CN112322536A - Bacillus belgii Z002, and acquisition method and application thereof - Google Patents
Bacillus belgii Z002, and acquisition method and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus beiLeisi Z002, an acquisition method and an application thereof, wherein the bacillus is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2020567. The obtaining method comprises the working procedures of separation, screening and purification, and the method is characterized in that firstly, endophytic bacteria are separated from tobacco plant tissues, and screened after dark culture, and the endophytic bacteria which antagonize rhizopus oryzae of tobacco mildew germs are finally purified. The application comprises the working procedures of fermentation, inoculation and modulation, firstly, the strain is cultured to obtain a zymophyte agent, the zymophyte agent is sprayed on the tobacco leaf stalks by a sprayer, the tobacco leaves are put into a curing barn, and the tobacco leaves are cured according to the normal curing process. The strain Bacillus belgii Z002 can effectively control the tobacco mildew disease in the baking period, and the relative prevention effect is above 80%. Secondly, the bacillus also has good bacteriostatic action on tobacco ralstonia solanacearum, phytophthora parasitica, botrytis cinerea, sclerotinia sclerotiorum, target spot pathogen, rhizoctonia rot, alternaria alternata and the like. The invention has good prevention and treatment effect and higher popularization and application value.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and relates to a Bacillus belgii Z002, an acquisition method and application thereof, in particular to a Bacillus belgii Z002 capable of realizing high-efficiency prevention and control of mildew disease in a baking period, an acquisition method and application thereof in prevention and control of the mildew disease of tobacco in a baking room.
Background
The tobacco mildew disease in the curing period is a fungal disease caused by infection of Rhizopus oryzae (Rhizopus oryzae), can be harmful to tobacco leaf stalks and mesophyll tissues, mainly takes a rotten type of leaf base, has the disease rate of more than 30 percent and the loss of about 20 percent. Therefore, the tobacco mildew disease has serious harm to the tobacco production.
The method for preventing and treating the tobacco mildew disease in the baking period comprises chemical agents, baking equipment improvement, biological prevention and treatment and the like.
Although the chemical prevention and control method has good prevention and control effect, the application of the chemical prevention and control method is limited because the chemical prevention and control method has the problems of pesticide residue, easy generation of drug resistance and the like and influences the safety of tobacco leaves.
The improvement of the baking equipment is mainly based on the improvement of poor ventilation and dehumidification effects of the existing baking room or the establishment of a novel baking room, the measure cost is high, and the promotion and demonstration driving within a certain time is needed.
Biological control is favored because of its high control efficiency, strong specificity, environmental friendliness, and low tendency to produce drug resistance.
At present, few microorganisms for preventing and controlling the tobacco mildew disease in the baking period are reported, and only Bacillus mucilaginosus (CN110384247A) and a combined microbial agent (CN110250209A) are reported as biocontrol products for preventing and controlling the tobacco mildew disease in the baking period.
Disclosure of Invention
The first purpose of the invention is to provide a bacillus beiLeisi Z002 capable of realizing high-efficiency prevention and control of mildew disease in the baking period. The second object of the present invention is to provide a method for obtaining the Bacillus belgii Z002. The third purpose of the invention is to provide the application of the bacillus beilesensis Z002 in preventing and treating the tobacco mildew disease in the baking period. The fourth purpose of the invention is to provide the application of the Bacillus belgii Z002 in the prevention and treatment of tobacco bacterial wilt, black shank, gray mold, sclerotinia, target spot, root rot and brown spot.
The first object of the present invention is achieved by:
a strain of Bacillus velezensis (Bacillus velezensis) is named as Z002, is identified as a strain of Bacillus velezensis (Bacillus velezensis) through biological characteristic detection and 16S rDNA sequence analysis, is separated from tobacco strain tissues, is preserved in China center for type culture Collection (CCTCC for short, with the address: Wuhan university No. 299 in Wuchang district, Wuhan City, Hubei province, China center for type culture Collection, zip code: 430072) in 10 months and 5 days in 2020, and has the preservation number of CCTCC No: m2020567.
