CN113337416B

CN113337416B - Biocontrol strain ZL-1 and application thereof

Info

Publication number: CN113337416B
Application number: CN202110209600.9A
Authority: CN
Inventors: 寇艳君; 时焕斌; 王聪聪; 邱结华
Original assignee: China National Rice Research Institute
Current assignee: China National Rice Research Institute
Priority date: 2021-02-25
Filing date: 2021-02-25
Publication date: 2022-03-18
Anticipated expiration: 2041-02-25
Also published as: CN113337416A

Abstract

A biocontrol strain ZL-1 and application thereof, belonging to the technical field of biology. The invention aims to provide a biocontrol strain ZL-1, and also aims to provide a biocontrol microbial inoculum containing the biocontrol strain ZL-1 and application of the biocontrol microbial inoculum in preventing and treating rice fungal diseases. The biocontrol strain ZL-1 and the biocontrol microbial inoculum thereof provided by the invention have broad-spectrum inhibition effect on various rice pathogenic fungi, have good antibacterial effect and high control effect, and overcome the defect that chemical bactericides are easy to generate resistance.

Description

Biocontrol strain ZL-1 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a biocontrol strain ZL-1 and an application thereof.

Background

With the increasing population, the demand of increasing the yield of food is more urgent. More and more pesticides are used at present, so that the crop yield is improved, and a plurality of environmental pollution problems are caused. The antagonistic microorganism in the crop growth environment is utilized to prevent and control the disease occurrence and develop into a green and environment-friendly way. The presence of many microorganisms, including both endophytic and exogenic bacteria, around food crops helps the plants to acclimatize and promote growth. Recent studies have reported that many endogenous bacteria play important roles in nutrient utilization, growth promotion and regulation of soil-borne and systemic pathogen infestation, such asGluconacetobacter diazotrophicus, Herbaspirillum, Burkholderia, Azospirillum, Bacillusspp.

Rice is a worldwide important food crop, and lives more than half of the world's population. The perennial occurrence of rice diseases threatens the stable and high yield of rice. At present, main fungal diseases in the rice planting process comprise rice blast, false smut, rice sheath blight, rice flax spot, rice ear rot, rice bakanae disease and the like. The method is an advantageous disease control strategy for preventing pathogenic fungi of rice by digging and utilizing beneficial microorganisms in the growing environment of rice.

KlebsiellaThe genus bacteria are reported to have various beneficial effects on host plants such as salt tolerance, nitrogen fixation and growth promotion. One strainKlebsiellaBelongs to salt-tolerant rhizobacteria MBE02, and has stimulation effect on the growth of peanuts. Can regulate and control the expression of a plurality of genes in a plant signal path, including jasmonic acid, ethylene and genes related to a pathogen defense signal path, thereby regulating and controlling plant immunity. Identification of one strain in stomach of citrus fruit flyKlebsiellaThe strain contains 213 unique genes, participates in amino acid metabolism and cofactor and vitamin metabolism, and can provide essential amino acids such as phenylalanine, tryptophan and methionine, and two vitamins for a host. According to the literature query, the genus has no report of the biocontrol effect on rice diseases.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a biocontrol strain ZL-1 and a technical scheme for application thereof.

The invention solves the technical problems by the following technical means:

the invention aims to provide a biocontrol strain ZL-1, wherein the biocontrol strain ZL-1 isKlebsiellaBelongs to the genus Lopa, which has been preserved in China center for type culture Collection at 26.1.1.1, with the preservation address of eight-way Lopa mountain in Wuchang district, Wuhan City, Hubei province and the preservation name of eight-way Lopa mountainKlebsiella sp.The preservation number of the strain is CCTCC M2021159.

Another purpose of the invention is to provide a biocontrol microbial inoculum containing the biocontrol strain ZL-1.

The invention also aims to provide the application of the biocontrol microbial inoculum in preventing and controlling rice fungal diseases.

Further, the rice fungal diseases described in the present invention are diseases caused by Pyricularia oryzae, Ustilaginoidea virens, Rhizoctonia solani, Ustilaginoidea virens, bakanae bacteria or Sesamum indicum, and preferably diseases caused by Pyricularia oryzae.

