CN112725220B - Lysobacter xylosus JZ3-4-7 and application thereof - Google Patents

Abstract

The invention discloses a polylysine bacillus xylosus JZ3-4-7 and application thereof, wherein the polylysine bacillus xylosus JZ3-4-7 is preserved in Guangdong province microorganism strain preservation center in 6-28 months in 2020, and the preservation number is GDMCC NO: 5363 and 60694, which has the sequence shown in SEQ ID No.1 by 16S rDNA sequence detection. The strain is separated from highland barley vinasse, and a fermentation product of the strain is proved by experiments to have the effect of efficiently inhibiting the pathogenic bacteria of the potato dry rot, and has the potential and the application prospect of being used as a biocontrol bacterium preparation for preventing and treating the potato dry rot.

Description

Lysobacter xylosus JZ3-4-7 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a polylysine bacillus xylosus JZ3-4-7 and application thereof.

Background

Potatoes (the name of the science is Solanum tuberosum), solanaceae Solanum, annual herbaceous plants and polyploidy crops, and the potatoes can be subjected to sexual reproduction by seeds in berries and can also be subjected to vegetative propagation by tubers. Potatoes are also rich in nutrient proteins, which have a better nutrient protein content than soy, most closely related to animal proteins. Potatoes are called the third most important grain crop in the world, mainly because of high nutritive value, strong adaptability and high yield. The potato is a tuber propagation, can be used as a medicine, and has the pharmaceutical effects of regulating the middle warmer, nourishing the stomach, invigorating the spleen, promoting diuresis, relaxing bowels, reducing blood sugar and blood fat, inducing diuresis and relieving swelling.

Potatoes are easy to be attacked by fungi and bacteria in a cellaring process, and are often infected by more than a single kind of bacteria, on one cellaring potato, the bacteria and the fungi usually exist at the same time, but not only one kind of fungi or bacteria, and the damage of cellaring diseases to the potatoes is increased year by year due to the fact that the causes of the potato diseases are various. The potato pathogenic bacteria have serious harmful diseases as follows: potato late blight, potato black nevus, potato wilt, potato dry rot, potato early blight, potato canceroma, potato silver rot, potato yellow wilt, potato powdery scab, potato gray mold, potato gangrene and potato anthracnose, and the bacterial diseases of potatoes mainly comprise: ring rot, soft rot, bacterial wilt, black shank, and scab. The measures to be prevented and controlled aiming at the cellar-stored diseases of the potatoes are currently researched: chemical control, disease-resistant breeding and biological control.

The distiller's grains are waste residue material obtained after grains are used as raw materials and subjected to fermentation, distillation, alcohol extraction and other processing processes, and the distiller's grains have high nutritive value, contain rich protein and amino acid, mineral elements such as phosphorus and potassium and various vitamins. In addition, trace amounts of beneficial components such as ribonucleic acid and purine produced by microbial cells may also be present. Various bacteria extracted from the vinasse flora have high application value, and have various applications in biological prevention and control and important application in the aspect of producing high-protein feed.

At present, most researches on control of potato cellaring diseases by methods such as fertilization and scientific germination acceleration are carried out in China, but the researches on biological control are few, and with the development of domestic biotechnology, domestic researchers begin to pay attention to biological control of soil-borne diseases, but due to the fact that the soil-borne diseases are caused by various reasons and complex floras, the reported biological extracting solution and the effect of the floras are unstable. The use of pesticides not only causes pollution to the environment and human bodies, but also pathogenic microorganisms are easy to generate drug resistance along with the increase of the use times, so that the control effect is influenced, and the high efficiency, the reasonability, the diversity and the low price of biological control are witnessed, so that the research on the biological control in the aspect of the cellar diseases of the potatoes is necessary.

Disclosure of Invention

The invention aims to provide a polylysine bacillus (Lysinibacillus pakistanensis) JZ3-4-7, which has a remarkable inhibiting effect on pathogenic fungi of the cellar diseases of potatoes and can be used for preventing and treating the cellar diseases of the potatoes.

The invention is realized by the following technical scheme:

the invention separates and purifies a strain JZ3-4-7 from highland barley vinasse, and the bacterial colony of the strain JZ3-4-7 is light yellow, smooth, moist and milk white after being cultured for 24 hours on an LB culture medium, the single bacterial colony is round or oval, and the edge is neat. After gram staining, it appeared bluish purple, rod-like, approximately 0.4. Mu. M.times. (1.5-3) μm in size.

