CN107136122B - Biocontrol microbial inoculum for preventing and treating potato late blight - Google Patents

Abstract

The invention discloses a biocontrol microbial inoculum for preventing and treating potato late blight, belonging to the technical field of crop disease prevention and treatment. The biocontrol microbial inoculum is prepared from streptomyces scabiosus (Streptomyces scabiosus)Streptomyceseurop aeiscabiei) St19, wherein the strain has been registered and preserved in China general microbiological culture Collection center at 24/3/2014, with the preservation number of CGMCCNO.8962. The biocontrol microbial inoculum can inhibit the growth of potato late blight bacteria and has good control effect on the potato late blight; can be used by root irrigation in the seedling stage or adult stage of the potato or mixed with organic fertilizer to prepare bacterial fertilizer. The St19 strain is obtained from potato rhizosphere soil, is harmonious and compatible with soil ecology, is nontoxic and free of pathogenicity, and therefore has great application potential in biological control of potato late blight.

Description

Biocontrol microbial inoculum for preventing and treating potato late blight

Technical Field

The invention relates to a biocontrol microbial inoculum for preventing and treating potato late blight, belonging to the technical field of crop disease prevention and treatment.

Background

From late blight bacteria (Phytophthora infestans The late blight caused by (Mont) de Bary) is a destructive disease in potato production, occurs and prevails in all main potato production areas in the world, seriously threatens the production of potatoes, causes harm, control difficulty and social influence exceeding rice blast and wheat stripe rust, and is regarded as the first international crop disease. The late blight is common and serious in China, and the economic loss of 80 billion yuan RMB caused by the late blight every year becomes the first obstacle limiting the production and the realization of industrialization of the potatoes in China.

Until now, the prevention and treatment of late blight mainly depends on the cultivation of disease-resistant varieties and the use of a large amount of chemical pesticides. The breeding of disease-resistant varieties is the most successful measure for preventing and treating epidemic diseases, but the toxic composition of late blight bacteria has the characteristic of variability, so that the resistance of the disease-resistant varieties bred by using main resistance genes is lost, and the screening and breeding of new varieties with epidemic disease resistance in a short time are very difficult. Although chemical pesticides play an important role in the development of agriculture, long-term use of chemical pesticides can have many negative effects. The use of chemical pesticides not only lacks long-term effectiveness, but also has the problems of ecological environment destruction, poisoning of people and livestock and beneficial organisms, great exceeding of the residual quantity of chemical substances in food and feed, influence on the living quality of people and livestock and the like, and the production and use of some high-toxicity and high-residue chemical pesticides are forbidden in many countries and regions. In the times that people pay more and more attention to their living environment and advocate green life, the exploration of effective biocontrol measures undoubtedly has great theoretical and practical significance.

Actinomycetes (Actinomycetes) are wide in distribution, various in types and different in metabolic function, and are a microbial resource with great value. At present, about 8000 bioactive substances are found from microorganisms, nearly 70 percent of the bioactive substances are generated by actinomycetes, particularly antibiotics generated by streptomyces are widely applied in agricultural production, and the antibiotic has the advantages of long control effect time, no toxicity, harmlessness, no residue, no pollution and the like, has great environmental benefit and economic benefit in agricultural production, and has wide development prospect. Therefore, screening and utilization of antagonistic bacteria to achieve the purpose of high-efficiency and environment-friendly biological control has become one of the hot spots of current agricultural science research.

Disclosure of Invention

The invention aims to: aiming at the problems that the potato late blight seriously occurs at present, the area is enlarged year by year, the chemical control effect is not ideal, and the environmental pollution is caused, the biocontrol microbial inoculum for controlling the potato late blight is provided.

The purpose of the invention can be realized by the following technical scheme:

a biological antibacterial agent for preventing and treating potato late blight is prepared from Streptomyces scabies (Streptomyces scabies)Streptomyceseuropaeiscabiei) St19, and the preparation method comprises:

1) preparing a seed solution: inoculating the streptomyces scabies St19 into a Gao's No. I liquid culture medium, culturing at 28 ℃ and 150 r/min for 5 d to prepare a seed solution;

2) fermentation: inoculating the seed liquid into a fermentation culture medium, wherein the fermentation conditions are as follows: the inoculation amount is 1-2% vol, the bottling amount is 100 mL/250 mL, the initial pH value of a fermentation medium is 7.0-7.2, the temperature is 28 ℃, 150 r/min and the fermentation time is 9 d, so that fermentation liquor is obtained;

3) preparation of sterile filtrate: and centrifuging the fermentation liquor at 4000 r/min for 5 min, and filtering and sterilizing by using a 0.45 mu m sterilization microporous filter membrane to obtain sterile filtrate, namely the biocontrol microbial inoculum.