The biocontrol strain ZL-1 and the biocontrol microbial inoculum thereof provided by the invention have broad-spectrum inhibition effect on various rice pathogenic fungi, have good antibacterial effect and high control effect, and overcome the defect that chemical bactericides are easy to generate resistance.

Drawings

FIG. 1 is a BLAST alignment analysis chart of a biocontrol strain ZL-1 of the present invention;

FIG. 2 is a strain morphology test chart of the biocontrol strain ZL-1 of the present invention;

FIG. 3 is a graph showing the results of a plate confrontation experiment of the biocontrol strain ZL-1 of the present invention;

FIG. 4 is a graph showing the inhibitory effect of the biocontrol strain ZL-1 of the present invention on Pyricularia oryzae.

Detailed Description

The invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1: screening of biocontrol strains

Collecting samples: the sample used in this example was collected from the Zhejiang Fuyang rice base and sampled by a random sampling method. 5 rice plants were harvested at different locations while retaining the soil of the rice root system. The sample is put into a sterilized sealing bag and taken back to a laboratory for biocontrol bacteria screening.

Preparing a bacterial suspension: taking 0.1g of collected tissues of rice plant root system soil, rice root system, rice stem, rice leaf, rice grain and the like, putting the collected tissues into a sterilized 1.5mL centrifuge tube, adding 1 mL of sterilized ddH2O and a small steel ball, and putting the sterilized ddH2O and the small steel ball into a grinder for oscillation and crushing. And standing the mixture on a centrifuge tube rack for 10-20 min. Taking the supernatant into a new centrifuge tube, and mixing the supernatant with the supernatant according to the proportion of 1: 10 (V/V), 1: 100 (V/V), 1: the bacterial suspension is diluted by 1000 (V/V) to prepare bacterial suspensions with different concentrations.

And (3) separating bacteria: 200 mul of each diluted bacterial suspension with different concentrations is sucked and evenly coated on a NA (beef extract 5 g, peptone 10 g, cane sugar 10 g, agar powder 15 g and deionized water 1L) solid culture medium, the culture dish is placed in a 25 ℃ incubator for dark inversion culture after being dried, a single bacterial colony is selected to a new NA culture dish after 24 hours for purification, and all separated bacterial strains are numbered according to different positions.

Screening of biocontrol strains: screening by adopting a confrontation culture method. The obtained monoclonal bacteria were cultured in an antibody-free LB liquid and were then placed in an incubator at 37 ℃ for shaking culture. 2 mu L of the overnight cultured bacteria were dropped on one side of a potato glucose medium (PDA potato 200g, glucose 20g, agar powder 20 g), and rice blast fungus blocks were placed symmetrically on the other side, as control DH5 alpha. Culturing in dark at 25 deg.C for 7 days, and measuring the longest growth diameter of Magnaporthe grisea as the index of antagonistic action of the strain to be tested. Through the experimental screening, a strain with obvious inhibition effect on rice blast germs is obtained, and the strain is numbered ZL-1, and 50% of glycerol and a bacterial liquid 1 of the strain: the 1 (V/V) mixture was stored in a refrigerator at-80 ℃.

Example 2: identification of biocontrol strains

DNA extraction: genomic DNA of ZL-1 was extracted using a Tiangen genome extraction kit. 2ml of overnight-cultured bacterial liquid is collected, centrifuged at 10000rpm for 1 min, and the supernatant is discarded. 200. mu.L of buffer GA was added to the pellet, and the pellet was shaken until the pellet was completely suspended. Add 20. mu.L of protease K solution to the tube and mix by inversion. Then adding 220 mu L of buffer solution B, shaking and mixing uniformly, standing at 70 ℃ for 10 min, adding 220 mu L of absolute ethyl alcohol, shaking and mixing uniformly, and quickly centrifuging to remove water drops on the inner wall of the tube cover. The solution in the tube was transferred to an adsorption column, centrifuged at 12000rpm for 30s, the waste liquid was discarded, and the adsorption column was returned to the collection tube. Add 500. mu.L of buffer GD (without adding absolute ethanol in advance) to the adsorption column, centrifuge at 12000rpm for 30s, discard the waste solution, and place the adsorption column back into the collection tube. Adding 600 μ L of rinsing solution PW (anhydrous ethanol is added in advance in appropriate proportion), centrifuging at 12000rpm for 30s, discarding waste liquid, and placing the adsorption column back into the collecting tube. The rinsing was repeated once. Discard the waste liquid, put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2 min. Standing the adsorption column at room temperature for 2 min, transferring into a clean centrifuge tube, adding 100 μ L deionized water, and standing at room temperature for 2 min. Centrifuging at 12000rpm for 2 min, and collecting the solution into a centrifuge tube, wherein the solution is ZL-1 genomic DNA.

