CN108315282B - Bacillus methylotrophicus and application thereof in preventing and treating cercospora - Google Patents
Bacillus methylotrophicus and application thereof in preventing and treating cercospora Download PDFInfo
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Classifications
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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
-
- 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
- C12R2001/07—Bacillus
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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
-
- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The invention discloses a methylotrophic bacillus and application thereof in preventing and treating cercospora cerifera. The strain is separated from Laizhou gulf sea mud of Taiwan, and has been preserved in China general microbiological culture Collection center in 26.9.2017 with the preservation number of CGMCC No. 14675. The biological control preparation prepared from the methylotrophic bacillus can efficiently control cercospora albilineans diseases (the control effect of 14 days after the last application reaches over 75 percent), can promote the growth of cercospora albilineans, and can be used as a biological pesticide or a biological fertilizer to control cercospora albilineans.
Description
Technical Field
The present invention belongs to the field of biological plant disease preventing and controlling technology. Specifically, the invention relates to a bacillus methylotrophicus and application thereof in preventing and treating cercospora cerifera, and a biological prevention and treatment preparation prepared from the bacillus methylotrophicus.
Background
The white wax is a good tree species for forestation of coastal wetlands, shelters, scenic forests and the like in China due to the characteristics of strong adaptability, fast growth, strong resistance, good material quality, high economic value, drought resistance, saline-alkali resistance, barren resistance and the like. In recent years, with the wide popularization and use of urban landscaping, diseases of fraxinus chinensis caused by a plurality of factors such as the regulation and transportation of seedlings of tree species in regions, the deterioration of environmental conditions and the like are increased, wherein Cercospora fraxinites Ell. Cercospora fragrans is caused by Cercospora fragilis el.et Ev, which damages the leaves of Cercospora fragilis, and erupts in 6-7 months. The cercospora cerifera can cause early leaf drop of cercospora cerifera, so that the growth of the cercospora cerifera is reduced, and the landscaping and beautifying effects are seriously affected.
At present, chemical agents are often adopted in production to prevent cercospora brown spot. Chemical control is easy to cause pesticide residue and environmental pollution, and long-term use of chemical pesticides causes pathogenic bacteria to generate drug resistance, so that the control effect is reduced and even fails. Biological control, safety to human and livestock, good environmental compatibility, difficult generation of drug resistance of pathogenic bacteria, and the like, has the advantages of chemical bactericides and gradually becomes the best mode for replacing the chemical bactericides. And the acquisition of the high-efficiency antagonistic bacteria is the basis of biological prevention and control.
The methylotrophic bacillus is a mesophilic, aerobic and spore-producing rod-shaped bacterium which widely exists in the internal organs such as the nature, plant roots and stems and the like, is safe and nontoxic, and has broad-spectrum antibacterial activity and extremely strong stress resistance. In recent years, domestic scholars have more application researches on bacillus methylotrophicus in the aspects of plant biocontrol, growth promotion and the like. Researches indicate that the bacillus subtilis has a strong inhibiting effect on rice bacterial wilt, banana wilt and the like and can promote the growth of crops. Therefore, the bacillus methylotrophicus has wide application prospect in the aspects of preventing and treating diseases, promoting crop growth and the like. As such, Bacillus methylotrophicus is being viewed by more and more enterprises as a functional bacterium for producing biological agents.
Disclosure of Invention
The invention aims to provide a novel microorganism, namely a bacillus methylotrophicus strain TJ2, for efficiently preventing and treating cercospora cerifera. The bacillus methylotrophicus strain has the characteristics of high growth speed, high yield, strong stress resistance, capability of fast and massively colonizing on the surface of a plant (especially white wax) and the like, thereby having good application prospect.
The above purpose of the invention is realized by the following technical scheme: the strain provided by the invention is a Bacillus methylotrophicus strain TJ2, is separated from Laizhou gulf sea mud in Taiwan, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 Xilu No.1 of the sunward area of Beijing, China academy of sciences microbiological research institute) in 26 months and 26 days in 2017, and the preservation number is CGMCC No. 14675. It has the following biological properties: the strain is in a rod shape on a beef extract peptone agar medium or an LB (Luria-Bertani) medium, the flagellum characteristic is obvious, a single colony is circular, the stain is white, the stain is opaque, the center is raised, the edge is neat, and wrinkles exist in the later period; culturing on beef extract peptone agar medium at 28 deg.C for two days, and examining under microscope to obtain rod-shaped thallus cell capable of moving. Gram-positive (E.coli control). And observing the spores to be oval or columnar after the spores are dyed.
