CN111893051A - Microbial agent for sweet potato seedling and rapid propagation and preparation method and application thereof - Google Patents
Microbial agent for sweet potato seedling and rapid propagation and preparation method and application thereof Download PDFInfo
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- CN111893051A CN111893051A CN202010045608.1A CN202010045608A CN111893051A CN 111893051 A CN111893051 A CN 111893051A CN 202010045608 A CN202010045608 A CN 202010045608A CN 111893051 A CN111893051 A CN 111893051A
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- sweet potato
- microbial agent
- weissella
- seedling
- potato seedlings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- Bioinformatics & Cheminformatics (AREA)
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- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
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- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
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Abstract
The invention belongs to the field of agricultural cultivation, and particularly relates to a microbial agent for sweet potato seedling raising and rapid propagation, and a preparation method and application thereof, wherein the active ingredients are pediococcus pentosaceus WS3 and Weissella YN 12; the Pediococcus pentosaceus WS3 is classified as Pediococcus pentosaceus with the preservation number of CGMCC NO. 15345; the classification name of Weissella YN12 is Weissella sp. preservation number CGMCC NO. 7.32. The compound microbial agent with good bacteriostatic effect and antibiotic sensitivity is selected, and the initial propagation speed of the microbial agent in the sweet potatoes is accelerated by virtue of the assistance effect among strains, the acid production capability is enhanced, and the adverse environment tolerance capability is improved, so that the activity of pathogenic bacteria in the seed potatoes is effectively inhibited; the sweet potato seedlings are quickly propagated, so that the quality and the quantity of the sweet potato seedlings are further improved, and the use amount of raw materials for growing the sweet potato seedlings is reduced.
Description
Technical Field
The invention belongs to the field of agricultural cultivation, and particularly relates to a microbial agent for sweet potato seedling culture and rapid propagation, and a preparation method and application thereof.
Background
Sweet potatoes (Ipomoea batatas (L.) and Sweet Potato) have the advantages of rich nutrition, good stability, high yield and the like, have the characteristics of food crops, economic crops, vegetable crops and forage crops, are widely planted in more than 100 countries in the world, and become the 5 th food energy supply plants in the world in the century. Sweet potatoes are important low-input, high-output, drought-resistant, barren-resistant and multipurpose grain, feed and industrial raw material crops and novel biological energy crops in China, and are only inferior to rice, wheat and corn in China and live at the 4 th position. In 2012, the planting area of the Shandong sweet potato is 24.5 kilohm2The sweet potato seedling growing medium accounts for 3.40% of the sowing area of grain crops in the whole province, the planted variety is mainly starch type, the total yield of the sweet potatoes is 185.79 ten thousand tons, and the sweet potato seedling growing medium accounts for 4.12% of the total yield of the grain crops in the whole province; the planting area of the sweet potato in 2015 China is 427 kilohm2About 24.48 tons/hm per unit yield2Total yield is 1.0664 million tons. In recent years, the demand of the market for sweet potatoes is increasingly rising, and the planting area is continuously expanded. The sweet potato seedlings are asexual propagation crops which germinate through the root tubers, and the seedling raising method has three defects in modern agriculture, namely, the sweet potatoes used for seedling raising are large in quantity, and the investment of raw materials is increased when the sweet potatoes are planted in a large area, so that the difficulty in storing the sweet potatoes is increased, and the sales income of the sweet potatoes is reduced; secondly, the sweet potato seedlings are directly planted in the field and have hidden danger of diseases, and if the sweet potato seedlings carry viruses and pathogenic bacteria, the diseases can be spread when the sweet potato seedlings are planted in a large area; thirdly, sweet potato seedlings are not cultured to be strong, viruses are easy to infect in large-area planting, and once the infected viruses are transmitted by generations, the yield of the sweet potatoes is reduced, so that the quality of the sweet potatoes is reduced, the sweet potatoes are difficult to store and the like.
Chemical bactericides are effective methods for controlling crop diseases, but have the problems of residual toxicity, environmental pollution, ecological balance damage and the like, so that biological disease control is increasingly regarded as important. The probiotics can provide nitrogen source, hormone and other nutrient substances for the plants, can participate in the defense function of the plants, and can enhance the capability of the plants for resisting stress, viruses and animal harm. The biological control effect of plant probiotics on plant diseases and the development and application of the biological control effect have been widely noticed and valued by plant scientists, plant pathologists, microbiologists and ecologists. The probiotics as natural additive is biological prevention and control preparation, and the metabolite of the probiotics can prevent and control fungi, bacteria and viruses, can promote the absorption of N, P, K by plants, and can prevent abiotic injury and the like.
In the prior art, probiotics are not applied to seedling and propagation of sweet potatoes, and in view of the problems and characteristics of sweet potato planting, lactobacillus with good bacteriostatic effect and antibiotic sensitivity is needed to be selected to prepare a microbial agent for seedling and rapid propagation of sweet potatoes so as to inhibit or kill potential or existing pathogenic bacteria in seed potatoes.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a microbial agent for sweet potato seedling raising and rapid propagation and a preparation method and application thereof, wherein the compound microbial agent with good bacteriostatic effect and antibiotic sensitivity is selected, and the initial propagation speed of the microbial agent in sweet potatoes is accelerated by virtue of the assistance among strains, the acid production capability is enhanced, and the adverse environment tolerance capability is improved, so that the activities of fungi and other adverse microorganisms in the seed potatoes are effectively inhibited; the sweet potato seedlings of the seed potatoes are quickly propagated so as to further improve the quality and the quantity of the sweet potato seedlings and reduce the consumption of raw materials for growing the sweet potato seedlings.
The technical scheme of the invention is as follows:
a microbial agent for sweet potato seedling and rapid propagation comprises active ingredients of pediococcus pentosaceus WS3 and Weissella YN 12; the Pediococcus pentosaceus WS3 is classified as Pediococcus pentosaceus and has been preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation date is 02/06/2018, the address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC NO. 15345; the Weissella YN12 is classified as Weissella sp, and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date of 2018, 03 and 14 days and the preservation number of CGMCC NO. 7.32.
The strain selected by the invention has the following properties:
the inhibitor has obvious inhibiting effect on escherichia coli and saccharomycetes, and can obviously inhibit the growth of fungi on the sweet potatoes which are rotten and blackened; the acid production capacity can be improved by growing strains sensitive to antibiotics such as streptomycin, ciprofloxacin, drazotocin, amoxicillin, erythromycin and cefradine on a heme culture medium.
