CN115093987B - Lactobacillus glutamicum and application thereof - Google Patents
Lactobacillus glutamicum and application thereof Download PDFInfo
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- CN115093987B CN115093987B CN202210519065.1A CN202210519065A CN115093987B CN 115093987 B CN115093987 B CN 115093987B CN 202210519065 A CN202210519065 A CN 202210519065A CN 115093987 B CN115093987 B CN 115093987B
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Abstract
The invention belongs to the field of biotechnology, and discloses a lactobacillus sitz and application thereof, wherein the lactobacillus sitz Lf-8 is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) for 12 months and 6 days in 2021, the address is North Chen West Lu No. 1, 3 in the Chaoyang area of Beijing, and the preservation number is CGMCC No.24043. The greenhouse experiment result shows that the Lf-8 has good inhibition effect on the tumorigenesis of blueberry root canceration bacteria, the inhibition effect is more than 68%, the prevention and treatment effect on white vegetable soft rot bacteria is also good, and the field prevention and treatment effect of the Lf-8 on blueberry root canceration bacteria reaches 50%. The lactobacillus sitophilum Lf-8 biological preparation can be applied in a root irrigation and spray mode, has remarkable effects of preventing and treating blueberry root cancer diseases and cabbage soft rot, has the effect of increasing yield, is safe and residue-free, has high efficiency and good disease control effect, and can be applied to field production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a lactobacillus situm and application thereof.
Background
At present, the root cancer is one of four problems in the field of plant protection in the world, the root cancer mainly damages the root, small protrusions appear at affected parts of main roots and side roots, the main roots and the side roots are gradually expanded into tumor-shaped objects, a plurality of tumors are aggregated into large tumors, the overground parts of the damaged plants grow poorly, the plants are short and the whole plants die seriously. The root cancer is taken as a serious bacterial disease, the pathogenic bacteria is agrobacterium tumefaciens (Agrobacterium tumefaciens), the host range is wide, and the plant can infect 643 dicotyledonous plants and few gymnosperms of the genus 331 of 93 families.
The control is extremely difficult due to the special pathogenic mechanism of agrobacterium tumefaciens. In the blueberry production practice, agricultural cultivation management is generally adopted, and disease-resistant varieties and chemical agent control methods are selected to control the occurrence of blueberry root cancer, wherein chemical control is widely used with the advantages of quick response, simple and convenient use, convenience and rapidness. In recent years, the research and development of biological prevention and treatment of the root cancer diseases are faster, such as radioactive agrobacterium K84 and MI-15, non-pathogenic agrobacterium vitis E26 and the like, have good prevention and treatment effects on the root cancer diseases of crops, and the biological prevention and treatment become the popular research field of plant disease prevention and treatment due to the characteristics of environmental friendliness, low cost and no pollution.
The vegetable bacterial soft rot has wide hosts, and the germs are hidden in plant tissues, and are easy to rot inwards from the root injury of the outer leaf stem during the storage period. Therefore, prevention and treatment of soft rot of vegetables are urgent.
At present, a method for combining breeding disease-resistant varieties and chemical agent control is mainly adopted for controlling the soft rot of vegetables. In the konjak planting process, the konjak is commonly intercropped with crops such as corn and the like, and biological bacterial manure is combined to control konjak soft rot, but the disease control effect is not ideal, and an efficient biocontrol microbial inoculum is extremely needed to be sought.
Through literature search, no public report on preparation of biological agents by using lactobacillus albedii and application of biological agents to fields for preventing and treating blueberry root cancer and cabbage soft rot has been found yet.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) At present, the root cancer disease occurs in different degrees in a plurality of blueberry planting bases in Yunnan, so that larger economic loss is caused, and the problem of the blueberry root cancer disease needs to be solved urgently. The method for combining the breeding of disease-resistant varieties and chemical agent control adopted for controlling the soft rot of vegetables has poor disease control effect.
(2) In the prior art, the public report of preparing biological agents by using lactobacillus albedii and applying the biological agents to fields for preventing and treating blueberry root cancer and cabbage soft rot is not seen.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides lactobacillus glutamicum (Lactobacillus farraginis) Lf-8 and application thereof.
The invention is realized in such a way that the lactobacillus gluteus Lf-8 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2021, the address is 1 th national institute of microbiology, china academy of sciences of China, and the preservation number is 24043.
The invention further aims to provide an application of the lactobacillus alfa-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot.
Further, the preparation method of the lactobacillus chaff Lf-8 biocontrol microbial agent comprises the following steps:
lactobacillus situm Lf-8 strain is cultured by conventional solid or liquid culture, shake-amplified culture, and fermentation culture to obtain biological preparation.