The culture method or the propagation method of the bacillus methylotrophicus strain TJ2 comprises the following steps:
(1) an LB culture medium is adopted for ordinary culture and preservation, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15-20 g of agar and 1000mL of distilled water, and the pH value is adjusted to 7.0-7.2.
(2) The laboratory liquid culture adopts an LB liquid culture medium, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.0-7.2.
(3) The solid culture medium formula comprises: comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid material comprises, by mass, 53: 27: 15: 5 of rice hulls, corn flour, soybean meal and bran; the inorganic salt comprises the following components in percentage by mass: 16% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 69% of light calcium carbonate.
(4) The formula of the mass fermentation culture comprises: the formula of the solid culture medium in the step (3).
The invention also provides a biological control preparation for controlling cercospora cerifera, which comprises the bacillus methylotrophicus strain TJ 2.
Also, the present invention provides a biocontrol method for controlling cercospora cerifera, which comprises applying the above-mentioned bacillus methylotrophicus (b.methylotrophicus) strain TJ2 or the above-mentioned biocontrol agent to cercospora cerifera plants having cercospora.
The invention also provides the application of the bacillus methylotrophicus strain or the biological control preparation in preventing and controlling cercospora cerifera.
The preparation method of the biological control preparation comprises the following steps:
(1) preparing a seed solution of the bacillus methylotrophicus strain TJ 2;
(2) inoculating the seed liquid prepared in the step (1) into a solid culture medium, and culturing at a constant temperature of 28-30 ℃;
(3) and (3) adding sterile water into the culture cultured in the step (2), mixing, filtering by using sterile gauze, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 28-30 ℃ and the relative humidity of more than 85%.
In one embodiment of the present invention, the preparation method comprises the steps of:
(1) transplanting spores of the bacillus methylotrophicus strain TJ2 into an LB liquid culture medium, and performing shake culture on a shaking table at 28-30 ℃ for 3-5 days to obtain seed liquid;
(2) inoculating the seed solution prepared in the step (1) into a solid culture medium according to the mass ratio of 10%, and performing shake culture at 28-30 ℃ for 3-5 days;
(3) mixing the culture cultured in the step (2) with sterile water according to the mass ratio of 1: 15, filtering with sterile gauze, inoculating the filtrate to a large amount of fermentation medium according to the volume ratio of 1: 6, and fermenting and culturing for 8-9 days in a fermentation chamber with the room temperature of 28-30 ℃ and the relative humidity of more than 85%.
Wherein the LB liquid culture medium in the step (1) has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.0-7.2.
The solid culture medium in the step (2) comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid material comprises, by mass, 53: 27: 15: 5 of rice hulls, corn flour, soybean meal and bran; the inorganic salt comprises the following components in percentage by mass: 16% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 69% of light calcium carbonate.
The mass fermentation medium in the step (3) is synchronous with the solid medium in the step (2).
Experiments show that the bacillus methylotrophicus strain has the characteristics of high growth speed, high yield, strong stress resistance, capability of fast and massively colonizing on the surface of a plant (especially on white wax) and the like, and therefore, the bacillus methylotrophicus strain has a good application prospect. The biological control preparation prepared from the methylotrophic bacillus can efficiently control the cercospora brown spot disease and promote the growth of cercospora wax, and is a biological control preparation with a great application prospect. The microbial preparation can be used as a biological pesticide or a biological fertilizer for preventing and treating cercospora cerifera. The test result shows that: the bacillus methylotrophicus strain TJ2 solid fermentation culture has obvious effect of preventing cercospora cerifera, the prevention effect reaches 75.93%, and the cercospora cerifera grows well without causing phytotoxicity.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications are within the scope of the invention.
Example 1
1. Isolation and purification of Bacillus methylotrophicus TJ2
The bacillus methylotrophicus TJ2 is obtained by separating sea mud by a dilution plate method and a plate marking method, and the separation method comprises the following steps:
(1) isolation of Bacillus strains
The sea mud sample was taken 11 months and 2 days 2013 from lauan bay, leizhou, tai city. Weighing 1g sea mud in 100mL sterile water, placing in 30 deg.C shaking table with 150rpm shaking for 10min, placing in 60 deg.C water bath, incubating for 30min, and collecting 100 μ L10-2、10-3、10-4The diluted solution was spread on LB medium plates, three in parallel per gradient, and cultured at 30 ℃ for 2dAnd then picking microbial colonies with different forms on the LB culture medium, streaking the microbial colonies on an LB culture medium flat plate, and observing the growth condition of the colonies regularly. Then adopting a plate marking method to purify the bacillus strains, numbering and storing.
