CN116240136B - Leuconostoc mesenteroides WZ-44 for antagonizing erwinia amylovora and Asian erwinia amylovora and application thereof - Google Patents
Leuconostoc mesenteroides WZ-44 for antagonizing erwinia amylovora and Asian erwinia amylovora and application thereof Download PDFInfo
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Abstract
The invention discloses an intestinal leuconostoc mesenteroides WZ-44 for antagonizing erwinia amylovora and Asian erwinia amylovora and application thereof, wherein the intestinal leuconostoc mesenteroides WZ-44 is preserved in China general microbiological culture collection center (CGMCC) with a strain preservation number of 23157 in the period of 08 and 19 of 2021; the novel recombinant strain has high antagonistic activity on both erwinia amylovora and Asian erwinia amylovora, and the secondary metabolite has high antagonistic activity on both erwinia amylovora and Asian erwinia amylovora and has good stability; the bactericide has stronger tolerance to some bactericides for preventing and treating pear fire blight in production; the biocontrol microbial inoculum prepared by the strain of the invention has better biocontrol effect on pear fire blight, can replace or partially replace chemical pesticides and/or agricultural antibiotics, and has better economic, social and ecological benefits.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to leuconostoc mesenteroides WZ-44 for antagonizing erwinia amylovora and application thereof.
Background
The pear fire epidemic disease comprises pear fire epidemic disease and Asian pear fire epidemic disease, is caused by pear fire epidemic bacteria (Erwinia amylovora) and Asian pear fire epidemic bacteria (Erwinia pyrifoliae) respectively, and is a destructive bacterial disease on rosaceous plants such as pears, apples and the like. The disease mainly damages flowers, fruits, leaves and young shoots, and soon withers and blackens after damage, just like fire burning, but still hangs on the tree without falling, thus obtaining the name. Currently, pear fire blight and Asian pear fire blight occur in more than 60 countries and regions of the world, and two pathogenic bacteria have been classified as quarantine pests by many countries and organizations such as China, european Union, and the like. The pear fire epidemic disease has strong destructiveness, greatly reduces the fruit yield, influences the fruit quality, and can cause death of fruit trees and even destroy gardens. In addition, strict quarantine measures limit international trade of fruits such as apples, pears and the like in epidemic areas, and cause huge economic loss to fruit farmers.
The pear fire epidemic disease occurs in individual pear gardens in Xinjiang pears and apple producing areas and in Gansu Hexi corridor, and the risk of spreading to main apple and pear producing areas in China exists; asian pear fire blight occurs sporadically in part of the orchard in the northwest of Zhejiang. The pear fire epidemic disease and Asian pear fire epidemic disease form serious threat to the healthy development and the fruit export trade of rosaceous fruit trees such as pears, apples and the like in China, so that the agricultural rural areas list the pear fire epidemic disease and the Asian pear fire epidemic disease in the 'one kind of crop disease and insect pest directory', thereby enhancing the prevention and control of the two diseases. At present, the prevention and control of the pear fire blight and the Asian pear fire blight mainly depend on chemical bactericides and agricultural antibiotics, including zinc thiazole, benziothiazolinone, copper hydroxide, kasugamycin, zhongshengmycin and the like. However, the long-term unreasonable use of chemical pesticides and agricultural antibiotics leads to gradual enhancement of pathogen resistance, blind increase of application dosage, unsatisfactory control effect, 50% -70% of comprehensive control effect on diseases, high medicament cost of 20-40 yuan/mu, easy generation of phytotoxicity during flowering phase, and serious threat of residual pesticides on food safety and ecological environment. In addition, since the cost of developing safe and efficient chemical bactericides is high, the investment risk is huge, and in recent years, few novel chemical bactericides for controlling crop bacterial diseases are registered.
In recent years, the biopesticide industry with the advantages of safety, no residue, environmental friendliness and the like is continuously growing, and the biopesticide industry becomes an important point of international green pesticide development. Biological control techniques based on microorganisms are considered to be one of the main directions of development for controlling bacterial diseases of crops in the future. Currently, there are two registered products (Blossom protection and Agriphage) using Aureobasidium pullulans (Aureobasidium pullulans) and phages as active ingredients abroad TM -Fire blank), has better control effect on pear Fire Blight. In China, the Klebsiella sp has a good control effect (CN 112322561A) on the pear fire epidemic disease, and the combination of bacillus sp and lactobacillus plantarum Lactobacillus plantarum has an obvious inhibition effect (CN 112725215A) on the pear fire epidemic disease bacteria, but no registered biocontrol product for controlling the pear fire epidemic disease exists in China.