The strain JZ3-4-7 has a remarkable inhibiting effect on cellar diseases of potatoes caused by fusarium (Fusarium sp.). The fusarium pathogenic fungi are particularly green 9B-4-6, green 9D-2-6, 65C-4-3 and 65D-5-2, pathogenic fungi green 9B-4-6 and green 9D-2-6 of potato dry rot are separated from a potato tuber 9 disease sample, and pathogenic fungi 65C-4-3 and 65D-5-2 are separated from a potato tuber 65 disease sample.

Furthermore, the sequence of the strain JZ3-4-7 is shown in SEQ ID No.1 by the sequence determination of 16SrDNA, and the strain is determined to be Lysinibacterium xylostellus (Lysinibacillus pakistanensis) by comparing the website https:// www.ezbiocloud.net.

Furthermore, the applicant reserves the JZ3-4-7 of the Lysinibacillus xylinus (Lysinibacillus pakistanensis) in the Guangdong province microbial strain reservation center; the preservation address is as follows: experiment large building No. 5, building of Jieli Zhonglu No. 100, junior, junxiu, guangdong province, guangzhou city; the preservation date is as follows: 28/6/2020, with deposit number GDMCC NO:60694.

In another aspect of the invention, the application of the polylysine xylophilus JZ3-4-7 in preventing and treating cellar diseases of potatoes is provided.

Specifically, the application of the bacterial suspension of the polylysine bacillus xylinum JZ3-4-7, or the culture solution or the fermentation product thereof in preventing and treating the cellar diseases of potatoes is provided.

Preferably, the cellaring disease is potato dry rot.

Meanwhile, the application of the polylysine bacillus xylosus JZ3-4-7 or the bacterial suspension thereof or the culture solution thereof or the fermentation product thereof in the preparation of the biological preparation for preventing and treating the cellar diseases of potatoes is also within the protection range of the invention.

Specifically, a thallus culture containing the xylem polylysine bacillus JZ3-4-7 is obtained through conventional liquid or solid culture, and liquid fermentation liquor is obtained through conventional liquid fermentation production; or adding one or more of surfactants such as dispersing agent, stabilizer, wetting agent, binder, defoaming agent, disintegrating agent, anti-freezing agent, etc. into the active ingredient obtained by liquid fermentation, or mixing the active ingredients and the adsorption carriers according to a certain proportion to prepare wettable powder, water dispersible granules, suspending agent, suspoemulsion, emulsion in water or microemulsion.

In another aspect of the invention, the biological agent for preventing and treating the cellar potato diseases is provided, and the biological agent takes polylysine JZ3-4-7 or a bacterial suspension thereof, or a culture solution thereof, or a fermentation product thereof as an active ingredient.

The beneficial effects of the invention are as follows:

the invention provides a polylysine bacillus xylosus JZ3-4-7, which is obtained by separating from fermented grains of highland barley white spirit, has inhibitory activity on pathogenic fungi green 9B-4-6, green 9D-2-6, 65C-4-3 and 65D-5-2, and has better control effect on dry rot of potatoes as proved by tests, and has potential and application prospect of being used as a biocontrol bacterium preparation for controlling the dry rot of potatoes.

Drawings

FIG. 1 is a culture characteristic of the strain JZ3-4-7 of the present invention;

FIG. 2 is the gram-staining result of the strain JZ3-4-7 of the present invention.

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 obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

EXAMPLE 1 isolation and identification of Strain JZ3-4-7

1) Isolation of Strain JZ3-4-7

The strain JZ3-4-7 is separated from highland barley vinasse, and a vinasse sample is collected from a cellar of a Qinghai mutual-aid highland barley wine factory. Taking 5g of a vinasse sample, placing 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. Sucking 1ml of sample stock solution, adding into 9ml of sterile water, and making into 10 ^-1 The solution of (1). Successively, the dilution is gradually reduced to 10 ^-2 、10 ^-3 . Spreading 0.2ml of the solution on PDA culture medium (potato 200g, glucose 15g, agar 15-20g, distilled water 1000ml.100mg/L chloramphenicol), starch culture medium (beef extract 5g, peptone 10g, naCl 5g, soluble starch 2g, agar 15-20g, distilled water 1000ml,100mg/L chloramphenicol, pH 7.0-7.2), and Chachi culture medium (sucrose 30g, nitro, nicotine, etc.)Sodium 2g, dipotassium hydrogen phosphate 1g, potassium chloride 0.5g, magnesium sulfate 0.5g, ferrous sulfate 0.01g, agar 15-20g, distilled water 1000ml,100mg/L chloramphenicol) and beef extract peptone medium (beef extract 3g, peptone 10g, naCl 5g, agar 20g, distilled water 1000ml, pH 7.0-7.2). The plate was placed 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 by using liquid paraffin, and storing the strains in a refrigerator at 4 ℃ for later use.

