CN113278551A

CN113278551A - Burkholderia, bacterial agent comprising Burkholderia, bacterial fertilizer, preparation method and application

Info

Publication number: CN113278551A
Application number: CN202110551427.0A
Authority: CN
Inventors: 刘裴清; 翁启勇; 李本金; 王荣波; 陈庆河
Original assignee: Institute of Plant Protection of FAAS
Current assignee: Institute of Plant Protection of FAAS
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2021-08-20
Anticipated expiration: 2041-05-20
Also published as: CN113278551B

Abstract

The invention provides Burkholderia, a microbial inoculum containing Burkholderia, a bacterial fertilizer, a preparation method and application, and belongs to the technical field of microbial preparations. Burkholderia sp K2h of the present invention has a deposit number of: CGMCC NO.22001, which can inhibit the growth of phytophthora capsici by generating active substances, shows better control effect on phytophthora capsici, and has the highest inhibition rate of 90.01 percent. Meanwhile, the method can effectively colonize roots, stems and leaves of the peppers, and improve the micro-ecological environment of crops. The pesticide effect result of the indoor pot culture shows that the control effect of the K2h microbial inoculum on pepper diseases is 88.63 percent, and the pesticide resistance is not easy to generate. When in use, the root irrigation is carried out at the seedling stage or the adult stage of the pepper. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.

Description

Burkholderia, bacterial agent comprising Burkholderia, bacterial fertilizer, preparation method and application

Technical Field

The invention relates to the technical field of microbial preparations, in particular to Burkholderia, a microbial inoculum comprising the Burkholderia, a bacterial fertilizer, a preparation method and application.

Background

The pepper phytophthora blight is a destructive soil-borne disease, and pepper can be diseased at any time and any part in the growth process. In recent years, the dead seedling rate of a heavily diseased field with pepper phytophthora blight reaches 30-100%. At present, the phytophthora blight of the pepper is one of the key factors which prevent the stable yield and the yield increase of the pepper. The chemical bactericide plays a vital role in preventing and treating the pepper phytophthora blight. However, with the large amount and unscientific use of chemical bactericides, many drawbacks are increasingly highlighted, such as drug resistance of pathogenic bacteria, environmental pollution, ecological system destruction and the like, which become major problems of the currently applied chemical bactericides. The biocontrol microbial inoculum has good compatibility with the environment, high efficiency, low toxicity, low residue and small selection pressure on pathogenic bacteria, and is one of the popular areas in the research and development of pesticides at present. The core of microbial control is screening effective biocontrol bacteria. At present, no record or report about the control of phytophthora capsici by using Burkholderia is available.

Disclosure of Invention

The invention aims to provide a burkholderia, a microbial inoculum containing the burkholderia, a bacterial fertilizer, a preparation method and application.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides Burkholderia (Burkholderia sp.) K2h with the preservation number as follows: CGMCC NO. 22001.

The invention also provides a bacterial agent containing the Burkholderia in the scheme.

Preferably, the efficiency of Burkholderia in the microbial inoculumThe number of viable bacteria is 1 × 10⁸～5×10¹⁰CFU/ml。

The invention also provides a preparation method of the microbial inoculum in the scheme, which comprises the following steps:

and inoculating the burkholderia to an LB liquid culture solution, and performing fermentation culture to obtain the microbial inoculum.

Preferably, the conditions of the fermentation culture include: the OD value of the seed liquid of the Burkholderia is 1.2, the inoculation amount of the Burkholderia is 5% -10% of the volume percentage of the LB liquid culture liquid, the pH value of the LB liquid culture liquid is 7.0, the culture temperature is 28-30 ℃, the shaking table oscillation rate is 180-200 rpm, and the culture time is 60-80 h.

The invention also provides a bacterial fertilizer which comprises an organic fertilizer and the microbial inoculum of the scheme or the microbial inoculum obtained by the preparation method.

The invention also provides the application of the Klebsiella pneumoniae, the microbial inoculum obtained by the preparation method or the bacterial manure in preventing and treating pepper phytophthora blight and/or promoting pepper growth.