Lactic acid bacteria (lactic acid bacteria, LAB) are a general term for a group of bacteria capable of producing large amounts of lactic acid using fermentable carbohydrates, and are generally considered as a group of microorganisms beneficial to humans, capable of producing beneficial substances and antimicrobial actives, and are widely used in food fermentation and food preservation. Leuconostoc mesenteroides (Leuconostoc mesenteroides) are important strains of the genus Leuconostoc in lactic acid bacteria, are gram-positive in staining, widely exist on the surface of plants, can ferment saccharides to produce various acids and alcohols, have the capabilities of high acid production, oxidation resistance, pathogen antagonism and the like, and are commonly used for fermenting dairy products, silage, pickle and fruit wine. To date, research reports on leuconostoc mesenteroides for preventing and treating plant bacterial diseases are still few, and biological characteristics, antibacterial spectrum and the like of different strains are obviously different, so that the leuconostoc mesenteroides has remarkable strain specificity. The invention patent CN 113308408A discloses an leuconostoc mesenteroides capable of producing bacteriocin, and the sugarcane juice raw material separated by the leuconostoc mesenteroides has obvious antibacterial effect on several food-borne pathogenic bacteria such as staphylococcus aureus, escherichia coli, salmonella and the like; the invention patent CN 114262666A discloses a Leuconostoc mesenteroides strain with better antagonistic activity on aquatic pathogenic bacteria, and pickle juice separated from the strain. The leuconostoc mesenteroides strain with higher antagonistic activity on the erwinia amylovora and the asian erwinia amylovora is found, and the stable, economic and efficient biocontrol microbial inoculum is developed, so that the method has important significance for green and efficient prevention and control of the erwinia amylovora.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide leuconostoc mesenteroides WZ-44 for antagonizing pathogens, in particular to erwinia amylovora and Asian erwinia amylovora and application thereof. The invention screens and provides the leuconostoc mesenteroides strain WZ-44 from the surface of young Korla pears, has strong growth inhibition activity on pathogenic bacteria such as erwinia amylovora, asian erwinia amylovora and the like, and has the characteristics of environmental friendliness, strong antagonistic activity, good biocontrol effect, stable produced antibacterial active substances, simple production and the like compared with chemical bactericides and agricultural antibiotics.
The aim of the invention is achieved by the following technical scheme:
the invention aims to provide a biocontrol strain for antagonizing pathogenic bacteria, which is named as leuconostoc mesenteroides (Leuconostoc mesenteroides) WZ-44 and is obtained by separating and screening from the surface of young fruit of kurla bergamot pear in Xinjiang.
Preservation information of leuconostoc mesenteroides (Leuconostoc mesenteroides) WZ-44: the strain is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (address: north Chen Xili No.1, 3 of the area of Chaoyang in Beijing, national academy of sciences of China, and post code 100101) at the date of 19 in 2021, and the strain preservation number is CGMCC No.23157.
Specifically, the leuconostoc mesenteroides WZ-44 disclosed by the invention is cultured on an MRS solid culture medium plate for 36 hours at the temperature of 28 ℃ to obtain colony characteristics: the single colony is milky white, smooth in surface, round or bean-shaped, and the diameter is 0.8-1.2 mm; the form of the thallus is spherical, and the diameter is about 0.5-0.7 mu m.
Specifically, the DNA sequence of the 16S rRNA of leuconostoc mesenteroides WZ-44 is shown as SEQ ID NO.1 in a sequence table.
The second object of the present invention is to provide a fermentation product of Leuconostoc mesenteroides WZ-44. The fermentation product of the present invention may be in any form of post-fermentation product conventional in the art, and for example, includes, but is not limited to, fermentation broth containing thallus, sterile fermentation filtrate after filtration and sterilization, or active ingredient obtained by extraction after fermentation of strain.
In some embodiments of the invention, the invention provides a fermentation broth of Leuconostoc mesenteroides WZ-44, where the fermentation broth may refer to a fermentation broth containing a thallus; in other embodiments of the invention, the invention provides a sterile fermentation filtrate of Leuconostoc mesenteroides WZ-44, which may be referred to as a broth after filtration and/or centrifugation of the broth to remove the cells.
The invention also provides a bacterial suspension comprising said leuconostoc mesenteroides WZ-44, which can be prepared as desired according to methods conventional in the art.
The invention also provides a biocontrol preparation containing the leuconostoc mesenteroides WZ-44 or the leuconostoc mesenteroides WZ-44 fermentation product or the bacterial suspension.
The fermentation product, fermentation liquor, sterile fermentation filtrate and biocontrol agent can be prepared according to a conventional method in the field, for example, after leuconostoc mesenteroides WZ-44 is prepared into seed liquor, the seed liquor is inoculated into a fermentation medium for fermentation to obtain fermentation liquor; obtaining sterile fermentation filtrate after centrifugal filtration of fermentation liquor; adding an auxiliary agent allowed in the field into the fermentation liquor to form a biocontrol preparation; and (3) centrifuging the fermentation liquor, collecting thalli, adding a protective agent and an auxiliary agent allowed in the field, and freeze-drying to form the freeze-dried powder microbial inoculum. For example, in one particular embodiment, the biocontrol agent further includes tween 80 and xanthan gum, which tween 80 and xanthan gum can be adjusted as is conventional in the art.
In a specific embodiment, the invention provides a preparation method of a biocontrol agent comprising the leuconostoc mesenteroides WZ-44 and/or leuconostoc mesenteroides WZ-44 fermentation product, which comprises the steps of:
(1) And (3) activating and culturing: taking the glycerol strain of the leuconostoc mesenteroides WZ-44, streaking and inoculating the glycerol strain to a fresh NA solid flat plate, and culturing for 24-36 h at 25-35 ℃;
(2) Seed liquid preparation: selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and standing and culturing for 16-24 hours at 25-35 ℃;
(3) Preparing biocontrol bacteria liquid: inoculating the seed liquid obtained in the step (2) into MRS liquid culture medium in a proportion of 1% -10% (v/v) for fermentation, wherein the fermentation temperature is 25-35 ℃, the loading amount is 200-900 mL/1000mL, the initial pH of the culture medium is 6.5-7.2, standing culture is carried out, shaking is carried out for 1 time every 4-12 hours, the fermentation time is 24-48 hours, and the bacterial density of the fermentation liquid is greater than 1.50X10 by counting by a flat plate coating method 9 cfu/mL；
(4) Preparing a fermentation liquor filtrate: centrifuging the WZ-44 fermentation liquor prepared in the step (3) for 10-15 min, taking supernatant, and filtering by using a 0.22 mu m bacterial filter to obtain a fermentation sterile filtrate.