Taking ZL-1 genome DNA as a template, a primer 63F: CAGGCCTAACACATGCAAGTC (as shown in SEQ ID NO. 2) and 13897R: GGGCGGTGATGTACAAGGC (as shown in SEQ ID NO. 3) amplify the 16S rRNA sequence.

PCR reaction (50. mu.L): comprises 25 mu L of 2 xPCR mix, 2 mu L of 100 ng/mu L DNA template, 2.5 mu L of 10 mu M primer 63F/13897R, and the balance ddH₂And O. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 30 sec, amplification for 35 cycles, and final extension at 72 ℃ for 5 min. After the reaction, a band of about 1300bp in size was detected by 1% agarose gel electrophoresis. And the remaining amplification products were subjected to sequencing analysis (Johngzhou health Biotech Co., Ltd.). A DNA fragment of 1306 bp in size is obtained by sequencing, and the nucleotide sequence of the DNA fragment is shown as SEQ ID NO. 1.

The sequence was analyzed by BLAST alignment using NCB site, and as shown in FIG. 1, it was found to be related to Klebsiella pneumoniae (K.sp.) (B.sp.) (B: (B.sp.))Klebsiellasp.) highest homology.

And (3) detecting the morphology of the strain ZL-1: streaking on LB medium without antibiotics showed the colony characteristics shown in FIG. 2: on an LB plate culture medium, the colony is smooth in shape, neat in edge, milky white in the initial stage, yellowish in the later stage and opaque. Microscopic observation shows that the thalli are round or thick and short rod-shaped, the two sides of the rod-shaped thalli are straight, some thalli are slightly expanded, and the thalli exist singly.

Meanwhile, the ZL-1 strain is preserved in China center for type culture collection with the preservation number of CCTCC M2021159 for the preservation time: 26/1/2021, named by classificationKlebsiella sp.。

Example 3: plate confrontation experiment of ZL-1 strain and other rice pathogenic fungi

The ZL-1 strain grown overnight was pipetted in 2. mu.L drops 2 cm from the edge of PDA solid medium and in another PDA solid medium in the same position as a drop DH 5. alpha. as a control. Activated colony edge hypha blocks of rice blast germs, rice false smut germs, rice sheath blight germs, rice bakanae germs, rice ear rot germs and rice flax spot germs are beaten by a puncher with the inner diameter of 6mm and are respectively inoculated at the symmetrical positions of a strain ZL-1. The growth of colonies was observed after 7 days of culture at 25 ℃ and the maximum growth diameter was measured. Hypha growth inhibition (%) = (control colony maximum diameter-treated colony maximum diameter)/control colony maximum diameter x 100%, each experiment was repeated 3 times, and 3 parallel experiments were performed. The results are shown in FIG. 3 and Table 1.

TABLE 1 inhibition of ZL-1 against a number of important pathogenic rice fungi

Pathogenic bacteria

Magnaporthe grisea

Ustilaginoidea virens

Rhizoctonia solani

Ear rot fungi

Bakanae disease

Flax germs

Inhibition ratio (%)

63.47±1.30

48.57±3.71

13.08±0.79

19.73±1.31

19.55±1.67

17.73±2.57

The confrontation results are shown in figure 3 and table 1, the Klebsiella pneumoniae ZL-1 has obvious inhibition effect on various important plant pathogenic fungi, wherein the inhibition efficiency on rice blast is the highest, and the inhibition rate can reach more than 63%, so that the ZL-1 strain is a strain with broad-spectrum inhibition effect on various rice pathogenic fungi.