(2) Screening of cercospora cerifera high-efficiency antagonistic bacillus strain
① preliminary screening, preparing PDA (Potato Dextrose agar) plate by using a contraposition culture method, taking fungus cakes with diameter of 5mm from edges of pathogenic bacteria of Bacillus cereus and cercospora leucovora by using a puncher, respectively transplanting the fungus cakes into the centers of two opposite sides of the plate, culturing at constant temperature of 25 ℃, and observing the inhibition effect of the Bacillus on the pathogenic bacteria day by day.
② rescreening the screened bacillus strains with high-efficiency antagonistic activity, mainly screening strains with better tolerance through temperature resistance, acid and alkali resistance and drug resistance tests, performing pot control tests and field tests, and identifying the screened strains TJ 2.
The inventor obtains a bacillus methylotrophicus TJ2 capable of efficiently preventing and treating cercospora cerifera by a large amount of screening work. Experiments prove that the bacillus methylotrophicus solid fermentation culture shows a very high-efficiency prevention and treatment effect in preventing and treating cercospora cerifera and enables cercospora cerifera to grow well. Therefore, the bacillus methylotrophicus is a new bacillus methylotrophicus strain with wide application prospect, and can be used for preparing a biological control preparation for controlling cercospora cerifera.
2. Identification of strains
(1) Microbiological characteristics: the strain is rod-shaped on a beef extract peptone agar medium or an LB medium, the flagellum characteristic is obvious, a single colony is round, the stain is white, the single colony is opaque, the center is raised, the edge is neat, and wrinkles are formed in the later period; culturing on beef extract peptone agar medium at 28 deg.C for two days, and examining under microscope to obtain rod-shaped thallus cell capable of moving. Gram-positive (E.coli control). And observing the spores to be oval or columnar after the spores are dyed.
(2) Molecular biological Properties
The sequence determination result of the 16s rDNA gene of the strain is as follows (SEQ-1):
AAGGTTACCTTACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGGCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGT。
the strain TJ2 is identified as Bacillus methylotrophicus, which is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu 1 of the Beijing area of the morning sun, institute of microbiology, China academy of sciences) in 26.9.2017, and the preservation number is CGMCC No. 14675.
Example 2
1. Fermentation process of methylotrophic bacillus strains
The LB liquid culture medium formula is: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.0-7.2.
Solid medium: the solid material, inorganic salt and water, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid material comprises, by mass, 53: 27: 15: 5 of rice hulls, corn flour, soybean meal and bran; the inorganic salt comprises the following components in percentage by mass: 16% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 69% of light calcium carbonate. The preparation method comprises the following steps: adding a proper amount of water (10-15 times of the mass) to dissolve inorganic salt to obtain an inorganic salt solution; mixing inorganic salt solution and solid material, adding water to adjust water content of the solid culture medium to 40-60%, and grasping the solid culture medium in hand with water in finger joint without dripping.
The mass fermentation culture medium is the same as the solid culture medium.
Bulk solid fermentation process of bacillus methylotrophicus (b. methylotrophicus) strain TJ 2:
① culture of strain seed liquid
A small amount of spores are picked from a test tube inclined plane by a Bacillus methylotrophicus strain (B.methylotrophicus) TJ2, transferred into an LB liquid culture medium, and shake-cultured for 3-5 days at 30 ℃ in a shaking table, wherein the spores are used as seed liquid.
② cultivation of solid producer strains
Inoculating the seed solution into a solid culture medium (500mL triangular flask) according to the mass ratio of 10%, culturing at constant temperature of 30 ℃ for 3-5 d, and shaking for multiple times in the middle.
③ fermentation of large amount of solid
Diluting a culture obtained by solid culture in ② with sterile water according to a ratio of 1: 15, filtering with sterile gauze, removing coarse residues to obtain a production bacterial liquid, inoculating the production bacterial liquid into a large amount of fermentation culture medium according to a volume ratio of 1: 6, placing the inoculated raw materials into a fermentation chamber (30 ℃ and relative humidity of more than 85%) for fermentation culture for 8-9 days, wherein the active bacteria can reach 20 hundred million/g.