Preferably, the application comprises the following steps:

and applying a microbial inoculum or bacterial fertilizer containing the Klebsiella pneumoniae during the seedling stage and/or the adult stage of the pepper.

Preferably, when the microbial inoculum is applied, the application mode comprises root irrigation, the application amount of each pepper is 50-100 mL of the microbial inoculum, the application times are 2-3 times, and the interval time between every two applications is 8-12 days.

Preferably, the microbial inoculum is applied for 2-3 times in the seedling stage and the adult stage of the pepper.

The invention has the beneficial effects that: the invention provides Burkholderia (Burkholderia sp.) K2h with the preservation number as follows: CGMCC NO. 22001. The Burkholderia K2h has a good control effect on phytophthora capsici by generating active substances to inhibit the growth of phytophthora capsici, and the inhibition rate can reach 90.01 percent at most. Meanwhile, the plant can be effectively colonized on roots, stems and leaves of the peppers, the micro-ecological environment of crops is improved, and the growth of the peppers is promoted. The pesticide effect result of the indoor pot culture shows that the control effect of the K2h microbial inoculum on pepper diseases is 88.63 percent, and the pesticide resistance is not easy to generate. When in use, the root irrigation is carried out at the seedling stage or the adult stage of the pepper. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use. The strain K2h is obtained from the soil around the roots of plants, is harmonious and compatible with the soil ecology, is nontoxic and free from pathogenicity, and therefore has good practical application value.

Biological preservation Instructions

Burkholderia (Burkholderia sp.) K2h, deposited at the China general microbiological culture Collection center on 12.03.2021, and addressed to the institute of microbiology, China academy of sciences, No. 3, West Lu No. 1, North Cheng, the open-faced area, Beijing, with the deposit numbers: CGMCC NO. 22001.

Drawings

FIG. 1 shows the colony morphology of Burkholderia K2 h;

FIG. 2 shows the microstructure of Burkholderia K2 h;

FIG. 3 is a phylogenetic tree of Burkholderia K2h and the 16S rDNA sequence of related species; adopting MEGA5.0 software, adopting an ortho-position connection method to display a systematic development tree of K2h and a 16S rDNA sequence of a related species, carrying out similarity repeated calculation for 1000 times, wherein the development tree nodes only display Bootstrap values larger than 50% and the superscript 'T' represents a model strain;

FIG. 4 is a graph showing the effect of Burkholderia K2h on the growth of Phytophthora capsici hyphae; note: wherein A is CK, B is K2h fermentation liquor treatment;

FIG. 5 shows the colonization of Burkholderia K2h on the pepper roots;

FIG. 6 shows that Burkholderia K2h significantly promoted pepper growth.

Detailed Description

In the invention, the Burkholderia K2h is cultured in a TSA culture medium at 36 ℃ for 24 hours, colonies are yellow, round, opaque and neat in edge, thalli are rod-shaped, are 0.4-0.6 mu m multiplied by 0.6-1.8 mu m, are arranged singly or in pairs and are gram-negative.

The burkholderia K2h is separated from the root and stem junction of the green soy beans planted in suburbs of Zhangzhou dragon, Fujian, China, and is ecologically compatible with soil, thereby being beneficial to fully exerting the advantages of the strains.

The invention also provides a bacterial agent containing the Burkholderia in the scheme. In the present invention, the effective viable count of Burkholderia in the microbial agent is preferably 1X 10⁸～5×10¹⁰CFU/ml。

In the present invention, the conditions of the fermentation culture preferably include: the OD value of the seed liquid of the Burkholderia is 1.2, the inoculation amount of the Burkholderia is 5% -10% of the volume percentage of the LB liquid culture liquid, the pH value of the LB liquid culture liquid is 7.0, the culture temperature is 28-30 ℃, the shaking speed of a shaking table is 180-200 rpm, and the culture time is 60-80 h. In the present invention, the time for the culture is preferably 72 hours.

In the invention, the organic fertilizer preferably comprises an agricultural waste organic fertilizer and/or a livestock and poultry manure organic fertilizer; the mass ratio of the microbial inoculum to the organic fertilizer is preferably (5-10): 1000, more preferably 8: 1000. the bacterial fertilizer is obtained by mixing an organic fertilizer and a microbial inoculum, and the invention has no special requirement on the mixing mode and takes uniform mixing as the standard.

