseases and the research and development of microorganism antagonism strains and products.
Description
Technical Field
The invention belongs to the technical field of plant disease prevention and control, relates to a biocontrol bacterium for mulberry bacterial diseases and application thereof, and in particular relates to pseudomonas L40-14 and application thereof in preventing and controlling mulberry diseases.
Background
The mulberry is taken as a arbor or shrub plant for providing main nutrition matters for the growth and development of silkworms, and is endangered by various diseases and insect pests throughout the year, such as bacterial wilt, sang Yibing and the like of the mulberry, which often occur in mulberry planting industry areas in various places, often cause large-area infection of a mulberry field, seriously influence the yield and quality of the mulberry leaves, and further influence the healthy and stable development of the mulberry industry (the identification and prevention of several common mulberry diseases, xu Weifang and the like, 2015).
Sang Xijun bacterial wilt is a disease of infection of mulberry by Sang Bingyuan bacteria Laurencia pseudosolanaceae, which causes dehydration wilt of the mulberry, bacterial pus on the cross section of branches, and fast transmission of mulberry infection of dyeing and finishing plants or whole mulberry fields, and is called Sang Wen by the masses. The silkworm area in Guangdong before liberation did not find Sang Qing blight, and the Sang Qing blight was found earliest in the sandy, luxuriant and other towns in Shunde city before and after 1968, and then found successively in the cities of south China sea, zhongshan and the like. The agricultural parlor in Guangdong province in 1978 has a census result of 164 towns and 8064 production teams, the area of Sang Qing withers is 1220.6 hectares, which accounts for 10.07% of 12174 hectares of cences in the census mulberry field (97.3% of the total area of the mulberry field in Guangdong province in the current year), and the diseases of Guangdong Sang Qing (the occurrence and prevention and control of the withers in Guangdong Sang Qing, mao Kengzu, 1996) are most serious.
Lawsonia solanaceae (Ralstonia solanacearum) is the second most serious phytopathogenic bacteria in the world, causing destructive bacterial wilt in many important crops. At the initial stage of bacterial wilt of the mulberry, the root phloem of the mulberry plant is normal, the phloem is peeled off to see the yellowing of the xylem, and the longitudinal brown streaks are accompanied with the development of the disease course, the longitudinal brown streaks are also seen on the branch xylem of the disease plant, the root xylem is blackened and finally rotted and shed (study outline of bacterial diseases of the mulberry, li Lei and the like, 2012).
At present, a traditional method for preventing and treating bacterial wilt of mulberries is mainly adopted, (1) high-quality disease-free seedlings are bred, (2) soil disinfection is carried out in a disease area, and the mulberries soil disinfectant can adopt medicines such as lime nitrogen, trichloroisocyanuric acid, chlorine dioxide, sodium hypochlorite, fresh lime and the like, (3) scattered disease plants are excavated in time, and soil in the disease area is disinfected, (4) bacterial wilt-resistant varieties are selected, the disease area is updated and reformed, (5) farm operation tools are strictly managed, (6) the disease area is subjected to reinforced quarantine management, (7) in the aspects of medicine prevention and treatment, 150 ml-250 ml of each plant can be subjected to root irrigation prevention and treatment by adopting a 20% thiazuril copper suspending agent 500 times, 30% copper succinate wettable powder 400 times, 80% ethylalliin emulsifiable concentrate 800 times and the like (Sang Weinian disease prevention and control technologies, she Weiqing and the like, 2020). The disease point and the surrounding soil are sterilized by scattering cover with quicklime or by sterilizing with formalin solution of 1:100 times or by irrigating with available chlorine bleaching powder solution. The cuprammonium mixture (3 kg of cuprammonium plus 0.5kg of copper sulfate) for healthy trees around the disease plant is mixed and stirred uniformly, and is filled into a plastic bag and sealed for 24 hours) for pouring 1:200 diluent, and the cuprammonium mixture can be used for root irrigation of thiabendazole, kasugamycin, cuaminosulfate and the like at the initial stage of disease occurrence, and can be used for preventing and controlling 2-3 times continuously for 7-10 days (mulberry bacterial disease research profile, li Lei and the like, 2012).