The Pediococcus pentosaceus WS3 and Weissella YN12 can obviously promote the proliferation speed and enhance the acid production capacity when growing on a culture medium prepared from sweet potatoes; when the protein is grown on a culture medium containing heme, the proliferation speed can be obviously promoted, and the acid production capacity can be enhanced.
The dependence of the strain on heme can promote the utilization of oxygen in the sweet potato contact surface by lactic acid bacteria, thereby reducing the damage of oxygen to sweet potatoes and lactic acid bacteria; the use of the strain for heme can promote the rapid mass multiplication of the strain on the contact surface of seed potatoes and improve the acid production rate and acid production amount of strain fermentation; the rapid mass propagation of lactic acid bacteria, which will inhibit the activity of undesirable microorganisms to prevent sweet potato decay; the symbiotic cooperation among the strains can ensure that the strains survive on the sweet potatoes, thus solving the problem of sweet potato decay during the germination of the sweet potatoes; under the condition that the lactic acid bacteria inhibit the harmful microorganisms, the sweet potato seedlings thrive; metabolites such as pyruvic acid and the like generated by growth and reproduction of lactic acid bacteria in the sweet potatoes promote the acquisition of absorbable nutrients of sweet potato seedlings; after the sweet potato seedlings are cut, the addition of the microbial agent is favorable for controlling the propagation of undesirable microorganisms and promoting lactic acid bacteria to become dominant strains of the sweet potato seedlings; the stem or root soaking before the sweet potato seedlings are planted in the greenhouse and the field is beneficial to enriching a large amount of lactic acid bacteria in the stems or roots of the sweet potato seedlings so as to resist the infection of harmful microorganisms in the soil and be beneficial to the healthy growth of the sweet potato seedlings in the greenhouse soil and the field soil.
Further, the pediococcus pentosaceus WS3 and Weissella YN12 are cultured by adopting an MRS liquid culture medium, and the microbial agent consists of liquid cultures of the pediococcus pentosaceus WS3 and Weissella YN12 according to a cfu ratio of 1: 2.
Further, the microbial agent contains at least 1 × 10 per 1ml7cfu Pediococcus pentosaceus WS3 and Weissella YN 12.
A preparation method of a microbial agent for sweet potato seedling culture and rapid propagation comprises the following steps:
(1) activating pediococcus pentosaceus WS3 and Weissella YN12 by adopting an MRS solid culture medium, and culturing by adopting an MRS liquid culture medium to obtain first-level culture bacteria of the two strains;
(2) mixing the cultured primary culture bacteria of pediococcus pentosaceus WS3 and Weissella YN12 according to the cfu ratio of 1:2, putting the mixed bacteria liquid into a fermentation liquid culture medium according to the inoculation amount of 10 percent for fermentation culture, and culturing for 10-12h at the temperature of 37 ℃ to obtain the fermented microbial agent. When in use, the fermented microbial agent fermentation liquor is diluted by 10 times to culture sweet potato seedlings. Rapid propagation and infiltration of the stem or root.
Further, the specific operation of the step (1) is as follows: taking out pediococcus pentosaceus WS3 and Weissella YN12 stored at-20 ℃, dipping a small amount of bacterial liquid by using inoculating loops respectively, streaking on an MRS solid culture medium, and culturing for 10-12h at 37 ℃; picking a single colony on an MRS solid culture medium by using an inoculating loop, inoculating the single colony in a 100mL triangular flask filled with 40mL MRS liquid culture medium, and culturing for 8-10h at 37 ℃ to obtain activated lactic acid bacteria; at this time, MRS liquid medium contains at least 1X 10/ml7cfu Pediococcus pentosaceus WS3 and Weissella YN 12.
Further, the fermentation liquid culture medium in the step (2) comprises the following components in percentage by liter: 5g/L of yeast powder, 3g/L of sweet potato powder, 15g/L of lactose, 2g/L, L g/L of diammonium hydrogen citrate-cysteine hydrochloride, 0.5g/L of heme and 10g/L, K of calcium carbonate2HPO42 g/L、MgSO4·7H2O 0.58g/L、MnSO4·H2O0.25g/L, Tween-801 ml/L, pH 6.4, and sterilizing at 121 deg.C for 15 min.
The application of the microbial agent for sweet potato seedling and rapid propagation in the seedling and rapid propagation of the sweet potato. When the microbial agent is specifically applied, other auxiliary materials can be added into the microbial agent according to the needs.
Further, the method comprises the following steps:
(1) seed potato selection and treatment: selecting sweet potatoes without disease insect spots as seed potatoes, putting the seed potatoes into a mesh bag, measuring microbial agent fermentation liquor, diluting the seed potatoes by 10 times with tap water, immersing the mesh bag filled with the seed potatoes into 10 times of diluent of a microbial agent, soaking for 30min, and taking out the mesh bag for later use;
(2) potato discharging: adding tap water into the sand after being exposed to the sun and cooled, stirring uniformly, wherein the adding amount of the tap water is that the sand is wet and is scattered by hand holding, and filling the wet sand into a foam box; discharging the seed potatoes soaked in the step (1) into a foam box filled with sand in an inclined arrangement mode, covering the seed potatoes with the sand, and completely pouring the sand again by using tap water; placing the foam box containing the seed potatoes into an illumination incubator and keeping ventilation, wherein the illumination time of a lamp in one day is 16 hours, and the dark time is 8 hours; after the seed potatoes in the illumination incubator are subjected to the high temperature of 32 ℃ for 2 days in the early stage, keeping the temperature of 25 ℃ all the time for growing seedlings at a flat temperature; when the sweet potato seedlings grow to 8-9 leaves, cutting the sweet potato seedlings with 5-6 leaves for transplanting; watering is not carried out within 2 days after the first seedling collection, bacteria are not watered, topdressing is not carried out, water is used for thorough watering on the 3 rd day after the seedling collection, after 1 hour, bacterial liquid diluted by 10 times of microbial agent is sprayed on sand, and the seedling collection is carried out again when the seedling grows to 8-9 leaves; the management after seedling picking is the same as that of the first seedling picking;
(3) and (3) rapid propagation of sweet potato seedlings: when the sweet potato seedlings are cut, thick white juice is secreted, the sweet potato seedlings are soaked in 10 times of diluent of a microbial agent for 40min and then transplanted into soil of a plastic greenhouse for rapid propagation, the temperature in the greenhouse is set to be more than or equal to 12 ℃, and when the sweet potato seedlings grow for 35-45 days, the growth condition of the sweet potato seedlings treated by the microbial agent is observed;
(4) and (3) field planting and sowing of the fast-propagation sweet potato seedlings: when sweet potato seedlings in the greenhouse are cut, thick white juice is secreted, the sweet potato seedlings are soaked in 10 times of diluent of a microbial agent for 40min and then transplanted into soil of a field; in the whole growth period, effective rainfall or irrigation is carried out for only 1-2 times; the sweet potato seedlings treated by the microbial inoculum grow sweet potato root tubers, and the yield of the sweet potato seedlings cultured by the microbial inoculum can be increased.