(5) Preparation of a biocontrol microbial agent: adding tween 80 with the total volume of 0.05-0.15% (v/v) and xanthan gum with the total mass of 0.1-0.2% (m/m) into the fermentation liquor prepared in the step (3). The present invention is not limited to this method of adding the adjuvants, and other methods of forming a microbial inoculum may be used.
In some implementations of the invention, the MRS liquid media composition is: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 5g of sodium acetate and MgSO 4 ·7H 2 O 0.58g，MnSO 4 ·4H 2 Adding water to 900mL of 0.25g of O, 1mL of Tween 80, sterilizing for 15min at the temperature of 121 ℃ with the pH of 6.8-7.0, adding 100mL of 20% glucose solution which is sterilized by filtration into the sterilized culture medium, and shaking and mixing uniformly.
In some implementations of the invention, the MRS solid medium composition is: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 5g of sodium acetate and MgSO 4 ·7H 2 O 0.58g，MnSO 4 ·4H 2 0.25g of O, 1mL of Tween 80, 16g of agar powder, adding water to 900mL, sterilizing at the pH of 6.8-7.0 and 121 ℃ for 15min, adding 100mL of 20% glucose solution which is sterilized by filtration into the sterilized liquid culture medium, and shaking and mixing uniformly.
In some embodiments of the invention, the NB media components are: 5g of peptone, 10g of sucrose, 1g of yeast extract, 3g of beef extract, adding water to 1000mL, sterilizing for 15min at 121 ℃ under the condition of pH of 6.8-7.2.
In some embodiments of the invention, the NA solid medium components are: 5g of peptone, 10g of sucrose, 1g of yeast extract, 3g of beef extract and 15g of agar powder, adding water to 1000mL, sterilizing for 15min at 121 ℃ and pH of 6.8-7.2.
In some implementations of the invention, the PDA solid medium components are: cutting 200g peeled potatoes into small pieces, adding water, boiling for 20-30 min, filtering with 6-8 layers of gauze, removing residues, adding 20g of glucose and 16g of agar powder into the filtrate, adding water to 1000mL, sterilizing at 121 ℃ for 15min.
The antibacterial activity of the WZ-44 fermented sterile filtrate provided by the invention has higher stability to high temperature, ultraviolet, trypsin, proteinase K and pepsin treatment, and has better stability in an acidic environment with a pH value less than or equal to 5.0.
The invention also aims to provide the application of the leuconostoc mesenteroides WZ-44, or a fermentation product thereof, or a bacterial suspension thereof, or a biocontrol agent thereof and the like in preventing and treating diseases caused by pathogenic bacteria.
The pathogenic bacteria are one or more of Pyricularia pyrifolia (Erwinia amylovora), pyricularia asiatica (Erwinia pyrifoliae), pyricularia pyrifolia (Dickeya fangzhongdai), cucumber bacterial angular leaf spot bacteria (Pseudomonas syringae pv. Lachrymans), chinese cabbage soft rot bacteria (Pectobacterium carotovorum subsp. Carotovorum), pear rot bacteria (Valsa pyri), pyricularia pyrifolia (Alternaria alternata) and Pyricularia pyrifolia (Colletotrichum gloeosporioides).
In particular, the inventor finds that the leuconostoc mesenteroides WZ-44, or a fermentation product thereof, or a bacterial suspension thereof, or a biocontrol agent thereof and the like have good control effect in controlling pear fire blight and/or Asian pear fire blight.
In one embodiment, the application of the leuconostoc mesenteroides WZ-44 microbial inoculum for controlling pear fire blight is specifically as follows: spraying treatment is carried out before pear infection with erwinia amylovora, and then spraying treatment is carried out for 1-2 times at intervals of 4-6 d.
In some specific embodiments, the leuconostoc mesenteroides WZ-44 in the liquid used for the spray treatment has a cell density of about 1.0X10 8 ～5.0×10 8 cfu/mL。
The fourth object of the invention is to provide the application of leuconostoc mesenteroides WZ-44, or the fermentation product, or the bacterial suspension, or the biocontrol agent and the like in the production of antibacterial active substances.
The antibacterial active substances disclosed by the invention are active substances capable of resisting one or more of Pyricularia pyrifolia (Erwinia amylovora), pyricularia pyrifolia (Erwinia pyrifoliae), pyricularia pyrifolia (Dickeya fangzhongdai), cucumber bacterial angular leaf spot (Pseudomonas syringae pv. Lachrymans), chinese cabbage soft rot fungus (Pectobacterium carotovorum subsp. Carotovorum), pear tree rot fungus (Valsa pyri), pyricularia pyrifolia (Alternaria alternata) and Pyricularia pyrifolia (Colletotrichum gloeosporioides). Preferably against Pyricularia pyrifolia (Erwinia amylovora) and/or Asian Pyricularia pyrifolia (Erwinia pyrifoliae).
