CN114480166B - Bacillus mojavensis and application thereof - Google Patents
Bacillus mojavensis and application thereof Download PDFInfo
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- CN114480166B CN114480166B CN202111578112.1A CN202111578112A CN114480166B CN 114480166 B CN114480166 B CN 114480166B CN 202111578112 A CN202111578112 A CN 202111578112A CN 114480166 B CN114480166 B CN 114480166B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
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Abstract
The invention provides bacillus mojavensis and application thereof, and relates to the technical field of microorganisms. The bacillus mojavensis provided by the invention is bacillus mojavensis (Bacillus mojavensis) FTB-P3, and is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 23349 in the year 09 and 02 of 2021. The bacterium has stronger capability of decomposing organic phosphorus and inorganic phosphorus at the same time; in addition, the fungus has an inhibiting effect on plant pathogenic bacteria such as wheat scab, corn big spot, rice sheath blight, cotton fusarium wilt, rape sclerotinia rot, watermelon fusarium wilt and tomato gray mold, and can be applied to control of related diseases.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus mojavensis capable of dissolving phosphorus and preventing and controlling crop diseases and application thereof.
Background
Phosphorus is one of the essential nutrient elements for plant growth, and the photosynthesis and in vivo biochemical process of the plant are all necessary to participate in the phosphorus, but the phosphorus exists in the soil mainly in a insoluble mineral state, and the insoluble mineral phosphorus cannot be directly absorbed and utilized by crops. To increase crop yield, over 90kg/hm 2 The phosphate fertilizer is applied to the soil, part of the phosphate fertilizer is absorbed and utilized by crops, and most of the phosphate fertilizer is converted into insoluble phosphorus to be applied back to the soil. The application of large amounts of phosphate fertilizers is increasingly serious with soil hardening, soil acidification, and soil impoverishment. Therefore, the utilization efficiency of phosphorus in soil is improvedThe method has very important significance in reducing the application amount of the chemical phosphate fertilizer. A large number of microorganisms exist in the soil, and can convert phosphorus which is difficult to be absorbed and utilized by plants into an absorbable and utilized form, and the microorganisms with the capability are called phosphate solubilizing bacteria or phosphate solubilizing bacteria. The development and utilization of phosphorus-dissolving microorganisms as microbial fertilizers has been the focus of attention. The phosphorus-dissolving microorganisms in the soil can convert insoluble phosphorus into soluble phosphorus through self growth and metabolism, so that crops can be promoted to absorb and utilize the insoluble phosphorus. However, microorganisms are only able to solubilize poorly soluble phosphorus, and cannot be considered as phosphate solubilizing bacteria, which also have to take into account the crop growth promoting effect. Therefore, reasonable utilization of phosphorus-dissolving microorganisms has great prospect for improving crop yield, reducing chemical fertilizer use and reducing environmental soil pollution.
In the growth process of crops, various diseases are easy to generate due to the influence of a plurality of factors such as growth environment, the diseases cause huge losses to agricultural production, chemical control methods are generally adopted by people in the past, and chemical pesticides have the advantages of high efficiency, quick response, simple and convenient application, large-scale application and the like, but due to the fact that the chemical pesticides are used for a large amount for a long time, the drug-resistant population of plant diseases and insect pests is increased, human health is threatened, the balance of a natural agricultural ecological system is damaged, and the like. 20. Since 90 th century, biological control of plant diseases has been widely focused on the enhancement of environmental awareness and the importance of food safety problems, and has been effective in the control of diseases of crops such as rice, wheat, corn, cotton, fruit trees, vegetables, etc. Bacillus is an important group of biocontrol microorganisms, is one of microorganisms with the widest distribution in nature, has the advantages of quick propagation, strong colonization capability and simple nutrition requirement, and has the functions of inhibiting various plant diseases and promoting plant growth.
Disclosure of Invention
Therefore, the invention aims to provide the bacillus mojavensis which has strong capability of decomposing organic phosphorus and inorganic phosphorus, has obvious inhibition effect on 7 common crop pathogenic bacteria such as wheat gibberella, corn maculosis, rice sheath blight and the like, and can be applied to control of related diseases.
In order to achieve the above object, the present invention provides the following technical solutions:
bacillus mojavensis (Bacillus mojavensis) FTB-P3 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 23349 in the year 09 and 02 of 2021; the nucleotide sequence of the 16S rDNA of the bacillus mojavensis is shown as SEQ ID NO. 1.