Further, the preparation method of the lactobacillus chaff Lf-8 biocontrol microbial agent comprises the following steps:
transferring the lactobacillus chaff Lf-8 to a solid flat plate culture medium, culturing at the constant temperature of 37 ℃ for 48 hours, and continuously subculturing for 2 generations to obtain strains;
inoculating the obtained strain into a 100mL triangular flask liquid culture medium, filling 50mL of the strain into each flask, and standing at a constant temperature of 37 ℃ for 18 hours to obtain seed liquid;
step three, inoculating the seed solution into 1000mL triangular flask MRS liquid culture medium according to the inoculum size of 2%, filling 500mL each time, and standing at a constant temperature of 37 ℃ for 36h to obtain a culture solution which is a biological preparation;
and step four, transferring the biological agent culture solution into a fermentation tank according to the inoculation amount of 2% for fermentation production, and obtaining a liquid preparation.
Further, the formula of the solid plate culture medium in the first step is as follows in percentage by weight: 1% of tryptone, 1% of beef extract, 0.5% of yeast powder, 2% of glucose, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 1.7% of agar, and 90.58% of water content and pH of 6.2-6.5.
Further, the formula of the liquid culture medium in the second step is as follows in percentage by weight: 1% of tryptone, 1% of beef extract, 0.5% of yeast powder, 2% of glucose, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, the water content of which is 92.28% and the pH value of which is 6.2-6.5.
Further, the formula of the liquid fermentation medium in the third step is as follows in percentage by weight: 1.5% of soybean peptone, 1% of beef extract, 0.5% of yeast powder, 2.5% of glucose, 0.1% of tween 80, 0.2% of monopotassium phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 0.01% of defoamer, 92.27% of water content and 5.0 of pH value.
Further, the liquid fermentation conditions in the fourth step include:
tank temperature: the tank temperature of the fermentation tank is controlled above 30 ℃, the temperature is regulated by a method of introducing cold water or hot water into the interlayer by measuring through a thermometer inserted into the culture medium.
Tank pressure: the pressure of the fermentation tank is controlled to be 0.5/cm 2 。
Stirring: adopts a static fermentation mode, and does not need stirring.
Sampling and checking: sampling from sampling tube 1 time every 2 hours, stirring at 180rpm for 5min before sampling, measuring pH, and smear to examine thallus morphology with crystal violet staining microscope, counting, and other culture conditions are unchanged.
Culture period: the fermentation tank has a culture period of 48 hours, the cultivation and tank placing are stopped, the fermentation tank is stirred for 5 minutes at 180rpm before the fermentation tank is placed, and the living bacteria are counted by a dilution plate method after sampling.
Further, the bacterial content was measured by dilution plate count, and the bacterial content of the liquid fermentation preparation was 30 hundred million CFU/mL.
Further, the lactobacillus chaff Lf-8 biocontrol microbial inoculum is applied by root irrigation and spraying.
In combination with the above technical solution and the technical problems to be solved, please analyze the following aspects to provide the following advantages and positive effects:
first, aiming at the technical problems in the prior art and the difficulty of solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
the invention relates to a lactobacillus sitalis and application thereof, belonging to the field of biotechnology, wherein the production strain is lactobacillus sitalis (Lactobacillus farraginis), named as Lf-8, and is preserved in China general microbiological culture Collection center (CGMCC) No.24043 at 12-month-6 of 2021. The strain has wide antibacterial spectrum and strong antibacterial capability. The plant extract has broad-spectrum antibacterial effect on pathogenic bacteria of different host root cancer pathogens such as blueberries, roses, chrysanthemum and the like, other vegetable soft rot pathogens, tobacco bacterial wilt and the like, and the inhibition rate is over 70 percent; in addition, the compound has an inhibiting effect on the root rot fungi of the pseudo-ginseng. The greenhouse experiment result shows that the Lf-8 has good inhibition effect on the tumorigenesis of blueberry root canceration bacteria, the inhibition effect is more than 68%, the prevention and treatment effect on white vegetable soft rot bacteria is also good, and the field prevention and treatment effect of the Lf-8 on blueberry root canceration bacteria reaches 50%. The results show that the lactobacillus chaff Lf-8 has good disease prevention effect, and particularly has great application potential on crop bacterial diseases. The invention can be used for preparing the biocontrol preparation for preventing and treating diseases of various cash crops such as blueberries, vegetables, pseudo-ginseng and the like, and has the characteristics of safety and high efficiency.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the invention has the characteristics of high efficiency, safety, no residue and good effect of controlling live bacteria of the Lf-8 strain, is easy for industrial production, and the produced strain of the Lf-8 strain (Lactobacillus farraginis) can be used for preparing biological agents for preventing and treating blueberry root cancer and vegetable soft rot in fields.
