CN113046281A - Streptomyces castochromosis JA58 for preventing and treating potato late blight and application thereof - Google Patents

Streptomyces castochromosis JA58 for preventing and treating potato late blight and application thereof Download PDF

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CN113046281A
CN113046281A CN202110549060.9A CN202110549060A CN113046281A CN 113046281 A CN113046281 A CN 113046281A CN 202110549060 A CN202110549060 A CN 202110549060A CN 113046281 A CN113046281 A CN 113046281A
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李本金
王荣波
刘裴清
林金龙
陈庆河
翁启勇
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Abstract

The invention discloses a streptomyces badius auberkii JA58 for preventing and treating potato late blight and application thereof, belonging to the technical field of crop disease prevention and treatment. Streptomyces castaneae (C.), (B.), (CStreptomyces badius) JA58 strain has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 21.9.2020, with the preservation number of CGMCC No. 20691. A biocontrol microbial inoculum for preventing and treating potato late blight is prepared from Streptomyces castaneae JA58 fermentation broth or Streptomyces castaneae JA58 thallus-free fermentation broth, can inhibit the growth of potato late blight bacteria, and has good prevention and treatment effects on potato late blight. The Streptomyces castochromogenes JA58 strain has wide application potential in biological control of potato late blight。

Description

Streptomyces castochromosis JA58 for preventing and treating potato late blight and application thereof
Technical Field
The invention belongs to the technical field of crop disease control, and particularly relates to streptomyces badius aubergiensis JA58 for controlling potato late blight and application thereof.
Background
The potato is the fourth major food crop in the world after rice, wheat and corn, China has become the first major country for producing and consuming the potato, and the planting area of the potato is more in a trend of increasing year by year in recent years. However, caused by late blightphytophthora infestansThe 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 about 20 hundred million dollars 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 cultivation 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 diversity, so that the disease-resistant varieties cultivated by using the main resistance genes lose resistance quickly. Due to the lack of durable and effective potato resistant varieties, spraying of bactericides is always the most effective means for preventing and controlling late blight, chemical pesticides play an important role in agricultural development, pathogenic bacteria can generate drug resistance after long-term use of the chemical pesticides, the problems that the ecological environment is damaged, poisoning of people and livestock and beneficial organisms, the residual quantity of chemical substances in food and feed exceeds the standard greatly, the living quality of people and livestock is affected and the like can occur after excessive use of the pesticides, 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, are the most main antibiotic producing bacteria in the nature, and the research of antagonistic Actinomycetes plays an important role in screening antagonistic bacteria. 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 is seriously generated, the area is enlarged year by year, the chemical control effect is not ideal, and the environmental pollution is caused at the same time, the streptomyces badius faeci JA58 for controlling the potato late blight and the application thereof are provided.
In order to achieve the purpose, the invention adopts the following technical scheme:
streptomyces aubergiensis JA58 for preventing and treating potato late blight, wherein the strain is Streptomyces aubergiensis (Streptomyces aubergiensis)Streptomyces badius) JA58, deposited in the China general microbiological culture Collection center, with the address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is 2020, 9 and 21 days, and the preservation number is CGMCC number 20691.
A biocontrol microbial inoculum for preventing and treating potato late blight comprises streptomyces badius flavidus (A.flavidus)Streptomyces badius) JA58 fermentation broth, or Streptomyces castochromogenes (II)Streptomyces badius) JA58 sterile broth.
Streptomyces castaneae (see below)Streptomyces badius) The preparation method of the JA58 fermentation liquor comprises the following steps:
1) preparing a seed solution: inoculating Streptomyces castochromogenes JA58 in Gao's I liquid culture medium, culturing at 28 deg.C and 180 r/min for 7 d to obtain seed liquid;
2) fermentation: inoculating the seed liquid into a fermentation culture medium, wherein the fermentation conditions are as follows: the inoculation amount is 1vol%, the bottling amount is 200 mL/250 mL, and the fermentation medium is initially inoculatedThe initial pH value is 7.0-7.2, the temperature is 28 ℃, the fermentation time is 10 days, and the streptomyces castanea (S. flavus) is obtainedStreptomyces badius) JA58 fermentation broth.