The physiological and morphological characteristics of the bacillus mojavensis (Bacillus mojavensis) FTB-P3 are as follows: gram-negative bacteria, aerobic bacteria; the thallus is in a rod shape, and the size is (0.5-0.8) mu m x (2.0-4.0) mu m; the spores are elliptical and 0.5-1.0 mu m long; the colony is regular, the edge is smooth and moist, and is opaque and yellowish.
Another object of the present invention is to provide a microbial agent, to which the above Bacillus mojavensis (Bacillus mojavensis) FTB-P3 is added.
Preferably, the effective bacterial count of the bacillus mojavensis in the microbial agent is 4.0-8.0x10 9 cfu/g。
Preferably, the bacillus mojavensis is immobilized on a porous carrier material; the porous carrier material comprises one or more of zeolite, activated carbon, diatomite and porous ceramsite.
Preferably, the bacillus mojavensis culture process comprises the following steps: activating the bacillus mojavensis, inoculating the bacillus mojavensis into a fermentation culture medium, and culturing for 12-24 hours at 28-32 ℃.
More preferably, the fermentation medium comprises the following components in parts by weight: 8-12 parts of peptone, 4-6 parts of beef extract powder, 4-6 parts of sodium chloride, 9-15 parts of agar, 800-1200 parts of distilled water and pH=7.2-7.4.
The invention also provides application of the bacillus mojavensis or the microbial agent in soil phosphate solubilizing and/or plant disease control; the plant disease pathogenic bacteria include: wheat scab Fusarium graminearum schw, corn big spot germ Exserohilum turcicum, rice sheath blight germ Rhizoclonia solani, cotton fusarium wilt germ Fusarium oxysporum f.sp.gasnfectum, rape sclerotinia sclerotiorum Sclerotinia sclerotiorum, watermelon fusarium wilt germ Fusarium oxysporum f.sp.niveum, tomato gray mold germ Botrytis cinerea Pers.
The invention has the beneficial effects that:
the invention provides bacillus mojavensis and application thereof, and the bacillus mojavensis has the effects of phosphate dissolving and crop pathogen inhibiting. The bacillus mojavensis FTB-P3 is applied to farmlands in the form of a microbial inoculum, so that the utilization rate of phosphorus by crops can be obviously improved; has obvious inhibition effect on the growth of wheat scab Fusarium graminearum schw, corn big spot germ Exserohilum turcicum, rice sheath blight germ Rhizoclonia solani, cotton fusarium wilt germ Fusarium oxysporum f.sp.gasnfectatum, rape sclerotinia germ Sclerotinia sclerotiorum, watermelon fusarium wilt germ Fusarium oxysporum f.sp.niveum and tomato gray mold germ Botrytis cinerea Pers, can be used for the control of related diseases, and has better control effect than that of conventional chemical preparations.
Drawings
FIG. 1 is a colony morphology of FTB-P3;
FIG. 2 is a diagram of FTB-P3 cells under a microscope.
Detailed Description
The invention provides bacillus mojavensis, the strain number is FTB-P3, and the bacillus mojavensis is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) in 09 month 02 of 2021, the preservation address is North Chen Xili No.1, 3 of the Korean region of Beijing, and the preservation number is CGMCC No.23349.
The bacillus mojavensis FTB-P3 is obtained by screening farmland soil of villages and villages of Yutian Zhenjia county of Tangshan city in Hebei province. The FTB-P3 strain obtained by screening was identified as Bacillus mojavensis (Bacillus mojavensis) by morphological identification and 16S rDNA sequencing analysis. The bacteria can grow in large quantity by aerobic culture for 18 hours at 30 ℃ on a nutrient agar culture medium, and the colony morphology result is shown as figure 1, and the colony is regular, smooth and moist at the edge, opaque and yellowish. As a result of observation under a microscope, the cells were found to be rod-like and were generally (0.5-0.8) μm X (2.0-4.0) μm in size, and the spores were oval in shape and 0.5-1.0 μm in length, as shown in FIG. 2. The sequence of the bacterial 16s rDNA universal primer is shown as SEQ ID NO.2 and SEQ ID NO. 3:
27F(5’-AGTTTGATCMTGGCTCAG-3’)
1492R (5'-GGTTACCTTGTTACGACTT-3'), wherein M represents degenerate base, M=A/C, namely M is A or C, the strain FTB-N1 is amplified and forward sequenced, the length of the obtained sequence is 1060bp, and the specific sequence is shown as SEQ ID NO. 1.
