CN110484478B

CN110484478B - Bacillus subtilis JZ2-1-12 and application thereof

Info

Publication number: CN110484478B
Application number: CN201910936675.XA
Authority: CN
Inventors: 沈硕; 李玮; 王舰
Original assignee: Qinghai Academy of Agricultural and Forestry Sciences
Current assignee: Qinghai Academy of Agricultural and Forestry Sciences
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2021-04-09
Anticipated expiration: 2039-09-29
Also published as: CN110484478A

Abstract

The invention discloses bacillus subtilis JZ2-1-12 and application thereof, wherein the bacillus subtilis JZ2-1-12 is preserved in Guangdong province microorganism strain preservation center in 2019, 6 and 17 days, and the preservation number is GDMCC No. 60696. The strain is separated from the fermented grains of the highland barley white spirit, and the fermentation product of the strain is proved by experiments to have the effects of efficiently inhibiting the pathogenic bacteria of the potato dry rot and the potato Y virus, and has the potential and the application prospect of being used as a biocontrol bacterium preparation for preventing and treating the potato dry rot and the potato Y virus.

Description

Bacillus subtilis JZ2-1-12 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis JZ2-1-12 and application thereof.

Background

The potato dry rot is a disease caused by the infection of tubers by fusarium solani, and the pathogenic types of the disease comprise 9 types and varieties. Affected tubers recover slowly and the diseased site is further enlarged in lower temperature, higher humidity environments. In addition, although potatoes have a high disease resistance during harvesting, they are susceptible to disease if there are many wounds. The potato dry rot is very common in production areas in various parts of China, and the occurrence of the dry rot seriously reduces the yield of potatoes in China. Generally, the yield loss of potatoes caused by dry rot during the cellaring process is 6-25%, and up to 60% in severe cases. Therefore, the control, especially the biological control, of the potato dry rot is of great significance. Generally speaking, the biological agent is susceptible to external environmental factors (such as temperature, soil pH value and ultraviolet intensity), so that the utilization rate of effective components in practical application is reduced. Therefore, whether the stability of the biocontrol preparation is good or not directly influences the popularization and development of the biological preparation.

Potato Virus Y (PVY) can be transmitted both non-persistently by over 40 aphid mediators and by host asexual propagation and mechanical abrasion. Is widely distributed in all parts of the world, and can infect various crops of solanaceae, chenopodiaceae, leguminosae, cucurbitaceae and the like. Among them, in the field production of Potato (Solanum tuberosum), the yield of the plant is generally reduced by about 50% after being infected with PVY, but when the plant is infected with other Potato viruses such as Potato Virus X (PVX) or Potato Virus A (PVA), the Potato can generate more serious disease symptoms, and sometimes the yield is reduced by even 80%. In addition, economic losses are also large for hosts such as tobacco (Nicotiana spp.), tomato (Solanum lycopersicum) and pepper (Capsicum spp.).

In recent years, a plurality of active bacteria separated from the fermented grains of the highland barley white spirit are bacillus, have higher grass inhibiting, bacteriostatic and antiviral activities, and have the potential of being developed as a biocontrol microbial inoculum and a microbial source biocontrol preparation. At present, reports about the inhibition of phytopathogens by biocontrol bacteria are more and more, but the reports about the inhibition of the activity of potato dry rot pathogenic bacteria and potato virus Y by biocontrol bacteria are few.

Disclosure of Invention

The invention aims to provide a strain JZ2-1-12 which has higher inhibitory activity on pathogenic bacteria of the potato dry rot disease, lays a foundation for separation and purification and structural identification of active substances for inhibiting the pathogenic bacteria of the potato dry rot disease, and thus provides a new theoretical basis and technical support for development of biological control preparations of the potato dry rot disease from microorganisms.