The formula of the fermentation medium in the step 2) is as follows: 0.5wt% of soluble starch, 1.5wt% of glucose, 1wt% of soybean meal, 0.2wt% of peptone, 0.2wt% of yeast extract and MgSO4·7H2O 0.05wt% 、K2HPO4 0.05wt% 、NaCl 0.1wt% 、CaCO30.2wt%, the balance being distilled water.
Streptomyces castaneae (see below)Streptomyces badius) Preparation of JA58 thallus-free fermentation broth: streptomyces castaneae (V.lutescens) (V.lutescens)Streptomyces badius) After the fermentation liquor of JA58 is centrifuged at 6000 r/min for 7 min, a 0.45 mu m sterilization microporous filter membrane is used for filtration sterilization to obtain streptomyces castanea (S. flavus) ((S. castanea))Streptomyces badius) JA58 sterile broth.
The application of the biocontrol microbial inoculum for preventing and treating the potato late blight in preventing and treating the potato late blight.
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 tomato rhizosphere soil, are harmoniously compatible with soil ecology, 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.
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FIG. 1 shows the hyphal morphology of strain JA58 (Electron microscope 2000X).
FIG. 2 shows the inhibitory effect of strain JA58 on the growth of the hyphae of potato late blight fungus from Fujian.
FIG. 3 shows the inhibitory effect of strain JA58 on the growth of hyphae of potato late blight fungus from Ningxia.
FIG. 4 shows the inhibition effect of the strain JA58 thallus-free fermentation liquid on the growth of potato late blight bacteria hyphae.
FIG. 5 shows the disease preventing effect of strain JA58 fermentation liquid on potato tuber slices.
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 with depths of 5-10 cm from Fujian city 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 in a gradient manner, 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 (3) uniformly coating on a plate, culturing and observing at 28 ℃, and picking different single colonies after 7 d and streaking for purification. Transferring the purified strain to a Gao's first slant culture medium for culture, storing at 4 ℃ for later use, and co-separating to obtain 32 actinomycetes.
EXAMPLE identification of the second Strain JA58
1. Morphological feature observation
The isolated strain JA58 was streaked on Gao's first medium, cultured at 28 ℃ for 10 days, and then the morphology of colonies was observed, while a sterile cover glass was obliquely inserted at 45 ℃ and cultured at 28 ℃ for 7 days, after which the cover glass was removed and the morphological characteristics of mycelia were observed under an electron microscope (FIG. 1).
2. Culture characteristic Observation
The strain JA58 is cultured for 7-10 days at 28 ℃ by adopting 8 culture media recommended by Streptomyces appraisal handbook, the growth condition of the strain is observed, and the color of aerial hyphae and hyphae in the medium and whether soluble pigment is generated are recorded. The strain JA58 grows well on a Gao's I culture medium, a Kjeldahl 1 culture medium, a Cnbeck culture medium, an ammonium starch culture medium and a PDA culture medium, and hyphae and aerial hyphae in the medium grow well; on the other hand, the growth on oat medium was general, the growth on glucose aspart medium and glucose yeast extract medium was poor, and no soluble pigment was produced on the above medium (Table 1). Based on the above morphological characteristics analysis, JA58 was preliminarily identified as a chestnut brown chain by referring to the identification manual of streptomycete
TABLE 1 culture characteristics of Strain JA58 on different media
Figure DEST_PATH_IMAGE002
3. 16S rDNA sequence analysis
After extracting the genome DNA of the strain JA58 by 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 JA58-16S rDNA, connecting, converting, identifying, and sequencing the positive clone to bioengineering (Shanghai) Co., Ltd to obtain nucleotide sequence information with a sequence total length of 1539 bp and a nucleotide sequence shown in SEQ DI No. 1. The obtained sequences were submitted to GenBank database and aligned by BLAST analysis, and it was found that all the strains having a higher homology to JA58 belong to the genus StreptomycesStreptomyces badiusThe similarity reaches 99.93 percent, and the strain JA58 is identified as the streptomyces aureofuscus (Streptomyces aubergiensis) by combining morphological characteristics and culture characteristicsStreptomyces badius）。
