CN108913618B - Bacillus amyloliquefaciens JSPB14 and application thereof - Google Patents
Bacillus amyloliquefaciens JSPB14 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological control of crop diseases, and particularly relates to bacillus amyloliquefaciens JSPB14 and application thereof, wherein the bacillus amyloliquefaciens is preserved in the China general microbiological culture Collection center in 11-20 months in 2017, and the strain preservation number is CGMCC No. 14934; the biocontrol microbial inoculum prepared by the strain has higher biocontrol effects on the post-harvest pleurotus eryngii ulceration germs, the rice bacterial streak germs and the rice bacterial streak germs, can avoid or reduce the residues of chemical pesticides and agricultural antibiotics, improves the food and environmental safety, and has better economic and social benefits.
Description
Technical Field
The invention belongs to the technical field of biological control of crop diseases, and particularly relates to bacillus amyloliquefaciens JSPB14 and application thereof.
Background
Pleurotus eryngii (Pleurotus eryngii) belongs to Agaricales (Aaicales) edible fungi, and has high nutritional and medicinal values. At present, the yield of pleurotus eryngii in China reaches more than 50 million tons per year, and the annual output value is 45-60 million yuan. The post-harvest pleurotus eryngii ulceration disease is a bacterial disease caused by infection of Lactococcus lactis subsp. Because the application standard of the picked fruit and vegetable bactericide is higher, the types and the application dosage of the bactericide which can be selected are strictly limited, and most countries forbid the use of agricultural antibiotics on picked fruits and vegetables, the types of the selected medicines are few, and the prevention and treatment effect is not ideal. The low temperature (1-5 ℃) is an effective measure for controlling the disease, but the cold chain control from the whole process of the picked pleurotus eryngii to the dining table needs advanced technical equipment and expensive logistics cost, and the economic benefit of the pleurotus eryngii industry is greatly reduced.
The bacterial diseases of rice mainly comprise bacterial leaf spot of rice (Xanthomonas oryzae pv. oryzae zicola) and bacterial leaf blight of rice (Xanthomonas oryzae pv. oryzae), are important diseases in rice production in China, and are 800-1500 ten thousand mu in annual occurrence area, 300-500 ten thousand mu in hazard area, so that the loss of rice yield is 5% -10%, and is more than 50% in serious cases, even in top-of-failure, which becomes one of important hidden dangers of rice safety production in China. At present, the prevention and control of rice bacterial diseases mainly depend on chemical pesticides and agricultural antibiotics. However, long-term unreasonable use of chemical pesticides and antibiotics leads to gradual increase of drug resistance of pathogenic bacteria, blind increase of application dosage, unsatisfactory prevention and treatment effect, and serious threat to food safety and ecological environment caused by residual pesticides.
The cost for developing safe and efficient novel chemical bactericides is high, and the investment risk is huge. Microbial-based biocontrol technologies are considered to be one of the major development directions for controlling crop bacterial diseases in the future. Bacillus spp belongs to gram-positive bacteria, is widely distributed in various habitats such as soil, plant surfaces, water bodies, agricultural wastes and the like, has broad-spectrum antibacterial activity, higher spore yield and stronger stress adaptability, and is considered to be one of beneficial microorganisms with application and development potentials. Among them, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a biocontrol microorganism widely recognized by scientists and consumers as safe to the human body, and a plurality of strains have been used for controlling plant diseases. However, the bacillus amyloliquefaciens strains from different sources have obvious differences in biological characteristics, antibacterial spectrum and the like and have strain specificity.
Disclosure of Invention
The invention solves the technical problems in the prior art and provides the bacillus amyloliquefaciens JSPB14 and application thereof.
In order to solve the problems, the technical scheme of the invention is as follows:
the invention provides Bacillus amyloliquefaciens JSPB14 which is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of Sui Luo No.1 of Beijing Korean district, Microbiol research institute of Chinese academy of sciences, zip code 100101) in 11.20.2017.20.A classification name of Bacillus amyloliquefaciens is CGMCC No. 14934. The Bacillus amyloliquefaciens strain is obtained by separating from a culture medium of pleurotus eryngii, is identified as Bacillus amyloliquefaciens (JSPB 14) by adopting a method of combining colony morphology observation, thallus morphology observation and gyrB gene sequence determination, and is named as JSPB 14.