In the present invention, the preferred application comprises the following steps:

In the invention, when the microbial inoculum is applied, the application mode comprises root irrigation, the application amount of each pepper is 50-100 mL, the application frequency is 2-3, and the microbial inoculum is preferably applied for 2-3 times in the seedling stage and the adult stage of the pepper. The interval time between every two applications is 8-12 days, and further preferably 10 days.

In the invention, when bacterial manure is applied, the application mode of the bacterial manure is preferably hole application, the application amount of the bacterial manure per pepper is 1-1.5 jin per pepper, the application frequency is preferably 2 times, and the bacterial manure is further preferably applied once in each seedling stage and each adult stage of the pepper.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. 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, screening and characterization of Burkholderia K2h Strain

Isolation of the Strain

Test materials: collected from the root-stem junction of green soy beans planted in suburbs of Zhangzhou dragon sea city, Fujian, China.

Soaking the joint of the roots and stems of the green soy beans in 75% alcohol for 30s on an ultra-clean workbench, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 1min, soaking with 75% alcohol for 30s, and washing with sterile water for 5 times. Weighing 1g of tissue blocks with sterilized surfaces, soaking the tissue blocks in L0mL phosphate buffer solution, grinding the tissue blocks in a sterile mortar, standing for 5min, taking supernatant, performing gradient dilution by 10 times by using sterile water, respectively taking 100 mu L of stock solution and diluent, coating the stock solution and the diluent on an LB culture medium plate, and culturing at 28 ℃ for 2-3 d until single colonies grow out. After the colony grows out, strain purification is carried out on an LB culture medium plate by a line drawing method.

II, identification of the strains

(I) morphological characteristics of the Strain

Colony morphology feature observation (see fig. 1 and 2): when the TSA culture medium is cultured for 24 hours at 36 ℃, bacterial colonies are yellow, round, opaque and neat in edges, thalli are rod-shaped, 0.4-0.6 mu m multiplied by 0.6-1.8 mu m, are arranged singly or in pairs and are gram-negative.

(II) analysis of 16S rDNA sequence of Strain

16S rDNA sequence analysis of bacterium K2 h: using bacterial 16S Universal primer 27F

(AGAGAGTTTGATCCTGGCTCAG, shown in SEQ ID NO: 1) and 1492R (GGTTACCTTGTTACGACTT, shown in SEQ ID NO: 2) amplify the 16S rDNA gene of the bacteria. The PCR reaction program is: pre-denaturation at 94 deg.C for 3min, then performing PCR amplification at 94 deg.C for 55s, 50 deg.C for 50s, 72 deg.C for 1min for 35 cycles, and finally performing 1.2% agarose gel electrophoresis detection on the PCR amplification product at 72 deg.C for l0 min. After the PCR product is recovered, cloning the PCR product to a T vector, selecting positive clone and sending the positive clone to Shanghai biological engineering company for sequencing, and logging in a GenBank and comparing and analyzing the sequencing result.

The experimental result shows that the 16S rDNA fragment of the strain K2h is 1371bp, and the BLAST comparison result shows that the homology with the 16S rDNA sequence of most Burkholderia strains reaches more than 99%. Therefore, by integrating the colony morphology of the strain, the analysis of the 16S rDNA sequence and the like (using MEGA5.0 software, the phylogenetic tree of K2h and the 16S rDNA sequence of the related species is shown in FIG. 3 by the ortho-ligation method), the strain of the present invention is identified as Burkholderia (Burkholderia sp.), the strain is numbered as K2h, and the strain is deposited as Burkholderia (Burkholderia sp.) CGMCC No.22001, the preservation unit: china general microbiological culture Collection center, preservation Address: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing.