The invention has the beneficial effects that:
(1) the microbial agent provided by the invention can inhibit or kill potential or existing pathogenic bacteria in seed potatoes, and through the compounding of the two strains, the initial propagation speed of the microbial bacteria in the sweet potatoes is accelerated by virtue of the cooperative action between the strains, the acid production capability is enhanced, and the adverse environment tolerance capability is improved, so that the activities of fungi and other adverse microorganisms in the sweet potatoes are effectively inhibited; the microbial agent is applied to the rapid propagation of the sweet potato seedlings, the quality and the quantity of the sweet potato seedlings can be further improved, and compared with a method for sprouting the sweet potato seedlings through the root tubers in the conventional sweet potato seedling culture, the method can greatly reduce the use amount of the raw materials for the sweet potato seedling culture.
(2) The invention effectively utilizes oxygen in the sweet potato environment and heme in the sweet potato, promotes the proliferation capability, acid production capability and bacteriostatic capability of the strain in the microbial agent, and simultaneously, metabolites of the strain such as organic acid, small peptide, amino acid and the like can inhibit the propagation and growth of harmful bacteria, promote the growth of sweet potato seedlings, improve the quality of the sweet potato seedlings and prolong the germination time of the sweet potato.
Drawings
FIG. 1 is a diagram showing the growth state of sweet potato seedlings produced from Jishu 26 after treatment with a microbial agent;
FIG. 2 is a diagram showing the growth state of sweet potato seedlings produced by treating watermelon red with a microbial agent;
FIG. 3 is a diagram showing the growth state of sweet potato seedlings produced by the tobacco potato 25 after being treated with a microbial agent;
FIG. 4 is a state diagram of sweet potato seedlings just planted and sowed in a greenhouse;
FIG. 5 is a diagram showing the state of sweet potato seedlings growing in a greenhouse for 40 days;
FIG. 6 shows the potato type of Jishu 26 after treatment with microbial inoculum;
FIG. 7 shows the potato type of watermelon red treated with microbial agents;
FIG. 8 shows the potato type of potato 25 after treatment with the microbial agent.
Detailed Description
The technical solutions of the present invention will be described in detail and fully with reference to the following specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
For a further understanding of the present invention, reference will now be made in detail to the following examples. In the following examples, unless otherwise specified, all methods are conventional; the reagents and biological materials used, unless otherwise specified, are commercially available in the stated percentages or concentrations, unless otherwise specified, are mass percentages.
The composition of each liter of MRS liquid culture medium used in the following examples was as follows: 5g/L of yeast powder, 3g/L of sweet potato powder, 15g/L of lactose, 2g/L, L g/L of diammonium hydrogen citrate-cysteine hydrochloride, 0.5g/L of heme and 10g/L, K of calcium carbonate2HPO42 g/L、MgSO4·7H2O 0.58g/L、MnSO4·H2O0.25g/L, Tween-801 ml/L, pH 6.4, and sterilizing at 121 deg.C for 15 min.
The adopted MRS solid culture medium comprises the following components per liter: 0.5 percent of agar powder by mass is added into the MRS liquid culture medium, the pH value is 6.4, and the mixture is sterilized for 15 minutes at 121 ℃.
Example 1
(I) isolation and purification of the Strain
Collecting water near Qingdao Shengli bridge and goat milk sold in the square hain plaza of Qingdao city, adding into sterile water according to the volume ratio of 1:10, uniformly mixing, continuously diluting, respectively coating 100 mu L of bacterial liquid with each dilution in MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 48 h; selecting single colonies with larger transparent circles, continuously streaking on an MRS solid culture medium for 3 times to obtain pure single colonies; selecting a single bacterial colony in an MRS liquid culture medium without calcium carbonate, and observing whether gas is produced, wherein the gas producing strain is heterotype fermentation lactic acid bacteria, and the non-gas producing strain is homotype fermentation lactic acid bacteria; 17 purified homotypic and heterotypic fermentation lactic acid bacterial strains are separated out; the strains are inoculated into MRS liquid culture medium and cultured for 20h at 37 ℃, 700 mu L of bacterium liquid is taken and added with 300 mu L of sterilized glycerol, and then the mixture is uniformly mixed and put into a refrigerator at minus 80 ℃ for long-term storage and a refrigerator at minus 20 ℃ for strain preparation.
The MRS liquid culture medium comprises the following components per liter: 10g/L of peptone, 5g/L of yeast powder, 10g/L of glucose, 15g/L of sucrose, 10g/L of beef extract, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.18g/L, L g/L of sodium pyruvate-cysteine hydrochloride, 0.5g/L of heme, 10g/L, K g/L of calcium carbonate2HPO42 g/L、MgSO4·7H2O 0.58g/L、MnSO4·H2O0.25g/L, Tween-801 ml/L; it has pH of 6.4, and is sterilized at 121 deg.C for 15 min.
(II) test of pathogenic bacteria of Yeast, Escherichia coli, and sweet Potato
Activation of lactic acid bacteria: taking out the lactic acid bacteria stored at the temperature of minus 20 ℃, injecting 5 mu L of the lactic acid bacteria into an MRS solid culture medium, coating the MRS solid culture medium, and culturing for 10 to 12 hours at the temperature of 37 ℃; and (3) picking a single colony on the MRS solid culture medium by using an inoculating loop, inoculating the single colony in a 10mL test tube filled with a 5mLMRS culture medium, and culturing at 37 ℃ for 8-10h to obtain activated lactic acid bacteria.