The colony characteristics of the bacillus amyloliquefaciens strain JSPB14 after being cultured for 48 hours on a 2YT solid medium plate at the temperature of 30 ℃ are as follows: the single colony is milky white, flat, round or nearly round, the diameter is 2.5 mm-4.0 mm, the colony surface is shriveled, and the edge is serrated; the shape of the cell is rod-shaped, the diameter is about 0.5 μm, the length is about 1 μm-2 μm, and the spore is in a silkworm cocoon shape. The 2YT solid medium used had the following composition: 17g of tryptone, 10g of yeast extract, 5g of NaCl, 15g of agar and distilled water, wherein the volume is 1000mL, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 121 ℃.
The invention provides a fermentation product of the bacillus amyloliquefaciens JSPB 14.
The invention provides a biocontrol microbial inoculum containing the fermentation product of the bacillus amyloliquefaciens JSPB14 and/or the bacillus amyloliquefaciens JSPB 14.
The invention provides a preparation method of a biocontrol microbial inoculum containing a fermentation product of bacillus amyloliquefaciens JSPB14 and/or bacillus amyloliquefaciens JSPB14, which comprises the following steps:
(1) Activation culture: taking a glycerol strain of the bacillus amyloliquefaciens strain JSPB14, streaking and inoculating the glycerol strain to a fresh 2YT solid plate, and culturing for 24-36 h at the temperature of 30 ℃;
(2) preparing a seed solution: selecting a single colony to inoculate in a 2YT liquid culture medium, and carrying out shake culture at 30 ℃ for 16-36 h, wherein the oscillation frequency is 100-200 r/min;
(3) preparing biocontrol bacterium liquid: inoculating the seed liquid obtained in the step (2) into a Landy culture medium for fermentation according to the ratio of 1-5% (v/v), wherein the fermentation temperature is 26-31 ℃, the initial pH of the culture medium is 6.5-7.2, the fermentation aeration is carried out according to the aeration ratio of 0.5: 1-2.5: 1, the stirring speed is 75-200 r/min, and the fermentation time is 36-48 h;
(4) preparing a biocontrol microbial inoculum A: and (4) centrifuging the biocontrol bacterium liquid fermented in the step (3), taking the sterile supernatant, adding potassium sorbate, then performing sterile filling, and storing at room temperature.
Preferably, in the step (4), the final concentration of the potassium sorbate added is 1 g/L.
Preparation method II
(1) Activation culture: taking a glycerol strain of the bacillus amyloliquefaciens strain JSPB14, streaking and inoculating the glycerol strain to a fresh 2YT solid plate, and culturing for 24-36 h at the temperature of 30 ℃;
(2) preparing a seed solution: selecting a single colony to inoculate in a 2YT liquid culture medium, and carrying out shake culture at 30 ℃ for 16-36 h, wherein the oscillation frequency is 100-200 r/min;
(3) preparing biocontrol bacterium liquid: inoculating the seed liquid obtained in the step (2) into a Landy culture medium for fermentation according to the ratio of 1-5% (v/v), wherein the fermentation temperature is 26-31 ℃, the initial pH of the culture medium is 6.5-7.2, the fermentation aeration is carried out according to the aeration ratio of 0.5: 1-2.5: 1, the stirring speed is 75-200 r/min, and the fermentation time is 36-48 h;
(4) preparing a biocontrol microbial inoculum B: adding the coco oil-based esterification compound and sodium benzoate into the fermentation liquor prepared in the step (3), adjusting the pH value to about 4.5, then carrying out sterile filling, and storing at room temperature.
Preferably, the final concentration of the cocoyl esterification complex in step (4) is 8% (v/v) and the final concentration of sodium benzoate is 1 g/L.
The 2YT culture medium comprises the following components: 17g of tryptone, 10g of yeast extract, 5g of NaCl and distilled water are added to a constant volume of 1000mL, the pH value is 7.0, and the mixture is sterilized for 20min at 121 ℃.
The components of the 2YT solid medium are as follows: 17g of tryptone, 10g of yeast extract, 5g of NaCl, 15g of agar and distilled water are added to a constant volume of 1000mL, the pH value is 7.0, and the mixture is sterilized at 121 ℃ for 20 min.
The Landy medium comprises the following components: 5g/L of L-glutamic acid, 1g/L, L g of yeast extract, 2mg/L of phenylalanine and 20g/L, KH g of glucose2PO41g/L、KCI 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H2O 0.15g/L、CuSO4·5H2O 0.16mg/L、MnSO45mg/L, pH 6.8-7.2, sterilizing at 121 ℃ for 20 min.
The invention provides application of the bacillus amyloliquefaciens JSPB14 or a fermentation product thereof or the biocontrol microbial inoculum in preventing and treating post-harvest pleurotus eryngii ulceration and rice bacterial streak.