The second object of the present invention is achieved by:
the method for obtaining the Bacillus belgii comprises the working procedures of separation, screening and purification, and specifically comprises the following steps:
A. separation: taking 0.4-0.6 g of tobacco plant tissue with thoroughly sterilized surface to be detected, putting the tobacco plant tissue and the sterilized steel balls into a 2mL sterilized centrifuge tube, adding 400-700 mu L of sterile water, and grinding for 2-4 min by using a tissue grinder, wherein the frequency is 20-40 r/s. Diluting the ground tissue fluid to 10-4100-300. mu.L of homogenate per gradient was applied to a separation medium and repeated 4 times per treatment. After culturing for 36-72 h in the dark at 25-30 ℃, selecting single colonies according to the difference of colony morphology, color and the like, streaking and purifying on the isolated medium again, and selecting single colonies subjected to streaking culture and transferring the single colonies into the test tube inclined plane of the isolated medium for preservation;
the culture conditions were: the temperature is 25-30 ℃, and the time is 40-55 h;
the components of the separation culture medium in g/L are as follows: 3.0-7.0 parts of peptone, 2-4.0 parts of beef extract, 6.0-10.0 parts of sodium chloride, 15-18 parts of agar and 6.8-7.5 parts of pH;
B. screening: a flat plate confronting method is adopted, a puncher with the diameter of 0.5-0.8 cm is used for attaching the rhizopus oryzae filament blocks of the tobacco mildew germs to the center of a screening culture medium plate, endophytic bacteria are inoculated on two sides in a symmetrical streak mode, and the treatment of only inoculating the rhizopus oryzae filament blocks of the tobacco mildew germs is used as a control. Carrying out constant-temperature culture, checking the opposite culture result when the contrast is full of all dishes, recording the width of an inhibition zone, judging whether the separated endophytic bacteria have antagonistic action on pathogenic bacteria according to the growth speed of two bacteria, the existence of the inhibition zone between bacterial colonies, the phenomena of sparse hyphae at the edges of the bacterial colonies, atrophy and the like, and repeating each treatment for 3 times;
the culture conditions were: the temperature is 26-30 ℃, and the time is 48-60 hours;
the screening culture medium comprises the following components in g/L: 180-220 parts of potato, 18-25 parts of glucose, 15-18 parts of agar, and adding distilled water to 1000mL, wherein the pH value is 6.8-7.4;
C. and (3) purification: carrying out streak purification on the screened strains by using a purification culture medium plate to obtain a single colony which grows vigorously, namely Bacillus velezensis Z002;
the culture conditions were: the temperature is 25-30 ℃, and the time is 40-55 h;
the components of the purification culture medium in g/L are as follows: 8.0-12.0 parts of peptone, 2.0-4.0 parts of beef extract, 4.0-6.0 parts of sodium chloride, 15.0-19.0 parts of agar and 7.0-7.5 parts of pH.
The third object of the present invention is achieved by:
the application of the Bacillus belgii Z002 in prevention and treatment of tobacco mildew disease comprises the processes of fermentation, inoculation and prevention and treatment, and specifically comprises the following steps:
a. fermentation: firstly, inoculating Bacillus velezensis Z002 to a slant culture medium to obtain a fermentation seed;
the culture conditions were: the temperature is 25-30 ℃, and the time is 48-72 hours;
the slant culture medium comprises the following components in g/L: 8.0-12.0 parts of peptone, 2.0-4.0 parts of beef extract, 4.0-6.0 parts of sodium chloride, 15.0-19.0 parts of agar and 7.0-7.5 parts of pH;
then inoculating the fermentation seeds into a seed culture solution, wherein the inoculation amount is 3-5 v/v%, and the shake flask liquid loading amount is 25-35 v/v%, so as to obtain a fermentation microbial inoculum;
the culture conditions were: the temperature is 25-30 ℃, and the time is 48-72 hours;
the components of the seed culture solution in g/L are as follows: 8.0-12.0 parts of tryptone, 4.0-6.0 parts of yeast extract, 4.0-6.0 parts of sodium chloride and 7.0-7.5 parts of pH;
b. inoculation: before or after the tobacco leaves are woven into rods, spraying 50-200 mL of fermentation inoculum per rod of tobacco leaves to the cut of the tobacco leaf stalks by using a sprayer;
c. modulation: and (3) baking the tobacco leaves sprayed with the fermentation inoculant for 7-11 days according to a normal modulation program, so that the tobacco mildew disease of the baking room can be effectively prevented and controlled.
The fourth object of the present invention is achieved by:
the application of the Bacillus belgii Z002 in tobacco bacterial wilt, black shank, gray mold, sclerotinia, target spot, root rot and brown spot specifically comprises the following steps:
screening: a plate confronting method is adopted, a puncher with the diameter of 0.5-0.8 cm is used for attaching tobacco mildew pathogen rhizopus oryzae hypha blocks to the center of a screening culture medium plate, endogenous bacteria are inoculated on two sides in a symmetrical streak mode, and the treatment of only inoculating tobacco ralstonia solanacearum, phytophthora parasitica, botrytis cinerea, sclerotinia sclerotiorum, target spot pathogen, rhizoctonia rot pathogen and alternaria alternate hypha blocks is used as a contrast. Carrying out constant-temperature culture, checking the opposite culture result when the contrast is full of all dishes, recording the width of an inhibition zone, judging whether the separated endophytic bacteria have antagonistic action on pathogenic bacteria according to the growth speed of two bacteria, the existence of the inhibition zone between bacterial colonies, the phenomena of sparse hyphae at the edges of the bacterial colonies, atrophy and the like, and repeating each treatment for 3 times;
the culture conditions were: the temperature is 26-30 ℃, and the time is 48-60 hours;
the screening culture medium comprises the following components in g/L: 180-220 parts of potato, 18-25 parts of glucose, 15-18 parts of agar, and distilled water till the volume is 1000mL and the pH value is 6.8-7.4.
The invention has the beneficial effects that:
the Bacillus belgii Z002 can be applied to prevention and treatment of tobacco mildew in the baking period, and has the following advantages:
1. the Bacillus velezensis Z002 is a high-efficiency biocontrol bacterium for the tobacco mildew in the curing period, and the relative control effect on the mildew in the tobacco leaf modulating process is more than 80%;
2. the Bacillus velezensis Z002 can grow in a culture medium containing 0-85 g/L NaCl and having a pH value of 3.0-10.0, has a growth temperature range of 4-60 ℃, has good stress resistance, can fully adapt to temperature and humidity environment changes and water and heat exchange characteristics in a tobacco leaf preparation process, and has a good colonization effect and high activity;
3. the Bacillus velezensis Z002 exists in tobacco plant tissue and tobacco planting soil in great amount, and has wide source, easy separation and culture;
4. the Bacillus velezensis Z002 has the advantages of simple production process, low cost and convenient application, and the tobacco leaves treated by the Bacillus velezensis Z002 have no pesticide residue and are harmless to people and livestock, thereby being beneficial to industrial production and application popularization of the strain;
5. the Bacillus velezensis Z002 has broad-spectrum bactericidal action, and has strong bacteriostatic ability on Ralstonia pseudomonad (Ralstonia pseudomonad), tobacco black shank (Phytophthora nicotianae), Botrytis cinerea (Botrytis cinerea), tobacco Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), tobacco target spot (Thanatephora cumeras), tobacco root rot (Fusarium oxysporum) and tobacco brown spot (Alternaria alternata), namely has good biocontrol potential on the 7 tobacco diseases.