Liquid culture of yeast and E.coli: static culturing Saccharomyces cerevisiae in YPD liquid culture medium at 28 deg.C for 48 hr; the Escherichia coli is statically cultured in LB liquid culture medium at 28 ℃ for 24 h.
And (3) determining the bacteriostatic ability of the lactobacillus by a double-layer agar plate punching method:
injecting 20mL of MRS solid culture medium into a culture dish with the diameter of 25cm, and cooling for 1 h; adding 6mL of yeast culture solution into 100mL of YPD semi-solid culture medium cooled to 45 ℃ or adding 6mL of escherichia coli culture solution into 100mL of LB semi-solid culture medium cooled to 45 ℃, shaking up, and quickly pouring on a bottom layer MRS culture medium; 3 cut-short l mL pipette Tip heads are placed on the bottom MRS culture medium at equal intervals; the height of the cut short Tip is 1cm, and the diameter is 0.9 cm; injecting YPD or LB semi-solid culture medium, and cooling for 1 hr to obtain double-layer culture medium; taking out the Tip to form a hole for placing the antibacterial; adding 150 μ L of the lactobacillus with the same concentration into two holes; the remaining one well was used as a control, and 150. mu.L of sterilized Russian water was added; culturing at 37 deg.C for 48h, and measuring and recording the diameter of inhibition zone of lactobacillus on yeast and Escherichia coli.
The YPD liquid culture medium comprises per liter components and contents as follows: 10g of yeast powder, 20g of peptone and 20g of glucose; the liquid culture medium of the escherichia coli, wherein each liter of LB liquid culture medium comprises the following components in percentage by weight: 10g of tryptone, 5g of yeast extract and 10g of NaCl.
The yeast culture solution is added into 100mL of semi-solid culture medium cooled to 45 ℃, and each liter of YPD semi-solid culture medium comprises the following components in percentage by weight: 10g of yeast powder, 20g of peptone, 20g of glucose and 0.7% agar powder; each liter of LB semisolid culture medium comprises the following components in percentage by weight: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 0.7% of agar powder.
The inhibitory ability of the above lactic acid bacteria against pathogenic bacteria causing sweet potato rot was measured: diluting the lactic acid bacteria liquid by 10 times, filling the diluted lactic acid bacteria liquid into a sprinkling can, spraying the lactic acid bacteria liquid on rotten sweet potatoes, continuously putting the rotten sweet potatoes on a sweet potato cellar, and recording the inhibition effect of lactic acid bacteria on pathogenic bacteria causing sweet potato rotting.
(III) susceptibility test of strains
Preparation of antibiotic filter paper sheet concentration: streptomycin, ciprofloxacin and gentamicin are prepared into three concentration gradients of 80 mug/mL, 320 mug/mL and 1280 mug/mL; amoxicillin, erythromycin and cefradine are all prepared into three concentration gradients of 0.625 mug/mL, 20 mug/mL and 80 mug/mL; a circular filter paper sheet with a diameter of 0.6cm was prepared from an F4-1 filter paper of Biotechnology engineering (Shanghai) Co., Ltd, and was put into the above antibiotic solution to obtain antibiotic filter paper sheets with different concentrations.
Culturing lactic acid bacteria: taking out lactobacillus stored at-20 deg.C, and collectingInjecting 5 mu L of the mixture into MRS solid culture medium, coating the mixture, and culturing the mixture for 10 to 12 hours at the temperature of 37 ℃; picking a single colony on an MRS solid culture medium by using an inoculating loop, inoculating the single colony in a 10mL test tube filled with a 5mLMRS culture medium, and culturing for 8-10h at the temperature of 37 ℃ to obtain activated lactic acid bacteria; inoculating activated lactobacillus into 250mL triangular flask containing MRS liquid culture medium at an inoculation amount of 10%, loading liquid volume of 100mL, and culturing at 37 deg.C for 10 hr to obtain lactobacillus solution for determining drug sensitivity, wherein the lactobacillus solution contains at least 1 × 10 per 1mL of MRS liquid culture medium7cfu lactic acid bacteria; the concentration of the bacterial liquid diluted with sterile water per 1mL of lactic acid bacteria is 1X 107cfu。
The drug sensitivity of the lactic acid bacteria is measured by a filter paper diffusion method: injecting 20mL of MRS solid culture medium without calcium carbonate into a culture dish with the diameter of 20cm, and cooling for 1 h; aspirate 100. mu.L of 1X 10 per mL7Putting the lactic acid bacteria liquid of cfu in the center of the culture medium, and coating a plate; taking 4 filter paper sheets, wherein the 4 filter paper sheets are respectively 3 antibiotic filter paper sheets with the same antibiotic but different concentrations, and the 4 th filter paper sheet is a contrast without antibiotic; after the surface of the agar absorbs the lactobacillus, the 4 filter paper sheets with even and moderate intervals are stuck on the surface of the agar of each culture dish; standing for 5min, turning over the plate, performing static culture at 37 ℃ for 24h, and measuring and recording the diameter of the inhibition zone of the antibiotic on the lactic acid bacteria.
As a result, 6 strains sensitive to antibiotics and having a good inhibitory effect on pathogenic bacteria on yeasts, Escherichia coli and rotten sweet potatoes were obtained.
(IV) Co-culture test of strains
Activation of lactic acid bacteria: taking out the 6 strains of lactic acid bacteria stored at the temperature of minus 20 ℃, injecting 5 mu L of lactic acid bacteria into an MRS solid culture medium, coating, and culturing for 10-12h at the temperature of 37 ℃; picking a single colony on an MRS solid culture medium by using an inoculating loop, inoculating the single colony in a 10mL test tube filled with a 5mLMRS culture medium, and culturing at 37 ℃ for 8-10h to obtain activated lactic acid bacteria; at this time, at least 1X 10 of the culture medium is contained in 1 mM MRRS liquid medium7cfu lactic acid bacteria.
Co-culturing lactic acid bacteria: performing mixed culture of different strains on the 6 cultured lactobacillus solutions according to the cfu ratio of 1:2, wherein the number of the strains in the mixed culture is respectively the mixed culture of two strains, three strains, four strains, five strains, six strains and the like, finally obtaining a seed solution for co-culture of the strains, inoculating the mixed seed solution into a fermentation liquid culture medium according to the inoculation amount of 10%, performing static culture at 37 ℃, measuring the concentration of the strain solution and the pH value of the culture medium at intervals of 1 day, and continuously measuring for 7 days.
