CN108977383B

CN108977383B - Paenibacillus for decomposing chitin and application thereof

Info

Publication number: CN108977383B
Application number: CN201810888980.1A
Authority: CN
Inventors: 田春杰; 吉丽; 田磊; 张建峰; 谢金宏; 陈德国
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2018-08-07
Filing date: 2018-08-07
Publication date: 2021-09-10
Anticipated expiration: 2038-08-07
Also published as: CN108977383A

Abstract

A chitin-decomposing Paenibacillus and its application, the present invention relates to a chitin-decomposing Paenibacillus and its application. The purpose of the present invention is to solve the existing problem that there is a hidden danger of pesticide residues in the control of botrytis cinerea. A chitin-decomposing Paenibacillus of the present invention is Paenibacillus sp. A10, which is preserved in China The Ordinary Microbiology Center of the Microbial Culture Collection Management Committee, the deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 02, 2018, and the deposit number is CGMCC No.15336. The application of the chitin-decomposing Paenibacillus of the present invention in the prevention and control of plant diseases. Strain A10 had a good antagonism to Botrytis cinerea, and the inhibitory effect of A10 fermentation broth on Botrytis cinerea reached 91.8%. The invention is applied to the technical field of environmental microorganisms.

Description

Paenibacillus for decomposing chitin and application thereof

Technical Field

The invention relates to a paenibacillus strain for decomposing chitin and application thereof.

Background

Paenibacillus is an aerobic or facultative anaerobic bacillus, can produce stress-resistant spores and has unique physiological characteristics. According to previous reports, paenibacillus can generate various antibacterial active substances in the metabolic process, and has important application value in the fields of medical treatment, environment, plant biological control and the like. The research is directed to using the cell wall of the microorganism as an action point, chitin is an important component of the cell wall of the fungus, and pathogenic microorganisms are killed by decomposing the cell wall of pathogenic fungi and destroying the integrity of the cell membrane, so that intracellular solutes leak out or interact with target molecules in the cell. Gray mold is a typical air-borne disease, can be spread along with air, water flow and agricultural operations, and can cause diseases of various fruits, vegetables and crops. In the actual disease control process, it is difficult to take effective measures to thoroughly cut off the infection source, and also difficult to thoroughly eliminate pathogenic bacteria in the case of invasion of the pathogenic bacteria. Therefore, the development of new, highly effective, low-toxicity agricultural antibiotics is imminent.

Disclosure of Invention

The invention aims to solve the problem of pesticide residue hidden danger in the existing main control method of gray mold, and provides a paenibacillus for decomposing chitin and application thereof.

The bacillus subtilis for decomposing chitin is bacillus subtilis A10, is preserved in China general microbiological culture Collection center, has a preservation address of No. 3 Xilu-Beijing Wenyu No.1 of the sunward region in Beijing, has a preservation date of 2018, 2 months and 02 days, and has a preservation number of CGMCC No. 15336.

The invention discloses application of paenibacillus for decomposing chitin, and relates to application of paenibacillus in preventing and treating rice diseases.

The invention adopts the separation and purification of the paenibacillus strain decomposing chitin in the wild rice rhizosphere soil:

primary screening: weighing 5g of rhizosphere soil sample from wild rice in east and rural areas of Jiangxi province in 45ml of sterile water by adopting a plate dilution coating method, standing at 120rpm for 20min, and taking supernatant for 10-time concentration gradient dilution. Respectively take 10^-1、10^-2、10^-3Dilutions were run at 80 μ L each, and each gradient was plated onto 3 plates of chitin medium, using sterile water instead of dilutions as a blank. The culture medium is placed in an incubator at 25 ℃ for 2-3 days in an inverted mode, and the growth of bacterial colonies and the generation of transparent rings are observed. (chitin Medium: K)₂HPO₄1g，MgSO₄·7H₂O0.5g，NaCl 0.5g，NH₄Cl 1Adding 15g of agar and distilled water to the volume of 1000ml, wherein 20ml of 1% colloidal chitin is added into each 80ml of chitin culture medium. )