The lactobacillus sitophilus Lf-8 biological preparation can be applied in root irrigation and spray modes, has remarkable effect of preventing and treating blueberry root cancer and cabbage soft rot, has the effect of increasing yield, and can be applied to field production.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
the blueberry is used as an emerging fruit with high economic value, high nutritional value and health care function, and has wide development prospect. Blueberry root cancer is the main disease in the current production of Yunnan blueberries, and the development of the blueberry root cancer is greatly limited. At present, the blueberry root cancer is mainly controlled chemically, but a large amount of pesticides are used for a long time to cause the problems of pesticide residue, environmental pollution, fruit quality reduction and the like, so that the biological control of the blueberry root cancer is a main research direction. As the K84 microbial inoculum is widely used at present, but K84 only selectively inhibits the protoplasm type I and type II agrobacterium containing nopaline type Ti plasmid, and is ineffective for the Yunnan blueberry root cancer virus.
At present, a method for combining breeding disease-resistant varieties and chemical agent control is mainly adopted for controlling the soft rot of vegetables. In the konjak planting process, the konjak is commonly intercropped with crops such as corn and the like, and biological bacterial manure is combined to control konjak soft rot, but the disease control effect is not ideal, and an efficient biocontrol microbial inoculum is extremely needed to be sought. Through literature search, no public report on preparation of biological agents by using lactobacillus albedii and application of biological agents to fields for preventing and treating blueberry root cancer and cabbage soft rot has been found yet. The lactobacillus alfa-8 has a broad antibacterial spectrum, can effectively inhibit various bacterial disease pathogenic bacteria, comprises various host root cancer bacteria (blueberries, chrysanthemum, roses and the like) and cabbage soft rot bacteria, can colonize in the rhizosphere soil of the blueberries, has strong adaptability to the ecological environment, and can more effectively control the blueberry root cancer diseases and the cabbage soft rot. The technical scheme of the invention is expected to solve the occurrence hazard of blueberry root cancer and cabbage soft rot, reduce economic loss, have higher commercial value and have good development prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a preparation method of the lactobacillus glutamicum Lf-8 biocontrol agent provided by the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides lactobacillus glutamicum and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
In order to fully understand how the invention may be embodied by those skilled in the art, this section is an illustrative embodiment in which the claims are presented for purposes of illustration.
The lactobacillus situm (Lactobacillus farraginis) Lf-8 strain provided by the embodiment of the invention is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 24043 in 12 th year of 2021. The China general microbiological culture Collection center, address: north and West road No. 1, 3 in the Korean area of Beijing, national institute of microbiology, postal code: 100101, telephone: 010-64807355, fax: 010-64807288, E-mail: cgmcc@im.ac cn, http:// www.cgmcc.net; biological materials (strain) referred to as L-8; the suggested classification name is lactobacillus albefactitius Lactobacilus farraginis; the biological material (strain) was received from the present collection on day 12 and 06 of 2021 and registered. In accordance with your request, the sample is stored for three decades from the day, and for five years after receiving the request to provide a sample of biological material before expiration. Viability of the biological material (strain) was measured by the present collection at day 2021, 12 and 06, and as a result, survival was observed.
The separation and colony characteristics and bacteria forms of the lactobacillus glutamicum Lf-8 provided by the embodiment of the invention are as follows:
the invention provides a production strain lactobacillus sitaglycone (Lactobacillus farraginis) Lf-8 which is separated from organic fertilizer applied to the vegetables in the Shanghai county of Yuxi city of Yunnan province, and is identified to be lactobacillus sitaglycone (Lactobacillus farraginis) Lf-8 by morphological, culture property, conventional physiological and biochemical, biolog full-automatic identification system determination and 16S rDNA sequence determination and phylogenetic analysis thereof.
The strain has the following characteristics:
(1) The bacterial colony is round, milky white and convex, the surface is smooth, moist and opaque, and the edge is neat; the bacterial cells are short rod-shaped, single, gram-positive, paired or short-chain arranged, and have no spores.
(2) Has the following physiological and biochemical characteristics: gram-positive staining, facultative anaerobic, can utilize glucose, fructose to produce acid and gas.