The chemical pesticide has the advantages of quick response, and can quickly relieve the illness state, but the soil environment is worsened, the drug resistance of the pathogeny is enhanced, and the residue of the chemical pesticide possibly causes poisoning or drug resistance of silkworms and affects the health of human beings when being used on a large scale. At present, related research on biological control of the Lawsonia of the Solanaceae is gradually increased, including separation and identification of antagonistic bacteria, antagonistic fungi, phage and the like. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) which has obvious antagonism effect on the tobacco bacterial wilt is researched and separated, and both thalli and secretion can inhibit the growth of the bacterial wilt. In field experiments, compared with a control, the microbial inoculum and the organic fertilizer added with the microbial inoculum can obviously reduce the infection rate of tobacco bacterial wilt. (isolation of tobacco-saving and field-effect bacterial wilt RS-22 in Yunnan and screening and identification of antagonistic bacteria thereof, li Junying, et al, 2021). Zhang Yuxia and the like are combined with organic fertilizers to prepare biological organic fertilizers, and have certain prevention and control effects on tobacco bacterial wilt (research on prevention and control effects of pseudomonas fluorescens and organic fertilizers on tobacco bacterial wilt in Qianjiang, zhang Yuxia and the like, 2022 years). Liu Zhengzheng and the like, and antagonistic actinomycetes which have a strong inhibition effect on tobacco bacterial wilt bacteria are screened out by a plate counter method (the inhibition effect and preliminary identification of the antagonistic actinomycetes F7-2 on the tobacco bacterial wilt bacteria, liu Zhengzheng and the like, 2020). Hu Ronghua and the like are separated from Jian tobacco field soil to obtain 6 bacterial wilt phages, and research on the influence of conditions such as temperature, pH, ultraviolet light, diethyl ether, chloroform and the like on the phages is developed (bacterial wilt phages separation and biological characteristic research thereof, hu Ronghua and the like, 2019).
At present, the biological prevention and treatment of the bacterial wilt of the mulberry are less studied, and chemical pesticides are mainly used for preventing and treating the bacterial wilt of the mulberry in the mulberry field.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a pseudomonas L40-14; the second aim is to provide a preparation for preventing and controlling plant diseases, which comprises the strain or/and fermentation liquor thereof; the third object is to provide the application of the Pseudomonas L40-14 or the preparation in preventing and controlling Lawsonia bacteria and/or preparing the preparation for preventing and controlling Lawsonia bacteria; the fourth aim is to provide the application of the pseudomonas L40-14 or the preparation in preventing and controlling plant diseases and/or preparing products for preventing and controlling plant diseases. The Pseudomonas sp has a strong antagonism effect on a plurality of strains of the Lawsonia of the Solanaceae preserved in a laboratory, and provides assistance for green prevention and control of the bacterial wilt of the mulberry field.
In order to achieve the above purpose, the invention adopts the following specific scheme:
in a first aspect, a Pseudomonas L40-14, classified as Pseudomonas sp, has been deposited under the accession number GDMCC No:62950 the collection unit is the microorganism strain collection center of Guangdong province, the collection address is the microbiological institute of Guangdong province, the collection date is 2022, 11 and 07.
In a second aspect, a plant disease control agent comprising the strain and/or a fermentation broth thereof.
Preferably, the plant disease is Morus wilt and/or Morus bacterial wilt.
More preferably, the mulberry wilt is bacterial wilt of mulberry.
In a third aspect, the use of the above Pseudomonas L40-14 or formulation for controlling Pseudomonas sp or for preparing a formulation for controlling Pseudomonas sp.
In a fourth aspect, the use of the Pseudomonas L40-14 or formulation described above for controlling plant diseases or for the preparation of a product for controlling plant diseases.
Preferably, the plant disease is caused by a bacterium of the genus Lawsonia. More preferably, the bacteria of the genus Lawsonia are Lawsonia pseudosolanaceae (Ralstonia pseudosolanacearum).
Preferably, the plant disease is a mulberry disease.
Preferably, the plant disease is mulberry wilt and/or mulberry bacterial wilt.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains the antagonistic bacteria with good antagonistic/preventing and controlling effects on a plurality of pathogenic bacteria of Sang Qing blight for the first time. The strain or the fermentation liquor thereof provided by the invention is used for biological means prevention and control of the bacterial wilt of the mulberry, is safe and effective, can avoid various problems of soil environment deterioration caused by chemical pesticides, drug resistance enhancement of pathogens, silkworm poisoning caused by pesticide residues, influence on human health and the like, lays a foundation for biological prevention and control of the mulberry diseases, has good application prospect, and is worthy of being widely popularized.
In the invention, a bacterial Pseudomonas (Pseudomonas sp.) with antagonistic effect on a plurality of strains of Pseudomonas (Ralstonia pseudolanacearum) is separated, the antagonistic effect on one strain of Pseudomonas of Moraceae is slightly lower than that of streptomycin sulfate, the antagonistic effect on the other five strains of Pseudomonas of Solanaceae is higher than that of streptomycin sulfate, the bacterial Pseudomonas (Pseudomonas sp.) can replace antibiotic medicines, and the invention meets the aim of green prevention and control. The potted plant experiment result shows that the prevention and treatment effect of the pseudomonas L40-14 on the potted plant of the fusarium wilt caused by a strain of pseudomonas solanaceae Lawsonia (RSM) reaches 71.4 percent, and the method has the potential of popularization and application to fields.