The combination of strains WS3 and YN12 showed the best proliferation advantage and survival advantage, and the concentration of bacterial liquid per ml was at least 0.993X 10 on day 17cfu, pH 4.5; on day 2, the concentration of the bacterial liquid per ml is at least 0.907X 107cfu, pH 3; on day 3, the concentration of bacterial liquid per ml is at least 0.902X 107cfu, pH 3.5; on day 4, the concentration of bacterial liquid per ml is at least 0.839 × 107cfu, pH 4; on day 5, the concentration of bacterial liquid per ml is at least 0.827 × 107cfu, pH 4; on day 6, the concentration of bacterial liquid per ml is at least 0.832 × 107cfu, pH 4; on day 6, the concentration of the bacterial liquid per ml is at least 0.842 × 107cfu, pH 4; this shows that the co-culture of the strains can promote the growth of the strains and obviously improve the survival ability of the strains.
(V) identification of the Strain
The optimal combination strains of lactic acid bacteria WS3 and YN12 obtained by screening in the above steps are respectively subjected to biological characteristics and 16S rDNA sequence identification.
Biological characteristics of the strains: the strain WS3 is gram-positive bacterium, the colony is milky white, the colony surface is smooth, the cell is rod-shaped, the growth temperature is 8-40 ℃, the optimum temperature is 30-34 ℃, the catalase is negative, the optimum carbon source is glucose, and the optimum inorganic phosphate is K2HPO4(ii) a The strain YN12 is a gram-positive bacterium, the colony is milky white, the surface of the colony is smooth, the cell is in a short rod shape, the growth temperature is 8-45 ℃, the optimum temperature is 30-34 ℃, the catalase is negative, the optimum carbon source is glucose, and the optimum inorganic phosphate is K2HPO4。
16S rDNA sequence: the nucleotide sequence of 16S rDNA of the strain WS3 is shown as a sequence 1 in a sequence table, and the nucleotide sequence of 16S rDNA of the strain YN12 is shown as a sequence 2 in the sequence table. The nucleotide sequence of the 16S rDNA of the strain WS3 has the highest homology with Pediococcus pentosaceus SL4 (sequence number NC-022780.1), and the homology is 98%; the nucleotide sequence of 16S rDNA of strain YN12 has the highest homology with Weissella convusa strain VTT E-90392 (SEQ ID NO: NZ _ CP027565.1), which is 98%.
Based on the above characteristics, the screened strain WS3 was identified as pediococcus pentosaceus (pediococcus pentosaceus), the screened strain YN12 was identified as Weissella (Weissella sp.), and deposited.
Example 2
Heme increases the proliferation density of pediococcus pentosaceus WS3 and Weissella YN 12.
Activation of lactic acid bacteria: taking out pediococcus pentosaceus WS3 and Weissella YN12 stored at-20 ℃, respectively injecting 5 mu L of the pediococcus pentosaceus WS3 and Weissella YN12 into an MRS solid culture medium, coating, and culturing at 37 ℃ for 10-12 h; picking a single colony on an MRS solid culture medium by using an inoculating loop, inoculating the single colony in a 10mL test tube filled with a 5mLMRS liquid culture medium, and culturing at 37 ℃ for 8-10h to obtain activated lactobacillus, wherein each milliliter of the MRS liquid culture medium at least contains 1 multiplied by 107cfu lactic acid bacteria.
Co-culture of pediococcus pentosaceus WS3 and weissella YN 12:
and (2) mixing the two cultured lactic acid bacteria liquids according to the cfu ratio of 1:2 to obtain a seed liquid for carrying out the co-culture of the strains, respectively inoculating the mixed seed liquid into an MRS liquid culture medium without calcium carbonate and heme and an MRS liquid culture medium without calcium carbonate and heme according to the inoculation amount of 10%, carrying out static culture at 37 ℃, and respectively measuring the thallus density for 10 hours in the culture medium. The test was carried out in 3 replicates, the average of the 3 replicates was the final value, and the cell densities per ml of the two media were 1.007X 106cfu and 1.06X 107cfu. The results show that the heme can improve the proliferation density of pediococcus pentosaceus WS3 and Weissella YN 12.
Example 3
Preparation and application of microbial agent
(1) Activation of lactic acid bacteria: taking out the pediococcus pentosaceus WS3 and Weissella YN12 screened in the above example 1 and stored at-20 ℃, respectively injecting 5 mu L of the pediococcus pentosaceus WS3 and Weissella YN12 into an MRS solid culture medium, coating, and culturing at 37 ℃ for 10-12 h; picking single colony on MRS solid culture medium with inoculating loop, inoculating into 10mL test tube containing 5mL MRS liquid culture medium, and culturing at 37 deg.C for 10 hr to obtain activated lactobacillus containing at least 1 × 10 per mL MRS liquid culture medium7cfu lactic acid bacteria.
(2) The preparation of the microbial agent for sweet potato seedling and rapid propagation comprises the following steps: mixing the bacterial liquids of the pediococcus pentosaceus WS3 and the Weissella YN12 cultured in the step (1) according to the cfu ratio of 1:2 to obtain a co-cultured seed liquid, inoculating the mixed seed liquid into an MRS liquid culture medium without calcium carbonate and containing heme according to the inoculation amount of 10%, and statically culturing for 10 hours at 37 ℃ to ensure that the thallus density in each milliliter of the liquid culture medium reaches 1 x 107cfu～0.832×1010cfu, the fermentation broth adjusted to a cell concentration of 1X 107cfu/mL, and diluted 10-fold for use. When the sweet potato seedlings are raised, 10 liters of diluent is added into every 5 kilograms of sweet potatoes.
(3) Seed potato selection: selecting three varieties of sweet potatoes, namely watermelon red, tobacco potato 25 and Jishu potato 26; within one week after the first two months, 5 kilograms of the three varieties of sweet potatoes without disease and insect spots are weighed respectively as seed potatoes, the seed potatoes are put into a mesh bag, 1 liter of microbial agent fermentation liquor is weighed and diluted to 10 liters by tap water, the mesh bag filled with the seed potatoes is immersed into 10 times of diluent of the microbial agent, and the mesh bag is taken out for standby after 30 minutes.