Re-screening: on a chitin culture medium, 60 colonies with larger transparent circles but different shapes such as shapes, colors and the like are respectively selected, plate streaking is carried out until the colonies are single, bacillus like (Panenibacillus sp) A10 which decomposes chitin is selected, and the physiological and biochemical characteristics of the A10 strain are identified according to part of culture media and methods recommended in a common bacteria system identification manual and a Berger system identification manual, wherein the A10 strain is positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquifying gelatin, free from hydrogen sulfide and negative in indole, and 10% in NaCl tolerance.

The genome of the strain was extracted using a genome extraction Kit (Fast DNA Spin Kit for Soil) and 16SrDNA was amplified. After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and the result showed an obvious characteristic band at 1500 bp. The amplified product is then sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequence is uploaded to Genbank. Homology alignment with sequences in a gene bank is carried out through Blast analysis, and a phylogenetic evolutionary tree is constructed by Neihbor-Joinhing in MEGA5.1 software. The A10 strain has the highest homology with Paenibacillus, reaching 99 percent, and is finally named as Paenibacillus sp A10.

The bacillus like A10 strain which can decompose chitin and has the inhibiting effect on gray mold is obtained by screening in wild rice rhizosphere soil, the preservation number is CGMCC NO.15336, and the bacillus like A10 strain is obtained by primarily selecting in wild rice rhizosphere soil in the west, east and rural areas of the river to obtain a strain with strong ability of decomposing chitin, and then repeatedly screening the strain with good gray mold antagonistic effect. A large number of experiments prove that the bacterial strain A10 can decompose important component chitin of a botrytis cinerea cell wall, and the inhibition effect of fermentation liquor of the bacterial strain A10 on the botrytis cinerea can reach 91.8%, so that the bacterial strain A10 has a good antagonistic effect on the botrytis cinerea.

The invention utilizes the means of biological control, namely, utilizes organisms or metabolites thereof to effectively control plant diseases, is more environment-friendly compared with other traditional chemical control, has no pesticide residue and no drug resistance, and provides theoretical basis for the development and effective utilization of microorganism resources for control. Thereby laying a long-term development for green organic agriculture.

Drawings

FIG. 1 is a map of the A10 strain on a PDA plate;

FIG. 2 is a molecular identification clustering map of A10 strain;

FIG. 3 is a graph showing the antagonistic effect of A10 strain on gray mold;

FIG. 4 is a graph showing the effect of A10 fermentation broth and Botrytis cinerea specific on Botrytis cinerea inhibition;

FIG. 5 is a graph showing the comparison of the effect of A10 fermentation broth and Botrytis cinerea specific on Botrytis cinerea inhibition; wherein a is A10 fermentation liquor, and b is Botrytis cinerea specific element.

Detailed Description

The first embodiment is as follows: the paenibacillus for decomposing chitin is paenibacillus A10 which is preserved in China general microbiological culture Collection center, the preservation address is No. 3 of No.1 Siro-Shih-1 of the Kyochen of the sunward area in Beijing, the preservation date is 2018, 2 and 02 days, and the preservation number is CGMCC No. 15336.

In the embodiment, the separation and purification of the paenibacillus strain decomposing chitin in wild rice rhizosphere soil are adopted:

primary screening: weighing 5g of rhizosphere soil sample from wild rice in east and rural areas of Jiangxi province in 45ml of sterile water by adopting a plate dilution coating method, standing at 120rpm for 20min, and taking supernatant for 10-time concentration gradient dilution. Respectively take 10^-1、10^-2、10^-3Dilutions were run at 80 μ L each, and each gradient was plated onto 3 plates of chitin medium, using sterile water instead of dilutions as a blank. The culture medium is placed in an incubator at 25 ℃ for 2-3 days in an inverted mode, and the growth of bacterial colonies and the generation of transparent rings are observed. (chitin Medium: K)₂HPO₄ 1g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，NH₄Adding distilled water into Cl 1g and agar 15g to reach a constant volume of 1000ml, wherein 20 is added into each 80ml chitin culture mediumml 1% colloidal chitin. )