Drawings
FIG. 1: the comparison of the colony morphology of Bacillus beiLeisi Z002 and Rhizopus oryzae of example 1.
FIG. 2: the colony morphology of Bacillus belgii Z002 of example 2 was 24h and 48 h.
FIG. 3: phylogenetic trees constructed using MEGA7 software.
Detailed Description
The present invention is further illustrated but not limited in any way by the following description and any alterations or modifications based on the teachings of the present invention are intended to fall within the scope of the present invention.
The Bacillus velezensis strain Z002 has been preserved in China center for type culture Collection, and the preservation number is CCTCC No: m2020567.
The strain is gram-positive, has elliptical or terminal spores which are not expanded, has smaller spores than bacteria, exists in the bacteria, has a circular bacterial colony with the diameter of 2-4 mm on a culture medium, is flat, has no bulge in the middle, is milky white, glossy and irregular in edge, and is aerobic bacteria. The strain can grow in a culture medium containing 0-85 g/L NaCl and having a pH of 3.0-10.0, and the growth temperature range is 4-60 ℃.
The strain has strong inhibition capability on tobacco mildew bacteria (Rhizopus oryzae), tobacco Ralstonia solanacearum (Ralstonia pseudomonaca), tobacco black shank bacteria (phytophthora nicotianae), tobacco Botrytis cinerea (Botrytis cinerea), tobacco Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), tobacco tarmac (Thanatephora cucumeris), tobacco root rot bacteria (Fusarium oxysporum) and tobacco Alternaria alternata (Alternaria alternata) bacteria.
The relative prevention effect of the strain on tobacco mildew in the baking period is more than 80%.
The method for obtaining the strain comprises the working procedures of separation, screening and purification, and specifically comprises the following steps:
A. separation: taking 0.4-0.6 g of tobacco plant tissue with thoroughly sterilized surface to be detected, putting the tobacco plant tissue and the sterilized steel balls into a 2mL sterilized centrifuge tube, adding 400-700 mu L of sterile water, and grinding for 2-4 min by using a tissue grinder, wherein the frequency is 20-40 r/s. Diluting the ground tissue fluid to 10-4And taking 100-300 mu L of homogenate per gradient and coating the homogenate on a separation medium, and repeating the treatment for 4 times. After culturing for 36-72 h in the dark at 25-30 ℃, selecting single colonies according to the difference of colony morphology, color and the like, streaking and purifying on the isolated medium again, and selecting single colonies subjected to streaking culture and transferring the single colonies into the test tube inclined plane of the isolated medium for preservation;
the culture conditions were: the temperature is 25-30 ℃, and the time is 40-55 h;
the components of the separation culture medium in g/L are as follows: 3.0-7.0 parts of peptone, 2.0-4.0 parts of beef extract, 6.0-10.0 parts of sodium chloride, 15-18 parts of agar and 6.8-7.5 parts of pH;
B. screening: a plate confronting method is adopted, a puncher with the diameter of 0.5-0.8 cm is used for attaching the mycelial blocks of the tobacco mildew germs to the center of a screening culture medium plate, endophytic bacteria are inoculated on two sides in a symmetrical streak mode, and the treatment of only inoculating the mycelial blocks of the tobacco mildew germs, namely rhizopus oryzae is used as a control. Carrying out constant-temperature culture, checking the opposite culture result when the contrast is full of all dishes, recording the width of an inhibition zone, judging whether the separated endophytic bacteria have antagonistic action on pathogenic bacteria according to the growth speed of two bacteria, the existence of the inhibition zone between bacterial colonies, the phenomena of sparse hyphae at the edges of the bacterial colonies, atrophy and the like, and repeating each treatment for 3 times;
the culture conditions were: the temperature is 26-30 ℃, and the time is 48-60 hours;
the screening culture medium comprises the following components in g/L: 180-220 parts of potato, 18-25 parts of glucose, 15-18 parts of agar, and adding distilled water to 1000mL, wherein the pH value is 6.8-7.4;
C. and (3) purification: carrying out streak purification on the screened strains by using a purification culture medium plate to obtain a single colony which grows vigorously, namely Bacillus velezensis Z002;
the culture conditions were: the temperature is 25-30 ℃, and the time is 40-55 h;
the components of the purification culture medium in g/L are as follows: 8.0-12.0 parts of peptone, 2.0-4.0 parts of beef extract, 4.0-6.0 parts of sodium chloride, 15.0-19.0 parts of agar and 7.0-7.5 parts of pH.
And B, the tobacco plant tissue in the step A is a leaf or a stem of the tobacco plant.