Compared with the prior art, the invention has the following advantages and effects:
the leuconostoc mesenteroides WZ-44 provided by the invention is a newly discovered lactobacillus biocontrol resource, has stronger antagonistic activity on erwinia amylovora, asian erwinia amylovora, cucumber bacterial angular leaf spot bacteria and celery cabbage soft rot bacteria, and has a certain antagonistic activity on pear rust water bacteria, pear tree rot bacteria, pear black spot bacteria and pear anthracnose bacteria; the antibacterial activity of the WZ-44 metabolite shows higher stability to high temperature, ultraviolet, trypsin, proteinase K and pepsin treatment, and shows better stability in an acidic environment with the pH value less than or equal to 5.0; the biocontrol microbial inoculum prepared by the strain has higher biocontrol effect on pear fire blight, can replace or reduce the dosage of chemical pesticides and/or agricultural antibiotics, and has better economic, social and ecological benefits.
Drawings
FIG. 1 is an antibacterial spectrum assay for biocontrol bacteria WZ-44;
FIG. 2 is a graph showing antagonistic circle diameters of biocontrol bacterium WZ-44 against 5 plant pathogenic bacteria;
FIG. 3 shows colony and thallus morphology of Leuconostoc mesenteroides WZ-44 on MRS solid medium plates;
FIG. 4 is a phylogenetic tree analysis of 16S rRNA gene sequences based on Leuconostoc mesenteroides WZ-44;
FIG. 5 shows the growth dynamics of Leuconostoc mesenteroides WZ-44 under different temperature conditions;
FIG. 6 is an antimicrobial activity assay of sterile filtrates of Leuconostoc mesenteroides WZ-44 at various fermentation times;
FIG. 7 is an antibacterial activity stability analysis of Leuconostoc mesenteroides WZ-44 fermented sterile filtrate;
FIG. 8 is a reversibility analysis of the antibacterial activity of the Leuconostoc mesenteroides WZ-44 fermented sterile filtrate under acid-base conditions;
FIG. 9 is a comparison of bacteriostatic activity of Leuconostoc mesenteroides WZ-44 fermented sterile filtrate against 3 bactericides;
FIG. 10 is a graph showing the tolerance of Leuconostoc mesenteroides WZ-44 to 2 bactericides for controlling pear fire blight.
Detailed Description
The following examples are illustrative of the present invention, but are not intended to be limiting. Unless otherwise specified, the technical methods used in the following examples are all conventional; unless otherwise specified, the experimental materials used in the examples below are conventional chemical and biochemical reagents.
The following examples are only examples of the present invention, which are preferred in part, and are merely illustrative of the present invention, and are not to be construed as limiting the scope of the present invention. It should be noted that any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The leuconostoc mesenteroides WZ-44 related by the invention is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms (address: north Xielu No.1, 3 of the university of China, microbiological institute of sciences of China, post code 100101) at the year 08 and the year 19, and is classified and named as Leuconostoc mesenteroides and the culture collection number is CGMCC No.23157. The erwinia amylovora (Erwinia amylovora) strain KL20-28, asian erwinia amylovora (Erwinia pyrifoliae) strain WZ20-3, erwinia amylovora (Dickeya fangzhongdai) strain B19, cucumber bacterial angular leaf spot bacteria (Pseudomonas syringae pv. Lachrymans) strain PSL, celery cabbage soft rot fungus (Pectobacterium carotovorum subsp. Carotovora) strain Pcc-3, pear rot fungus (Valsa pyri) strain VP94, pear black spot fungus (Alternaria alternata) strain HB55 and pear anthracnose fungus (Colletotrichum gloeosporioides) strain NC40 used in the invention are all laboratory isolated preservation strains, and the inventors and applicant promise to permanently provide the public.
Example 1: isolation and identification of Leuconostoc mesenteroides WZ-44
(1) Separating strain resources to be screened: 44 parts of flowers, young fruits, tender tips and the like are collected from a Korla pear garden, each part of sample is sheared and uniformly mixed by using sterile scissors, 10g of the sample is taken and put into a 250mL triangular flask containing 50mL of sterile water and 20 fine glass beads, and the triangular flask is placed on a shaking table for shake culture for 30min at the temperature of 160r/min and 28 ℃; standing for 10min, taking 1mL of supernatant, carrying out 10-time gradient dilution by using sterilized deionized water, absorbing 100 mu L of each concentration gradient dilution, coating on an NA solid flat plate, and standing and culturing for 36h at 28 ℃. And (3) picking single colonies with different forms by using an inoculating loop, streaking and purifying the single colonies on the NA solid culture medium plate for 2 times, transferring the purified single colonies onto the NA solid culture medium plate, culturing the single colonies for 36 hours at 28 ℃, and storing the single colonies at 4 ℃ for later use.
(2) Screening of biocontrol strains: culturing each strain purified on NA solid plate in NB liquid culture medium, and culturing at 28deg.C for 24 hr at 150r/min as seed solution; in addition, the glycerol strains of the Pyricularia oryzae KL20-28 and the Pyricularia oryzae WZ20-3 are respectively streaked and inoculated on a NA solid plate, cultured for 36 hours at 28 ℃, then single colony is selected and inoculated in a 100mL conical flask containing 25mL NB liquid culture medium, cultured for 12 hours to 16 hours at 150r/min and 28 ℃, and the culture solution is adjusted to OD 600 About 1.0, adding into liquid low temperature NA agar medium according to 1% (v/v), and rapidly mixing to obtain NA solid nutritional tablet containing bacteriaA plate; 2 mu L of strain seed liquid to be detected is spotted on the prepared NA solid nutrition plate; placing the treated antagonistic flat plate at 28 ℃ for culturing for 36 hours, and evaluating antagonistic activity of the strain to be tested on erwinia amylovora and Asian erwinia amylovora through the diameter of the antagonistic ring; then, selecting a strain with the antagonistic circle diameter larger than 1.5cm, and determining the antagonistic activity of the primary antagonistic strain on the erwinia amylovora KL20-28, the Asian erwinia amylovora WZ20-3, the erwinia amylovora B19, the cucumber bacterial angular leaf spot PSL and the celery cabbage soft rot fungus Pcc-3 according to the method.