Example 4: spray inoculation of rice

The wild type strain B157 of rice blast fungus which has been cultured for 7 days is washed off surface hyphae by sterile water under aseptic conditions, the surface hyphae are collected in a 1.5mL centrifuge tube after being filtered by sterilized three-layer mirror paper, the concentration of sporophyll is adjusted to 1 × 105/mL, ZL-1 strain on a non-anti LB culture medium is dipped by a pipette tip and added into spore liquid, and the spore liquid without the strain ZL-1 and the sterile water are used for spray inoculation.

Selecting a disease-sensitive rice variety CO39 growing to 4-leaf stage, rolling a PVC film into a cylinder shape to cover the rice, and sealing with water to prevent air leakage. The spore liquid of the rice blast germs with the adjusted concentration is uniformly sprayed on the surfaces of the rice leaves by a pneumatic pump, and the upper opening is quickly sealed by a preservative film after the spore liquid is sprayed. Placing in incubator, dark treating at 22 deg.C for 2 days, light treating at 25 deg.C for 6 days alternately, taking off rice leaf, and sticking on A4 paper with double-sided adhesive tape. And photographing, scanning and storing. And calculating the inhibition effect according to the lesion area of the rice leaf. As a result, as shown in FIG. 4, the ZL-1 strain had a significant inhibitory effect against Pyricularia oryzae.

Sequence listing

<110> institute of Rice research in China

<120> biocontrol strain ZL-1 and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Klebsiella sp (Klebsiella sp.)

<400> 1

gactatcgag agcttgctct cgggtgacga gcggcggacg ggtgagtaat gtctgggaaa 60

ctgcctgatg gagggggata actactggaa acggtagcta ataccgcata acgtcgcaag 120

accaaagtgg gggaccttcg ggcctcatgc catcagatgt gcccagatgg gattagctgg 180

taggtggggt aacggctcac ctaggcgacg atccctagct ggtctgagag gatgaccagc 240

cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg gaatattgca 300

caatgggcgc aagcctgatg cagccatgcc gcgtgtgtga agaaggcctt cgggttgtaa 360

agcactttca gcggggagga aggcggtgag gttaataacc tcatcgattg acgttacccg 420

cagaagaagc accggctaac tccgtgccag cagccgcggt aatacggagg gtgcaagcgt 480

taatcggaat tactgggcgt aaagcgcacg caggcggtct gtcaagtcgg atgtgaaatc 540

cccgggctca acctgggaac tgcattcgaa actggcaggc tagagtcttg tagagggggg 600

tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg aggaataccg gtggcgaagg 660

cggccccctg gacaaagact gacgctcagg tgcgaaagcg tggggagcaa acaggattag 720

ataccctggt agtccacgct gtaaacgatg tcgatttgga ggttgtgccc ttgaggcgtg 780

gcttccggag ctaacgcgtt aaatcgaccg cctggggagt acggccgcaa ggttaaaact 840

caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgatgcaacg 900

cgaagaacct tacctggtct tgacatccac agaactttcc agagatggat tggtgccttc 960

gggaactgtg agacaggtgc tgcatggctg tcgtcagctc gtgttgtgaa atgttgggtt 1020

aagtcccgca acgagcgcaa cccttatcct ttgttgccag cggttcggcc gggaactcaa 1080

aggagactgc cagtgataaa ctggaggaag gtggggatga cgtcaagtca tcatggccct 1140

tacgaccagg gctacacacg tgctacaatg gcatatacaa agagaagcga cctcgcgaga 1200

gcaagcggac ctcataaagt atgtcgtagt ccggattgga gtctgcaact cgactccatg 1260

aagtcggaat cgctagtaat cgtagatcag aatgctacgt atgagg 1306

<210> 2

<211> 21

<212> DNA

<213> primer (primer)

<400> 2

caggcctaac acatgcaagt c 21

<210> 3

<211> 19

<212> DNA

<213> primer (primer)

<400> 3

gggcggtgat gtacaaggc 19

Claims

1. A biocontrol strain ZL-1 with a preservation number of: CCTCC M2021159.

2. A biocontrol microbial inoculum comprising the biocontrol strain ZL-1 as defined in claim 1.

3. The use of the biocontrol microbial inoculum of claim 2 in controlling rice fungal diseases.

4. The use according to claim 3, wherein the fungal disease of rice is a disease caused by Pyricularia oryzae, Ustilaginoidea virens, Rhizoctonia solani, Ustilaginoidea virens, Miao germ or Sesamum indicum.