The invention is realized by the following technical scheme:

the invention separates strain JZ2-1-12 from the fermented grains of highland barley white spirit, extracts 16S rDNA of the strain JZ2-1-12, amplifies 16S rDNA fragments, and the measured 16S sequence is shown as SEQ ID NO. 1. The measured 16S sequence is compared with a National Center for Biotechnology Information (NCBI) database, JZ2-1-12 and a known strain Bacillus subtilis subsp. subtilis (ABQL01000001) are clustered in the same branch, the genetic relationship is nearest, and the similarity is up to 99.93 percent, so that the JZ2-1-12 is determined to belong to Bacillus subtilis in classification.

Based on the characteristics, the invention provides a Bacillus subtilis JZ2-1-12, wherein the strain is preserved by a preservation unit: guangdong province microorganism strain preservation center, preservation address: the preservation date of the five storied building of the experimental building of the microbiological institute of hundred province, the first reign in Guangzhou city of China: and 6, 17.2019, with the preservation number GDMCC No. 60696.

The invention provides application of bacillus subtilis JZ2-1-12 in preventing and treating potato dry rot.

Preferably, the application of the fermentation product of the bacillus subtilis JZ2-1-12 in preventing and controlling the dry rot of potatoes is also within the protection scope of the invention.

In another aspect of the invention, the application of the bacillus subtilis JZ2-1-12 in preparing a biological agent for preventing and treating potato dry rot is provided.

Preferably, the application of the fermentation product of the bacillus subtilis JZ2-1-12 in preparing the biological preparation for preventing and controlling the potato dry rot is also within the protection scope of the invention.

In another aspect of the invention, the application of the bacillus subtilis JZ2-1-12 in preventing and treating diseases caused by the potyvirus is provided.

Preferably, the application of the fermentation product of the bacillus subtilis JZ2-1-12 in preventing and treating diseases caused by the potato virus Y is also within the protection scope of the invention.

The invention has the beneficial effects that:

the invention provides bacillus subtilis JZ2-1-12, which is obtained by separating from fermented grains of highland barley white spirit and has inhibitory activity on pathogenic bacteria of potato dry rot, and in addition, the extract of the fermentation liquor of the strain JZ2-1-12 has good thermal stability and acid-base stability and can adapt to different pH value environments when being used as a biological pesticide for development. Experiments prove that the strain JZ2-1-12 fermentation liquor extract has stronger inhibitory activity on the pathogenic bacteria of the potato dry rot, and has the potential and application prospect of being used as a biocontrol bacterial preparation for preventing and treating the potato dry rot.

Drawings

FIG. 1 shows the culture characteristics and gram-stain characteristics of strain JZ 2-1-12; A. morphological features on beef extract peptone medium; B. gram stain results under light microscope (400 ×);

FIG. 2 is a phylogenetic tree of strain JZ2-1-12 based on the 16S r DNA sequence;

FIG. 3 shows the hypha morphology of the pathogenic bacteria of dry rot of potato treated with the extract of the fermentation broth of strain JZ 2-1-12; A. normal potato dry rot pathogens; B. treating pathogenic bacteria of the potato dry rot disease by using the n-butyl alcohol extract; C. treating pathogenic bacteria of potato dry rot with ethyl acetate extract; D. treating pathogenic bacteria of potato dry rot with ethyl acetate extract;

FIG. 4 is the effect of temperature on the bacteriostatic activity of the fermentation broth extract of strain JZ 2-1-12;

FIG. 5 is the effect of pH on the bacteriostatic activity of the fermentation broth extract of strain JZ 2-1-12;

FIG. 6 shows the effect of UV irradiation on the bacteriostatic activity of the fermentation broth extract of strain JZ 2-1-12.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 isolation of Strain JZ2-1-12