Respectively inoculating pear rot germ VP94, pear black spot germ HB55 and pear anthracnose germ NC40 mycelium blocks at the center of a PDA solid culture medium flat plate, and culturing for 4-5 d at 25 ℃; then, mycelium blocks with the diameter of 4mm are respectively picked from the edges of the colony of the flat plate by using a sterile puncher, are placed at the center of a novel PDA solid flat plate, are cultivated for 2 days at the temperature of 25 ℃, are inoculated with 2 mu L of seed solution of the strain to be detected at the position of 2.5cm away from the center of the flat plate, are cultivated for 4 days in a counter-opposing way at the temperature of 25 ℃, and the antagonistic activity of the strain to be detected on 3 pear pathogenic fungi is observed.
As a result of the above plate antagonism test, it was found that the strain numbered WZ-44 exhibited the best among 746 strains tested, and it exhibited strong antagonistic activity against Pyricularia pyriformis, pyricularia asiatica, horseradish cucurbita pepo, and Brassica rapa soft rot, and antagonistic circles had diameters of 1.77cm, 2.47cm, 1.80cm and 1.77cm, respectively. Has certain antagonistic activity to pear rust water pathogen, pear rot pathogen, pear black spot pathogen and pear anthracnose pathogen, as shown in figures 1 and 2. Each strain was set up in 3 replicates.
(3) Morphology observation of biocontrol strain WZ-44: picking single colony from WZ-44 plate stored at 4 deg.C by aseptic inoculating loop, streaking culturing on MRS solid plate, culturing at 28 deg.C for 36 hr, wherein single colony is milky white, smooth, round or bean-shaped, and has diameter of 0.8-1.2 mm, as shown in figure 3; the cells cultured for 24 hours were picked up and observed by a scanning electron microscope, and the cells were spherical in shape and had a diameter of about 0.5 μm to 0.7. Mu.m, as shown in FIG. 3.
(4) Physicochemical experimental analysis of biocontrol strain WZ-44
The results of the physicochemical experiment analysis of biocontrol strain WZ-44 are shown in Table 1.
TABLE 1 physicochemical experimental analysis results of biocontrol strain WZ-44
(5) Molecular identification of biocontrol strain WZ-44: the single colony of WZ-44 is picked by using a sterilized toothpick, cultured for 24 hours in a 250mL conical flask containing 50mL NB liquid culture medium at 150r/min and 28 ℃, and the total genome DNA of WZ-44 is extracted by using a bacterial genome DNA extraction kit. The DNA fragment was PCR amplified and sequenced using the common primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') of the bacterial 16S rRNA gene, and the gene sequence of the 16S rRNA of the WZ-44 strain is shown as SEQ ID NO. 1. Based on the obtained 16S rRNA gene sequence and GenBank database, the strain was polymerized with Leuconostoc mesenteroides (Leuconostoc mesenteroides) by performing a treelike analysis on WZ-44 using MEGA 7.0 software, as shown in FIG. 4.
Based on the colony morphology, the thallus morphology, the physicochemical experimental analysis and the molecular identification results, the biocontrol strain WZ-44 belongs to Leuconostoc mesenteroides (Leuconostoc mesenteroides).
Example 2: growth dynamics of Leuconostoc mesenteroides WZ-44 in MRS liquid medium
Taking out the WZ-44 strain stored in glycerol from the ultralow temperature refrigerator at the temperature of-70 ℃, streaking and inoculating the strain onto an NA solid plate, and culturing for 36 hours at the temperature of 28 ℃; selecting single colony of WZ-44 by using a sterilized toothpick, inoculating the single colony into a 250mL conical flask containing 50mL MRS liquid culture medium, and culturing for 24 hours at 28 ℃ to obtain seed liquid; inoculating the seed solution into 250mL conical flask containing 50mL MRS liquid culture medium at 2% (v/v), culturing at 10deg.C, 15deg.C, 20deg.C, 25deg.C, 30deg.C, 35deg.C, and 40deg.C for 60 hr, and measuring absorbance (OD) of the fermentation broth at 0 hr, 4 hr, 8 hr, 12 hr, 24 hr, 36 hr, 48 hr, 60 hr 600 ). As a result, it was found that WZ-44 had a suitable growth temperature of 25℃to 35℃and an OD of 12 hours after inoculation 600 Reaching 2.178-2.409 with a wide range of suitable growth temperatures, e.gFig. 5 shows; each treatment was repeated three times.
