CN107760640B

CN107760640B - Bacillus methylotrophicus B18 and preparation and application of liquid preparation thereof

Info

Publication number: CN107760640B
Application number: CN201711289844.2A
Authority: CN
Inventors: 李姝江; 朱天辉; 方馨玫; 何倩倩; 黎肇家; 谯天敏; 韩珊
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2020-06-09
Anticipated expiration: 2037-11-24
Also published as: CN107760640A

Abstract

The invention discloses a bacillus methylotrophicus B18 and preparation and application of a liquid preparation thereof, wherein the bacillus methylotrophicus B18 is preserved in China general microbiological culture collection center in 2017, 9 and 18 months, and the preservation number is CGMCC No. 14641. The liquid preparation can be prepared by taking bacillus methylotrophicus B18 as a strain and performing seed culture and fermentation culture. The bacillus methylotrophicus B18 provided by the invention has special effect on the bark rot of phellodendron, has stress resistance, good storage property, high temperature resistance, low temperature resistance and drying resistance, is suitable for commercial production, can avoid the serious consequence of environmental pollution caused by chemical drugs, and has the characteristics of sustainability and low cost.

Description

Bacillus methylotrophicus B18 and preparation and application of liquid preparation thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus methylotrophicus B18 strain, a liquid preparation thereof and application thereof in prevention and treatment of phellodendron bark rot.

Background

The cortex phellodendri is a main raw material for extracting berberine, is also an important economic tree species and traditional Chinese medicinal materials for bark in China, has more than 2000 years of medicinal history up to the present, contains various alkaloids, has special fragrance and bitter taste, has the effects of purging fire, removing steam, purging fire, detoxifying, reducing swelling, removing putrefaction and the like, and is a precious medicinal resource. The phellodendron bark is a clinically common traditional Chinese medicine for clearing heat and drying dampness, has wide application and large market demand, can be prepared into various Chinese patent medicines, cosmetics, biological pesticides and the like, and has important value for life health of people. In addition, the phellodendron is a precious tree, the wood is light yellow, has straight grains and a thin structure, is not cracked, has good material quality, is tough and elastic, has strong water-moisture resistance and corrosion resistance, is easy to process, and is a superior industrial material. In addition, the tree crown is wide, the tree form is tall and big, and the branches and leaves are luxuriant in summer, so that a good shade effect can be formed, and the tree can also be used as a garden tree species. The wide planting not only beautifies the environment, but also is active and economical. The yield mainly comes from wild resources, in recent years, the demand is large, and in addition, the golden cypress is a perennial woody medicinal material, the growth cycle is long, and the supply of the medicinal material is short.

Bark rot easily occurs in the growing process of phellodendron amurense, which is characterized in that the branches and the trunks generate dark brown spots from round to oval, the diseased parts become soft and water stain-like, and the later stage of drying shrinkage due to water loss generates cracks, which cause the leaves to yellow, fall off and the branches and the trunks to die seriously. When the humidity in the forest is higher, a large number of orange filaments grow out on the trunk and become conidia of germs. Currently, there is no special research on this aspect. At present, the prevention and treatment are mainly carried out by removing the disease source through forest culture measures, generally, chemical pesticides and chemical fertilizers are taken as the main measures, but the chemical pesticides (fertilizers) are applied for a long time, so that the environment is easily polluted, the ecology is damaged, and the research on the comprehensive disease prevention and treatment measures is still needed.

Disclosure of Invention

The invention aims to solve the technical problem of the prior art in preventing and treating the bark rot of golden cypress, and provides the application of the bacillus methylotrophicus B18 and the liquid preparation thereof in preventing and treating the bark rot of golden cypress.

The invention is realized by the following technical scheme:

the Bacillus methylotrophicus B18 strain is separated from healthy eucommia bark in Sichuan Mali invertebrate drug field by adopting a plate dilution coating method in 2016, 11 and 20 days, and is preserved in China general microbiological culture Collection center (CGMCC) of China institute of microbiological culture Collection, China academy of sciences, institute of microbiology, 9 and 18 days in 2017, wherein the address of the Bacillus methylotrophicus B18 strain is No. 3 of the West Lu No.1 of the North Cheng of the sunward region in Beijing, and the preservation number is CGMCC No. 14641.