The embodiment utilizes a biological control means, namely, organisms or metabolites thereof are utilized to effectively control plant diseases, and compared with other traditional chemical control methods, the method is more environment-friendly, has no pesticide residue and no drug resistance, and provides a theoretical basis for development and effective utilization of microbial resources for control. Thereby laying a long-term development for green organic agriculture.

The second embodiment is as follows: the application of the paenibacillus for decomposing chitin in the embodiment refers to the application in plant disease control.

The bacillus like A10 strain which can decompose chitin and has an inhibiting effect on gray mold is obtained by screening in wild rice rhizosphere soil, the preservation number is CGMCC NO.15336, the bacillus like A10 strain is a strain which has strong ability of decomposing chitin and is obtained by primary selection in wild rice rhizosphere soil in the east and rural areas of the west of the river, and then the strain with good antagonistic effect on gray mold is repeatedly screened, a large number of experiments prove that the strain A10 can decompose important component chitin of gray mold cell walls, and the inhibiting effect of fermentation liquor of the strain A10 on gray mold can reach 91.8 percent, so that the strain A10 has good antagonistic effect on gray mold.

The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the bacillus like bacteria for decomposing chitin is applied to preventing and treating gray mold. The rest is the same as the second embodiment.

The fourth concrete implementation mode: the method for screening paenibacillus sp.a10 for decomposing chitin in the embodiment comprises the following steps: primary screening: weighing 5g of rhizosphere soil sample from wild rice in east and rural areas of Jiangxi province in 45ml of sterile water by adopting a plate dilution coating method, standing at 120rpm for 20min, and taking supernatant for 10-time concentration gradient dilution. Respectively take 10^-1、10^-2、10^-3Dilutions were run at 80 μ L each, and each gradient was plated onto 3 plates of chitin medium, using sterile water instead of dilutions as a blank. The culture medium is placed in an incubator at 25 ℃ for 2-3 days in an inverted mode, and the growth of bacterial colonies and the generation of transparent rings are observed. (chitin Medium: K)₂HPO₄1g，MgSO₄·7H₂O0.5g，NaCl 0.5g，NH₄Cl 1g, agar 15g, distilled water to 1000ml, wherein 20ml 1% colloidal chitin is added into each 80ml chitin culture medium. )

Re-screening: on a chitin culture medium, 60 colonies with larger transparent circles but different shapes such as shapes, colors and the like are respectively selected, plate streaking is carried out until the colonies are single, a morphological diagram of a paenibacillus sp A10 and a10 strain on a PDA plate is shown in figure 1, and the physiological and biochemical characteristics of the A10 strain are identified by referring to partial culture medium and methods recommended in a common bacteria system identification manual and a Bergey system identification manual, wherein the A10 strain is positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquefied in gelatin, free of hydrogen sulfide, negative in indole and 10% in NaCl tolerance.

The fifth concrete implementation mode: the identification of paenibacillus sp.a10 of the embodiment:

1. physiological and biochemical identification

According to the parts of culture media and methods recommended in the handbook for identifying common bacteria systems and the handbook for identifying Bergey systems, the physiological and biochemical characteristics of the A10 strain are identified, and the A10 strain is positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquefied in gelatin, free of hydrogen sulfide, negative in indole and 10% in NaCl tolerance.

TABLE 1 physiological and biochemical Properties of Strain A10

Note: "+" indicates positive, and "-" indicates negative.