The tobacco plant tissue in the step A is preferably the leaves and/or stems of the cured tobacco 'Honghuadajinyuan', and the tobacco planting soil is the tobacco planting soil of the cured tobacco 'Honghuadajinyuan'.
Preferably, 0.4g of the tobacco plant tissue and the sterilized steel balls are put into a 2mL sterilized centrifuge tube together, 600 mu L of sterile water is added, and the mixture is ground for 3min by a tissue grinder with the frequency of 30 r/s. Obtaining a tissue homogenate.
The room temperature in the step A is 20-25 ℃.
The culture condition of the step A is preferably 28 ℃ and the time is 48 h.
The pathogen of the tobacco mildew disease in the step B is Rhizopus oryzae (Rhizopus oryzae).
The culture conditions in the step B are preferably 28 ℃ and 48 h.
The culture conditions in the step C are preferably 28 ℃ and 48 hours.
The application of Bacillus belgii Z002 in preventing and treating tobacco mildew disease comprises the processes of fermentation, inoculation and prevention, and specifically comprises the following steps:
a. fermentation: firstly, inoculating Bacillus velezensis Z002 to a slant culture medium to obtain a fermentation seed;
the culture conditions were: the temperature is 25-30 ℃, and the time is 48-72 hours;
the slant culture medium comprises the following components in g/L: 8.0-12.0 parts of peptone, 2.0-4.0 parts of beef extract, 4.0-6.0 parts of sodium chloride, 15.0-19.0 parts of agar and 7.0-7.5 parts of pH;
then inoculating the fermentation seeds into a seed culture solution, wherein the inoculation amount is 3-5 v/v%, and the shake flask liquid loading amount is 25-35 v/v%, so as to obtain a fermentation microbial inoculum;
the culture conditions were: the temperature is 25-30 ℃, and the time is 48-72 hours;
the components of the seed culture solution in g/L are as follows: 8.0-12.0 parts of tryptone, 4.0-6.0 parts of yeast extract, 4.0-6.0 parts of sodium chloride and 7.0-7.5 parts of pH;
b. inoculation: before or after the tobacco leaves are woven into rods, diluting the fermentation inoculum by 1-4 times, and spraying 50-200 mL of fermentation inoculum per rod;
c. modulation: and (3) baking the tobacco leaves sprayed with the fermentation inoculant for 7-11 days according to a normal modulation program, so that the tobacco mildew disease can be effectively prevented and controlled.
And c, fermenting in the step a, wherein the rotating speed of a shake flask is 180-225 r/min.
The culture conditions in step a are preferably 28 ℃ and the time is 24 h.
The effective viable count of the fermentation inoculum in the step a is 1 multiplied by 1010～1×1012CFU/mL。
The inoculation in step b can also be carried out during the preparation process, during the tobacco filling process or before baking.
The tobacco leaves in the steps b and c comprise any one of flue-cured tobacco, cigar tobacco, burley tobacco and sun-cured tobacco.
Preferably, the tobacco leaves in the steps b and c are flue-cured tobaccos.
The modulation in the step b refers to a process from fresh tobacco leaves to dry tobacco leaves, and specifically relates to a baking process of flue-cured tobacco, an airing process of sun-cured tobacco and a post-modulation aging process of tobacco leaves.
And c, for the modulation in the step c, special modulation conditions do not need to be set for the tobacco leaves in the weaving process, the spraying amount of the microbial inoculum needs to be controlled not to exceed 150 mL/rod for the tobacco leaves inoculated in the tobacco loading process or before baking, and the tobacco leaves are baked for 7-11 days according to normal modulation conditions.
And c, after the tobacco leaves sprayed with the fermentation inoculant are kept for 7-11 d of modulation time, the relative control effect of the tobacco mildew is over 80%.
The following describes embodiments of the present invention in more detail with reference to specific examples:
example 1
Acquisition of Bacillus velezensis Z002
A. Separation: 0.4g of tobacco stem tissue with thoroughly sterilized surface is taken, and is put into a 2mL sterilizing centrifuge tube together with a sterilizing steel ball, 600 mu L of sterile water is added, and the mixture is ground for 3min by a tissue grinder with the frequency of 30 r/s. Diluting the ground tissue fluid to 10-4mu.L of each homogenate was applied to a separation medium (peptone 5.0g, beef extract 3.0g, sodium chloride 8.0g, agar 15.0g, pH 7.0) per gradient and repeated 4 times per treatment. After culturing in the dark at 28 ℃ for 48h, selecting single colonies according to the differences of colony morphology, color and the like, streaking and purifying on a separation culture medium again, and moving the single colonies subjected to streaking culture into a test tube inclined plane of the separation culture medium for preservation;
B. screening: a plate confronting method is adopted, a puncher with the diameter of 0.8cm is used for attaching tobacco mildew germ hypha blocks to the center of a plate of a screening culture medium (200 g of potatoes, 20g of glucose and 15g of agar, distilled water is added to the plate to reach 1000mL, and the pH value is 7.2), endogenous bacteria are inoculated on two sides of the plate which are 3.5cm away from the center of a fungus cake in a symmetrical streak mode, and the treatment of only inoculating the tobacco mildew germ rhizopus oryzae hypha blocks is used as a control. And (3) carrying out constant-temperature culture at 28 ℃, checking the opposing culture result when the total culture dish is full of contrast, recording the width of a bacteriostasis zone, judging whether the separated endophytic bacteria have antagonistic action on pathogenic bacteria according to the growth speed of two bacteria, the existence of the bacteriostasis zone between bacterial colonies, whether hyphae at the edges of the bacterial colonies are sparse and withered and the like, and repeating the treatment for 3 times.