The lees sample is collected from the cellar of Qinghai mutual aid highland barley winery. Taking 5g of a vinasse sample, putting the vinasse sample in 100ml of sterile water, and fully oscillating for 30min at normal temperature of 200r/min to obtain a vinasse sample stock solution. Aspirate 1mL of sample stock solution and add to 9mL of sterile water to make 10^-1The solution of (1). Successively, the dilution is gradually reduced to 10^-2、10^-3. 0.2mL of the solution was spread on a beef extract peptone medium (beef extract 3g, peptone 10g, NaCl 5g, agar 20g, distilled water 1000mL, pH 7.0-7.2) plate. The plate was incubated in an incubator at 28 ℃ for a certain period of time and observed. When bacterial colonies appear on the culture medium plate, selecting a small amount of hyphae or spores, inoculating the hyphae or spores on a new culture medium plate, storing the strains on a culture medium inclined plane in a test tube until single bacterial colonies appear on the culture medium plate, sealing the strains with liquid paraffin, and storing the strains in a refrigerator at 4 ℃ for later use.

EXAMPLE 2 identification of Strain JZ2-1-12

1) Morphological identification of Strain JZ2-1-12

Taking the activated strain JZ2-1-12, and observing the colony morphology characteristics (including shape, size, luster, raised shape, transparency, edge and the like) and thallus morphology characteristics (including gram stain, spore morphology and motility) of the growth of the strain.

As shown in FIG. 1, the strain JZ2-1-12 was purple in gram stain and was a gram-positive bacterium, and the cells were straight rod-shaped and moved with periphytic flagella. The colony has regular edge, is milk white, opaque, flat and has surface wrinkles.

2) Physiological and biochemical identification of strain JZ2-1-12

With reference to Bergey's Manual of bacteriological identification (ninth edition), chapter fourteenth of Biochemical characterization, the strain JZ2-1-12 was subjected to citrate utilization test, catalase, glucose oxidative fermentation, sugar, alcohol fermentation, methyl red (M.R), V-P test, O-nitrophenyl-beta-D galactopyranoside (ONPG) test, nitrate reduction, gelatin liquefaction and litmus milk test.

As shown in Table 1, strain JZ2-1-12 can liquefy gelatin and can use citrate, sucrose and mannitol. The litmus milk is in a coagulated state in the test. Glucose can be decomposed with or without the participation of oxygen molecules. Wherein the catalase test, the nitrate reduction test and the V-P test are all positive. Both methyl red and ONPG tests were negative.

TABLE 1 physiological and biochemical identification results of strain JZ2-1-12

3) Molecular biological identification of Strain JZ2-1-12

The strain DNA is extracted by adopting the procedure of a column type bacteria DNA extraction kit of Shanghai biological engineering (Shanghai) corporation.

PCR amplification System: 10 XPCR Buffer 2.5. mu.L, dNTP (10 mmol. multidot.L)^-1) mu.L of 2. mu.L, 1. mu.L of each of primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGA CTT-3'), 1. mu.L of template DNA, 0.5. mu.L of Taq DNA polymerase, ddH₂Make up to 25. mu.L of O.

The PCR reaction conditions are as follows: 5min at 94 ℃; at 94 ℃ for 10s, at 54 ℃ for 30s, at 72 ℃ for 80s, for 35 cycles; 10min at 72 ℃.

And after the reaction is finished, taking 5 mu L of PCR product to carry out 1% agarose gel electrophoresis detection, and sending the PCR product to Shanghai bioengineering GmbH for sequencing, wherein the sequence is shown as SEQ ID NO. 1. And submitting the sequencing result to a website http:// www.ezbio closed. net for 16S rDNA sequence alignment, and determining the classification status of the strains. And performing strain sequence analysis and phylogenetic tree construction by MEGA 7.0 software.

16S rDNA sequence (1379bp) obtained by sequencing strain JZ2-1-12(MN083310) is submitted to https:// www.ezbiocloud.net website, and is compared with known 16S rDNA sequence in database, strain sequence with homology of more than 95% with 16S rDNA sequence of strain JZ2-1-12 is downloaded, and phylogenetic tree is constructed by MEGA 7.0 software (figure 2). The strain preservation number is as follows: GDMCC No. 60696, deposited in 2019 at 17.6.9 to Guangdong province culture Collection. As can be seen from FIG. 2, the strain JZ2-1-12 and the known strain Bacillus subtilis (ABQL01000001) cluster in the same branch, and have the closest genetic relationship and the similarity is as high as 99.93%. Finally determining the strain JZ2-1-12 as Bacillus subtilis by combining the morphological, physiological and biochemical characteristics and molecular biological identification results of the strain JZ 2-1-12.