The single colony of the bacillus methylotrophicus B18 cultured on the NA plate culture medium for 2d is white and round, has irregular edges, dry surface, folds, no stickiness and is opaque.

The sequence of the 16S rDNA of the bacillus methylotrophicus B18 is as follows: 1 in SEQ ID NO.

The liquid preparation with the bacillus methylotrophicus as the active component is prepared by taking bacillus methylotrophicus B18 as a strain through seed culture and fermentation culture.

The liquid preparation using the bacillus methylotrophicus B18 as the strain contains live spores with the number not less than 1 multiplied by 10⁹cfu/g。

The liquid preparation taking the bacillus methylotrophicus B18 as the strain is prepared by the following method:

1) first-order inclined plane seeds: preparing a beef extract peptone slant culture medium, inoculating bacillus methylotrophicus, and culturing to prepare a first-level seed;

2) secondary liquid seeds: sterilizing the nutrient meat culture solution under high pressure, placing in a triangular flask under the control of oxygen, inoculating Bacillus methylotrophicus slant seeds in an aseptic state, and performing shake culture to obtain Bacillus methylotrophicus liquid seeds;

3) liquid fermentation: the nutrient meat culture solution is sterilized under high pressure, placed in a triangular flask under the control of oxygen supply, inoculated with bacillus methylotrophicus liquid seeds in an aseptic state, the inoculum size is 10 percent of the total volume of the liquid, subjected to shaking culture to prepare a bacillus methylotrophicus liquid preparation, and then stored.

The beef extract peptone slant culture medium comprises the following components: 7g of beef extract, 10g of peptone, 5g of NaCl, 18g of agar and 1000mL of water.

The nutrient meat culture solution comprises the following components: 7g of beef extract, 10g of peptone, 5g of NaCl, and 1000mL of phellodendron twig immersion liquid.

The liquid preparation containing the active ingredient of the bacillus methylotrophicus B18 obtained by the preparation method is also within the protection scope of the invention.

The bacillus methylotrophicus B18 disclosed by the invention is applied to prevention and treatment of the bark rot of golden cypress.

The application of the bacillus methylotrophicus B18 liquid preparation in preventing and treating the bark rot of golden cypress is to scrape disease spots clean, dip liquid preparation diluent by using a sterile cotton ball and smear the diseased parts at intervals of 7d once for 5 times.

The liquid preparation is applied to the treatment of the skin rot disease, and the diluent of the liquid preparation is prepared by preparing the liquid preparation into the liquid preparation with the concentration of 1 x 10⁹cfu/mL, then diluted 400-fold.

Of course, the application of the bacterial suspension or the secondary metabolite of the bacillus methylotrophicus B18 in the preparation of the medicament for preventing and treating the phellodendron bark rot is also within the protection scope of the invention.

The method for preventing and treating the rotten skin disease of the golden cypress by the bacillus methylotrophicus B18 also belongs to the protection range of the invention, and the method is to apply the golden cypress in a smearing way in the growth process of the golden cypress; the smearing is bacterial suspension or fermentation liquor or metabolite or preparation of the bacillus methylotrophicus B18.

The invention has the beneficial effects that:

the bacillus methylotrophicus B18 provided by the invention has a special effect on phellodendron bark rot, tests show that the inhibition rate of the bacillus methylotrophicus B18 on the phellodendron bark rot reaches 98.36%, the bacillus methylotrophicus B18 is prepared into a liquid preparation, has a good prevention and treatment effect, is suitable for commercial production, can avoid the serious consequence of environmental pollution caused by chemical drugs, has the characteristics of stress resistance, good storage property, high temperature resistance, low temperature resistance and drying resistance, conforms to the national sustainable development strategy, has the characteristic of low cost compared with the chemical drugs, and has a wide application prospect.

Drawings

FIG. 1 is an electrophoretogram of PCR amplification products of B18rDNA-ITS sequences of Bacillus methylotrophicus of the present invention;

FIG. 2 is a phylogenetic tree of strain B18 constructed based on the 16S rDNA sequence.