2) Identification of strains

(1) Morphological identification of Strain JZ3-4-7

The activated active strain JZ3-4-7 is inoculated on an LB culture medium, the colony morphological characteristics (including shape, luster, raised shape, transparency, edge and the like) and the thallus morphological characteristics (including gram stain and spore morphology) of the growth of the strain are observed, and the strain is morphologically identified according to Bergey's Manual of identification of bacteria (ninth edition) and the Manual of identification of common bacteria systems.

As shown in figure 1, after the strain JZ3-4-7 is cultured on an LB culture medium for 24 hours (figure 1), the colony is light yellow, smooth, moist and milky white, the single colony is circular or elliptical in shape, and the edge is neat. The blue-purple color, rod-like shape, and about 0.4. Mu. M.times. (1.5-3) μm in size after gram staining, indicates that this strain JZ3-4-7 is a gram-positive bacterium (FIG. 2).

(2) Physiological and biochemical identification of strain JZ3-4-7

The physiological and biochemical indicators of the active strain JZ3-4-7, such as catalase, oxidase, beta-galactosidase, nitrate reduction, gelatin liquefaction, starch hydrolysis, casein, arginine double hydrolase, arginine decarboxylase, H2S production, saligenin, indole, hemolysis, urease and the like, were determined according to Bergey' S Manual of bacteria identification (ninth edition) and Manual of general bacteria System identification.

The results showed that strain JZ3-4-7 has catalase, oxidase, β -galactosidase, nitrate reductase, amylolytic, and caseinase activities, but not arginine dihydrolase, arginine decarboxylase, saligenin, indole, hemolytic, and anaerobic enzyme activities.

3) Molecular biological identification of strain JZ3-4-7

The strain DNA was extracted according to the procedure of the column type bacterial DNA extraction kit of Shanghai Biotechnology engineering (Shanghai) Ltd.

The reaction conditions are as follows: denaturation at 94 ℃ 45sec, annealing at 50 ℃ 45sec, extension at 72 ℃ 75sec, and reaction at 50L for 30 cycles. And detecting the PCR product by agarose gel electrophoresis, and obtaining a sequence result by cloning and sequencing.

After the reaction is finished, 5 mu L of PCR product is taken to carry out 1% agarose gel electrophoresis detection, the PCR product is sent to Shanghai bioengineering GmbH to carry out sequencing, the sequence of the strain JZ3-4-7 measured by a 16S rDNA sequence is shown as SEQ ID No.1, and the strain is determined to be the polylysine xylophila (Lysinibacillus pakistanensis) by comparison on the website https:// www.ezbiocloud.net/.

Meanwhile, the applicant reserves the strain JZ3-4-7 in Guangdong province microbial strain collection center in 28 th 6 th 2020, wherein the preservation number is GDMCC NO:60694.

example 2 inhibitory Activity against Potato pathogenic fungi

Activating the strain JZ3-4-7 by adopting a plate marking method: the bacterial inoculating loop is held by hand, the metal wire of the inoculating loop is erected at the outer flame of the alcohol lamp, the inoculating loop is burnt till the metal wire is red and transparent, then the inoculating loop is slightly inclined, the metal rod is burnt, and the connecting part of the metal wire and the metal rod needs to be fully burnt during burning. And (3) opening a culture dish cover of the left-handed bacteria solid culture dish, making an opening angle smaller than 45 degrees, marking the strains on the inoculating loop on a flat plate, continuously marking from top to bottom, firing the inoculating loop after each marking, and requiring the latter area to be connected with the former area end to end but not to be lapped with other areas. The inoculated plate is placed in a constant temperature incubator at 37 ℃ for inverted culture for 48h.

The pathogenic bacteria are activated by adopting an inoculation method: selecting a small amount of potato pathogenic fungi stored in a liquid Dan Lafeng by using a fungus inoculating needle, and inoculating the strain of the potato pathogenic fungi on the inclined surface of the culture medium into a freshly prepared solid culture medium. And (4) putting the inoculated culture medium into an incubator, carrying out inverted culture, and culturing for 7d at the temperature of 28 ℃.