C. And (3) purification: the screened strains are streaked and purified by a purification medium (10.0 g of peptone, 3.0g of beef extract, 5.0g of sodium chloride, 15g of agar and pH 7.2) and cultured at 28 ℃ for 48h to obtain vigorous single colonies.
And (3) test results: and screening a strain with good bacteriostatic ability on the tobacco mildew disease from 208 strains of tobacco endophytic bacteria, and naming the strain as Z002. When the strain Z002 is cultured in opposition to Rhizopus oryzae (Rhizopus oryzae), aerial mycelia of Rhizopus oryzae are sparse, and mycelia are formed only around the fungus cake, while the culture dish is full of mycelia in the control plate; the growth of Rhizopus oryzae in the plates was also limited, and the average zone of inhibition that strain Z002 could form against the growth of Rhizopus oryzae in the medium was 6.67mm (Table 1 and FIG. 1). The strain Z002 can inhibit the growth of rhizopus oryzae aerial hyphae and the growth of hyphae in a culture medium, and the fact that the strain Z002 can inhibit the rhizopus oryzae through various mechanisms is shown.
TABLE 1 antagonistic Effect of Z002 Strain on tobacco mildew disease test results
Example 2
Identification and preservation of Bacillus velezensis Z002
(1) Identification of Bacillus velezensis Z002
The selected strain Z002 is subjected to biological analysis and molecular biological method identification by a conventional method. The molecular identification method comprises the following steps: the kit for extracting the bacterial genome DNA and the method are shown in the specification of the kit. And (3) performing PCR (polymerase chain reaction) amplification by using a primer F27/R1492 under conventional conditions, recovering an amplification product by using glue, connecting the amplification product to a vector pEAZY-T5 Zero vector, thermally shocking and transforming escherichia coli competent cells DH5 alpha, and selecting colonies to perform colony PCR identification by using M13F/M13R as a primer. Positive clones were sent to Shanghai Yingjun Biotech Co., Ltd for sequencing.
The test results are reported below:
1. the culture characteristics are as follows: the strain is cultured on an NA plate culture medium at 28 ℃, and the colony is light milky, purulent, round, neat in edge and moist in surface at the initial culture stage; the bacterial colony at the later stage of culture is light yellow, the flat center has no bulge, the edge is irregular, the surface is dry, and the wrinkles are not obvious (see figure 2); the white mycoderm is formed on the surface of the culture medium by static culture in the liquid culture medium. The morphological characteristics are basically consistent with those of the bacillus described in the handbook of identification of common bacteria systems (Dongxu bead et al, science Press, 2001), and the Z002 strain is preliminarily judged to be bacillus.
2. Morphological characteristics: the strain is cultured at 28 ℃, spores are mesogenic or terminal, the strain is oval and does not expand, and the spores are smaller than bacteria under a microscope. Negative in acid-fast staining, no parasit crystal, capable of moving, and periflagellar. The average size of the cells is 0.50 to 0.70 μm × 2.1 to 2.9 μm, and gram staining is positive.
3. The stability is characterized in that: the strain can grow in a culture medium containing 0-85 g/L NaCl and having a pH value of 3.0-10.0, the growth temperature range is 4-60 ℃, the optimum growth temperature is 28-35 ℃, and the optimum pH value is 7.0-7.2.
4. 16S rDNA sequence analysis: the sequence comparison of the 16S rDNA sequence of the strain and an Ezbiochoud database (https:// www.ezbiocloud.net /) includes that the 16S rDNA sequence homology of the strain and the Bacillus reaches 99.71%, 99.51%, 99.52% and 99.39% with the B-16 sequence homology of a Bacillus nematocida mode strain, NCIB 3610 and KCTC 13613 sequence homology of a Bacillus subtilis mode strain and DSM 7 sequence homology of the Bacillus amyloliquefaciens mode strain. Comparing rRNA/ITS database by using NCBI online analysis tool BLASTn, finding that the homology between the Z002 strain 16S rDNA sequence and the Bacillus velezensis strain FZB42 sequence is up to 99.60%. The phylogenetic tree was constructed using MEGA7 software, and as a result (see FIG. 3) the Z002 strain was clustered with Bacillus beijerinckii FZB42, indicating that Z002 was Bacillus velezensis and was a new strain.
The strain identifies the Z002 strain as Bacillus belgii (Bacillus velezensis) through sequence comparison and biological characteristics.
The 16S rDNA sequence of the Z002 strain is shown in a sequence table.
(2) Deposit of Bacillus velezensis Z002
From the above identification results, it was confirmed that the strain Z002 was a strain of Bacillus subtilis (Bacillus velezensis) and named as Z002. Has been preserved in China center for type culture Collection (CCTCC for short, the address is Wuhan university No. eight one way 299 in Wuchang district, Wuhan city, Hubei province, China center for type culture Collection, zip code: 430072) in 10 months and 5 days in 2020, and the preservation number is CCTCC No: m2020567. The 16S rDNA sequence of bacillus belgii Z002 was submitted to GenBank under sequence accession No. MW 064126.