Detailed Description

The technical solutions of the present invention are further described with reference to the following embodiments, which are merely preferred embodiments of the present invention, and not limiting the present invention in other forms, and any person skilled in the art may change or modify the technical contents disclosed above into equivalent embodiments with equivalent changes. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the scope of the present invention, unless they depart from the technical spirit of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation and characterization of Bacillus methylotrophicus B18

1.1 isolation, purification and preservation of endophytic bacteria

Taking healthy phellodendron amurense bark in a Sichuan Mali hupehensis Uygur pit medicated field for treating bark rot. The collected sample is washed cleanly by sterile water, 1.0g of tissue is weighed, the tissue is soaked in 70% alcohol for 1-2min, then sterilized by 1-3% sodium hypochlorite solution for 3-5min, after being washed for a plurality of times by sterile water, 100 mu L of the last washing liquid is absorbed to smear an NA flat plate (3 g of beef extract, 10g of peptone, 5g of sodium chloride, 15-20g of agar powder, 1000mL of distilled water and pH 7.0, the mixture is evenly mixed and subpackaged and then is sterilized at 121 ℃ for 30min under high pressure), and the mixture is cultured in the dark at 27 ℃ for 24h for comparison of surface sterilization and checking whether the surface sterilization is complete or not.

Cutting the surface-sterilized bark tissue, placing into a sterile mortar, adding sterilized quartz sand and 10mL sterile water, grinding, standing for 30min, and diluting for 10 min^-1，10^-2，10^-3，10^-4，10^-5Total 5 gradients, 100. mu.L 10 aspirated^-3，10^-4，10^-5The 3 gradient slurries were applied to NA plates (beef extract 3g, peptone 10g, sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7) respectively0, uniformly mixing, subpackaging, sterilizing at 121 ℃ for 30min under high pressure), repeating each treatment for 3 times, culturing at 27 ℃ for 48h, and selecting single colonies with obvious colony morphology difference to enter a primary screen.

According to the difference of colony size, color, protrusion, edge characteristic, smooth surface, transparency, etc., selecting single colony, streaking and purifying on NA plate, and transferring the NA slant to 4 deg.c for storage. A total of 31 endophytic bacteria were obtained.

1.2 plate antagonism of endophytic bacteria against Cork-tree Pityrosporum ovale

Plate confrontation method: inoculating 7d of phellodendron bark rotten skin disease (Nectria haematococca) cake (diameter 6mm) in the center of a culture dish, dipping and culturing 2d of endophyte at a position about 3cm away from the cake by using inoculating rings, drawing a thin line on each symmetrical side of the pathogen, culturing at 27 ℃, taking a plate only inoculated with the pathogen as a control, measuring the colony growth diameter of the pathogen when the control is full of the dish, and repeating the treatment for 3 times. The colony growth inhibition was calculated as follows:

the colony growth inhibition rate (control colony net growth diameter-treated colony net growth diameter)/control colony net growth diameter × 100%.

Table 1 shows that the 31 endophytic bacteria have a good antagonistic effect on phellodendron amurense (Nectria haematococca), and the inhibition rate of B18 reaches 98.36%.

TABLE 1 antagonistic action of endophytic bacteria against the Scleroderma destructor (7d)

1.3 identification of the species of endogenous antagonistic bacteria

The endophyte B18 is identified as bacillus methylotrophicus by morphological observation and combination of physiological and biochemical indexes (table 2) and molecular biology.

TABLE 2 physiological and biochemical indices of Strain B18

A single colony of 2d B18 strain cultured on NA plate medium was white, round, irregular in edge, dry in surface, wrinkled, non-sticky, and opaque.

Molecular biological identification (16S rDNA): extracting bacterial DNA to perform PCR amplification and electrophoresis, and recycling a product to a biotechnology company for sequencing; the homology BLAST analysis of the determined sequence with the sequences already reported in the GenBank database was carried out, and multiple sequence comparisons were carried out with Clustalx (1.83) software, and phylogenetic trees were constructed by the adjacency method in Mega4.0 software to determine the phylogenetic status of the B18 strain in microorganisms. Molecular biology identification to obtain a DNA fragment of 959bp in length (figure 1), submitting the 16S rDNA sequence of the B18 strain to a GenBank database for BLAST analysis, selecting a bacterial 16S rDNA sequence with higher homology, performing multiple-match array analysis by using Clustalx software, and constructing a phylogenetic tree by using Mega analysis software (figure 2). It can be seen that the B18 strain supports a poly of 1 branch with 99% of nucleotide sequence similarity of the 16s rrna gene and a higher abduction value with HQ662592 and KC790268, and is far from other Bacillus, indicating that B18 is closest to the genetic relationship with Bacillus methylotrophicus.