Wherein, the formula of the fermentation medium in the step 2) is as follows: 2 percent of soluble starch, 1.5 percent of glucose, 2 percent of soybean meal and MgSO₄·7H₂O 0.05% wt、K₂HPO₄ 0.1% wt、NaCl 0.1% wt、CaCO₃0.2% wt, the balance being distilled water.

The streptomyces scabicus St19 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 3 months and 24 days 2014, and the preservation number is CGMCC NO. 8962.

The application modes include the following two modes:

1) irrigating roots at the seedling stage or adult stage of the potatoes for use: continuously using for 2-3 times in the seedling stage or adult stage of the potatoes, wherein the interval is 7-10 d each time;

2) mixing with organic fertilizer to obtain bacterial fertilizer.

The invention has the beneficial effects that:

the biocontrol microbial inoculum for preventing and treating the potato late blight can obviously inhibit the growth of the potato late blight bacteria. Because the potato seed extract is a biological agent, a series of problems caused by the use of chemical pesticides do not exist, the farmland pollution can be reduced, and the potato late blight can be effectively prevented and treated.

The biocontrol actinomycetes used by the invention are separated from potato rhizosphere soil, are ecologically compatible with the soil, and are beneficial to fully exerting the advantages of the strains.

The biocontrol actinomycete strain used in the invention has simple culture conditions, is easy for industrial production and has good development and application prospects.

Drawings

FIG. 1 is the colony morphology of the strain St19 on the Gao's first medium;

FIG. 2 shows aerial hyphae and spore filament morphologies (400X under light microscope) of strain St 19;

FIG. 3 shows the hyphal morphology of strain St19 (Electron microscope 5000X);

FIG. 4 is a plate bacteriostasis effect diagram of the antagonistic bacteria sterile filtrate of the strain St19 on potato late blight bacteria.

Detailed Description

For a better understanding of the present invention, reference is made to the following examples and accompanying drawings which are set forth to illustrate, but are not to be construed as the limit of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Example isolation of antagonistic Actinomycetes

The specific process is as follows:

1. collection of soil samples

Collecting rhizosphere soil in 5-10 cm depths of different potato plots from Zhongning county of Fujian province, subpackaging and marking, bringing back to a laboratory, and separating after natural air drying.

2. Isolation of Actinomycetes

The separation was performed by plate dilution. Weighing 10 g of soil sample, pouring into a triangular flask filled with small glass beads and 90ml of sterile water, shaking for 30 min, standing for 5 min, sequentially diluting by 10 times, and respectively preparing into 10^-2、10^-3、10^-4、10^-5、10^-60.1 ml of each of the suspensions was added to the culture medium of Gao's No. one (K was added to the medium to a final concentration of 50 mg/L)₂Cr₂O₇) And uniformly coating the plate, culturing and observing at 28 ℃, and selecting different single colonies after 5-7 days for streaking and purifying. Transferring the purified strain to a Gao's first slant culture medium for culture, storing at 4 ℃ for later use, and performing co-separation to obtain 85 actinomycetes.

EXAMPLE two identification of Strain St19

1. Morphological feature observation

Strain St19 was streaked on a Gao's first medium, and cultured at 28 ℃ for 10 days, and then the morphology of colonies was observed (FIG. 1), while a sterile cover glass was inserted obliquely at 45 ℃ and cultured at 28 ℃ for 7 days, and then the cover glass was removed and the morphological characteristics of mycelia were observed under an optical microscope and an electron microscope (FIGS. 2 and 3).

2. Culture characteristic Observation

And (3) adopting 8 culture media recommended by Streptomyces appraisal handbook, culturing the strain St19 at 28 ℃ for 7-10 days, observing the growth condition of the strain, and recording the colors of aerial hyphae and hyphae in the substrate and whether soluble pigments are generated. The strain St19 grows well on a Gao's first culture medium, a Cbeck culture medium, an ammonium starch culture medium, a PDA culture medium, a glucose yeast extract culture medium and an oat culture medium, and the mycelia and aerial mycelia in the medium grow well; while the growth was poor on the Klebsiella medium and the Glucochinine medium, yellow-fading or brown-fading soluble pigments were produced on the Gauss-I medium and the PDA medium (Table 1). According to the above morphological feature analysis, St19 was preliminarily identified as Streptomyces scabies, in contrast to the manual for identification of Streptomyces.