Example 3
The test of the control test of the Bacillus velezensis Z002 on the tobacco mildew disease in the curing period is carried out in the Phoenix town of GuanCity under the State of Dali of Yunnan province, the tobacco variety is 'Honghuadajinyuan', and the tobacco leaves are the middle leaves.
The test method comprises the following steps:
a. fermentation: activating the strain Z002 in a slant culture medium (10.0 g of peptone, 3.0g of beef extract, 5.0g of sodium chloride, 15.0g of agar and pH 7.0) from an ultralow temperature refrigerator at-80 ℃, and culturing in a constant temperature incubator at 28 ℃ for 48 h; selecting a strain of armillaria, inoculating the strain into a 50mL centrifugal tube containing 10mL seed culture solution (10.0 g of peptone, 3.0g of beef extract, 5.0g of sodium chloride, 15.0g of agar and pH 7.0), and placing the tube in a constant-temperature incubator at 28 ℃ for 24 hours; then transferring the seed liquid to a fermentation medium containing 1000mL (peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar 15.0g, pH 7.0), and culturing in a constant-temperature incubator at 28 ℃ for 72h to obtain a fermentation microbial inoculum;
b. inoculation: the test is carried out in a Phoenix town of Guanhou city under Dalizhou, after tobacco leaves are woven into rods, the fermentation inoculum is sprayed to the wound of a petiole by a sprayer according to 200mL of tobacco leaves per rod, the treatment of spraying the petiole by a fermentation culture medium is used as a reference, and the treatment is repeated for 3 times when 10 tobacco leaves are treated;
c. modulation: and (3) baking the tobacco leaves sprayed with the fermentation inoculant according to a normal modulation program for 9d, investigating disease grades of various treatments after modulation, and calculating disease indexes and relative prevention effects. The disease degree of tobacco leaves is classified into 0, 1, 3, 5, 7 and 9 grades according to disease grades (table 2), the disease index is 100 × Σ (number of disease plants of each grade × representative value of each grade)/(total number of investigation plants × highest representative value), and the relative control effect of biocontrol bacteria is 100 × (control group disease index-treatment group disease index)/(control group disease index).
TABLE 2 grading of the disease states of rotten tobacco leaves
| Grade of disease condition | Occurrence of mildew |
| 0 | Normal tobacco leaf |
| 1 | The mildew on tobacco stalk is less than 1cm, and the leaf is not attacked |
| 3 | The mildew on tobacco stalk is more than 1cm, and the leaf is not attacked |
| 5 | The tobacco stalk is mildewed, and the mildewed area of the leaves reaches more than 1 percent to 25 percent |
| 7 | The tobacco stalk is mildewed, and the mildewed area of the leaves reaches more than 26 percent to 50 percent |
| 9 | The tobacco stalk is mildewed, and the mildewed area of the leaves reaches more than 50 percent |
And (3) test results: as can be seen from the data in Table 3, the strain Z002 has a good effect of preventing and treating the tobacco mildew disease in the baking period. Weaving tobacco leaves into rods, spraying Z002 fermentation liquor, and checking the disease index to be 7.40 after baking; and the disease index of the spraying fermentation liquid culture medium is 62.96. The results show that the petiole of the tobacco leaf is treated by the Z002 microbial inoculum fermentation liquor, the mildew disease of the tobacco can be effectively prevented and controlled, and the relative prevention effect reaches 88.23%.
TABLE 3 comparative test results of the prevention of tobacco mildew by the strain Z002
| Treatment of | Index of disease condition | Relative control effect (%) |
| Z002 liquid preparation | 7.40b | 88.23％ |
| CK | 62.96a | - |
Example 4
Test for controlling tobacco mildew during baking period by using Bacillus velezensis Z002
Example 4 the procedure was the same as in example 3 except for the following tests and results.
1. The test is carried out in Nanjian town of Nanjian county, Dalizhou, Yunnan province, and the tobacco leaves are the upper leaves of a flue-cured tobacco variety 'Honghuadajinyuan'.
2. In the test, after tobacco leaves are woven into rods, 2 times of diluent of strain Z002 fermentation liquor is adopted, and 75 mL/rod is sprayed on petioles by a sprayer.
3. In the test, 80 tobacco rods are co-processed by the strain Z002 fermentation liquor; controls were not treated to develop spontaneously.
TABLE 4 comparative test results of the prevention of the mildew disease of tobacco by the strain Z002
And (3) test results: the test co-treats 80 tobacco rods, wherein 57 tobacco rods have no disease, 23 tobacco rods have disease grade of 1, the disease incidence rate is 28.75%, and the disease index is 3.19; a total of 81 controls with 11 non-diseased, 40 graded 1, and the remaining 30 graded 3, with a disease incidence of 86.42 and a disease index of 17.83. The results show that the 2-time diluent of the Z002 fermentation liquor has a good prevention and treatment effect on tobacco mildew in the baking period, and the relative prevention effect is 82.08%. The Bacillus belgii Z002 has good effect of preventing and treating the tobacco mildew disease in the baking period, and has good development potential.
Example 5
-test of inhibition of Bacillus velezensis Z002 against 7 tobacco pathogens
Example 5 the procedure was the same as in example 1 except for the following tests and results.
1. The bacterial wilt of tobacco (Ralstonia pseudolaricina) is diluted to 10-4200 mu L of the strain is coated on a screening culture medium plate, the screening culture medium plate is placed on a super clean bench to be dried, a single colony of the biocontrol bacteria to be detected is picked by using a sterile toothpick and inoculated at the position of the surface of the screening culture medium, which is 2.50cm away from the center of a culture dish, 4 points are symmetrically inoculated on each dish, and the screened biocontrol bacteria is directly placed in a 28 ℃ incubator to be cultured for 48 hours after being dried.