The inhibition rate of the strain JZ3-4-7 on pathogenic bacteria is determined by adopting the opposing growth:

inoculating a strain JZ3-4-7 which is subjected to activation culture for 24 hours to one side of a PDA (personal digital assistant) flat plate with the diameter of 9cm and is 2cm away from the edge, inoculating a potato pathogenic fungus cake with the diameter of 5mm to the other side of a circle center extending straight line, taking potato pathogenic fungi which is only inoculated as a contrast, repeating the treatment for 3 times, sealing the culture box at 28 ℃ and culturing for 7 days in the dark, observing whether a transparent inhibition zone appears between the strain JZ3-4-7 and the potato pathogenic fungi, measuring the diameter of a bacterial colony, calculating the inhibition rate, screening halophilic bacteria strains with the inhibition activity, and storing for later use.

Bacteriostatic rate (%) = (diameter of control bacterial colony-diameter of treated bacterial colony)/diameter of control bacterial colony x 100%.

Potato pathogenic bacteria are selected from: qing 9B-4-6, qing 9D-2-6, qing 65C-4-3 and Qing 65D-5-2 are all provided by the academy of agriculture and forestry, qinghai province.

As shown in Table 1, the strains JZ3-4-7 have different inhibiting effects on 4 pathogenic fungi, namely cyan 65C-4-3, cyan 9D-2-6, cyan 65D-5-2 and cyan 9B-4-6, and after the strains are cultured for 7 days, the inhibiting rates on the pathogenic fungi, namely cyan 65C-4-3, cyan 9D-2-6, cyan 65D-5-2 and cyan 9B-4-6, are 52.1%, 34.8%, 42.9% and 39.4%, respectively (Table 1).

TABLE 1 inhibitory Activity of Strain JZ3-4-7 on Potato pathogenic fungi

EXAMPLE 3 stability of Strain JZ3-4-7

By adopting the confronting culture method in the embodiment 2, after the strains JZ3-4-7 are confronted and cultured for 7d and 14d, the width of the bacteriostatic band is measured, and the reduction rate of the width of the bacteriostatic band of 14d is calculated.

The width reduction rate of the bacteriostatic band (%) = (7 d bacteriostatic band width value-14 d bacteriostatic band width value)/7 d bacteriostatic band width value is multiplied by 100 percent

Evaluation criteria for strain stability: the width reduction rate of the bacteriostatic zone is not less than 70 percent and is shown as "- -"; VF < 70% of 50% or more is relatively unstable and is represented by "-"; VF < 50% more stable than 20%, indicated by "+"; VF < 20% was stable as indicated by "+". "" indicates no data.

As shown in Table 2, the strain JZ3-4-7 has higher inhibitory activity on the pathogenic fungus blue 9B-4-6 of the potato dry rot, and the evaluation result of the stability of the inhibitory activity shows that: the inhibition effect on the pathogenic bacteria is stable, and the inhibition zone reduction rate of JZ3-4-7 is 25%.

TABLE 2 stability of the distillers' grains bacterial strains against Potato pathogen fungus inhibition

EXAMPLE 4 bacteriostatic Activity of extract of Strain JZ3-4-7

1) Fermentation culture and fermentation liquor pretreatment of strain JZ3-4-7

Fermentation culture:

(1) seed culture: inoculating the activated strain JZ3-4-7 to a triangular flask containing 1000mL of 800mL bacterial liquid culture solution by using a inoculating needle, and performing constant-temperature shaking culture at 37 ℃ and 200r/min for 2d to obtain the strain serving as a seed.

(2) Fermentation culture: absorbing a proper amount of seed culture solution into a triangular flask containing 1000mL of 800mL of bacterial liquid culture solution, carrying out shake culture on a constant temperature shaking table at 37 ℃ and 200r/min for 3-5 d, and taking out the culture from the shaking table machine for later use.

Treatment of the culture solution: primarily centrifuging the strain JZ3-4-7 fermentation liquor for 20min at 4 ℃ and 12000r/min, and taking supernatant in a low-temperature high-speed refrigerated centrifuge. Sequentially extracting the fermentation liquor with chloroform, ethyl acetate and n-butanol, and concentrating the extract under reduced pressure to obtain the extracts. Filtering under reduced pressure, placing the filtrate in a low-temperature high-speed refrigerated centrifuge again, collecting supernatant, and filtering with 0.22 μm microporous membrane to remove insoluble substances, and storing the filtrate at 4 deg.C for use.