TABLE 1 culture characteristics of the Strain St19 on different media

3. 16S rDNA sequence analysis

After extracting the genomic DNA of the strain St19 by using the genome extraction kit, the PCR amplification of 16S rDNA is carried out by adopting universal primers F: 5'-AGAGTTTGATCCTGGCTCAG-3' and R: 5'-GGTTACCTTGTTACGACTT-3'. Recovering PCR amplification product to obtain St19-16S rDNA, connecting, converting, and identifying, wherein the positive clone is sent to biological engineering (Shanghai) Co., Ltd to sequence to obtain nucleotide sequence information with whole sequence length of 1548 bp (please see sequence). The obtained sequences were subjected to BLAST analysis alignment in GenBank database, and it was found that all the strains having a high homology with St19 belong to the genus StreptomycesStreptomyceseuropaeiscabiei99% similarity, and combining morphological and cultural characteristics, strain St19 was identified as S.scabies (S.scabies) ((S.scabies))Streptomyceseuropaeiscabiei)。

Example optimization of fermentation Process of the third St19 Strain

Inoculating the strain into Gao's No. I liquid culture medium, culturing at 28 deg.C and 150 r/min for 5 d to obtain seed liquid, and inoculating into fermentation culture medium. The optimal fermentation medium formula of the strain is obtained by taking potato late blight bacteria as target bacteria, screening a carbon source and a nitrogen source, and performing a single factor test and an orthogonal test under nutritional conditions: 2 percent of soluble starch, 1.5 percent of glucose, 2 percent of soybean meal and MgSO₄·7H₂O 0.05% wt、K₂HPO₄ 0.1% wt、NaCl 0.1% wt、CaCO₃0.2% wt, the balance being distilled water. The optimal fermentation conditions are as follows: the inoculation amount is 1-2% vol, the bottling amount is 100 mL/250 mL, the initial pH value of the fermentation medium is 7.0-7.2, the temperature is 28 ℃, the fermentation time is 150 r/min, and 9 d is obtained. And centrifuging the fermentation liquor at 4000 r/min for 5 min, and filtering and sterilizing by using a 0.45 mu m sterilization microporous filter membrane to obtain sterile filtrate.

Example four Strain St19 sterile filtrate bacteriostasis assay

Measuring relative antibacterial rate by growth rate method, mixing sterile filtrate and rye culture medium cooled to about 40 deg.C at a volume ratio of 1:10, pouring into flat plate, and adding sterilized water into rye culture medium at the same ratio as control. The method comprises the steps of placing a late blight germ filament block with consistent bacterial age and diameter of 5mm in the center of a rye flat plate, placing the rye flat plate in a thermostat for culturing at 20 ℃, measuring the diameter of a bacterial colony at regular time after 15 days, calculating the relative bacteriostasis rate, and repeating the treatment for 5 times, wherein the result shows that the strain St19 has a good antagonistic effect on potato late blight germs (figure 4).

EXAMPLE five bacterial strain St19 fermentation liquor potted plant control effect test

Selecting 3-5 leaf-sized potato late blight high-sensitivity variety (Favorita) seedlings, and irrigating 50ml St19 strain fermentation liquor (about 10 ml) in each seedling⁹CFU/ml). After 24 h, the roots are irrigated and inoculated with zoospore suspension (5 multiplied by 10) of potato late blight bacteria⁴pieces/mL), 20 mL per plant. After 4 days of inoculation, 100ml of St19 strain fermentation liquor is poured once. The 1000-fold liquid irrigation of 50% Anke wettable powder is used as pesticide control, and the plant treated with clear water is used as blank control. Each treatment had 15 seedlings, and each treatment was repeated 3 times. Investigation of onset every day after onsetAnd (4) counting the morbidity and disease index until the clear water control morbidity reaches more than 80 percent, and ending the experiment.

The disease grading criteria are as follows:

level 0: no disease spots;

level 1: the area of the lesion spots accounts for less than 5% of the whole leaf area;

and 3, level: the lesion area accounts for 6-10% of the whole leaf area;

and 5, stage: the lesion area accounts for 11% -20% of the whole leaf area;

and 7, stage: the lesion area accounts for 21-50% of the whole leaf area;

and 9, stage: the lesion area accounts for more than 50% of the whole leaf area.

Disease index (%) = [. sigma. pathological stage number x representative number)/(most severe stage number x total leaf number) ] × 100%

Control effect (%) = [ (blank disease index-treatment disease index)/blank disease index ] × 100%

The experimental results are shown in table 2, and the results show that the actinomycete St19 fermentation liquid for test can obviously reduce the disease index of the potato late blight, the control effect is up to 74.65%, and the control effect is only slightly lower than 80.22% of that of 1000 times of that of 50% anc wettable powder serving as a chemical pesticide. The results of greenhouse pot experiments show that the St19 strain can effectively prevent and treat potato late blight and has good application potential.