2. In the test, tobacco black shank bacteria, tobacco botrytis cinerea, tobacco sclerotinia sclerotiorum, tobacco target spot bacteria, tobacco root rot bacteria and tobacco alternaria alternata are taken as indicator bacteria respectively, and the bacteriostasis capability of the Z002 strain on the pathogenic bacteria is detected.
And (3) test results: as can be seen from Table 5, Bacillus belgii Z002 has strong bacteriostatic action on 7 important tobacco disease pathogenic bacteria to be tested, and has super strong bacteriostatic action on alternaria alternate and sclerotinia sclerotiorum. The Z002 strain has strong inhibition capability on tobacco Ralstonia pseudomonad (Ralstonia pseudomonacea), tobacco black shank (Phytophthora nicotianae), tobacco Botrytis cinerea (Botrytis cinerea), tobacco Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), tobacco target spot (Thanatephora cuprinis), tobacco root rot (Fusarium oxysporum) and tobacco brown spot (Alternaria alternata), namely has good biocontrol potential on the 7 tobacco diseases.
TABLE 5 inhibitory potency of Bacillus belgii Z002 against 7 tobacco pathogens
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> Bacillus belgii Z002, and acquisition method and application thereof
<130> 2020-11-05
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1511
<212> DNA
<213> Bacillus velezensis
<400> 1
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ctgcctgtaa gactgggata actccgggaa accggggcta ataccggatg gttgtctgaa 180
ccgcatggtt cagacataaa aggtggcttc ggctaccact tacagatgga cccgcggcgc 240
attagctagt tggtgaggta acggctcacc aaggcaacga tgcgtagccg acctgagagg 300
gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg 360
aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc 420
ggatcgtaaa gctctgttgt tagggaagaa caagtgccgt tcaaataggg cggcaccttg 480
acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 540
tggcaagcgt tgtccggaat tattgggcgt aaagggctcg caggcggttt cttaagtctg 600
atgtgaaagc ccccggctca accggggagg gtcattggaa actggggaac ttgagtgcag 660
aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca 720
gtggcgaagg cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga 780
acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagggggt 840
ttccgcccct tagtgctgta gctaacgcat taagcactcc gcctggggag tacggtcgca 900
agactgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960
tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc tagagatagg 1020
acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080
gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca gcattcagtt 1140
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tcatgcccct tatgacctgg gctacacacg tgctacaatg gacagaacaa agggcagcga 1260
aaccgcgagg ttaagccaat cccacaaatc tgttctcagt tcggatcgca gtctgcaact 1320
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aggtaacctt ttaggagcca gccgccgaag gtgggacaga tgattggggt gaagtcgtaa 1500
caaggtaacc g 1511
Claims (13)
1. The Bacillus velezensis strain Z002 has been preserved in China center for type culture Collection, and the preservation number is CCTCC No: m2020567.
2. The method for obtaining Bacillus belgii Z002 according to claim 1, comprising the steps of:
a first step, separation, comprising:
(1) taking 0.4-0.6 g of tobacco plant tissues with thoroughly sterilized surfaces after inspection, putting the tobacco plant tissues and sterilizing steel balls into a 2mL sterilizing centrifuge tube, adding 400-700 mu L of sterile water, and grinding for 2-4 min by using a tissue grinder, wherein the frequency is 20-40 r/s;
(2) diluting the ground tissue fluid to 10-4Coating 100-300 mu L of homogenate liquid on a separation culture medium in each gradient, and repeating the treatment for 4 times;
(3) after dark culture, selecting single colonies according to the difference of colony morphology and color, streaking and purifying on a separation culture medium again, and moving the single colonies subjected to streaking culture into a test tube inclined plane of the separation culture medium for preservation;
and a second step, screening, comprising:
(1) adopting a plate confronting method, adhering the rhizopus oryzae filament blocks of the tobacco mildew germs to the center of a screening culture medium plate by using a puncher with the diameter of 0.5-0.8 cm, symmetrically scribing and inoculating endophytic bacteria at two sides, and taking the treatment of only inoculating the rhizopus oryzae filament blocks of the tobacco mildew germs as a contrast;
(2) carrying out constant-temperature culture, checking the opposite culture result when the two bacteria grow on the whole dish in a contrasting manner, recording the width of an inhibition zone, judging whether the separated endophytic bacteria have antagonistic action on pathogenic bacteria according to the growth speed of the two bacteria, the existence of the inhibition zone between bacterial colonies and the phenomena of sparseness and atrophy of hyphae at the edge of the bacterial colonies, and repeating the treatment for 3 times;
and step three, purification, comprising:
and (3) streaking and purifying the screened strain by using a purification medium plate to obtain a single colony which is exuberant, namely the Bacillus belgii Z002.
3. An acquisition method as claimed in claim 2, characterized in that in the first separation step:
the culture conditions are as follows: the temperature is 25-30 ℃, and the time is 40-55 h;
the components of the separation culture medium in g/L are as follows: 3.0-7.0 parts of peptone, 2-4.0 parts of beef extract, 6.0-10.0 parts of sodium chloride and 15-18 parts of agar; the pH value is 6.8-7.5.