3) The growth inhibition of the pathogenic fungi hypha is determined by adopting a hypha growth rate method, a perforating device with the diameter of 6mm is used for cutting fungus cakes on the outer edges of the bacterial colonies of each cultured pathogenic fungus, 4 kinds of extracts are diluted and added into a culture medium (sterilized at 121 ℃ for 20 min) at 50 ℃ according to the final concentration of 5mg/mL, 10mg/mL and 20mg/mL, the mixture is uniformly mixed and poured into a culture dish, the culture dish is placed in an incubator for constant-temperature culture at 37 ℃ for 7d, and 3 times of experiments are repeated. The colony diameter was measured every day according to the cross method, and the growth inhibition rate was calculated according to the following formula, taking the average.

Inhibition = (control colony diameter-treated colony diameter)/control colony diameter × 100%

As shown in Table 3, the results of the inhibitory activity of different organic solvent extracts of the liquid fermentation broth of the Saccharomyces cerevisiae strain JZ3-4-7 on the pathogenic fungi of the cellaring disease of potato show that: the n-butanol extract of the distillers' grains bacterial strain JZ3-4-7 has the highest inhibitory activity, and the inhibition rates to the potato dry rot pathogen bacterial blue 9B-4-6 are respectively 56.9% and 81.8% under the concentration of 10mg/mL and 20 mg/mL. Under the concentrations of 10mg/mL and 20mg/mL, the inhibition rates of the chloroform extract on the potato dry rot pathogenic bacterium green 9B-4-6 are respectively 52.9 percent and 56.6 percent; the inhibition rates of the ethyl acetate extract on the potato dry rot pathogen blue 9B-4-6 are respectively 62.5% at the concentration of 20 mg/ml. The ethyl acetate extract has inhibition rates of 63.5% on potato dry rot pathogen bacterial blue 9B-4-6 at the concentration of 20mg/ml respectively.

TABLE 3 inhibitory Activity of Strain JZ3-4-7 fermentation broth extract on Potato pathogenic fungus cyan 9B-4-6

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> polylysine xylophilus JZ3-4-7 and application thereof

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Lysinibacillus xylanilyticus (Lysinibacillus pakistanensis)

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ggcggtgtgt acaaggcccg ggaacgtatt caccgcggca tgctgatccg cgattactag 120

cgattccggc ttcatgtagg cgagttgcag cctacaatcc gaactgagaa cgactttatc 180

ggattagctc cctctcgcga gttggcaacc gtttgtatcg tccattgtag cacgtgtgta 240

gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttatcac 300

cggcagtcac cttagagtgc ccaactaaat gatggcaact aagatcaagg gttgcgctcg 360

ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc 420

accgttgccc ccgaagggga aactatatct ctacagtggt caacgggatg tcaagacctg 480

gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540

gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt 600

tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt ttacggcgtg 660

gactaccagg gtatctaatc ctgtttgctc cccacgcttt cgcgcctcag cgtcagttac 720

agaccagaaa gtcgccttcg ccactggtgt tcctccaaat ctctacgcat ttcaccgcta 780

cacttggaat tccactttcc tcttctgcac tcaagtcccc cagtttccaa tgaccctcca 840

cggttgagcc gtgggctttc acatcagact taaaggaccg cctgcgcgcg ctttacgccc 900

aataattccg gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960

cgtggctttc taataaggta ccgtcaaggt acagccagtt actactgtac ttgttcttcc 1020

cttacaacag agttttacga tccgaaaacc ttcttcactc acgcggcgtt gctccatcag 1080

gctttcgccc attgtggaag attccctact gctgcctccc gtaggagtct gggccgtgtc 1140

tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtgagc 1200

cattacctca ccaactagct aatgcgccgc gggcccatcc tatagcgaca gcgagatgcc 1260

gtctttcagt atgtcaccat gaggtgacat agattattcg gtattagccc cggtttcccg 1320

gagttatccc aaactatagg gtaggttgcc cacgtgttac tcacccgtcc gccgctaacg 1380

tcaaaggagc aagctccttc tctgttcgct cgactg 1416

Claims (2)

1. A L.xylanisolvens (A.) (Lysinibacillus pakistanensis) JZ3-4-7, wherein said polylysine xylophilus JZ3-4-7 has been deposited at the Guangdong province collection of microorganisms at 28.6.2020, with the deposit number GDMCC NO:60694.

2. a biological preparation for preventing and treating cellar potato diseases, which is characterized in that the biological preparation takes the polylysine bacillus xylinum JZ3-4-7 or fermentation liquor thereof as an active ingredient, and the cellar potato diseases are dry rot of potatoes.

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