In order to determine the control effect of the bacillus methylotrophicus strain TJ2 on cercospora leucovora, a basis is provided for popularization and application of the bacillus methylotrophicus strain. We performed the test in Hiden Shouguang City between 5 and 7 months in 2016, and the test results are reported as follows:
1 test materials and methods
1.1 test agent
A solid fermentation culture (20 billion/g) of bacillus methylotrophicus strain TJ2 produced by the fermentation method of example 2 of the present invention; 65% wettable powder of zineb (commercially available from Shandong Xinbang Biochemical Co., Ltd.).
1.2 test materials and control objects
The test material was white wax; the control object is Cercospora fragilis Ell.
1.3 conditions of the test
The test site is arranged in a saline-alkali soil afforestation test station of the scientific research institute of forestry in Shandong province. The test soil type is salinized moisture soil, the organic matter content is 0.52%, the pH value is 7.3, the cultivation conditions and management measures of all test districts are consistent, and the cercospora leaf spot is in the early stage of disease development when the pesticide is applied.
1.4 test design and arrangement
The experiment has 3 treatments, namely 10 times of liquid of a solid fermentation culture of a bacillus methylotrophicus strain TJ2, 600 times of liquid of 65% zineb wettable powder and no application of clear water as controls, wherein each treatment is repeated for 4 times and each treatment is 60 strains. Spraying once in 2016, 5-month 16 days, 6-month 16 days and 7-month 11 days, wherein the spraying device is a type I-nong-16 sprayer.
1.5 test investigation and calculation method
1.5.1 weather conditions
The medicine for the 1 st application (5 months and 16 days) is sunny in the day, the wind power is 3 grades, the highest air temperature is 27 ℃, the lowest air temperature is 16 ℃, and the relative humidity is 42%. The medicine is applied for the 2 nd time (16 days in 6 months), the day is sunny, the wind power is 3 grades, the highest air temperature is 33 ℃, the lowest air temperature is 22 ℃, and the relative humidity is 44%; the 3 rd application (7 months and 11 days) is cloudy in the same day, has wind power of 3 grades, and has a maximum air temperature of 35 ℃, a minimum air temperature of 24 ℃ and a relative humidity of 64 percent.
1.5.2 drug efficacy and safety investigation
And (3) drug effect investigation: investigation was performed 14d after the last application. Each treatment investigated 50 plants, investigated plant leaves, investigated morbidity, recorded disease progression and calculated disease index.
Safety investigation: and (5) observing the safety of the white wax at 7d and 14d after the first application, describing the phytotoxicity symptoms in detail and determining the phytotoxicity degree according to the phytotoxicity degree grading standard if the phytotoxicity occurs.
Classification standard of cercospora leaf spot:
level 0: the leaves have no scab.
Level 1: the lesion area is below 1/4 of the leaf area.
And 2, stage: the area of the lesion spots accounts for 1/4-1/2 of the area of the leaves.
And 3, level: the area of the lesion spots accounts for 1/2-3/4 of the area of the leaves.
4, level: the lesion area may account for more than 3/4% of the leaf area or cause defoliation.
1.5.3 method for calculating drug effect
The preventing and treating effect is calculated according to the formulas (1) and (2):
in the formula: CK (CK)0-pre-drug condition index in the placebo zone;
CK1-disease index after drug administration in the placebo zone;
PT0-disease index before application before treatment with the agent;
PT1disease index after application after medicament treatment.
1.5.4 direct Effect on white wax
Observing whether the pesticide has phytotoxicity on the white wax, and recording the type and the degree of the phytotoxicity. In addition, the effect on the growth of white wax was also recorded.
Phytotoxicity was recorded in the following manner:
(a) if the phytotoxicity can be measured or calculated, it is expressed in absolute terms, for example, as plant height.
(b) In other cases, the extent and frequency of phytotoxicity can be estimated in two ways:
① the phytotoxicity degree of each cell is recorded according to phytotoxicity grading method, and is represented by-, + + + + + + +, +.
The phytotoxicity grading method comprises the following steps:
and (2) preparing: no chemical injury;
+: mild phytotoxicity, no influence on plant growth;
++: moderate phytotoxicity, can be recovered, and does not reduce the biomass of plants;
+++: moderate phytotoxicity affects the normal growth of plants and reduces the biomass of the plants to a certain extent;
++++: serious phytotoxicity, plant growth retardation and serious reduction of plant biomass.
② the percentage of phytotoxicity is evaluated by comparing the chemical treatment area with the blank control area, and the phytotoxicity symptoms (dwarfing, chlorosis, malformation, etc.) of the plants are accurately described.