(4) Potato discharging: adding tap water into the sand after being exposed to the sun and cooled, stirring uniformly, wherein the adding amount of the tap water is that the sand is wet and is scattered by hand holding, and filling the wet sand into a foam box; discharging the seed potatoes soaked in the step (1) into a foam box filled with sand, wherein the seed potatoes are placed in an inclined arrangement mode, the seed potatoes are covered with the sand, and the sand is poured through tap water again; putting the foam box filled with the seed potatoes into an illumination incubator; the illumination incubator is kept ventilated, the illumination time of the LED lamp in one day is 16h, and the dark time is 8 h; after the seed potatoes in the illumination incubator are subjected to the early stage of high temperature of 32 ℃ for 2 days, the seedlings are grown at a flat temperature of 25 ℃; as shown in FIGS. 1 to 3, the sweet potato seedlings of all three sweet potato varieties thrive.
When the sweet potato seedlings grow to 8-9 leaves, cutting the sweet potato seedlings with 5-6 leaves for transplanting; watering is not carried out within 2 days after the first seedling collection, bacteria are not watered, topdressing is not carried out, watering is carried out on the 3 rd day after the seedling collection, after 1h, 1L of bacterial liquid of lactobacillus fermentation liquor diluted by 10 times is sprayed on sand, and the seedling collection is carried out again when the seedling grows to 8-9 leaves; the management after seedling picking is the same as that of the first seedling picking; under the culture mode, the sweet potato seedlings can always germinate, branch and grow and are picked for one year.
(5) And (3) rapid propagation of sweet potato seedlings: after the sweet potato seedlings are cut, soaking the sweet potato seedlings in lactobacillus fermentation liquor diluted by 10 times for 40min, transplanting the sweet potato seedlings into soil of a plastic greenhouse for rapid propagation, wherein the temperature in the greenhouse is more than or equal to 12 ℃, as shown in figures 4 to 5, when the sweet potato seedlings cultured by the microbial agent grow in the greenhouse for 30 to 40 days, the sweet potato seedlings treated by the microbial agent thrive; the following table 1 shows the growth characteristics of the microbial agent treated sweet potato seedlings in the greenhouse.
TABLE 1 growth characteristics of sweet potato seedlings in greenhouse
As can be seen from Table 1, when the greenhouse grows for about 1 month, the sweet potato seedlings treated by the microbial inoculum have the following advantages compared with the sweet potato seedlings not treated by the microbial inoculum: the length of the leaf pitch becomes shorter, the diameter of the stem becomes larger, the petiole length of the third leaf below the top end of the stem increases, and the number of branches increases.
(6) The rapid propagation effect of the sweet potato seedlings is as follows: the sweet potato seedlings are soaked in the lactobacillus fermentation solution diluted by 10 times for 40min and then transplanted into soil of a plastic greenhouse for rapid propagation, and the sweet potato seedlings are rapidly propagated in the greenhouse, grow continuously on the original basis and have 2-7 branches.
(7) The field effect of the sweet potato seedlings is as follows: when the fast-propagation sweet potato seedlings are picked, thick and white juice is secreted, and the fast-propagation sweet potato seedlings are soaked in lactobacillus fermentation liquid diluted by 10 times for 40min and then transplanted into soil of a field; in the whole growth period, the demand of water is less, fibrous roots are fresh on the tuberous roots of the sweet potatoes, and the yield of the sweet potatoes is increased. After the sweet potato seedlings cultured by the microbial agent are sowed into a field from a greenhouse, the sweet potato seedlings have obvious drought tolerance, which shows that the growth vigor of the sweet potato seedlings is good and fibrous roots are few on the tuberous roots of the sweet potato during the whole growth period and only 1-2 times of effective rainfall or irrigation; from FIGS. 6 to 8, it can be seen that the growth of the microbial agent-treated sweet potato tuber is good. The untreated sweet potatoes show slow growth, and fibrous roots grow on the tuberous roots of the sweet potatoes; the yield of the sweet potato root tuber of the sweet potato seedlings cultivated by the microbial agent is increased, and as can be seen from table 2, the table 2 shows the growth characters and yield characteristics of the rapidly-propagated sweet potato seedlings in the field.
TABLE 2 growth characteristics and yield characteristics of rapidly-propagated sweet potato seedlings in the field
As seen from Table 2, after the sweet potato seedlings cultured with the microbial additives of example 3 were grown in a 36-day field, the leaf pitch of the sweet potato seedlings became short and the petiole of the third leaf below the top of the stem became long; after 125 days of field growth, the yield was increased compared with the control.
From the results, the sweet potato seedlings treated by the microbial agent provided by the invention achieve good effects in the germination time period and the rapid propagation time period of the sweet potato seedlings. Compared with the sweet potato seedlings without the microbial inoculum treatment, the sweet potato seedlings have the excellent characteristics of more white juice in the seedlings, shorter leaf spacing, larger leaf area, no decay of potato seeds and the like during seedling raising, the sweet potato seedlings continuously maintain the excellent characters during rapid propagation, and the rapid propagation seedlings obtain the results of less water demand and improved yield after being planted in a field.
The above description is only for the preferred embodiment of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like made within the scope of the present invention should be included in the patent protection scope of the present invention.