EXAMPLE inhibitory Effect of the three Strain JA58 on the growth of the hyphae of potato late blight bacterium from Fujian
A plate confronting culture method is adopted, potato late blight bacteria (from Fujian) cakes with consistent ages and diameters of 7mm are inoculated on a plate culture medium formed by proportionally mixing a rye culture medium and a Gao's No.1 culture medium in the middle of a culture dish, bacterial colonies of a strain JA58 are inoculated at positions 2.5 cm away from two opposite sides of a hypha block, the potato late blight bacteria of a non-inoculated strain JA58 are used as a control, the steps are repeated for 3 times, and the size of a bacterial inhibition zone is measured after the potato late blight bacteria are placed in a constant-temperature culture box at 28 ℃ for culture for 10 days, so that the strain JA58 has a good antagonistic effect on the growth of the potato late blight bacteria hypha (figure 2).
Example inhibition of the growth of the hyphae of potato late blight bacterium from Ningxia by the four Strain JA58
A plate confronting culture method is adopted, potato late blight bacteria (from Ningxia) cakes with consistent bacterial age and diameter of 7mm are inoculated in the center of a mixed culture dish of a rye culture medium and a Gao's No.1 culture medium in equal proportion, bacterial strains JA58 are inoculated at positions 2.5 cm away from two opposite sides of a hypha block, the potato late blight bacteria of an unanswered bacterial strain JA58 are used as a control, the steps are repeated for 3 times, and after the potato late blight bacteria are placed in a constant-temperature incubator at 28 ℃ and cultured for 10 days, the size of a bacterial inhibition zone is measured, and the result shows that the bacterial strain JA58 has good antagonistic effect on the growth of the potato late blight bacteria hypha (figure 3).
EXAMPLES bacteriostatic assay of five strains JA58 sterile fermentation broth
The preparation of the strain JA58 sterile fermentation broth comprises the following steps:
1) preparing a seed solution: inoculating the strain JA58 into a Gao's I liquid culture medium, culturing at 28 ℃ and 180 r/min for 7 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% vol, the bottling amount is 200 mL/250 mL, the initial pH value of a fermentation medium is 7.0-7.2, the temperature is 28 ℃, 180 r/min, and the fermentation time is 10 days, so that fermentation liquor is obtained;
3) preparation of thallus-free fermentation liquor: and centrifuging the fermentation liquor at 6000 r/min for 7 min, and filtering and sterilizing by using a 0.45 mu m sterilization microporous filter membrane to obtain the sterile fermentation liquor.
The formula of the fermentation medium in the step 2) is as follows: 0.5wt% of soluble starch, 1.5wt% of glucose, 1wt% of soybean meal, 0.2wt% of peptone, 0.2wt% of yeast extract and MgSO4·7H2O 0.05wt% 、K2HPO4 0.05wt% 、NaCl 0.1wt%、CaCO30.2wt% ofThe rest is distilled water.
Measuring relative antibacterial rate by growth rate method, mixing strain JA58 thallus-free fermentation liquid and rye culture medium cooled to 45 deg.C at a volume ratio of 1:10, pouring into flat plate, and adding sterilized water into the rye culture medium at the same ratio as control. The late blight fungus cakes with consistent fungus age and diameter of 7mm are placed in the center of a rye plate and cultured in a thermostat at 20 ℃, the diameter of a bacterial colony is measured at regular time after 15 days, the relative bacteriostasis rate is calculated, and the treatment is repeated for 3 times, so that the result shows that the strain JA58 has good antagonistic effect on potato late blight germs (figure 4).