Based on the characteristics, the B18 strain is classified and named as Bacillus methylotrophicus (B18), and has been stored in the general microbiological culture Collection center of the institute of microbiology, China Committee for culture Collection of microorganisms (CGMCC) No.14641 in 2017, 9 and 18 days.

EXAMPLE 2 preparation of liquid formulation of Bacillus methylotrophicus B18

The bacillus methylotrophicus B18 obtained in example 1 was cultured by seed culture and fermentation to prepare a liquid preparation, specifically by the following method:

a beef extract peptone slant culture medium (7 g of beef extract, 10g of peptone, 5g of NaCl, 18g of agar and 1000mL of water) is prepared by a conventional method, inoculated with bacillus methylotrophicus and cultured to prepare a first-class seed.

The nutrient meat culture solution (7 g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of phellodendron twig soaking solution) is sterilized under high pressure, bottled in a 300mL triangular flask according to the proportion of 100mL of liquid under the control of introducing oxygen, inoculated with bacillus methylotrophicus slant seeds in an aseptic state, inoculated with 2 slant seeds in each flask, and subjected to shaking culture to prepare the bacillus methylotrophicus liquid seeds. Wherein 10g of phellodendron amurense twig is boiled in 1000mL of water for 10 minutes and filtered to obtain filtrate which is phellodendron amurense twig soaking solution.

The nutrient meat culture solution (7.0 g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of water) is sterilized under high pressure, bottled according to the proportion that 100mL of liquid is contained in a 300mL triangular flask (oxygen introduction control), inoculated with B18 liquid seeds in an aseptic state, the inoculation amount is 10 percent of the total volume of the liquid, the temperature is 27 ℃, the initial pH value is 7.0, and the mixture is subjected to shaking culture at 120r/min for 48 hours to prepare the B18 microbial inoculum.

And (3) preparation preservation: the bottled preparation can be stored at normal temperature for 1 year or at low temperature (4 deg.C) for 2 years without affecting the preventing and treating effects.

The liquid preparation prepared by the method and using Bacillus methylotrophicus B18 as strain contains viable spore number not less than 1 × 10⁹cfu/g。

When the microbial inoculum is used, the disease spots are scraped clean, and the disease parts are smeared by dipping liquid preparation diluent with a sterile cotton ball at intervals of 7d for 5 times.

Example 3 potted plant control efficacy test

Selecting 60 annual potted phellodendron amurense seedlings as experimental objects, and selecting conidia (spore concentration is 1 × 10) of PDA (potato spiraea sinensis) slant phellodendron amurense⁶cfu/mL)30mL of the shoot, treating the phellodendron seedlings by spraying the fungicide prepared in the example 2 at different concentrations by adopting a spraying method, spraying 1 time (3 times in total) every 7 times with 100mL of the fungicide in each pot, repeating the steps for 3 times by taking sterile water as a control, and counting the morbidity of the phellodendron. 10 phellodendron seedlings are treated each time.

And (3) after the pathogenic bacteria are inoculated for 30 days, disease investigation statistics is carried out according to the disease grading standard in the table 3, and the morbidity, disease index and prevention and treatment effect are calculated.

TABLE 3 grading Standard of the rotten skin disease of phellodendron

Incidence (%) — number of diseased plants/total number of plants × 100;

the disease index is [ sigma (number of branches at disease level x representing stage number)/(total number of branches x representing stage number with highest disease occurrence) ] × 100;

the preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index x 100.

Table 4 shows the control effect of B18 endophytic bacteria after 30 days of potting test, and the biocontrol bacteria B18 stock solution and diluent (50-800 times) show good protection effect on phellodendron bark rot bacteria after 30 days of pathogen inoculation. The control effect of the B18 fermentation stock solution is as high as 96.56%, the morbidity is obviously reduced to only 2%, the growth and the reproduction of phellodendron bark rot germs are inhibited, and the diseases can be basically controlled to be prevented. After the stock solution B18 is diluted, the control effect is reduced along with the increase of the dilution times to show different control effects, the control effect is more than 60% below 800 times of dilution, and the control effect is only 15.66% at 1200 times, so 50 times, 100 times, 200 times, 400 times and 800 times of dilution solution are selected for the determination of field experiments.