And comparing the sequencing results in NCBI database, and finally identifying and screening to obtain a sample which is bacillus mojavensis (Bacillus mojavensis) FTB-P3.
Another object of the present invention is to provide a microbial agent containing the Bacillus mojavensis, wherein the effective bacterial count of lysobacter in the microbial agent is preferably 4.0 to 8.0X10 9 cfu/g, the microbial agent also has the biological activity function of the FTB-P3 of the bacillus mojavensis (Bacillus mojavensis); further preferably, the effective bacterial count is 6.0 to 8.0X10 9 cfu/g. Preferably, the culturing process of the bacillus mojavensis comprises the following steps: activating the bacillus mojavensis (Bacillus mojavensis) FTB-P3, inoculating the bacillus mojavensis into a fermentation medium, and culturing for 12-24 hours at 28-32 ℃; further preferably, the cells are inoculated into a fermentation medium and cultured at 30℃for 18 hours.
Preferably, the fermentation medium comprises the following components in parts by weight: 8-12 parts of peptone, 4-6 parts of beef extract powder, 4-6 parts of sodium chloride, 9-15 parts of agar, 800-1200 parts of distilled water and pH=7.2-7.4; further, 10 parts of peptone, 5 parts of beef extract powder, 5 parts of sodium chloride, 12 parts of agar, 1000 parts of distilled water and ph=7.2-7.4 are preferable. The raw materials of the culture medium are not particularly limited, and the culture medium can be prepared by using products conventionally and commercially available in the field.
The invention preferably fixes the cultured bacillus mojavensis (Bacillus mojavensis) FTB-P3 on a porous carrier material, and more preferably the porous carrier material comprises one or more of zeolite, activated carbon, diatomite and porous ceramsite.
The invention also provides application of the bacillus mojavensis (Bacillus mojavensis) FTB-P3 or a microbial agent prepared from the bacillus mojavensis in soil phosphate solubilizing and/or crop disease control. Further preferably, the microbial inoculum is applied in an amount of 3 to 10% by mass of the soil, and more preferably, the microbial inoculum is applied in an amount of 5% by mass of the soil. The plant disease pathogenic bacteria of the present invention include: wheat scab Fusarium graminearum schw, corn leaf spot germ Exserohilum turcicum, rice sheath blight germ Rhizoclonia solani, cotton fusarium wilt germ Fusarium oxysporum f.sp.gasnfectum, rape sclerotinia sclerotiorum Sclerotinia sclerotiorum, watermelon fusarium wilt germ Fusarium oxysporum f.sp.niveum, tomato gray mold germ Botrytis cinerea Pers.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Inoculating bacillus mojavensis FTB-P3 into 10 parts of peptone, 5 parts of beef extract powder, 5 parts of sodium chloride, 12 parts of agar and 1000 parts of distilled water, and culturing for 18 hours at the temperature of 30 ℃ with the pH being regulated to be 7.3 to obtain bacillus mojavensis FTB-P3 fermentation liquor. Mixing the prepared fermentation liquor with porous carrier material zeolite to obtain Bacillus mojavensis FTB-P3 with effective viable count of 6.0X10 9 cfu/g microbial agent.
Example 2
Inoculating bacillus mojavensis FTB-P3 into 8 parts of peptone, 4 parts of beef extract powder, 4 parts of sodium chloride, 9 parts of agar and 800 parts of distilled water, and culturing for 24 hours at the temperature of 28 ℃ with the pH being regulated to be 7.2, so as to obtain bacillus mojavensis FTB-P3 fermentation liquor. Mixing the prepared fermentation broth with porous carrier material (such as active carbon and diatomite, etc.), to obtain Bacillus mojavensis FTB-P3 with effective viable count of 4.0X10 9 cfu/g of microbial agent.
Example 3
Bacillus mojavensis FTB-P3 is inoculated in 12 parts of peptone, 6 parts of beef extract powder, 6 parts of sodium chloride, 15 parts of agar and 1200 parts of distilled water, and the pH is regulated to be 7.4, and the mixture is cultured for 12 hours at the temperature of 32 ℃. Obtaining the bacillus mojavensis FTB-P3 fermentation liquor. Mixing the prepared fermentation broth with porous carrier material (zeolite and porous ceramsite, etc.), to obtain Bacillus mojavensis FTB-P3 with effective viable count of8.0×10 9 cfu/g microbial agent.