Example 2 inhibitory Activity of extract of fermentation broth of Strain JZ2-1-12 against pathogenic bacteria of Potato Dry rot disease

Inoculating the cultured strain JZ2-1-12 into 400mL beef extract peptone liquid culture medium, placing in a constant temperature shaking cabinet at 33 deg.C, and shake-culturing at 180r/min for 5d to obtain fermentation liquor. And filtering the fermentation liquor by a Buchner funnel under reduced pressure to remove thalli, thus obtaining the thallus-removed fermentation liquor.

Placing 500mL of the fermentation liquor without bacteria in a 3000mL separating funnel, sequentially and fully contacting and uniformly mixing with equal volume of ethyl acetate and n-butanol, respectively extracting for 3 times, standing until a small amount of water and an organic phase at the bottom layer of the fermentation product are layered, collecting and combining the organic phase. And concentrating the organic phase obtained by extraction under reduced pressure by a rotary evaporator to respectively obtain ethyl acetate and n-butanol extract. Dissolving the extract in dimethyl sulfoxide (v/v ≤ 2%) and preparing into solutions with concentrations of 6.25, 12.50, 25.00, 50.00 and 100.00mg/mL with sterile water, vacuum filtering the extract solution with 0.22 μm microporous membrane for three times, and repeating the treatment for three times with sterile water and dimethyl sulfoxide (v/v ≤ 2%) mixed solution as blank control. The antibacterial activity of the fermentation liquor extracting solution is measured by adopting an oxford cup method, and 200 mu L of extract solution subjected to suction filtration is injected into each oxford cup.

The calculation formula is as follows:

as can be seen from Table 2, when the concentration of the ethyl acetate extract solution is 12.50mg/mL, the inhibition activity on the pathogenic bacteria of the potato dry rot is the highest, and the inhibition rate reaches 75.00%; when the concentration of the n-butanol extract solution is 50.00mg/mL, the inhibition activity on the pathogenic bacteria of the potato dry rot is the highest, and the inhibition rate is 66.13%. By toxicityThe regression equation can be found: EC of ethyl acetate and n-butanol extract solution for inhibiting activity of pathogenic bacteria of potato dry rot₅₀0.19 and 12.96mg/mL, respectively.

TABLE 2 inhibitory Activity of the extract of the fermentation broth of the Strain on the pathogenic bacteria of Potato Dry rot

Example 3 teratogenic effect of fermentation broth extract on potato dry rot pathogen hyphae the potato dry rot pathogen hyphae in example 2 were picked up near the edge of the zone of inhibition and under normal conditions to prepare slide specimens, each treatment was repeated three times, and the hyphae morphology of the treatment group and the control group were observed under an optical microscope.

As shown in figure 3, both fermentation broth extracts have strong teratogenic effects on the hyphae of the pathogenic bacteria of potato dry rot. The normal potato dry rot pathogenic bacteria hyphae are uniform in thickness, smooth in surface, full and normal in extension growth all around. Hyphae at the edge of the bacteriostatic zone treated by the n-butanol extract are twisted and folded, and the phenomena of hyphae branching, breakage and uneven thickness also occur. The hyphal ends near the edges of the zone treated with the ethyl acetate extract expanded and also appeared to be entangled, twisted, misshapen and broken to varying degrees.

Example 4 Effect of fermentation broth extract on spore germination of pathogenic bacteria of Dry rot of Potato on perforated Potato cake of pathogenic bacteria of Potato with 6mm diameter puncher, inoculating into PDA liquid Medium, culturing in shaking flask cabinet at 28 deg.C and 180r/min for 3d, filtering with 4 layers of gauze and centrifuging at 12000r/min for 15min, collecting supernatant, formulating into 1 × 10 with sterile water⁶CFU/mL spore suspension was ready for use.