The application method of the biocontrol microbial inoculum A in preventing and treating the ulceration disease of the picked pleurotus eryngii comprises the following steps: diluting the biocontrol microbial inoculum A by 1-5 times, treating the harvested pleurotus eryngii by using a soaking mode, airing and placing according to the conditions of a harvested goods shelf.
The application method of the biocontrol microbial inoculum B in preventing and treating the bacterial leaf streak of rice comprises the following steps: diluting the biocontrol microbial inoculum B by 60-80 times, uniformly spraying at the initial generation stage of the bacterial leaf streak of the rice, and spraying for 1 time at intervals of 7-9 days.
Compared with the prior art, the invention has the advantages that,
the bacillus amyloliquefaciens strain JSPB14 provided by the invention is a newly discovered bacillus biocontrol resource, has higher antagonistic activity on postharvest pleurotus eryngii ulceration germs, rice bacterial streak germs and rice bacterial blight germs, has higher biocontrol effect on postharvest pleurotus eryngii ulceration diseases and rice bacterial streak diseases, can avoid or reduce the residues of chemical pesticides and agricultural antibiotics, improves the food and environmental safety, and has better economic and social benefits.
Drawings
FIG. 1 shows antagonistic activity of 4 strains of preliminarily identified biocontrol bacteria on Pleurotus eryngii ulceration bacteria;
FIG. 2 shows the colony, thallus and spore forms of Bacillus amyloliquefaciens JSPB14 on a 2TY culture medium;
FIG. 3 is a phylogenetic tree analysis based on the gyrB gene sequence of Bacillus amyloliquefaciens JSPB 14;
FIG. 4 shows the growth and sporulation dynamics of Bacillus amyloliquefaciens JSPB14 in Landy medium;
FIG. 5 shows the antibacterial activity of the secondary metabolites of Bacillus amyloliquefaciens JSPB14 at different fermentation times;
FIG. 6 is an antagonistic spectrum determination of Bacillus amyloliquefaciens JSPB 14;
FIG. 7 is the antagonistic activity of Bacillus amyloliquefaciens JSPB14 against different pathogenic bacteria;
FIG. 8 shows that Bacillus amyloliquefaciens JSPB14 biocontrol microbial inoculum can prevent and treat ulceration of picked eryngii and abalone;
FIG. 9 shows the prevention and treatment effect of the bacillus amyloliquefaciens JSPB14 biocontrol microbial inoculum on the postharvest eryngii abalone fester.
Detailed Description
The Bacillus amyloliquefaciens strain JSPB14 related by the invention has been preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of Suzuku No.1 of Beijing republic of facing-Yang, Microbiol research institute of Chinese academy of sciences, zip code 100101) in 11-20.2017, is classified and named as Bacillus amyloliquefaciens, and the strain preservation number is CGMCC No. 14934. The adopted pleurotus eryngii ulcerating pathogen Lactococcus lactis subsp lactis is obtained by separating and identifying the adopted pathogenic pleurotus eryngii in a research room; rice bacterial blight (Xanthomonas oryzae pv. oryzae), rice bacterial leaf spot (Xanthomonas oryzae pv. oryzae), Xanthomonas campestris wild rape pathogenic variants (Xanthomonas campestris pathogenic variants) (Xanthomonas campestris pathogenic pv. camptosris), Erwinia carotovora subspecies carotovora (Erwinia carotovora), solanaceous, Staphylococcus aureus (Staphylococcus aureus) are all the storage strains in the research room, and other units or individuals can ask for the above 7 pathogenic bacteria from the research room.
The following examples are provided to illustrate the present invention, but are not intended to limit the present invention. Unless otherwise specified, the technical methods used in the following examples are all conventional methods; unless otherwise specified, the test materials used in the following examples are conventional chemical reagents and biochemical reagents.
The 2YT medium components described below are: 17g of tryptone, 10g of yeast extract, 5g of NaCl and distilled water are added to a constant volume of 1000mL, the pH value is 7.0, and the mixture is sterilized for 20min at 121 ℃. Agar is added to the 2YT solid medium by 15 g/L.
The Landy medium described below consisted of: 5g/L of L-glutamic acid, 1g/L, L g of yeast extract, 2mg/L of phenylalanine and 20g/L, KH g of glucose2PO41g/L、KCI 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H2O 0.15g/L、CuSO4·5H2O 0.16mg/L、MnSO45mg/L, pH 6.8-7.2, sterilizing at 121 ℃ for 20 min.