Sequence listing
<110> Qingdao university of science and technology
<120> microbial agent for sweet potato seedling and rapid propagation and preparation method and application thereof
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aaagatggct ctgctatcac ttctggatgg acccgcggcgtattagctag ttggtgaggt 240
aaaggctcac caaggcagtg atacgtagcc gacctgagag ggtaatcggc cacattggga 300
ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttcca caatggacgc 360
aagtctgatg gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa agctctgttg 420
ttaaagaaga acgtgggtaa gagtaactgt ttacccagtg acggtattta accagaaagc 480
cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggatt 540
tattgggcgt aaagcgagcg caggcggtct tttaagtcta atgtgaaagc cttcggctca 600
accgaagaag tgcattggaa actgggagac ttgagtgcag aagaggacag tggaactcca 660
tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtctg 720
gtctgcaact gacgctgagg ctcgaaagca tgggtagcga acaggattag ataccctggt 780
agtccatgcc gtaaacgatg attactaagt gttggagggt ttccgccctt cagtgctgca 840
gctaacgcat taagtaatcc gcctggggag tacgaccgca aggttgaaac tcaaaagaat 900
tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc 960
ttaccaggtc ttgacatctt ctgacagtct aagagattag aggttccctt cggggacaga 1020
atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagttctcg 1080
cacgagcgca ccttatacta gcgcagcata gtggcacctc tagtgaagac ttgcggtgac 1140
taccggagag aggtggggac gacgtcaaat catcatgccc cttatgacct gggctacaca 1200
cgtgctacaa tggatggtac aacgagtcgc gaaaccgcga ggttaagcta atctcttaaa 1260
accattctca gttcggactg taggctgcaa ctcgcctaca cgaagtcgga atcgctagta 1320
atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380
accatgagag tttgtaacac ccaaagccgg tggggtaacc tttttagga 1429
<210>2
<211>2884
<212>DNA
<213> Weissella (Pediococcus pentosaceus)
<400>2
tgcaagtcga acgaacttcc gttaattgat tatgacgtac ttgtactgat tgagatttta 60
acacgaagtg agtggcgaac gggtgagtaa cacgtgggta acctgcccag aagtagggga 120
taacacctgg aaacagatgc taataccgta taacagagaa aaccgcatgg ttttctttta 180
aaagatggct ctgctatcac ttctggatgg acccgcggcg tattagctag ttggtgaggt 240
aaaggctcac caaggcagtg atacgtagcc gacctgagag ggtaatcggc cacattggga 300
ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttcca caatggacgc 360
aagtctgatg gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa agctctgttg 420
ttaaagaaga acgtgggtaa gagtaactgt ttacccagtg acggtattta accagaaagc 480
cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggatt 540
tattgggcgt aaagcgagcg caggcggtct tttaagtcta atgtgaaagc cttcggctca 600
accgaagaag tgcattggaa actgggagac ttgagtgcag aagaggacag tggaactcca 660
tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtctg 720
gtctgcaact gacgctgagg ctcgaaagca tgggtagcga acaggattag ataccctggt 780
agtccatgcc gtaaacgatg attactaagt gttggagggt ttccgccctt cagtgctgca 840
gctaacgcat taagtaatcc gcctggggag tacgaccgca aggttgaaac tcaaaagaat 900
tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc 960
ttaccaggtc ttgacatctt ctgacagtct aagagattag aggttccctt cggggacaga 1020
atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagttctcg 1080
cacgagcgca ccttatacta gcgcagcata gtggcacctc tagtgaagac ttgcggtgac 1140
taccggagag aggtggggac gacgtcaaat catcatgccc cttatgacct gggctacaca 1200
cgtgctacaa tggatggtac aacgagtcgc gaaaccgcga ggttaagcta atctcttaaa 1260
accattctca gttcggactg taggctgcaa ctcgcctaca cgaagtcgga atcgctagta 1320
atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380
accatgagag tttgtaacac ccaaagccgg tggggtaacc tttttaggaa tgcaagtcga 1440
acgctttgtg gttcaactga tttgaagagc ttgctcagat atgacgatgg acattgcaaa 1500
gagtggcgaa cgggtgagta acacgtggga aacctacctc ttagcagggg ataacatttg 1560
gaaacagatg ctaataccgt ataacaatga caaccgcatg gttgttattt aaaagatggt 1620
tctgctatca ctaagagatg gtcccgcggt gcattagcta gttggtaagg taatggctta 1680
ccaaggcgat gatgcatagc cgagttgaga gactgatcgg ccacaatggg actgagacac 1740
ggcccatact cctacgggag gcagcagtag ggaatcttcc acaatgggcg aaagcctgat 1800
ggagcaacgc cgcgtgtgtg atgaagggtt tcggctcgta aaacactgtt gtaagagaag 1860
aatgacattg agagtaactg ttcaatgtgtgacggtatct taccagaaag gaacggctaa 1920
atacgtgcca gcagccgcgg taatacgtat gttccaagcg ttatccggat ttattgggcg 1980
taaagcgagc gcagacggtt atttaagtct gaagtgaaag ccctcagctc aactgaggaa 2040
ttgctttgga aactggatga cttgagtgca gtagaggaaa gtggaactcc atgtgtagcg 2100
gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctttct ggactgtaac 2160
tgacgttgag gctcgaaagt gtgggtagca aacaggatta gataccctgg tagtccacac 2220
cgtaaacgat gagtgctagg tgtttgaggg tttccgccct taagtgccgc agctaacgca 2280
ttaagcactc cgcctgggag tacgaccgca aggttgaaac tcaaaggaat tgacggggac 2340
cgcacaagcg gtggagcatg tggtttaatt tcgaagcaac gcgaagaacc tttaccaggt 2400
cttgacatcc ctttgacacc tccagagatg gagcgttccc tttcgggaac aagtgacagg 2460
tgggtgcatg gttgtcgtca gctcgtggtc gtgagaatgt tgggtttagt cccgccaacg 2520
agcgtcaatc ctttatttac ctagttgtcc agcattcagg tggggcactc taatggagac 2580
cttgctgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 2640
tgacctgggc tacacacgtg ctacaatggc gtatacaacg agttgccaac ccgcgagggt 2700
gagctaatct cttaaagtac gtctcagttc ggattgtagg ctgcaactcg cctacatgaa 2760
gtcggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc cgggtcttgt 2820
acacaccgcc cgtcacacca tgagagtttg taacacccaa agccggtggg gtaaccttcg 2880
ggag 2884
Claims (8)
1. A microbial agent for sweet potato seedling and rapid propagation is characterized in that the active ingredients of the microbial agent are Pediococcus pentosaceus WS3 and Weissella YN 12; the Pediococcus pentosaceus WS3 is classified as Pediococcus pentosaceus and has been preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation date is 02/06/2018, the address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC NO. 15345; the Weissella YN12 is classified as Weissella sp, and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date of 2018, 03 and 14 days and the preservation number of CGMCC NO. 7.32.
2. The microbial agent for breeding and rapid propagation of sweet potatoes as claimed in claim 1, wherein the pediococcus pentosaceus WS3 and Weissella YN12 are cultured in MRS liquid medium, and the microbial agent is composed of liquid cultures of pediococcus pentosaceus WS3 and Weissella YN12 in a cfu ratio of 1: 2.