By adopting a concave glass culture method, ethyl acetate and n-butyl alcohol extracts of the fermentation liquor of the strain JZ2-1-12 are respectively prepared into solutions with the concentrations of 6.25, 12.50, 25.00, 50.00 and 100.00mg/mL, 30 mu L of potato dry rot pathogen spore suspension is absorbed into a groove of a concave glass, 30 mu L of extract solutions with different concentrations are respectively absorbed into the groove and are uniformly mixed, and sterile water is used as a reference. The cells were placed in a petri dish by covering with a cover glass, incubated in an incubator at 28 ℃ and counted under a microscope using a hemocytometer when the number of spores germinated (the length of the germ tube exceeds half the diameter of the spores) of the control reached 70 to 80%. Each treatment was repeated three times and the average was taken. The spore germination inhibition rate is calculated according to the following formula:

as shown in Table 3, the fermentation broth extract was effective in inhibiting germination of potato dry rot pathogen spores. When the concentration of the ethyl acetate extract solution is 12.50mg/mL, the number of spores is only about 7950 at least, and the germination inhibition rate of pathogenic bacteria spores reaches 69.64%; when the concentration of the n-butanol extract solution is 50.00mg/mL, the germination inhibition rate of pathogenic spore is 68.99%; the toxicity regression equation can be used for obtaining: EC of ethyl acetate and n-butanol extract solution for germination of pathogenic bacteria spores of potato dry rot₅₀0.93 and 9.36mg/mL, respectively.

TABLE 3 influence of the extract solution of the strain fermentation broth on spore germination of pathogenic bacteria of potato dry rot

Example 5 Effect of different conditions on the bacteriostatic Activity of the extract of fermentation broth of Strain JZ2-1-12

1) Thermal stability

Respectively carrying out water bath treatment at 40 ℃, 60, 80 and 100 ℃ and high-pressure steam sterilization treatment at 121 ℃ for 60min on an ethyl acetate extract solution with the concentration of 12.50mg/mL and an n-butanol extract solution with the concentration of 50.00mg/mL, cooling, carrying out suction filtration by using a 0.22 mu m sterile filter membrane, and detecting the inhibitory activity of the extract solution on the pathogenic bacteria of the potato dry rot by using the solution which is not subjected to heating treatment after suction filtration as a control.

As can be seen from FIG. 4, when the temperature is below 80 ℃, the bacteriostatic activity of the ethyl acetate and n-butanol fermentation broth extract solution reaches more than 50.00%; when the temperature is increased to 100 ℃, the bacteriostatic activity of the ethyl acetate extract is reduced by 14.99 percent compared with the control, and the n-butanol extract is only reduced by 13.75 percent compared with the control; when the temperature reaches 121 ℃, the bacteriostatic activity of the extract is still kept above 45.00 percent. This shows that the strain JZ2-1-12 fermentation liquor ethyl acetate and n-butanol extract still have higher antibacterial activity and better thermal stability at high temperature.

2) Stability to acid and base

Adjusting the pH values of an ethyl acetate extract solution with the concentration of 12.50mg/mL and an n-butanol extract solution with the concentration of 50.00mg/mL to 5, 6, 7, 8 and 9 by using 1mol/L hydrochloric acid and 1mol/L sodium hydroxide respectively, standing for 24 hours, carrying out suction filtration on the solution through a 0.22 mu m microporous filter membrane, and detecting the inhibitory activity of the solution on the potato dry rot pathogenic bacteria by using the solution without the pH value as a control.

As can be seen from fig. 5, at pH 6, the bacteriostatic activity of the ethyl acetate and n-butanol fermentation broth extract solutions was highest, 73.75% and 67.50%, respectively. The antibacterial activity of the extract solution is slightly reduced with the increase of the pH value, but still kept above 50.00 percent. This shows that the bacteriostatic activity of the ethyl acetate and n-butanol extracts of the fermentation liquor of the strain JZ2-1-12 is less influenced by pH, and the strain has good acid-base stability.