The MRS culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast extract, 2g/L of diammonium citrate, 20g/L of glucose and 10g/L, MgSO of beef extract4.7H2O 0.58g/L、MnSO4.4H2O 0.25g/L、K2HPO42g/L, 2g/L sodium acetate, 801 mL/L Tween, pH 6.8, and sterilization at 121 ℃ for 20 min. MRS solid medium is added with agar 15 g/L.
Hereinafter referred to as "coco esterification complex" under the trade designation "SF-98B", available from Hezhou Ruiyuan peat products, Inc.).
Example 1:
isolation and identification of Bacillus amyloliquefaciens strain JSPB14
(1) Separating strain resources to be screened: randomly sampling 50g of culture medium with high incidence of pleurotus eryngii canker after being picked, taking 5 parts of medium samples in total, uniformly mixing, putting 10g of medium into a 250mL conical flask containing 20 particles of fine glass beads and 100mL of sterilized deionized water, and placing the conical flask on a shaking bed for shake culture at 150r/min and 30 ℃ for 6 hours. Standing for 10min, taking 1mL of supernatant, performing gradient dilution with 10 times of sterilized deionized water, sucking 100 μ L of each concentration gradient diluent, coating on a 2YT solid plate, and standing and culturing at 30 ℃ for 36 h. Picking single colony by using inoculating loop, streaking and purifying for 2 times on a 2YT solid culture medium, transferring the purified single colony with different forms to a 2YT solid plate, standing and culturing for 36h at 30 ℃, and storing at 4 ℃ for later use.
(2) Plate antagonistic screening of biocontrol strains: culturing each purified strain stored on a 2YT solid plate in a 2YT liquid culture medium under the culture conditions of 150r/min, 30 ℃ and 36h to serve as seed liquid for later use; alternatively, the glycerol strain of lactococcus lactis subsp lactis strain SLPE1-3 was streaked on an MRS solid plate, cultured at 30 ℃ for 36 hours, then a single colony was picked and inoculated into a 250mL Erlenmeyer flask containing 50mL of MRS liquid medium, subjected to static culture at 30 ℃ for 12 hours, and the culture broth (OD) was prepared600About 1.0) is added into a liquid low-temperature 2YT agar culture medium, and is rapidly and uniformly mixed to prepare a 2YT solid nutrition plate; dropping 2 mu L of seed liquid of the strains to be detected on the prepared 2YT solid nutrition plate, and dropping 4 strains to be detected on each plate; 3 replicates per strain; and (3) placing the treated antagonistic flat plate at 30 ℃ for culturing for 48h, and evaluating the antagonistic activity of the strain to be tested on the lactococcus lactis subsp. As a result, it was found that among the 367 strains tested, the numbers JSPB3, JSPB14, JSPB106 and JSPB247 produced antagonistic loops with diameters of more than 10mm, wherein the diameters of the antagonistic loops of JSPB14 reached 13.0mm, as shown in fig. 1.
(3) Morphological observation of the biocontrol strain JSPB 14: performing dilution plating culture on JSPB14 on a 2YT solid plate under the culture condition of 30 ℃ and 48 hours, wherein a single colony is milky white, flat, round or nearly round, the diameter is 2.5 mm-4.0 mm, the surface of the colony is dried and shrunk, and the edge is serrated, as shown in figure 2A; observed under scanning electron microscope, the shape of the thallus is rod-shaped, the diameter is about 0.5 μm, the length is about 1 μm-2 μm, and the spore is cocoon-shaped, as shown in fig. 2B and 2C.
(4) Molecular identification of biocontrol strain JSPB 14: a single JSPB14 colony is picked by a sterilized toothpick and cultured in a 250mL conical flask containing 50mL of 2YT liquid culture medium at the temperature of 30 ℃ at the speed of 150r/min for 36h, and the total genomic DNA of JSPB14 is extracted by a bacterial genomic DNA extraction kit. PCR-amplifying DNA fragments by using universal primers UP-1(5 '-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP-2r (5 '-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3') of bacterial gyrB genes; the PCR product was sequenced using the sequencing primers UP-1S (5'-GAAGTCATCATGACCGTTCTGCA-3') and UP-2Sr (5'-AGCAGGGTACGGATGTGCGAGCC-3'). Based on the obtained gyrB gene sequence and GenBank database, the JSPB14 is subjected to evolutionary tree analysis by using ClustalX 1.83 software and MEGA 5.05 software, and the strain is determined to belong to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), as shown in FIG. 3.