Example disease prevention Effect of six Strain JA58 fermentation broth on Potato Tuber slices
Preparing potato susceptible variety (Versati) tuber slices (2.0 multiplied by 0.5 cm), dripping 40uL of strain JA58 fermentation liquor prepared in the fifth embodiment on the surfaces of the slices, taking a Gao's No.1 liquid culture medium with the same volume as the fermentation liquor as a control, 10 slices each time, inoculating late blight bacterium cakes with the diameter of 7mm on the surfaces of the slices coated with the fermentation liquor and the Gao's No.1 liquid culture medium after culturing in the dark at 20 ℃ for 24 h, observing the disease incidence after culturing in the dark at 20 ℃ for 10 days, and counting the disease prevention effect.
Disease index = [ Σdisease number × number of disease blocks)/(highest disease number × total number of investigation) ] × 100
The prophylactic effect = [ (control disease index-treatment disease index)/control disease index ] × 100%
The results (fig. 5) show that the fermentation broth treated tuber slices have sparse surface mycelia, even no mycelia, and the disease index of 4.5, while the control tuber slices have thick and abundant surface mycelia and spread and grow along the slice surfaces, almost cover the whole surfaces of the tubers, and the disease index reaches 100. Therefore, the strain JA58 fermentation liquor has a very obvious prevention effect on late blight on potato in-vitro tubers, and the prevention effect reaches 95.5%.
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
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Claims (6)

1. A streptomyces badius aubergiensis JA58 for preventing and treating potato late blight is characterized in that: the strain is streptomyces castanea (S.flavus) (S.castanea)Streptomyces badius) JA58, deposited in China general microbiological culture Collection center (CGMCC), with a date of 9 months and 21 days in 2020The preservation number is CGMCC number 20691.
2. The biocontrol microbial inoculum for preventing and treating potato late blight is characterized in that: the biocontrol microbial inoculum comprises Streptomyces castochromosus JA58 fermentation liquor or Streptomyces castochromosus JA58 sterile body fermentation liquor.
3. The biocontrol microbial inoculum for controlling potato late blight according to claim 2, which is characterized in that: the preparation method of the streptomyces castaneae JA58 fermentation liquor comprises the following steps:
1) preparing a seed solution: inoculating Streptomyces castochromogenes JA58 in Gao's I liquid culture medium, culturing at 28 deg.C and 180 r/min for 7 d to obtain seed liquid;
2) fermentation: inoculating the seed liquid into a fermentation culture medium, wherein the fermentation conditions are as follows: the inoculation amount is 1vol%, the bottling amount is 200 mL/250 mL, the initial pH value of a fermentation medium is 7.0-7.2, the temperature is 28 ℃, 180 r/min, and the fermentation time is 10 days, so that the Streptomyces castochromosis JA58 fermentation liquid is obtained.
4. The biocontrol microbial inoculum for controlling potato late blight according to claim 3, which is characterized in that: the formula of the fermentation medium in the step 2) is as follows: 0.5wt% of soluble starch, 1.5wt% of glucose, 1wt% of soybean meal, 0.2wt% of peptone, 0.2wt% of yeast extract and MgSO4·7H2O 0.05wt% 、K2HPO4 0.05wt% 、NaCl 0.1wt% 、CaCO30.2wt%, the balance being distilled water.
5. The biocontrol microbial inoculum for controlling potato late blight according to claim 2, which is characterized in that: the preparation of the streptomyces castanea JA58 thallus-free fermentation liquor comprises the following steps: and (3) centrifuging the Streptomyces castochromosis JA58 fermentation liquor for 7 min at 6000 r/min, and filtering and sterilizing by using a 0.45 mu m sterilization microporous filter membrane to obtain the thallus-free fermentation liquor of the Streptomyces castochromosis JA 58.
6. The use of the biocontrol bacterial agent for controlling potato late blight as claimed in claim 2 for controlling potato late blight.
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