3) UV stability

Placing ethyl acetate extract solution with concentration of 12.50mg/mL and n-butanol extract solution with concentration of 50.00mg/mL under 254nm ultraviolet lamp for 20cm irradiation for 1, 3, 5, 7, and 9h respectively, vacuum-filtering with 0.22 μm microporous membrane, and detecting the inhibitory activity of the extract solution without ultraviolet irradiation treatment on pathogenic bacteria of potato dry rot.

As shown in FIG. 6, after the strain JZ2-1-12 fermentation liquor ethyl acetate and n-butanol extract solution are respectively irradiated for 1, 3, 5, 7 and 9 hours at the ultraviolet position of 20cm under 254nm, the antibacterial activity is reduced by about 10.00%, but the antibacterial activity is still maintained at more than 55.00%. The antibacterial activity of the fermentation liquor ethyl acetate extract is higher than that of the n-butyl alcohol extract. This shows that the bacteriostatic active ingredients of the ethyl acetate and n-butanol extract solution of the fermentation liquor of the strain JZ2-1-12 have better stability under ultraviolet irradiation.

Example 6 Effect of bacterial Strain JZ2-1-12 fermentation broth extract on inhibition of PVY Activity by bacteriostasis

Carrying out seedling pot planting on the Sansheng tobacco in a sunlight greenhouse, when 4-5 leaves of a plant are completely expanded, respectively mixing a strain fermentation liquor ethyl acetate extract and an n-butanol extract with equal volume of PVY virus liquid for 30min according to a passivation mode to be used as treatment, taking the mixed PVY virus liquid with equal volume of a phosphate buffer solution as a positive control, taking only inoculated phosphate buffer solution as a blank control, taking the mixed PVY virus liquid with equal volume of an 8% Ningnanmycin aqueous solution as a negative control, inoculating the Sansheng tobacco by using a traditional friction inoculation method, and repeating the experiment for 3 times. And (4) after inoculation for 7d, taking leaves at the top of the plant, and screening active bacteria by adopting a Real-time PCR method.

According to the original detection result of Real Time qPCR, according to 2^-△△ctRelative quantitative calculation formula:

F＝2^{- { [ average CT value of target Gene of group to be tested-average CT value of housekeeping Gene of group to be tested]- [ average CT value of target Gene in control group-average CT value of Gene in control group]}}The expression level of the target gene PVY in each treatment was calculated.

The strain had PVY inhibition rate (expression level of PVY in control-expression level of PVY in treatment)/expression level of PVY in control × 100%.

As can be seen from Table 4, the n-butanol extract of the fermentation broth of strain JZ2-1-12 has higher inhibitory activity against PVY at concentrations of 50mg/mL and 100mg/mL, and the inhibitory rates are 80.96% and 87.03%, respectively. The ethyl acetate extract of the strain JZ2-1-12 fermentation liquor has higher inhibitory activity to PVY under the concentration of 100mg/mL, and the inhibition rate is 83.03%. In conclusion, the n-butanol extract has higher inhibitory activity on PVY.

TABLE 4 Activity of fermentation broth extract of strain JZ2-1-12 for inhibiting PVY

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> academy of agriculture and forestry of Qinghai province

<120> bacillus subtilis JZ2-1-12 and application thereof

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Claims

1. The bacillus subtilis JZ2-1-12 is characterized in that the bacillus subtilis JZ2-1-12 is preserved in Guangdong province microorganism culture collection center in 2019, 6 and 17 days, and the preservation number is GDMCC No. 60696.

2. The use of bacillus subtilis JZ2-1-12 of claim 1 for controlling diseases caused by potyvirus.

3. The use as claimed in claim 2, wherein the fermentation product of bacillus subtilis JZ2-1-12 is used for controlling diseases caused by potyvirus.