TABLE 2 prevention and treatment effects of actinomycete St19 fermentation broth on potato late blight

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> institute of plant protection of academy of agricultural sciences of Fujian province

<120> a biocontrol microbial inoculum for preventing and treating potato late blight

<130> 3

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> F

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 19

<212> DNA

<213> R

<400> 2

ggttaccttg ttacgactt 19

<210> 3

<211> 1548

<212> DNA

<213> St19-16S rDNA

<400> 3

agcttgcatg cctgcaggtc gacgattaga gtttgatcct ggctcaggac gaacgctggc 60

ggcgtgctta acacatgcaa gtcgaacgat gaaccacttc ggtggggatt agtggcgaac 120

gggtgagtaa cacgtgggca atctgccctt cactctggga caagccctgg aaacggggtc 180

taataccgga tacaacactc tcgggcatcc gatgagtgtg gaaagctccg gcggtgaagg 240

atgagcccgc ggcctatcag cttgttggtg aggtaacggc tcaccaaggc gacgacgggt 300

agccggcctg agagggcgac cggccacact gggactgaga cacggcccag actcctacgg 360

gaggcagcag tggggaatat tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtga 420

gggatgacgg ccttcgggtt gtaaacctct ttcagcaggg aagaagcgaa agtgacggta 480

cctgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcga 540

gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gtctgtcgcg tcgaatgtga 600

aagcccgggg cttaaccccg ggtctgcatt cgatacgggc agactagagt gtggtagggg 660

agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 720

aaggcggatc tctgggccat tactgacgct gaggagcgaa agcgtgggga gcgaacagga 780

ttagataccc tggtagtcca cgccgtaaac ggtgggaact aggtgttggc gacattccac 840

gtcgtcggtg ccgcagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct 900

aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggc ttaattcgac 960

gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aacggccaga gatggtcgcc 1020

cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1080

ggttaagtcc cgcaacgagc gcaacccttg ttctgtgttg ccagcatgcc cttcggggtg 1140

atggggactc acaggagact gccggggtca actcggagga aggtggggac gacgtcaagt 1200

catcatgccc cttatgtctt gggctgcaca cgtgctacaa tggcaggtac aatgagctgc 1260

gaagccgtga ggcggagcga atctcaaaaa gcctgtctca gttcggattg gggtctgcaa 1320

ctcgacccca tgaagtcgga gttgctagta atcgcagatc agcagtgctg cggtgaatac 1380

gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggtaaca cccgaagccg 1440

gtggcccaac cccttgtggg agggagctgt cgaaggtggg actggcgatt gggacgaagt 1500

cgtaacaagg taccaatctc tagaggatcc ccgggtaccg agctcgaa 1548

Claims (2)

1. The biocontrol microbial inoculum for preventing and treating potato late blight is characterized by comprising streptomyces scabiosus (Streptomyces scabiosus)Streptomyceseuropaeiscabiei) St19, and the preparation method comprises:

1) preparing a seed solution: inoculating the streptomyces scabies St19 into a Gao's No. I liquid culture medium, culturing at 28 ℃ and 150 r/min for 5 d to prepare a seed solution;

2) fermentation: inoculating the seed liquid into a fermentation culture medium, wherein the fermentation conditions are as follows: the inoculation amount is 1-2% vol, the bottling amount is 100 mL/250 mL, the initial pH value of a fermentation medium is 7.0-7.2, the temperature is 28 ℃, 150 r/min and the fermentation time is 9 d, so that fermentation liquor is obtained;

3) preparation of sterile filtrate: centrifuging the fermentation liquor at 4000 r/min for 5 min, and filtering and sterilizing by using a 0.45 mu m sterilization microporous filter membrane to obtain sterile filtrate, namely the biocontrol microbial inoculum;

the formula of the fermentation medium in the step 2) is as follows: 2 percent of soluble starch, 1.5 percent of glucose, 2 percent of soybean meal and MgSO₄·7H₂O 0.05% wt、K₂HPO₄ 0.1% wt、NaCl 0.1% wt、CaCO₃0.2 wt% and the balance of distilled water;

the streptomyces scabicus St19 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 3 months and 24 days 2014, and the preservation number is CGMCC NO. 8962.

2. The biocontrol microbial inoculum for controlling potato late blight according to claim 1, which is applied in the following two ways:

1) irrigating roots at the seedling stage or adult stage of the potatoes for use: continuously using for 2-3 times in the seedling stage or adult stage of the potatoes, wherein the interval is 7-10 d each time;

2) mixing with organic fertilizer to obtain bacterial fertilizer.

Images