Example 3: antibacterial active substance produced by fermentation of leuconostoc mesenteroides WZ-44 and stability analysis thereof
Seed solution of Leuconostoc mesenteroides WZ-44 was prepared as in the above-described example 2, inoculated in a proportion of 2% (v/v) into a 250mL Erlenmeyer flask containing 100mL MRS medium, and cultured at 28℃for stationary culture, and the antibacterial activity of the fermented sterile filtrate was measured at 24 hours, 36 hours and 48 hours after inoculation, respectively, by the following method: collecting samples at the above time points, centrifuging at 10,000r/min for 10min, filtering and sterilizing with 0.22 μm bacterial filter, collecting 1mL sterile filtrate, packaging into 1.5mL sterile centrifuge tube, and storing at-70deg.C for use. In order to determine the stability of WZ-44 secondary antibacterial substances, sterile filtrates fermented for 36h were treated as follows, respectively: (1) Taking the packaged sterile filtrate, respectively treating with water bath at 60deg.C for 30min, water bath at 80deg.C for 30min, and water bath at 100deg.C for 5min, and taking untreated fermented sterile filtrate as Control (CK); (2) Taking the split sterile filtrate, respectively treating with trypsin, proteinase K and pepsin with final concentration of 100 μg/mL in water bath at 37deg.C for 3 hr, and taking untreated fermented sterile filtrate as Control (CK); (3) Taking 5mL of aseptic filtrate, placing the aseptic filtrate into an aseptic culture dish, opening a dish cover, placing the dish cover at a position 60cm below an ultra-clean workbench 25W ultraviolet lamp, vertically and downwards irradiating the dish cover for 1h, and taking untreated fermented aseptic filtrate as a reference (CK); (4) Placing 5mL of fermentation sterile filtrate in a 10mL centrifuge tube, respectively adjusting pH values to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, standing at room temperature for 1h, and then filtering with a 0.22 μm bacterial filter, wherein untreated fermentation sterile filtrate is used as a Control (CK); (5) And (3) respectively regulating the pH value of 5mL of fermentation sterile filtrate to 3.0, 7.0 and 9.0 in a 10mL centrifuge tube, standing for 1h at room temperature, taking 2.5mL of sterile filtrate treated with the pH values of 7.0 and 9.0, regulating the pH value to 4.0 by using dilute hydrochloric acid, filtering each treated sterile filtrate by using a 0.22 mu m bacterial filter, taking sterile water with the pH value of 3.0 modulated by using dilute hydrochloric acid as a negative control (K-), taking untreated fermentation sterile filtrate as a positive control (K+), and taking the pH value of untreated fermentation sterile filtrate to be about 4.1. Seed liquid (OD) of pear fire epidemic fungus KL20-28 600 About 1.0) is added into a liquid low-temperature NA culture medium according to the proportion of 1% (v/v) and is quickly and evenly mixed to preparePreparing a solid nutrition plate containing bacterial NA; after the plate is solidified, uniformly punching holes at the center of the plate and the position 2.5cm away from the center by using a sterilization puncher, wherein the aperture is 4mm, adding 30 mu L of fermentation sterile filtrate subjected to different treatments into each hole, and culturing for 36h at 28 ℃. Each treatment was repeated 3 times. As a result, the WZ-44 fermented sterile filtrate for 24 hours, 36 hours and 48 hours has strong antagonistic activity on erwinia amylovora, wherein the antibacterial activity of the 36-hour fermented sterile filtrate is strongest, and the diameter of an antagonistic circle reaches 2.67cm, as shown in figure 6; antagonistic activity of WZ-44 fermentation sterile filtrate shows stronger stability under heat treatment, protease treatment, ultraviolet irradiation and acidic environment (pH value is less than or equal to 5.0), as shown in figure 7; in addition, the antibacterial activity of the WZ-44 fermentation sterile filtrate starts to be reduced at the pH of 5.0, the antibacterial activity is completely lost under the condition that the pH is more than or equal to 6.0, but the antibacterial activity is almost completely recovered when the pH value of the fermentation sterile filtrate with the antibacterial activity lost is adjusted back to the pH value of 4.0, which shows that the secondary antibacterial active substance of the WZ-44 shows better stability and reversibility, is beneficial to separation of the antibacterial active substance and development of subsequent biocontrol products, and is more convenient for industrial processing, as shown in figure 8.
Example 4: comparative test of antagonistic Activity of Leuconostoc mesenteroides WZ-44 fermented sterile filtrate and registered Bactericide
Preparing 36h fermented sterile filtrate of leuconostoc mesenteroides WZ-44 according to the description of the above example 3, and selecting the raw medicines of kasugamycin hydrochloride, zinc thiazole and benziothiazolinone registered in production for controlling the pyritic disease, respectively dissolving the kasugamycin hydrochloride and the benziothiazolinone in sterile water, and preparing 40 mug/mL of the kasugamycin hydrochloride and 100 mug/mL of the benziothiazolinone solution; a500. Mu.g/mL zinc thiazole solution was prepared using DMSO. NA medium plates containing Pyricularia pyrifolia KL20-28 were prepared as described in example 3 above, punched uniformly at the center of the plates and 2.5cm from the center, and had a pore diameter of 4mm, WZ-44 fermentation sterile filtrate and 3 kinds of bactericide solutions prepared as described above were added to each well, 30. Mu.L of each well was added, left to stand for 2 hours, and then transferred to 28℃for cultivation, and antagonistic circle diameters were measured after 36 hours, with sterile water as a control. As a result, it was found that the antibacterial activity of the WZ-44 fermented sterile filtrate and 40. Mu.g/mL kasugamycin hydrochloride was strongest, the antagonistic circle diameters were 2.60cm and 2.54cm, respectively, and significant (P < 0.05) was better than 100. Mu.g/mL of benziothiazolinone (antagonistic circle diameter: 1.39 cm) and 500. Mu.g/mL of zinc thiazole (antagonistic circle diameter: 0.74 cm), as shown in FIG. 9.