Strain preservation information: pseudomonas L40-14, classified as Pseudomonas sp, has been deposited under the accession number GDMCC No:62950 the collection unit is the microorganism strain collection center of Guangdong province, the collection address is the microbiological institute of Guangdong province, the collection date is 2022, 11 and 07.
Drawings
FIG. 1 is a graph showing the results of the antibacterial effect of L40-14 on L.pseudosolanaceae; a: lawsonia pseudosolanaceae (RSM); b: laugh of the family pseudosolanaceae (all-solanum lycoprotein); c pseudosolanaceae laugh bacteria (elephant state); d: lauterol pseudosolanaceae (RSH); e: lauteri (willow); f: lawsonia pseudosolanaceae (Indella).
FIG. 2 is a colony morphology characterization of the L40-14 strain LB medium.
FIG. 3 is a gram of L40-14 strain.
FIG. 4 is an electron microscope image of strain L40-14.
FIG. 5 is a phylogenetic tree of L40-14 strains constructed based on rRNA.
FIG. 6 is a biological control experiment of strain L40-14 on bacterial wilt caused by Laurencia pseudosolanaceae; a is a control group, B is an experimental group, and C is a blank control group.
FIG. 7 is an alignment plot based on L40-14 whole genome mapping.
Detailed Description
The invention separates a probiotic strain with different degrees of antagonism/prevention and control effects on Sang Qing cumosis pathogens from root tissues of mulberry, combines morphological characteristics, physiological and biochemical characteristics and 16s rRNA sequence analysis to identify the probiotic strain, determines the probiotic strain as Pseudomonas sp, and has good antagonism/prevention and control effects on a plurality of strains of pathogenic bacteria of Sang Qing cumosis.
The technical solutions in the embodiments of the present invention will be clearly and completely described below in connection with the embodiments of the present invention.
Test strain: the false Solanaceae Laurus is separated and stored in the laboratory.
The main reagent comprises: PCR reagents, proteinase K, etc. are purchased from Shanghai, inc.
(1) Experimental method
1. Separation and purification of rhizosphere biocontrol bacteria
And (5) collecting the rhizosphere soil of the mulberry by using a three-point sampling method. When in operation, firstly, the hoe is used for removing the dead branches, leaves and soil on the surface layer, then 10-20 cm of the mulberry root is deeply dug, the fibrous root part is cut off, the fibrous root part and the soil are filled into a sterile bag together, the sterile bag is numbered, the date is written, and the sterile bag is put into a laboratory for preservation at the temperature of minus 20 ℃ for standby.
After the fibrous roots are clamped by sterile forceps, shaking the fibrous roots to make the root soil fall, and collecting the fallen soil. Weighing 1.0g of mulberry root soil under aseptic condition, placing into a test tube filled with 9ml of sterile water beside flame, and making into 10 -1 Shaking at 36 deg.C for 30min to disperse the bacteria. 9 test tubes were taken and numbered 10 in sequence -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 9mL of sterile water was measured and added to the test tubes. 1mL10 was then pipetted using a sterile pipette -1 Suspension addition 10 -2 In the test tube, 1mL of the solution 10 is sucked after shaking -2 Adding the suspension to 10 -3 And so on.
Culturing and separating rhizosphere soil bacteria by plate smearing method, and selecting dilution of 10 -4 、10 -5 、10 -6 Is a suspension of (a). Then, 0.1ml of soil dilution is sucked up by a pipette, inoculated on the center of the plate with different dilution numbers, and uniformly smeared on the plate by a coating rod. Placing the smeared flat plate on a table top for 20-30min to allow bacteria liquid to permeate into the culture medium, and then inverting the flat plate. Sealing the culture dish with sealing film, and culturing at 28deg.C for 1-3d. After colony purification is carried out on the flat plate, single colony is moved to an inclined plane for culture for standby.
2. Preparation of antagonistic fermentation products
The purified single colony is selected and cultured in LB liquid medium at 28 ℃ and a shaking table of 140r/min for 3d, centrifuged at 8000r/min for 10min, and the supernatant is filtered by a bacterial filter (0.22 mu m microporous filter membrane) for bacteriostasis test.
3. Rhizosphere antagonistic bacteria screening
The antagonistic activity of endophytes is determined by a perforating method. 3 holes having a diameter of 5mm were uniformly punched in a culture dish on which the test strain had been uniformly spread with a punch. 50. Mu.L of the sterile filtrate was removed with a micropipette. A positive control was performed with 100ppm of streptomycin sulfate and a blank was performed with physiological saline. Each plate was replicated 3 times. The dishes were placed in a constant temperature incubator at 28℃and after 48 hours the zone of inhibition was observed and measured.