2 results
2.1 controlling Effect of the test agent on cercospora cerifera
The control effect of the test agent on cercospora cerifera is shown in tables 1-2. The results in table 2 show that the bacillus methylotrophicus (b. methylotrophicus) strain TJ2 solid fermentation culture and 65% zineb wettable powder both have good control effect on cercospora cerifera, wherein the control effect of the bacillus methylotrophicus (b. methylotrophicus) strain TJ2 solid fermentation culture is more than 75%.
From the disease index of cercospora leaf spot field test (table 2), 14 days after the last application, the control effect of bacillus methylotrophicus (b.methylotrophicus) TJ2 is 75.93%, which is obviously higher than the control effect of 58.78% of 65% zineb wettable powder.
TABLE 1 cercospora leaf spot disease status before three agent treatments
TABLE 2 prevention and control of cercospora leaf spot by three chemical treatments
2.2 white wax safety investigation
Through the observation of the application of the medicines of 7d and 14d, the growth of the ceresin wax is normal and no phytotoxicity is generated in each medicine treatment area compared with the control area, which indicates that the tested concentration of the bacillus methylotrophicus solid fermentation culture is safe to the ceresin wax.
3 small knot
3.1 from the view of disease index and prevention and treatment effect, the bacillus methylotrophicus strain TJ2 has better prevention and treatment effect on cercospora cerifera, and the prevention and treatment effect reaches more than 75% in 14 days after the last application.
3.2 from the growth vigor of the white wax, the bacillus methylotrophicus strain TJ2 has the growth promoting effect on the white wax, has no phytotoxicity, and is safe and reliable.
SEQUENCE LISTING
<110> scientific research institute of forestry in Shandong province
<120> bacillus methylotrophicus and application thereof in preventing and treating cercospora cerifera
<130>0
<160>1
<170>PatentIn version 3.3
<210>1
<211>1393
<212>DNA
<213> 16s rDNA Gene sequence of Bacillus methylotrophicus strain TJ2
<400>1
aaggttacct taccgacttc gggtgttaca aactctcgtg gtgtgacggg cggtgtgtac 60
aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg attccagctt 120
cacgcagtcg agttgcagac tgcgatccga actgagaaca gatttgtggg attggcttaa 180
cctcgcggtt tcgctgccct ttgttctgtc cattgtagca cgtgtgtagc ccagggcata 240
aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtcacct 300
tagagtgccc aactgaatgc tggcaactaa gatcaagggt tgcgctcgtt gcgggactta 360
acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac tctgcccccg 420
aaggggacgt cctatctcta ggattgtcag aggatgtcaa gacctggtaa ggttcttcgc 480
gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 540
gtttcagtct tgcgaccgta ctccccaggc ggagtgctta atgcgttagc tgcagcacta 600
aggggcggaa accccctaac acttagcact catcgtttac ggcgtggact accagggtat 660
ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agttacagac cagagagtcg 720
ccttcgccac tggtgttcct ccacatctct acgcatttca ccgctacacg tggaattcca 780
ctctcctctt ctgcactcaa gttccccagt ttccaatgac cctccccggt tgagccgggg 840
gctttcacat cagacttaag aaaccgcctg cgagcccttt acgcccaata attccggaca 900
acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg gctttctggt 960
taggtaccgt caaggtgccg ccctatttga acggcacttg ttcttcccta acaacagagc 1020
tttacgatcc gaaaaccttc atcactcacg cggcgttgct ccgtcagact ttcgtccatt 1080
gcggaagatt ccctactgct gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt 1140
ggccgatcac cctctcaggt cggctacgca tcgtcgcctt ggtgagccgt tacctcacca 1200
actagctaat gcgccgcggg tccatctgta agtggtagcc gaagccacct tttatgtctg 1260
aaccatgcgg ttcagacaac catccggtat tagccccggt ttcccggagt tatcccagtc 1320
ttacaggcag gttacccacg tgttactcac ccgtccgccg ctaacatcag ggagcaagct 1380
cccatctgtc cgt 1393
Claims (5)
1. Bacillus methylotrophicus strain TJ2, wherein the preservation number of the strain is CGMCC No. 14675.
2. Use of the bacillus methylotrophicus strain TJ2 of claim 1 for controlling cercospora cerifera.
3. A biocontrol formulation comprising the bacillus methylotrophicus strain TJ2 of claim 1.
4. Use of the biological control formulation of claim 3 for controlling cercospora leaf spot.
5. A biocontrol method for controlling cercospora, characterized in that a bacillus methylotrophicus strain TJ2 of claim 1 or a biocontrol formulation of claim 3 is applied to a cercospora plant having cercospora.
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