(3) The strain has wide antibacterial spectrum and strong antibacterial capability, can inhibit a plurality of host agrobacterium tumefaciens (Agrobacterium tumefaciens) such as blueberries, roses, chrysanthemums and the like, and has strong inhibition effects on pathogenic bacteria such as rice bacterial leaf spot bacteria (Xanthomonas oryzae pv. Oryzicola), rice bacterial leaf blight bacteria (Xanthomonas oryzae pv. Oryzae), cabbage black rot bacteria (Xanthomonas campestris pv. Campestris), citrus canker bacteria (Xanthomonas citri subsp. Citri), strawberry angular leaf spot bacteria (Xanthomonas fragariae), cabbage soft rot bacteria (Pectobacterium carotovorum subsp. Carotovorum), tobacco bacterial wilt bacteria (Ralstortia solonacearum) and the like; in addition, the composition has good inhibition effect on pathogenic bacteria of fungal diseases such as phytophthora capsici (Phytophthora capsic) and pseudo-ginseng damage post-spore (Cylindrocarpon destructans).
The lactobacillus sitophilus (Lactobacillus farraginis) Lf-8 strain is used for preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot. The lactobacillus alfa (Lactobacillus farraginis) Lf-8 strain is subjected to conventional solid or liquid culture, shake cultivation, preparation of biological preparation by fermentation culture, application of the biological preparation to blueberry and cabbage plants, and determination of the prevention and treatment effects of the biological preparation on blueberry root cancer and cabbage soft rot.
As shown in fig. 1, the preparation method of the lactobacillus glutamicum Lf-8 biocontrol microbial agent provided by the embodiment of the invention comprises the following steps:
s101, transferring the lactobacillus sitosteroni Lf-8 to a solid plate culture medium, culturing at the constant temperature of 37 ℃ for 48 hours, and continuously subculturing for 2 generations to obtain strains;
s102, inoculating the obtained strain into a 100mL triangular flask liquid culture medium, and carrying out stationary culture at a constant temperature of 37 ℃ for 18 hours for obtaining seed liquid in each flask of 50 mL;
s103, inoculating the seed solution into 1000mL triangular flask MRS liquid culture medium according to the inoculum size of 2%, filling 500mL each time, and standing at a constant temperature of 37 ℃ for 36h to obtain a culture solution which is a biological preparation;
s104, transferring the biological agent culture solution into a fermentation tank according to the inoculation amount of 2% for fermentation production, and obtaining a liquid preparation.
The biological preparation prepared by the production strain of the invention is applied by root irrigation and spraying. The prevention and control tests are carried out on vegetable test fields in the Panlong region of Kunming, yunnan province and field areas in Yuezhou, qujing, so that the biological agent has obvious and stable prevention and control effects, can effectively control blueberry root cancer and cabbage soft rot, has a field prevention and control effect on blueberry root cancer of more than 50%, and has a greenhouse prevention and control effect on white vegetable soft rot of 64.92%.
Example 1: preparation of biocontrol strain Lf-8 biocontrol microbial inoculum
The biocontrol strain is lactobacillus situm (Lactobacillus farraginis) Lf-8. The following media formulations are all weight percentages.
Lactobacillus chaff (Lactobacillus farraginis) Lf-8 was transferred to solid plate medium with the following formula: 1% of tryptone, 1% of beef extract, 0.5% of yeast powder, 2% of glucose, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 1.7% of agar, and 90.58% of water content and pH of 6.2-6.5. Culturing at 37 ℃ for 48h, and continuously subculturing for 2 generations to obtain the strain.
The obtained strain was inoculated into a 100mL triangular flask (50 mL per bottle) of liquid medium with the following formula: 1% of tryptone, 1% of beef extract, 0.5% of yeast powder, 2% of glucose, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, the water content of which is 92.28% and the pH value of which is 6.2-6.5. And (3) carrying out stationary culture at a constant temperature of 37 ℃ for 18 hours to obtain shake flask seeds, namely seed liquid. Inoculating the seed solution into 1000mL triangular flask (500 mL each flask) MRS liquid culture medium according to the inoculation amount of 2%, and performing stationary culture at the constant temperature of 37 ℃ for 36h to obtain the culture solution, namely the biological agent.
Transferring the biological agent culture solution into a fermentation tank according to the inoculation amount of 2% for fermentation production, and obtaining the liquid preparation. The formula of the liquid fermentation medium is as follows: 1.5% of soybean peptone, 1% of beef extract, 0.5% of yeast powder, 2.5% of glucose, 0.1% of tween 80, 0.2% of monopotassium phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 0.01% of defoamer, 92.27% of water content and 5.0 of pH value.
Liquid fermentation conditions
Tank temperature: the tank temperature of the fermentation tank is controlled above 30 ℃, and the temperature can be regulated by a method of introducing cold water or hot water into the interlayer by measuring through a thermometer inserted into the culture medium.
Tank pressure: the pressure of the fermentation tank is controlled to be 0.5/cm 2 。
Stirring: adopts a static fermentation mode, and does not need stirring.