3. The method of claim 1A microbial agent for sweet potato seedling and rapid propagation is characterized in that every 1ml of the microbial agent at least contains 1 x 107cfu Pediococcus pentosaceus WS3 and Weissella YN 12.
4. The preparation method of the microbial agent for sweet potato seedling culture and rapid propagation according to claims 1-3, characterized by comprising the following steps:
(1) activating pediococcus pentosaceus WS3 and Weissella YN12 by adopting an MRS solid culture medium, and culturing by adopting an MRS liquid culture medium to obtain first-level culture bacteria of the two strains;
(2) mixing the cultured primary culture bacteria of pediococcus pentosaceus WS3 and Weissella YN12 according to the cfu ratio of 1:2, putting the mixed bacteria liquid into a fermentation liquid culture medium according to the inoculation amount of 10 percent for fermentation culture, and culturing for 10-12h at the temperature of 37 ℃ to obtain the fermented microbial agent.
5. The preparation method according to claim 4, wherein the specific operation of the step (1) is as follows: taking out Pediococcus pentosaceus WS3 and Weissella YN12 stored at-20 ℃, respectively dipping a little bacterial liquid by using inoculating loops, streaking on an MRS solid culture medium, and culturing for 10-12h at 37 ℃; picking a single colony on an MRS solid culture medium by using an inoculating loop, inoculating the single colony in a 100mL triangular flask filled with 40mL of MRS liquid culture medium, and culturing for 8-10h at 37 ℃ to obtain activated lactic acid bacteria; at this time, MRS liquid medium contains at least 1X 10/ml7cfu Pediococcus pentosaceus WS3 and Weissella YN 12.
6. The method according to claim 4, wherein the fermentation liquid medium in the step (2) has a composition and content per liter of: 5g/L of yeast powder, 3g/L of sweet potato powder, 15g/L of lactose, 2g/L, L g/L of diammonium hydrogen citrate-cysteine hydrochloride, 0.5g/L of heme and 10g/L, K of calcium carbonate2HPO42 g/L、MgSO4·7H2O 0.58g/L、MnSO4·H2O0.25g/L, Tween-801 ml/L, pH 6.4, sterilizing at 121 deg.C for 15 min。
7. The application of the microbial agent for sweet potato seedling and rapid propagation in the seedling and rapid propagation of the sweet potato.
8. Use according to claim 7, characterized in that it comprises the following steps:
(1) seed potato selection and treatment: selecting sweet potatoes without disease insect spots as seed potatoes, putting the seed potatoes into a mesh bag, measuring microbial agent fermentation liquor, diluting the seed potatoes by 10 times with tap water, immersing the mesh bag filled with the seed potatoes into 10 times of diluent of a microbial agent, soaking for 30min, and taking out the mesh bag for later use;
(2) potato discharging: adding tap water into the sand after being exposed to the sun and cooled, stirring uniformly, wherein the adding amount of the tap water is that the sand is wet and is scattered by hand holding, and filling the wet sand into a foam box; discharging the seed potatoes soaked in the step (1) into a foam box filled with sand in an inclined arrangement mode, covering the seed potatoes with the sand, and completely pouring the sand again by using tap water; placing the foam box containing the seed potatoes into an illumination incubator and keeping ventilation, wherein the illumination time of a lamp in one day is 16 hours, and the dark time is 8 hours; after the seed potatoes in the illumination incubator are subjected to the high temperature of 32 ℃ for 2 days in the early stage, keeping the temperature of 25 ℃ all the time for growing seedlings at a flat temperature; when the sweet potato seedlings grow to 8-9 leaves, cutting the sweet potato seedlings with 5-6 leaves for transplanting; watering is not carried out within 2 days after the first seedling collection, bacteria are not watered, topdressing is not carried out, water is used for thorough watering on the 3 rd day after the seedling collection, after 1 hour, bacterial liquid diluted by 10 times of microbial agent is sprayed on sand, and the seedling collection is carried out again when the seedling grows to 8-9 leaves; the management after seedling picking is the same as that of the first seedling picking;
(3) and (3) rapid propagation of sweet potato seedlings: when the sweet potato seedlings are cut, thick white juice is secreted, the sweet potato seedlings are soaked in 10 times of diluent of a microbial agent for 40min and then transplanted into soil of a plastic greenhouse for rapid propagation, the temperature in the greenhouse is set to be more than or equal to 12 ℃, and when the sweet potato seedlings grow for 35-45 days, the growth condition of the sweet potato seedlings treated by the microbial agent is observed;
(4) and (3) field planting and sowing of the fast-propagation sweet potato seedlings: when sweet potato seedlings in the greenhouse are cut, thick white juice is secreted, the sweet potato seedlings are soaked in 10 times of diluent of a microbial agent for 40min and then transplanted into soil of a field; in the whole growth period, effective rainfall or irrigation is carried out for only 1-2 times; the sweet potato seedlings treated by the microbial agent grow sweet potato tuberous roots.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115232764A (en) * | 2022-06-11 | 2022-10-25 | 青岛科技大学 | Microbial agent |
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| KR20170032636A (en) * | 2015-09-15 | 2017-03-23 | 주식회사 웰빙엘에스 | Method of preparing fermented coffee using lactic acid bacteria isolated from Kopi Luwak |
| CN107455262A (en) * | 2017-09-26 | 2017-12-12 | 牛力立 | A kind of plant modification method of detoxification red heart" sweet potato |
| CN108265016A (en) * | 2018-01-29 | 2018-07-10 | 四川农业大学 | A kind of lactic bacteria additive and its application, preparation method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20170032636A (en) * | 2015-09-15 | 2017-03-23 | 주식회사 웰빙엘에스 | Method of preparing fermented coffee using lactic acid bacteria isolated from Kopi Luwak |
| CN107455262A (en) * | 2017-09-26 | 2017-12-12 | 牛力立 | A kind of plant modification method of detoxification red heart" sweet potato |
| CN108265016A (en) * | 2018-01-29 | 2018-07-10 | 四川农业大学 | A kind of lactic bacteria additive and its application, preparation method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115232764A (en) * | 2022-06-11 | 2022-10-25 | 青岛科技大学 | Microbial agent |
| CN115232764B (en) * | 2022-06-11 | 2023-06-06 | 青岛科技大学 | Microbial agent |
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