The nucleotide sequence of the 16S rDNA segment is shown in SEQ ID NO: 3, specifically:

example 2 preparation of biocontrol Burkholderia K2h microbial inoculum

Activating Burkholderia K2h preserved at 4 ℃ on an LB plate culture medium, and culturing at 28 ℃ for 24h to obtain the activated strain. The activated strain K2h was selected with an inoculating loop and inoculated into 100mL of LB medium, and cultured at 28 ℃ and 220rpm for 24 hours to prepare a seed solution. Inoculating Burkholderia K2h seed solution (OD value of the Burkholderia K2h seed solution is 1.2) into LB liquid culture solution according to the inoculation amount of 5% by volume fraction, culturing at 28 ℃, and culturing at 180rpm for 72h to obtain biocontrol bacteria fermentation liquor, namely the biocontrol bacteria agent, wherein the total viable bacteria concentration of the bacteria agent obtained by fermentation is 8 multiplied by 10⁸～5×10¹⁰CFU/ml。

Example 3 biocontrol Burkholderia K2h antagonistic assay

A plate confronting culture method is adopted to perform antagonistic determination on the phytophthora capsici by the strain K2h, firstly, a phytophthora capsici mycelium block is connected to the center of a PDA culture medium plate, when the colony of the phytophthora capsici grows to 3cm, the strain K2h is connected to 4 weeks of the colony, the width of a bacteriostatic band of the strain K2h on the phytophthora capsici is measured after the colony is cultured for 5 days at 28 ℃, the experimental result is shown in figure 4, and the structure of the figure 4 shows that the strain K2h has better antagonistic effect on the phytophthora capsici, and the inhibition rate reaches 90.00%.

Example 4 potted control of Phytophthora capsici by biocontrol Burkholderia K2h fermentation broth

The pepper variety is sweet pepper, and the experimental place is the perennial pepper epidemic disease occurrence place. After the seedlings are sown for 7-10 days, each seedling is irrigated with 50ml of K2h strain fermentation liquor (8 multiplied by 10)⁸CFU/ml), watering once more 100ml strain fermentation liquor in the flowering period. A50% anc wettable powder 800-fold liquid (purchased from Hanbang plant protection agent, Limited liability company, Tianjin) is used for root irrigation treatment as a pesticide control, and a fermentation medium and a clear water treated plant are used as a blank control. Each treatment was 10 seedlings, 3 replicates per treatment. And investigating the morbidity every day after the disease is developed, and counting the morbidity and disease index until the clear water control morbidity reaches more than 80 percent.

The disease grading criteria are as follows:

level 0: the inoculated part is unchanged or slightly browned;

level 1: the inoculation part generates scabs, and the area of the scabs accounts for less than 1/4 of the area of the stem;

and 3, level: the lesion area of the inoculation part accounts for about 1/4-1/2 of the total area of the stem part;

and 5, stage: the lesion at the inoculation part occupies more than 1/2 of the area of the stem part;

and 7, stage: the disease spots of the inoculated part are connected and form a stem winding phenomenon, but the plant does not wither or die;

and 9, stage: the plant will wither or die.

Disease index (%) [. sigma (number of disease plants at each stage x disease value)/(number of total investigated plants x highest value) ]. times.100%

The control effect (%) is [ (control disease index-treatment disease index)/control disease index ] × 100%

(control refers to clear water control treatment).

Results of the experiments referring to fig. 5, fig. 6 and table 1, fig. 5 shows the colonization of burkholderia K2h on pepper roots; FIG. 6 shows that Burkholderia K2h significantly promoted pepper growth, and in FIG. 6, the front row pepper seedlings were treated with CK2 (fermentation medium), and the rear row pepper seedlings were treated with Burkholderia K2 h. Table 1 shows that the actinomycete K2h fermentation liquid for test can obviously reduce the disease index of pepper phytophthora blight, the control effect is up to 89.21%, and the control effect is 80.81% higher than that of 800 times of chemical pesticide 50% Anke wettable powder. In addition, as can be seen from table 1, the disease index of the fermentation medium treatment is slightly lower than that of the clear water control treatment, but the difference between the two is not significant. The greenhouse pot experiment result shows that the K2h strain can effectively prevent and treat pepper phytophthora blight and shows good application potential.