Sampling and checking: sampling from sampling tube 1 time every 2 hours, stirring at 180rpm for 5min before sampling, measuring pH, and smear to examine thallus morphology with crystal violet staining microscope, counting, and other culture conditions are unchanged.
Culture period: the fermentation tank was incubated for about 48 hours, then the incubation was stopped and the tank was placed, stirred at 180rpm for 5 minutes before the placement, and samples were taken for viable count by the dilution plate method.
Liquid fermentation preparation: the bacterial content is measured by dilution plate counting, and the bacterial content reaches 30 hundred million CFU/mL, thus obtaining the biological agent for controlling diseases in the field.
Example 2: experiment of inhibiting different pathogenic bacteria by using Lactobacillus oryzanol Lf-8
1. Test material
1.1 test Strain biocontrol Strain
Lactobacillus situm (Lactobacillus farraginis) Lf-8.
1.2 test pathogenic strains
Phytopathogenic bacteria: blueberry (Agrobacterium tumefaciens), rose (Agrobacterium tumefaciens), chrysanthemum (Agrobacterium tumefaciens), rice stripe (Xanthomonas oryzae pv. Oryzicola), rice bacterial leaf blight (Xanthomonas oryzae pv. Oryzae), black rot of cabbage (Xanthomonas campestris pv. Campestris), citrus canker (Xanthomonas citri subsp. Citri), strawberry angular leaf spot (Xanthomonas fragariae), cabbage soft rot (Pectobacterium carotovorum subsp. Carotovorum), tobacco bacterial wilt (Ralstortia solonacearum).
Plant pathogenic fungi: phytophthora capsici (Phytophthora capsic) and pseudo-ginseng destroyed cyclosporin (Cylindrocarpon destructans). The above strains were all supplied by the university of agriculture, yunnan, bacterial laboratory.
1.3 test Medium
Lactobacillus gluteus (Lactobacillus farraginis) Lf-8 uses MRS culture medium, pathogenic bacteria such as Rhizoctonia cerealis uses YEM culture medium, cabbage soft rot bacteria uses LB culture medium, other pathogenic bacteria use NA culture medium, and pathogenic fungi use PDA culture medium.
2. Test method
The antagonism test uses a plate counter method.
(1) Preparation of bacterial suspension
Culturing the purified lactobacillus sitosterae Marinae Lf-8 on MRS solid culture medium, picking single colony, inoculating into a triangular flask (100 mL) filled with 50mL MRS liquid culture medium, and standing at 37deg.C for 24h; inoculating different agrobacterium tumefaciens in pathogenic bacteria into triangular flask filled with 50mL of YEM liquid culture medium, inoculating cabbage soft rot fungus into LB liquid culture medium, inoculating other pathogenic bacteria into NA culture medium, and shake culturing at 28deg.C and 150r/min for 24 hr. Sterile water is used to carry out OD on the bacterial suspension 600 Adjusting to 0.5 for standby.
(2) Determination of the bacteriostatic action on pathogenic bacteria
Taking the pathogenic bacteria suspension obtained in the last step, respectively inoculating the pathogenic bacteria suspension into YEM, LB, NA culture medium cooled to about 45 ℃ according to the proportion of 1%, and pouring the culture medium into a sterile ultra-clean workbench to form a bacteria-containing flat plate. After the culture medium is solidified, the oxford cup is symmetrically placed on a bacteria-containing culture medium, 200 mu L of Lf-8 bacterial suspension obtained in the previous step is added into the oxford cup, and the oxford cup is placed in a 28 ℃ incubator for 2-3 d, and experimental results are observed and recorded.
(3) Determination of the bacteriostatic action against pathogenic fungi
Pathogenic fungi bacterial cake (diameter 6 mm) is inoculated in the center of PDA flat plate, oxford cup is placed at 2cm position on two sides, and 200 mu L of Lf-8 bacterial suspension is taken and added into oxford cup. Placing the mixture in a 28 ℃ incubator for 5-7 days, observing the bacteriostasis condition, and recording the experimental result.
3. Antibacterial experiment results of chaff lactobacillus (Lactobacillus farraginis) Lf-8 on different plant pathogenic bacteria (see table 1)
TABLE 1 bacteriostatic Effect of biocontrol strain Lf-8 on plant pathogenic bacteria
As shown in Table 1, the Lactobacillus oryzanol (Lactobacillus farraginis) Lf-8 has strong antagonism to 12 pathogenic bacteria and fungi to be tested, wherein the Lactobacillus oryzanol Lf-8 has the strongest inhibition to rice bacterial leaf streak bacteria, bacterial blight bacteria and phytophthora capsici, and the weakest inhibition to white vegetable soft rot bacteria and tobacco bacterial wilt bacteria.