TABLE 4B 18 control Effect of endophytic bacteria after 30d of potting test

Example 4 field Effect control experiment

1) The liquid preparation of B18 (1X 10) obtained in example 2 was used⁹cfu/mL) are sequentially prepared into 50-fold, 100-fold, 200-fold, 400-fold and 800-fold diluents;

2) firstly, the diseased spots are scraped clean, the disease affected part is smeared with diluent dipped by a sterile cotton ball, and the disease affected part is smeared with sterile water as a contrast. Every treatment is provided with 200 lesions, and the treatment is performed once at intervals of 7d for 5 times. Each treatment was repeated 3 times.

Investigation and statistics are carried out before smearing and at the 30 th day after the last smearing, the number of plants with diseases, the disease degree and the number of cured spots are recorded, and the cure rate of the spots is calculated.

The lesion cure rate (%) — the number of cured lesions/total number of lesions × 100.

After applying the application method (table 5), the liquid preparation has obvious effect. The cure rate of the stock solution is as high as 95 percent, the cure rate is in a descending trend along with the increase of the dilution, wherein the cure rates of 50, 100, 200 and 400-time dilution solutions are all higher than 80 percent, and the difference is not obvious; the cure rate of 800 times of liquid is obviously reduced to 69 percent. In practical application of forestry production, under the condition of considering economic factors, the selection should be carried out in 400 times of liquid, and the concentration of 400 times is recommended.

TABLE 5 field treatment effect of applying method on bark rot of phellodendron

Sequence listing

<110> Sichuan university of agriculture

<120> preparation and application of bacillus methylotrophicus B18 and liquid preparation thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>959

<212>DNA

<213> Bacillus methylotrophicus (Bacillus methylotrophicus)

<400>1

actgcggcgt gctataatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 60

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120

accggggcta ataccggatg gttgtctgaa ccgcatggtt cagacataaa aggtggcttc 180

ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240

aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420

caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720

gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780

gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 840

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggt 959

Claims

1. A Bacillus methylotrophicus (Bacillus methylotrophicus) B18, wherein the strain is deposited, and the deposition unit: china general microbiological culture collection center with the collection number of CGMCC No. 14641.

2. A method for producing a liquid preparation comprising the Bacillus methylotrophicus B18 of claim 1 as an active ingredient, comprising the steps of:

1) first-order inclined plane seeds: preparing a beef extract peptone slant culture medium, inoculating bacillus methylotrophicus B18, and culturing to prepare a first-level slant seed;

2) secondary liquid seeds: sterilizing the nutrient meat culture solution under high pressure, placing in a triangular flask under oxygen control, inoculating the first-stage slant seeds in an aseptic state, and performing shaking culture to obtain second-stage liquid seeds;

3) liquid fermentation: sterilizing the nutrient meat culture solution under high pressure, placing in a triangular flask under the control of oxygen supply, inoculating secondary liquid seeds in an aseptic state, wherein the inoculation amount is 10% of the total volume of the liquid, performing shake culture to prepare a liquid preparation taking bacillus methylotrophicus B18 as an active ingredient, and then storing;

the beef extract peptone slant culture medium comprises the following components: 7g of beef extract, 10g of peptone, 5g of NaCl, 18g of agar and 1000mL of water;

the nutrient meat culture solution comprises the following components: 7g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of phellodendron twig immersion liquid.

3. A liquid preparation comprising Bacillus methylotrophicus B18 as an active ingredient, which is prepared by the method according to claim 2.

4. The liquid preparation according to claim 3, wherein the number of viable spores contained in the liquid preparation is not less than 1X 10⁹cfu/g。

5. The use of Bacillus methylotrophicus B18 according to claim 1 for preventing and treating cortex Phellodendri rot disease caused by the pathogen Nectria haematococca.

6. The use of the liquid preparation according to claim 3 for the control of phellodendron bark rot caused by the pathogenic bacterium Nectria haematococca, characterized in that the diseased spots are scraped clean, and the diseased parts are smeared with the liquid preparation diluent dipped with a sterile cotton ball 5 times at intervals of 7 d.