Example 5: tolerance of leuconostoc mesenteroides WZ-44 to 2 germicides for controlling pear fire blight
Zhongshengmycin and kasugamycin hydrochloride are two main bactericides for preventing and treating pear fire blight in current production. The experiment is used for measuring the tolerance of leuconostoc mesenteroides WZ-44 to 2 bactericides indoors, and takes the pear fire epidemic disease bacteria KL20-28 as a control, and the specific method is as follows: first, the final concentration of the mesogen and kasugamycin hydrochloride was 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, and the final concentration of the kasugamycin hydrochloride was 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL. Leuconostoc mesenteroides WZ-44 culture solution (OD 600 Approximately 1.0) and a culture solution (OD) of Pyricularia pyrifolia KL20-28 600 Approximately 1.0), 5-fold, 25-fold and 125-fold dilutions of WZ-44 and KL20-28 stock solutions, respectively, and 2. Mu.L of each of the gradient dilutions were incubated on NA solid plates containing the bactericide at 28℃for 36h. As a result, it was found that Leuconostoc mesenteroides WZ-44 exhibited a stronger tolerance to 2 bactericides than that of Pyricularia pyrifolia KL20-28, as shown in FIG. 10. The tolerance of leuconostoc mesenteroides WZ-44 to 2 bactericides enables the leuconostoc mesenteroides to be used alternately with the bactericides.
Example 6: prevention effect of leuconostoc mesenteroides WZ-44 microbial inoculum on pear fire blight in flowering phase
WZ-44 seed liquid was prepared according to the method of example 2, and inoculated into a 500mL Erlenmeyer flask containing 200mL of MRS liquid medium at a ratio of 2% (v/v), and cultured at 28℃for 36 hours, whereby the cell density was about 1.5X10 9 cfu/mL; adding 0.1% (v/v) Tween 80 and 0.15% (m/v) pretreated xanthan gum into the prepared WZ-44 fermentation broth, and fully stirring and uniformly mixing to obtain the WZ-44 biocontrol microbial agent for later use. Simultaneously, streaking and inoculating glycerol strain of Pyricularia oryzae KL20-28 onto NA solid plate, culturing at 28deg.C for 36 hr, then picking single colony, inoculating into 100mL conical flask containing 25mL NB liquid culture medium, culturing at 150r/min at 28deg.C for 24Collecting thallus by centrifugation at 10,000r/min and 4deg.C for 5min, and regulating thallus density to 1.0X10 by using sterile water 8 About cfu/mL, adding 0.1% (v/v) Tween 80 to obtain the pathogen inoculation liquid.
Collecting pear branches in the early flowering stage of an annual year, carrying out a prevention test of leuconostoc mesenteroides WZ-44 microbial inoculum on pear fire blight in the flowering stage, and setting 3 treatments: treatment 1, spraying the above-prepared biocontrol microbial inoculum 3-fold diluent (thallus density about 5.0X10) 8 cfu/mL), spraying the erwinia amylovora inoculation liquid after 24 hours; 2, spraying a erwinia amylovora inoculation liquid; and 3, spraying a 3-time diluent of the biocontrol microbial agent as a control. 3 replicates were set per treatment, 2 shoots per replicate. All treatments are placed in a temperature chamber with the temperature of 24-28 ℃ and the relative humidity of about 85 percent, and the photoperiod is 12h/12h. After inoculation of pathogenic bacteria for 4d, the disease flowers, disease leaves and leaf disease progression of each treatment are investigated, and the disease flower rate, disease leaf rate, disease index and WZ-44 biocontrol microbial inoculum control effect are counted.
The classification method of the damage of the blade comprises the following steps:
level 0: no disease spots;
stage 1: the area of the disease spots accounts for less than 5% of the whole leaf area;
3 stages: the area of the lesion accounts for 6-10% of the whole leaf area;
5 stages: the area of the lesion accounts for 11% -20% of the whole leaf area;
7 stages: the area of the lesion accounts for 21% -50% of the whole leaf area;
stage 9: the area of the disease spots accounts for more than 51% of the whole leaf area.
The results are shown in Table 2, the biocontrol effect of the 3-time diluent of the leuconostoc mesenteroides WZ-44 biocontrol agent on the pear fire blight in the flowering period is 76.1%, and the 3-time diluent of the WZ-44 biocontrol agent shows better safety on the pear flowers and leaves.