Example 3: greenhouse disease control effect of lactobacillus situm Lf-8 preparation on blueberry root cancer bacteria
Test strain: lactobacillus situm (Lactobacillus farraginis) Lf-8, blueberry root canker (Agrobacterium tumefaciens) LM11.
Inoculating beneficial lactobacillus sitosterae Lf-8 into MRS liquid culture medium, standing at 37deg.C for 36 hr, and adjusting concentration to 1.5X10 with sterile water 9 About CFU/mL Lf-8 bacterial suspension for later use.
Inoculating blueberry Rhizoctonia cerealis LM11 into YEM liquid culture medium, shake culturing at 28deg.C and 150rpm for 36 hr, and adjusting concentration to 3.0X10 with sterile water 8 About CFU/mL LM11 bacterial suspension, for use.
Blueberry variety: "Jewielry".
Test setup 3 treatments: (1) biocontrol bacteria treatment: uniformly pouring the Lf-8 bacterial suspension into root soil of potted blueberry seedling (6 month seedling) to obtain final concentration of 1.0X10 8 Inoculating the agrobacterium tumefaciens of the blueberry to the rhizosphere soil of the blueberry seedling by a root irrigation method after 24 hours with CFU/g to obtain the final concentration of 1.0x10 7 CFU/g. (2) pesticide treatment: uniformly pouring 80% ethylicin (1000 times liquid) with the same volume as Lf-8 bacterial suspension into root soil of potted blueberry seedling (6 month seedling), inoculating into Agrobacterium tumefaciens of blueberry by root irrigation method after 24 hr to obtain final concentration of 1.0X10 7 CFU/g. (3) control group: pouring the same volume of water into blueberry Miao Genji, and inoculating into agrobacterium tumefaciens of blueberry by root irrigation method after 24 hr to obtain final concentration of 1.0X10 7 CFU/g. The root and stem bases of blueberries were scratched and wounds were made on the roots when the pathogenic bacteria were inoculated, 15 basin blueberry seedlings were treated each, and the incidence and disease index of the blueberry seedlings were investigated after 6 months.
Grading according to the grading standard of blueberry root cancer (see table 2), and calculating the morbidity, disease index and prevention effect of each treatment according to the following formula.
Incidence = (number of survey plants/total number of survey plants) ×100%
Disease index = [ Σ (number of disease stages×number of stages represented)/(total number of strains×number of highest stages represented value) ]100
Control effect = (1-A/B) ×100%
Wherein A is the disease index of the control group, and B is the disease index of the test group.
Table 2 blueberry root cancer disease grading criteria
Test results and analysis: as shown in Table 3, compared with 80% ethylicin (1000 times liquid) and clear water control, the incidence rate and the disease index of the blueberry seedling root cancer treated by the Lf-8 microbial agent are obviously reduced, and the relative prevention effect is obviously better than that of ethylicin, so that the Lf-8 microbial agent has good and stable biocontrol effect and good greenhouse prevention effect on the blueberry root cancer.
Table 3 Lf-8 greenhouse control of blueberry root cancer
Note that: the different letters in the table from the same column indicate a significant level of difference of 0.05.
Example 4: greenhouse disease control effect of biocontrol bacterium Lf-8 preparation on white vegetable soft rot fungi
Test strain: lactobacillus oryzae (Lactobacillus farraginis) Lf-8, cabbage soft rot (Pectobacterium carotovorum subsp. Carotovorum) RF1.
Inoculating beneficial lactobacillus sitosterae Lf-8 into MRS liquid culture medium, standing at 37deg.C for 36 hr, and adjusting concentration to 1.0X10 with sterile water 8 About CFU/mL Lf-8 bacterial suspension for later use.
Inoculating cabbage soft rot fungus RF1 into LB liquid medium, and culturing at 28deg.C and 150rpm
Shaking culture for 36 hr, and adjusting concentration to 1.0X10 with sterile water 8 About CFU/mL of the RF1 bacterial suspension is used.
Cabbage variety: lu Chunbai 1 No. 1 (83-1), qingdao International seedling Co., ltd.