2. Molecular identification

The genome of the strain was extracted using a genome extraction Kit (Fast DNA Spin Kit for Soil) and 16SrDNA was amplified. The PCR universal primer is: 27F (5 '-agaggtttgattctcggctcag-3'), 1492R (5 '-ggttactctgttacgactt-3'), reaction 25 μ L, Premix version 2.012.5 μ L, 27F (10nM)1 μ L, 1492R (10nM)1 μ L, DNA template 1 μ L, sterile water deficient to 25 μ L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 50s, extension at 72 ℃ for 90s, and 35 cycles; final extension at 72 ℃ for 10 min.

After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and the result showed an obvious characteristic band at 1500 bp. The amplified product is then sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequence is uploaded to Genbank. The DNA sequence is shown as SEQ ID NO: 1 is shown. Homology alignment with sequences in the gene bank was performed by Blast analysis and phylogenetic evolutionary trees were constructed using Neihbor-joining in MEGA5.1 software (fig. 2). The A10 strain has the highest homology with Paenibacillus, up to 99%, and the final named circle is Streptomyces hanekiensis sp A10.

Functional verification of the paenibacillus sp.a10 of the embodiment on gray mold prevention and control:

test 1: antagonism of gray mold by plate confrontation method

And (3) determining the bacteriostatic activity of the antagonistic strain on the gray mold by adopting a plate confrontation method. Firstly, gray mold is activated on a PDA plate, and a pathogenic bacteria plate with vigorous growth is selected. Preparing a bacterial cake with a diameter of 7mm by a puncher, inversely placing the bacterial cake on a PDA (personal digital Assistant) plate, inoculating the bacterial cake of the antagonistic bacterial strain at a position about 2cm away from pathogenic bacteria, taking the plate without the antagonistic bacterial strain as a control, repeating 3 times in each group, placing the plate in a constant temperature incubator at 26 ℃ for culturing for 2-6 days, and observing the size of a bacteriostatic circle (figure 3). And recording the minimum radius of the colony of the pathogenic bacteria and the radius of the control colony at the time of 2, 4 and 6 days, and calculating the bacteriostasis rate.

Bacteriostatic ratio (%) - (control colony radius-pathogen colony radius)/control colony radius × 100

From the bacteriostatic rate data of different days, the A10 strain has the best inhibition effect on gray mold at the 4 th day, which reaches 85.9%, and has a descending trend at the sixth day, but still reaches 85%.

Test 2: inhibition effect of A10 fermentation liquor and Botrytis cinerea specific element on Botrytis cinerea

Preparation of a10 fermentation broth: 100ml of fermentation medium (fermentation medium: 10.0g of glucose, 7.5g of yeast extract, distilled water to a constant volume of 1000ml) is filled into a 250ml triangular flask, and the mixture is autoclaved at 121 ℃. Inoculating A10 bacterial cake into cooled liquid culture medium in a clean bench, fermenting and culturing in a constant temperature shaking table at 160rpm for 3 days to obtain fermentation liquid, centrifuging the fermentation liquid at 3500rpm for 5 minutes to collect thallus, washing with sterile water for 2 times, resuspending in sterile water, measuring concentration with a blood counting plate, and diluting to 10⁶cfu/ml for use.

Preparing a pesticide diluent: the commercial gray mold special effect element (North America agriculture large) is diluted by 500-fold and 1000-fold according to the use instruction, and the gray mold special effect element is diluted by 250-fold and 500-fold in order to verify the inhibiting effect of A10 on pathogenic bacteria.

Antagonistic test: 100 mu LA10 of fermentation filter, 250 times of gray mold specific element diluent and 500 times of gray mold specific element diluent are respectively taken and coated on a PDA flat plate, 7mm gray mold pathogenic bacteria cakes are respectively inoculated in the center of the flat plate, sterile water is used as a reference (figure 4), each group is repeated for 3 times, the constant temperature culture is carried out at 26 ℃, the diameters of the pathogenic bacteria are measured on 2 nd, 3 th, 4 th and 5 th days, and the bacteriostasis rate is calculated.