Example 4
And determining the effective content in the culture solution by using a molybdenum blue colorimetric method, and determining the phosphorus dissolving capacity.
Bacillus mojavensis FTB-P3 was activated and inoculated in 10g peptone, 5g beef extract powder, 5g sodium chloride, 12g agar, 1000ml distilled water, ph=7.3, and cultured at 30 ℃ for 18 hours to prepare a bacterial suspension. The plates were perforated with a punch on lecithin and phospholime plates, 3 plates per plate, 30. Mu.L of bacterial suspension was inoculated per well, 3 plates were repeated, and incubation was performed at 30 ℃. After 72 hours, the diameter of the lecithin flat-plate turbid ring and the diameter of the phospholime flat-plate transparent ring were measured.
Test results: the average diameter of a turbid ring formed by the bacillus mojavensis FTB-P3 on a lecithin flat plate is 2.566cm, and the diameter of a transparent ring formed on a phosphorus lime flat plate is 2.473cm, which shows that the bacillus mojavensis FTB-P3 has stronger capability of decomposing organic phosphorus and inorganic phosphorus.
Example 5
Bacillus mojavensis FTB-P3 is inoculated in PDA culture medium and cultured at 30 deg.c for 18 hr for activation. The plant pathogenic bacteria including wheat gibberella, corn big spot, rice sheath blight, cotton fusarium wilt, rape sclerotium germ, watermelon fusarium wilt and tomato gray mold germ are put in the center of a PDA culture medium by using an 8mm puncher, bacillus mojavensis FTB-P3 is inoculated at a position 2cm away from the edge of a plate, the plate is repeatedly used for 3 times, the plate is cultured for 5 days at 30 ℃, whether a bacteriostasis ring is generated around a colony is observed, and the bacterial inhibition rate is counted.
Antibacterial ratio = (control colony diameter-treated colony diameter)/control colony diameter x 100%
TABLE 1 antibacterial Rate of Bacillus mojavensis FTB-P3 against 7 pathogenic bacteria
As can be seen from Table 1, bacillus mojavensis FTB-P3 has various degrees of inhibition on 7 common crop pathogens.
Example 6
Activating Bacillus mojavensis FTB-P3, inoculating 10g peptone, 5g beef extract powder, 5g sodium chloride, 12g agar, 1000ml distilled water, adjusting pH=7.3, culturing at 30deg.C for 18 hr, fixing the activated strain on zeolite porous material, and preparing microbial agent 1-3 (effective bacterial count of Bacillus mojavensis FTB-P3 inoculated zeolite is 4.0X10) 9 cfu/g、6.0×10 9 cfu/g and 8.0X10 9 cfu/g)。
The cotton fusarium wilt bacteria NJ are cultured on a PSA culture medium for 7 days, and are beaten into bacterial blocks with the diameter of 6mm by a puncher.
Taking loam of a certain cultivated land in a back lake industrial aggregation area of Yutian county, sterilizing by damp heat for 2 times, 1 hour each time, and filling into pots, wherein each pot is 5kg. The zeolite and microbial agent are applied according to 5 per mill of soil mass for 1-3, in addition, a carbendazim treatment group is arranged, and 50% of carbendazim wettable powder cotton seeds are used for soaking seeds for 1h.
Inoculating 50 pathogenic bacteria in each pot by using a pathogen block seed dressing method. The test consisted of 5 treatments: firstly, blank control (fusarium wilt bacteria); 2. microbial agent 1 (microbial agent 1+ fusarium wilt); 3. microbial agent 2 (microbial agent 2+ fusarium wilt); 4. microbial inoculum 3 (microbial inoculum 3+fusarium wilt) and 5. Carbendazim (carbendazim+fusarium wilt) are repeated 3 times. 20 cotton seeds were sown in each pot, and the disease condition of cotton wilt was investigated 30 days after sowing.