The test method comprises the following steps: 1, a 1-cm-diameter wound with a depth of 2mm is pricked on the cabbage stalk by an inoculating needle, and after the wound is dried, the cabbage is treated and inoculated in a spraying mode, wherein the growth period of the cabbage is 45d. Test setup 3 treatments: (1) biocontrol bacteria treatment: uniformly spraying Lf-8 bacterial suspension on cabbage stems for 5mL and inoculating 5mL of cabbage soft rot disease bacteria RF1 bacterial suspension after 24h. (2) pesticide treatment: uniformly spraying 200 times of chlorothalonil on the cabbage stems for 5mL and inoculating 5mL of cabbage soft rot disease bacteria RF1 bacterial suspension after 24h. (3) control group: and (3) using an equal volume of sterile water as a control, and inoculating 5mL of cabbage soft rot disease bacteria RF1 bacterial suspension after 24 hours. Incubating cabbage in a greenhouse at 25deg.C with relative humidity of 35%, counting disease index of soft rot of cabbage after 2d, and calculating control effect of soft rot of white cabbage. Each treatment of 10 cabbages was repeated 3 times. Disease grading standard: grade 0-no disease; grade 1-invasive but not progressive; grade 2 lesions develop to a diameter of 2-3 mm; 3-stage-the lesion is enlarged to 3-4 mm in diameter; the diameter of the grade 4-disease spots is more than 4mm; grade 5-patch, forming massive rot.
Test results and analysis: the test results are shown in Table 4, and the table 4 shows that the control effect of the lactobacillus situm Lf-8 on the white vegetable soft rot is best compared with the aseptic water and the chlorothalonil, and the control effect reaches 64.92%, which indicates that the biocontrol microbial agent Lf-8 has good and stable control effect on the white vegetable soft rot.
Table 4 Lf-8 effects of controlling white cabbage soft rot fungi
Note that: the different letters in the table from the same column indicate a significant level of difference of 0.05.
Example 5: field control effect of lactobacillus situm Lf-8 on blueberry root cancer
Test material: lactobacillus chaff Lf-8 biocontrol microbial inoculum.
Blueberry variety: "Jewielry".
Test site: the mountain guard is a mountain guard, euclin district, the village Qu Jingjia of the mountain pear tree, the modern agriculture limited company.
The test method comprises the following steps: preparation of Lactobacillus situm Lf-8 biological preparation referring to example I, fermentation broth is obtained and used for root irrigation in blueberry base. The blueberry base is internally provided with 4 treatments, wherein the areas with moderate and consistent morbidity are selected for root irrigation: (1) root irrigation of Lf-8 liquid preparation diluted 20 times; (2) Root irrigation is carried out on bacillus subtilis fermentation broth (the inhibiting effect on blueberry root cancer bacteria is proved indoors) diluted by 20 times; (3) root irrigation with 200 times of 3% benziothiazolinone; and (4) root irrigation with clear water is used as a control. 30 blueberry seedlings per cell, 3 cells per treatment are repeated. Root irrigation is started on 1 day of 2 months, 2L of each blueberry seedling is irrigated, and root irrigation is continuously carried out for 3 times at 15d intervals. The fruit setting rate of blueberries is investigated in the beginning of 4 months, the hundred-grain weights of 3 blueberries are investigated in 5 months, and the incidence rate and the disease index of new tumors at the root of blueberry root cancer plants are investigated and treated and controlled in the end of 7 months.
Grading according to the grading standard of blueberry root cancer (see table 2), and calculating the fruit setting rate, morbidity, disease index and prevention effect of each treatment according to the following formula.
Fruit setting rate = (total number of ovary enlarged fruits/total number of single flowers) ×100%
Incidence = (number of survey plants/total number of survey plants) ×100%
Disease index = [ Σ (number of disease stages×number of stages represented)/(total number of strains×number of highest stages represented value) ]100
Control effect = [1- (A/B) × (D/C) ] ×100%
Wherein A is the disease index after the treatment of the test area, B is the disease index before the treatment of the test area, C is the disease index after the treatment of the control area, and D is the disease index before the treatment of the control area.
Test results and analysis: the test results are shown in Table 5, and compared with the clear water control, the test results show that after the disease area blueberry plants are continuously root-irrigated for 3 times by using Lf-8 bacterial liquid, the fruit setting rate and the hundred-grain weight of the blueberry plants are obviously increased, the occurrence rate and the disease index of the blueberry seedling neoplasia are obviously reduced, and compared with 3% of the thiazide pesticide and the bacillus subtilis commodity, the blueberry root cancer plant neoplasia inhibition and disease control are more advantageous. The plant root irrigation method has obvious field effect and yield increasing effect after 3 times of continuous root irrigation, and can be used for field prevention and treatment of blueberry root cancer. The biological control microbial inoculum lactobacillus sitalis Lf-8 has stable and better control effect and yield increasing effect on blueberry root cancer diseases, and also has the basic condition of mass popularization in production.
Table 5 Lf-8 effect of controlling blueberry root cancer in field and its effect on yield
Note that: the different letters in the table from the same column indicate a significant level of difference of 0.05.