4. An acquisition method as claimed in claim 2, characterized in that in the second step of screening:
the culture conditions are as follows: the temperature is 26-30 ℃, and the time is 48-60 hours;
the screening culture medium comprises the following components in g/L: 180-220 parts of potatoes, 18-25 parts of glucose and 15-18 parts of agar, and adding distilled water to 1000 mL; the pH value is 6.8-7.4.
5. The method for obtaining as claimed in claim 2, characterized in that in the third purification step:
the culture conditions are as follows: the temperature is 25-30 ℃, and the time is 40-55 h;
the components of the purification culture medium in g/L are as follows: 8.0-12.0 parts of peptone, 2.0-4.0 parts of beef extract, 4.0-6.0 parts of sodium chloride and 15.0-19.0 parts of agar; the pH value is 7.0-7.5.
6. The acquisition method according to any one of claims 2 to 5, characterized in that:
in the first step, the tobacco plant tissue is the leaves and/or stems of flue-cured tobacco Honghuadajinyuan.
7. Use of bacillus belgii according to claim 1 for the control of tobacco mildew disease, comprising the steps of:
step a, fermentation, comprising:
(1) inoculating Bacillus belgii Z002 to a slant culture medium to obtain a fermentation seed;
(2) inoculating the fermentation seeds into a seed culture solution, wherein the inoculation amount is 3-5 v/v%, and the shake flask liquid loading amount is 25-35 v/v%, so as to obtain a fermentation microbial inoculum;
step b, inoculation: before or after the tobacco leaves are woven into rods, diluting the fermentation inoculum by 1-4 times, and spraying 50-200 mL of fermentation inoculum to the tobacco leaf stalk wound of each rod;
step c, modulation: and (3) baking the tobacco leaves sprayed with the fermentation inoculant for 7-11 days according to a normal modulation program, so that the tobacco mildew disease of the baking room can be effectively prevented and controlled.
8. The use of Bacillus belgii in the control of tobacco mildew disease according to claim 7, wherein said inoculating Bacillus belgii Z002 onto a slant medium in step a fermentation further comprises:
the slant culture medium comprises the following components in g/L: 8.0-12.0 parts of peptone, 2.0-4.0 parts of beef extract, 4.0-6.0 parts of sodium chloride and 15.0-19.0 parts of agar; the pH value is 7.0-7.5;
the culture conditions are 25-30 ℃ and the time is 48-72 h.
9. The use of bacillus belgii for the control of tobacco mildew disease according to claim 7, wherein said inoculating the fermented seeds into a seed culture broth in step a fermentation further comprises:
the components of the seed culture solution in g/L are as follows: 8.0-12.0 parts of tryptone, 4.0-6.0 parts of yeast extract and 4.0-6.0 parts of sodium chloride; the pH value is 7.0-7.5;
the culture conditions are as follows: the temperature is 25-30 ℃, and the time is 48-72 hours.
10. Use of bacillus belgii in the control of tobacco mildew disease according to claim 7, wherein:
in the step a, the effective viable count of the zymophyte agent is 1 multiplied by 1010～1×1012CFU/mL。
11. Use of bacillus belgii in the control of tobacco mildew disease according to any one of claims 7 to 10, wherein:
and c, in the step a, inoculation is carried out in the tobacco leaf weaving process and the tobacco loading process.
12. Use of bacillus belgii in the control of tobacco mildew disease according to claim 11, wherein:
in the step c, special modulation conditions do not need to be set for the tobacco leaves in the weaving process, the spraying amount of the microbial inoculum needs to be controlled not to exceed 150 mL/rod for the tobacco leaves inoculated in the tobacco loading process or before baking, and the tobacco leaves are baked for 7-11 days according to normal modulation conditions.
13. Use of the Bacillus belgii of claim 1 for the control of tobacco bacterial wilt (Ralstonia pseudomonaceum), tobacco black shank (Photorhora nicotianae), tobacco gray mold (Botrytis cinerea), tobacco Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), tobacco target spot (Thanatephora cuumeris), tobacco root rot (Fusarium oxysporum) and/or tobacco brown spot (Alternaria alternata).
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| CN111440743A (en) * | 2020-04-09 | 2020-07-24 | 山东农业大学 | Bacillus belezii PEBA20 for disease prevention and growth promotion and soil improvement and application thereof |
| CN113969247A (en) * | 2021-11-09 | 2022-01-25 | 云南省烟草农业科学研究院 | Bacterium for inhibiting tobacco disease pathogenic bacteria and application thereof |
| CN114657097A (en) * | 2022-03-25 | 2022-06-24 | 青岛农业大学 | Bacillus beleisi LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof |
| CN114958640A (en) * | 2021-07-15 | 2022-08-30 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Bacillus velezensis EM-1 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114958640A (en) * | 2021-07-15 | 2022-08-30 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Bacillus velezensis EM-1 and application thereof |
| CN113969247A (en) * | 2021-11-09 | 2022-01-25 | 云南省烟草农业科学研究院 | Bacterium for inhibiting tobacco disease pathogenic bacteria and application thereof |
| CN113969247B (en) * | 2021-11-09 | 2023-02-28 | 云南省烟草农业科学研究院 | Bacterium for inhibiting tobacco disease pathogenic bacteria and application thereof |
| CN114657097A (en) * | 2022-03-25 | 2022-06-24 | 青岛农业大学 | Bacillus beleisi LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof |
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