The bacteriostatic ratio (%) - (control colony diameter-pathogen colony diameter)/control colony diameter × 100%

The results show that: since 500 times diluted Botrytis specific has almost no inhibition effect on Botrytis cinerea, the inhibition effects of A10 fermentation broth and 250 times Botrytis cinerea specific are only compared in the figure (FIG. 5), wherein the inhibition effect of A10 fermentation broth on Botrytis cinerea is gradually enhanced with the increase of days, the inhibition effect of A10 fermentation broth on Botrytis cinerea reaches 91.8% on day 5, the inhibition effect of Botrytis cinerea specific is gradually reduced, and the inhibition effect of Botrytis cinerea specific on day 2 is preferably 68%.

Sequence listing

<110> institute of geography and agroecology of northeast China academy of sciences

<120> paenibacillus for decomposing chitin and application thereof

<160> 3

<210> 1

<211> 1438

<212> DNA

<213> Paenibacillus sp (Panenibacillus sp.)

<400> 1

ggcctgtggg gtgctataca tgcaagtcga gcggaccctt cggggttagc ggcggacggg 60

tgagtaacac gtaggcaacc tgcctgtaag actgggataa ctaccggaaa cggtagctaa 120

gaccggataa gtgattctct cgcatgagag gatcaagaaa cacggggcaa cctgtggctt 180

acagatgggc ctgcggcgca ttagctagtt ggtggggtaa cggctcacca aggcgacgat 240

gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300

acgggaggca gcagtaggga atcttccgca atggacgcaa gtctgacgga gcaacgccgc 360

gtgagtgatg aaggttttcg gatcgtaaag ctctgttgcc agggaagaac gtcgtggaga 420

gtaactgctc tgcgaatgac ggtacctgag aagaaagccc cggctaacta cgtgccagca 480

gccgcggtaa tacgtagggg gcaagcgttg tccggaatta ttgggcgtaa agcgcgcgca 540

ggcggccgct taagtctggt gtttaagccc gaggctcaac ctcggttcgc actggaaact 600

gggtggcttg agtgcaggag aggaaagcgg aattccacgt gtagcggtga aatgcgtaga 660

gatgtggagg aacaccagtg gcgaaggcgg ctttctggcc tgtaactgac gctgaggcgc 720

gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780

gctaggtgtt aggggtttcg atacccttgg tgccgaagta aacacaataa gcactccgcc 840

tggggagtac gctcgcaaga gtgaaactca aaggaattga cggggacccg cacaagcagt 900

ggagtatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatccctct 960

gaatatccta gagatagggt aggccttcgg gacagaggag acaggtggtg catggttgtc 1020

gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgaactta 1080

gttgccagca ttgagttggg cactctaagt tgactgccgg tgacaaaccg gaggaaggtg 1140

gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtac tacaatggcc 1200

ggtacaacgg gaagcgaagt cgcgagatgg agccaatcct aagaaagccg gtctcagttc 1260

ggattgcagg ctgcaactcg cctgcatgaa gtcggaattg ctagtaatcg cggatcagca 1320

tgccgcggtg aatacgttcc cgggtcttgt acacaccgcc cgtcacacca cgagagttta 1380

caacacccga agtcggtggg gtaaccgcaa ggagccagcc gccgaagtgg ttgaatga 1438

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer 27F.

<400> 2

AGAGTTTGAT CCTGGCTCAG 20

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer 1492R nucleotide sequence.

<400> 3

GGTTACCTTG TTACGACTT 19

Claims

1. The application of the paenibacillus for decomposing chitin in preventing and treating gray mold is characterized in that the paenibacillus for decomposing the chitin is paenibacillus (Panenibacillus sp.) A10 and is preserved in the China general microbiological culture Collection center, the preservation address is No.1 Beijing West Luo No. 3 of the sunward district of Beijing, the preservation date is 2018, 2 months 02 and the preservation number is CGMCC No. 15336.