The overall result shows that the lactobacillus glutamicum Lf-8 biological agent can be applied by root irrigation and spraying, has remarkable effect of preventing and treating blueberry root cancer and cabbage soft rot, has the effect of increasing yield, and can be applied to field production.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. The lactobacillus sitaglycone Lf-8 is characterized in that the lactobacillus sitaglycone Lf-8 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2021, and has the address of North Chen West Lu No. 1, 3 in the Korean region of Beijing city and the preservation number of CGMCC No.24043.
2. Use of lactobacillus alfa-8 according to claim 1 in the preparation of a preparation for preventing and treating blueberry root cancer and vegetable soft rot.
3. The application of the lactobacillus alfa-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot as claimed in claim 2, wherein the preparation method of the lactobacillus alfa-8 biocontrol agent comprises the following steps:
lactobacillus situm Lf-8 strain is cultured by conventional solid or liquid culture, shake-amplified culture, and fermentation culture to obtain biological preparation.
4. The application of the lactobacillus alfa-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot as claimed in claim 2, wherein the preparation method of the lactobacillus alfa-8 biocontrol agent comprises the following steps:
transferring the lactobacillus chaff Lf-8 to a solid flat plate culture medium, culturing at the constant temperature of 37 ℃ for 48 hours, and continuously subculturing for 2 generations to obtain strains;
inoculating the obtained strain into a 100mL triangular flask liquid culture medium, filling 50mL of the strain into each flask, and standing at a constant temperature of 37 ℃ for 18 hours to obtain seed liquid;
step three, inoculating the seed solution into 1000mL triangular flask MRS liquid culture medium according to the inoculum size of 2%, filling 500mL each time, and standing at a constant temperature of 37 ℃ for 36h to obtain a culture solution;
and step four, transferring the culture solution into a fermentation tank according to the inoculation amount of 2% for fermentation production, and obtaining the liquid preparation.
5. The application of lactobacillus oryzae Lf-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot as claimed in claim 4, wherein the solid plate culture medium in the first step comprises the following formula in percentage by weight: 1% of tryptone, 1% of beef extract, 0.5% of yeast powder, 2% of glucose, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 1.7% of agar, and 90.58% of water content, wherein the pH value is 6.2-6.5.
6. The application of lactobacillus oryzae Lf-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot as claimed in claim 4, wherein the liquid culture medium in the second step comprises the following components in percentage by weight: 1% of tryptone, 1% of beef extract, 0.5% of yeast powder, 2% of glucose, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, the water content of which is 92.28%, and the pH value of which is 6.2-6.5.
7. The application of lactobacillus oryzae Lf-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot as claimed in claim 4, wherein the liquid fermentation medium in the third step comprises the following components in percentage by weight: 1.5% of soybean peptone, 1% of beef extract, 0.5% of yeast powder, 2.5% of glucose, 0.1% of tween 80, 0.2% of monopotassium phosphate, 0.5% of sodium acetate, 0.2% of diammonium hydrogen citrate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 0.01% of defoamer, 92.27% of water content and pH5.0.
8. The use of lactobacillus alf-8 as claimed in claim 4 in the preparation of a formulation for preventing and treating blueberry root cancer and soft rot of vegetables, wherein the liquid fermentation conditions in the fourth step include:
tank temperature: the tank temperature of the fermentation tank is controlled to be more than 30 ℃, the temperature is regulated by a method of introducing cold water or hot water into the interlayer by measuring through a thermometer inserted into a culture medium;
tank pressure: the pressure of the fermentation tank is controlled to be 0.5/cm 2 ；
Stirring: adopting a static fermentation mode, and stirring is not required;
sampling and checking: sampling from the sampling tube 1 time every 2 hours, stirring for 5min at 180rpm before sampling, measuring pH, and smearing to examine thallus morphology with crystal violet staining mirror, counting if there is any mixed bacterial contamination, and keeping other culture conditions unchanged;
culture period: the fermentation tank has a culture period of 48 hours, the cultivation and tank placing are stopped, the fermentation tank is stirred for 5 minutes at 180rpm before the fermentation tank is placed, and the living bacteria are counted by a dilution plate method after sampling.
9. The use of lactobacillus alfa-8 as claimed in claim 8 in the preparation of a formulation for preventing and treating blueberry root cancer and soft rot of vegetables, wherein the bacterial content is measured by dilution plate counting, and the bacterial content of the liquid fermentation formulation is 30 hundred million CFU/mL.
10. The application of the lactobacillus alfa-8 in preparing a preparation for preventing and treating blueberry root cancer and vegetable soft rot as claimed in claim 2, wherein the lactobacillus alfa-8 biocontrol agent is applied by root irrigation and spraying.
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| CN113308419A (en) * | 2021-07-22 | 2021-08-27 | 中国农业科学院农产品加工研究所 | Lactobacillus chaff for fermentation and application thereof |
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