TABLE 2 prevention effect of Leuconostoc mesenteroides WZ-44 microbial inoculum on pear fire blight in flowering phase
Example 7: prevention effect of leuconostoc mesenteroides WZ-44 microbial inoculum on pear fire blight
Selecting a seedling of a seedling-growing birthwort seedling of 45 days which grows robustly, and measuring the prevention effect of the leuconostoc mesenteroides WZ-44 bacterial agent on the pear fire blight. Leuconostoc mesenteroides WZ-44 microbial inoculum and pyriform epidemic KL20-28 pathogenic bacteria inoculum were prepared as described in example 6 above for use, and 1 wound was pricked at the basal petiole of each leaf of the pyrus pyrifolia seedling for use using an inoculating needle. Test set up 4 treatments: treatment 1, spraying the above-prepared biocontrol microbial inoculum 3-fold diluent (thallus density about 5.0X10) 8 cfu/mL), spraying the erwinia amylovora inoculation liquid after 2d, and spraying the 1-time biocontrol microbial inoculum 3-time diluent after 4 d; treatment 2, spraying the 6-fold dilution of the biocontrol microbial agent (cell density of about 2.5X10) 8 cfu/mL), spraying the erwinia amylovora inoculation liquid after 2d, and spraying the 1-time biocontrol microbial inoculum 6-time diluent after 4 d; treatment 3, spraying 15 times of the prepared biocontrol microbial inoculum (thallus density about 1.0X10) 8 cfu/mL), spraying the erwinia amylovora inoculation liquid after 2d, and spraying the 1-time biocontrol microbial inoculum 15-time diluent after 4 d; and 4, spraying sterile water firstly, spraying the erwinia amylovora inoculation liquid after 2d, and spraying sterile water for 1 time after 4 d. All the pear seedlings treated by the method are placed in a greenhouse under the conditions of 24-28 ℃ and the relative humidity of about 80 percent, and the photoperiod is 12h/12h. And 6d after inoculation of pathogenic bacteria, the number of disease leaves and the disease progression of each treatment are investigated, and the disease leaf rate, the disease index and the prevention effect of the WZ-44 microbial inoculum on the pear fire epidemic disease are counted. As shown in Table 3, the effect of continuously spraying the leuconostoc mesenteroides WZ-44 microbial inoculum for 3 times on preventing the pear fire epidemic disease reaches 84.3%, the effect of the diluent for 6 times on preventing the pear fire epidemic disease reaches 78.2%, the effect of the diluent for 15 times on preventing the pear fire epidemic disease reaches 68.7%, the application and development prospects are good, and the microbial inoculum can replace or partially replace chemical bactericides and/or agricultural antibiotics.
TABLE 3 prevention effect of Leuconostoc mesenteroides WZ-44 inoculant on Pyricularia oryzae
The above examples illustrate that leuconostoc mesenteroides WZ-44 and its secondary metabolites according to the present invention have strong antagonistic activity against Pyricularia pyriformis, pyricularia asiatica, cucumber bacterial angular leaf spot and Chinese cabbage soft rot, and their secondary antibacterial substances exhibit good stability. Leuconostoc mesenteroides WZ-44 shows strong tolerance to some bactericides used for controlling pear fire blight in production. The leuconostoc mesenteroides WZ-44 microbial inoculum has good prevention and control effects on pear fire blight, has high safety on pear trees, can replace or reduce the dosage of chemical pesticides and/or agricultural antibiotics, improves the safety of food and ecological environment, and has good development and application prospects.
Claims (9)
1. A leuconostoc mesenteroides WZ-44 for antagonizing bacteria is characterized by comprising the following componentsLeuconostoc mesenteroides) The preservation number is CGMCC No.23157.
2. A bacterial suspension comprising leuconostoc mesenteroides WZ-44 of claim 1.
3. Biocontrol formulations prepared from the leuconostoc mesenteroides WZ-44 of claim 1 or the bacterial suspension of claim 2.
4. Use of leuconostoc mesenteroides WZ-44 according to claim 1, the bacterial suspension according to claim 2 or the biocontrol agent according to claim 3 for the prevention and/or treatment of diseases caused by pathogenic bacteria, wherein the pathogenic bacteria are one or more of erwinia amylovora (Erwinia amylovora), erwinia amylovora (Erwinia pyrifoliae), erwinia amylovora (Dickeya fangzhongdai), cucumber bacterial angular leaf spot bacteria (Pseudomonas syringae pv. lachrymans), chinese cabbage soft rot bacteria (Pectobacterium carotovorum subsp.
5. The use according to claim 4, wherein the pathogenic bacteria is Pyricularia pyrifoliaErwinia amylovora) Or/and Asian erwinia amylovoraErwinia pyrifoliae）。
6. The use according to claim 5, wherein the use for preventing and/or treating a pear fire disorder or an asian pear fire disorder is: spraying treatment is carried out before or at the initial stage of the onset of the pear fire epidemic disease and the Asian pear fire epidemic disease, and then spraying treatment is carried out for 1-2 times at intervals of 4-6 days.
7. The method according to claim 6, wherein the leuconostoc mesenteroides WZ-44 has a cell density of 5.0X10% in the liquid used for the spray treatment 7 ～1.0×10 9 cfu/mL。
8. Use of leuconostoc mesenteroides WZ-44 according to claim 1, the bacterial suspension according to claim 2 or the biocontrol formulation according to claim 3 for the manufacture of an antibacterial active substance, wherein the antibacterial active substance is an active substance capable of combating one or more of erwinia amylovora (Erwinia amylovora), erwinia amylovora (Erwinia pyrifoliae), erwinia amylovora (Dickeya fangzhongdai), cucumber bacterial angular leaf spot bacteria (Pseudomonas syringae pv. lachrymans), chinese cabbage soft rot bacteria (Pectobacterium carotovorum subsp.
9. The use according to claim 8, wherein the antibacterial active substance is an antibacterial agent against erwinia amylovoraErwinia amylovora) Or/and Asian erwinia amylovoraErwinia pyrifoliae) Is an active substance of (a).
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| WO2014184410A1 (en) * | 2013-05-13 | 2014-11-20 | Universitat De Girona | Strain of lactobacillus plantarum for controlling fire blight |
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