TABLE 2 Effect of different treatments on the incidence of cotton wilt disease
| Treatment of | Disease plant rate% | Preventing and curing effect is% |
| Blank control | 71.67 | —— |
| Microbial agent 1 | 43.33 | 39.54 |
| Microbial agent 2 | 35.00 | 51.16 |
| Microbial agent 3 | 26.67 | 62.79 |
| Carbendazim | 53.33 | 25.59 |
As can be seen from Table 2, the Bacillus mojavensis FTB-P3 has good prevention and treatment effects on cotton wilt, can reach 39.54-62.79%, and is obviously superior to common bactericide carbendazim seed soaking treatment.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and variations could be made by those skilled in the art without departing from the principles of the present invention, and such modifications and variations should also be considered as being within the scope of the present invention.
Sequence listing
<110> Feng Tian Bao agricultural technologies Co., ltd
<120> a Bacillus mojavensis and application thereof
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gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta ctagcgattc 120
cagcttcacg cagtcgagtt gcagactgcg atccgaactg agaacagatt tgtgggattg 180
gcttaacctc gcggtttcgc tgccctttgt tctgtccatt gtagcacgtg tgtagcccag 240
gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg tcaccggcag 300
tcaccttaga gtgcccaact gaatgctggc aactaagatc aagggttgcg ctcgttgcgg 360
gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc tgtcactctg 420
cccccgaagg ggacgtccta tctctaggat tgtcagagga tgtcaagacc tggtaaggtt 480
cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc 540
ctttgagttt cagtcttgcg accgtactcc ccaggcggag tgcttaatgc gttagctgca 600
gcactaaggg gcggaaaccc cctaacactt agcactcatc gtttacggcg tggactacca 660
gggtatctaa tcctgttcgc tccccacgct ttcgctcctc agcgtcagtt acagaccaga 720
gagtcgcctt cgccactggt gttcctccac atctctacgc atttcaccgc tacacgtgga 780
attccactct cctcttctgc actcaagttc cccagtttcc aatgaccctc cccggttgag 840
ccgggggctt tcacatcaga cttaaggaac cgcctgcgag ccctttacgc ccaataattc 900
cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta gccgtggctt 960
tctggttagg taccgtcaag gtaccgccct attcgaacgg tacttgttct tccctaacaa 1020
cagagcttta cgatccgaaa accttcatca ctcacgcggc 1060
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Claims (7)
1. A bacillus mojavensis is characterized in that the bacillus mojavensis is named as bacillus mojavensis (Bacillus mojavensis) FTB-P3 and is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 23149 at the date of 09 and 02 of 2021.
2. A microbial agent comprising the Bacillus mojavensis according to claim 1.
3. The microbial agent according to claim 2, wherein the effective bacterial count of bacillus mojavensis in the microbial agent is 4.0-8.0x10 9 cfu/g。
4. The microbial agent of claim 2, wherein the bacillus mojavensis is immobilized on a porous carrier material; the porous carrier material comprises one or more of zeolite, activated carbon, diatomite and porous ceramsite.
5. The microbial agent according to claim 2, wherein the preparation method of the microbial agent comprises the following steps: activating the bacillus mojavensis, inoculating the bacillus mojavensis into a fermentation culture medium, and culturing for 12-24 hours at 28-32 ℃.
6. The microbial agent according to claim 5, wherein the fermentation medium comprises the following components in parts by weight: 8-12 parts of peptone, 4-6 parts of beef extract powder, 4-6 parts of sodium chloride, 9-15 parts of agar, 800-1200 parts of distilled water and pH=7.2-7.4.
7. Use of the bacillus mojavensis according to claim 1 or the microbial inoculum according to any one of claims 2 to 6 for soil phosphate solubilizing and/or for controlling plant diseases; the plant disease pathogenic bacteria are selected from the group consisting of: wheat scab Fusarium graminearumschw, corn big spot bacteria Exserohilum, rhizoctonia solani sp. Vasopressi, fusarium oxysporum sp. Vasobifidum, sclerotinia sclerotinia sclerotiorum, fusarium oxysporum sp. Niveum, botrytis cinerea Pers.
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Effective date of registration: 20240205 Address after: 064100 Huxing Road, Houhu industrial cluster, Yutian County, Tangshan City, Hebei Province 1966 Patentee after: FENG TIAN BAO AGRICULTURE TECHNOLOGY CO.,LTD. Country or region after: China Patentee after: Hebei Fenglingguan Agricultural Technology Co.,Ltd. Address before: 064100 Huxing Road, Houhu industrial cluster, Yutian County, Tangshan City, Hebei Province 1966 Patentee before: FENG TIAN BAO AGRICULTURE TECHNOLOGY CO.,LTD. Country or region before: China |