(2) Experimental results
Screening endophytic antagonistic bacteria, finding that one strain has antagonism on various strains of Lawsonia inermis of the Solanaceae, and naming the strain as L40-14, and performing biological strain preservation, wherein the preservation number is: GDMCC No:62950, antagonism of which is shown in FIG. 1 and Table 1.
Table 1: results of antagonism of L40-14 against various strains of L.pseudosolanaceae are shown.
The results show that the antagonistic bacteria show obvious antagonistic effect on various strains of the pseudosolanaceae La. sp (Ralstonia pseudosolanacearum), and besides streptomycin sulfate with the antagonistic effect on RSM pseudosolanaceae La. sp being lower than 100ppm, the antagonistic effect on other strains of the pseudosolanaceae La. sp is higher than 100ppm streptomycin sulfate.
EXAMPLE 2 identification of antagonistic bacteria
The culture characteristics of colony plates, gram staining and various physiological and biochemical index tests are important bases for determining the classification status of bacterial strains. 16S rRNA was used as a reference standard for evolution analysis. The nucleotide sequence of the 16S rRNA is shown as SEQ ID NO: 01.
Colony morphological characteristics of the L40-14 strain LB medium are shown in FIG. 2, gram staining results are shown in FIG. 3, and a strain electron microscope diagram is shown in FIG. 4. The phylogenetic tree constructed based on rRNA is shown in FIG. 5.
EXAMPLE 3 potted control of antagonistic bacteria L40-14
(1) Experimental method
Soil used in the potting test is humus soil, and a susceptible variety of Guisang 12 is selected as a mulberry seed variety. The pathogen of the mulberry wilt is a strain of Laurella multocida of the experimental separation and preservation. The mulberry seedlings are 50d seedlings. In the experimental group, root injury treatment of test mulberry seedlings is carried out by using sterile scissors, namely fibrous roots are sheared off, about 5cm main roots are reserved, and 10mL of 10 is added 6 cfu/mL of Lawsonia pseudosolanaceae, followed by 10mL of 10 6 cfu/mL of antagonistic bacteria L40-14. Control group, inoculated with pathogen alone plus 10mL of LB sterilized broth. The blank was added with 20mL of LB broth sterilized, 10 mulberry seedlings were treated each. The results are shown in FIG. 6. And (5) researching and recording the disease condition of the mulberry plants, and calculating the disease rate and the prevention effect.
(2) Experimental results
The results show that the incidence rate of the experimental group is 20%, the incidence rate of the control group is 70%, the incidence rate of the blank control group is 0%, and the calculated control efficiency is 71.4%.
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (10)
1. Pseudomonas L40-14, classified as Pseudomonas sp, has been deposited under the accession number GDMCC No:62950 the collection unit is the microorganism strain collection center of Guangdong province, the collection address is the microbiological institute of Guangdong province, the collection date is 2022, 11 and 07.
2. A formulation for controlling plant diseases, characterized in that: comprising the Pseudomonas L40-14 strain according to claim 1 and/or a fermentation broth thereof.
3. A formulation for controlling plant diseases according to claim 2, wherein: the plant diseases are mulberry wilt and/or mulberry bacterial wilt.
4. A formulation for controlling plant diseases according to claim 3, wherein: the mulberry wilt is bacterial wilt of mulberry.
5. Use of a formulation according to any one of claims 1, L40-14 or 2-4 for combating pseudomonas solanacearum or for preparing a formulation for combating pseudomonas solanacearum.
6. Use of a pseudomonas L40-14 according to claim 1 or a formulation according to any one of claims 2-4 for controlling plant diseases or for the preparation of a product for controlling plant diseases.
7. The use according to claim 6, characterized in that: the plant disease is caused by bacteria of the genus Lawsonia.
8. The use according to claim 7, characterized in that: the Lawsonia bacteria is Lawsonia pseudosolanaceae.
9. The use according to claim 6, characterized in that: the plant disease is mulberry disease.
10. The use according to claim 9, characterized in that: the plant diseases are mulberry wilt and/or mulberry bacterial wilt.
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| CN115478035A (en) * | 2022-10-20 | 2022-12-16 | 贵州省烟草公司黔西南州公司 | Pseudomonas bryostelii GT31, application thereof and obtained control agent |
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| CN108998389A (en) * | 2018-07-26 | 2018-12-14 | 上海交通大学 | There is pseudomonad and the application of antagonism to rice Xanthomonas campestris and Pyricularia oryzae |
| CN109988730A (en) * | 2019-04-09 | 2019-07-09 | 华南农业大学 | A kind of biocontrol microorganisms of mulberry tree disease and its application |
| CN114317356A (en) * | 2021-12-30 | 2022-04-12 | 华南农业大学 | Pseudomonas terricola Q1-7 strain and application thereof in preventing and treating bacterial wilt |
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