TABLE 1 potted plant control of phytophthora capsici by biocontrol Burkholderia K2h fermentation broth

Treatment of	Disease index%	The control effect is%
			K2h fermentation broth	11.27±2.18c	89.21％
CK1 (50% Anke wettable powder 800 times liquid)	16.56±3.21b	80.81％
			CK2 (fermentation medium)	80.23±2.12a	--
CK3 (Water)	81.25％±2.25a	--

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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<120> Burkholderia, microbial inoculum comprising Burkholderia, bacterial fertilizer, preparation method and application

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ggttaccttg ttacgactt 19

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gaaagcgggg gaccttcggg cctcgcgcta tagggttggc cgatggctga ttagctagtt 180

ggtggggtaa aggcctacca aggcgacgat cagtagctgg tctgagagga cgaccagcca 240

cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga attttggaca 300

atgggcgaaa gcctgatcca gcaatgccgc gtgtgtgaag aaggccttcg ggttgtaaag 360

cacttttgtc cggaaagaaa tccttggctc taatacagtc gggggatgac ggtaccggaa 420

gaataagcac cggctaacta cgtgccagca gccgcggtaa tacgtagggt gcaagcgtta 480

atcggaatta ctgggcgtaa agcgtgcgca ggcggtttgc taagaccgat gtgaaatccc 540

cgggctcaac ctgggaactg cattggtgac tggcaggcta gagtatggca gaggggggta 600

gaattccacg tgtagcagtg aaatgcgtag agatgtggag gaataccgat ggcgaaggca 660

gccccctggg ccaatactga cgctcatgca cgaaagcgtg gggagcaaac aggattagat 720

accctggtag tccacgccct aaacgatgtc aactagttgt tggggattca tttccttagt 780

aacgtagcta acgcgtgaag ttgaccgcct ggggagtacg gtcgcaagat taaaactcaa 840

aggaattgac ggggacccgc acaagcggtg gatgatgtgg attaattcga tgcaacgcga 900

aaaaccttac ctacccttga catggtcgga atcctgctga gaggtgggag tgctcgaaag 960

agaaccggcg cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 1020

agtcccgcaa cgagcgcaac ccttgtcctt agttgctacg caagagcact ctaaggagac 1080

tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcctcatggc ccttatgggt 1140

agggcttcac acgtcataca atggtcggaa cagagggttg ccaacccgcg agggggagct 1200

aatcccagaa aaccgatcgt agtccggatt gcactctgca actcgagtgc atgaagctgg 1260

aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggt cttgtacaca 1320

ccgcccgtca caccatggga gtgggtttta ccagaagtgg ctagtctaa 1369

Claims

1. Burkholderia (Burkholderia sp.) K2h with the collection number: CGMCC NO. 22001.

2. A bacterial agent comprising the Burkholderia bacterium according to claim 1.

3. The microbial preparation according to claim 2, wherein the effective viable count of Burkholderia in the microbial preparation is 1X 10⁸～5×10¹⁰CFU/ml。

4. A method for producing the microbial agent according to claim 2 or 3, comprising the steps of:

5. The method of claim 4, wherein the conditions of the fermentation culture comprise: the OD value of the seed liquid of the Burkholderia is 1.2, the inoculation amount of the Burkholderia is 5% -10% of the volume percentage of the LB liquid culture liquid, the pH value of the LB liquid culture liquid is 7.0, the culture temperature is 28-30 ℃, the shaking table oscillation rate is 180-200 rpm, and the culture time is 60-80 h.

6. A bacterial fertilizer, which comprises an organic fertilizer and the microbial inoculum according to claim 2 or 3 or the microbial inoculum prepared by the preparation method of any one of claims 4 to 5.

7. The use of the Klebsiella pneumoniae of claim 1, the microbial inoculum of claim 2 or 3, the microbial inoculum obtained by the preparation method of any one of claims 4 to 5 or the bacterial manure of claim 6 in controlling pepper phytophthora blight and/or promoting pepper growth.

8. The application according to claim 7, characterized in that it comprises the following steps:

9. The use of claim 8, wherein when the microbial inoculum is applied, the application mode comprises root irrigation, the application amount of each pepper is 50-100 mL microbial inoculum, the application times are 2-3 times, and the time interval between every two applications is 8-12 d.

10. The use as claimed in claim 9, wherein the fungicide is applied 2-3 times in a seedling stage and an adult stage of pepper.