Example 2:
growth dynamics of JSPB14 in Landy medium and antibacterial activity of secondary metabolite
Taking out the JSPB14 strain preserved by glycerol from an ultralow temperature refrigerator at-70 ℃, streaking and inoculating the strain onto a 2YT solid plate, and culturing for 36h at 30 ℃; selecting JSPB14 single colony with sterilized toothpick, inoculating into 250mL conical flask containing 50mL2YT liquid culture medium, and culturing at 30 deg.C for 36 hr at 150r/min to obtain seed solution; the seed solution was inoculated into a 250mL Erlenmeyer flask containing 50mL of Landy broth at a ratio of 5% (v/v), and cultured at 30 ℃ for 60 hours at 75 r/min. Respectively investigating the population density of the thalli at 0h, 4h, 8h, 12h, 18h, 24h, 36h, 48h and 60h after inoculation, and the specific method comprises the following steps: samples were taken at the above time points, and 10-fold dilution was performed with sterile water, and 100. mu.L of each concentration-gradient dilution was pipetted onto a 2YT solid plate, and cultured at 30 ℃ for 48 hours for colony counting. Investigating spore formation density at 0h, 4h, 8h, 12h, 18h, 24h, 36h, 48h and 60h after inoculation, wherein the specific method comprises the following steps: samples were taken at the above time points, and were subjected to 80 ℃ hot water bath for 30min, followed by 10-fold gradient dilution with sterilized water, 100. mu.L of each concentration gradient dilution was aspirated, and the resulting samples were spread on a 2YT solid plate, and cultured at 30 ℃ for 48 hours to count colonies. As a result, it was found that the cell density reached 3.87X 10 at 12 hours after inoculation8CFU/mL, the cell density reached 9.13 × 10 at 48h8CFU/mL, as shown in FIG. 4; a large amount of spores are formed 12h after inoculation, and the spore density reaches 8.07 multiplied by 10 at 24h7CFU/mL, spore density reached the highest 1.47X 10 at 48h8CFU/mL, as shown in FIG. 4. Three replicates of each treatment were performed.
Fermenting JSPB14 in Landy culture medium according to the above method, respectively investigating antibacterial activity of fermented secondary metabolite 24h, 36h, and 48h after inoculation, and specificallyThe method comprises the following steps: 2mL of each bottle was sampled at the above time points, centrifuged at 15,000g for 10min, sterilized by filtration using a 0.22 μm sterile filter, and the sterile filtrate was stored at-70 ℃ for further use. Culturing lactococcus lactis subsp lactis SLPE1-3 in culture medium (OD)600About 1.0) adding the mixture into a liquid low-temperature 2YT agar culture medium according to the proportion of 1% (v/v), quickly mixing uniformly, and preparing a 2YT solid nutrition plate; after the plate was solidified, 5 wells were uniformly punched on the plate by means of a sterilization punch, 40. mu.L of sterilized water (treatment number: CK, the same below), 40. mu.L of 100. mu.g/mL ampicillin (Amp100), 40. mu.L (24h) of 24h sterile fermentation broth, 40. mu.L (36h) of 36h sterile fermentation broth, and 40. mu.L (48h) of 48h sterile fermentation broth were added to each well, and the plate was incubated at 30 ℃ for 36h to examine the diameter of the antagonistic circle. As a result, the antibacterial activity of the sterile fermentation supernatant is enhanced with the increase of the fermentation time, the diameter of the inhibition zone of 48h of the sterile fermentation supernatant on the lactococcus lactis subspecies lactis reaches 21.8mm, the antagonistic activity (20.1mm) is obviously higher than that of 36h, and the antibacterial activity is not obviously different from that of a control medicament 100 mu g/mL of ampicillin (21.5mm), and the antibacterial activity is shown in figure 5.
Example 3:
antibacterial spectrum determination of biocontrol strain JSPB14
To evaluate the antibacterial spectrum of the secondary antibacterial substance of Bacillus amyloliquefaciens strain JSPB14, 6 main crop-pathogenic bacteria and 1 human opportunistic pathogenic bacteria were selected for plate antagonism experiments including Lactococcus lactis subsp. The specific method comprises the following steps: taking out the glycerol strains of the 6 pathogenic bacteria except lactococcus lactis subsp lactis from a-70 deg.C ultra-low temperature refrigerator, streaking on 2YT solid culture medium respectively, culturing at 30 deg.C for 36 hr, selecting single colony in 2YT liquid culture medium, and culturing at 30 deg.C and 150r/min to OD600Is approximately equal to 0.5 for standby; taking out glycerol strain of lactococcus lactis subspecies lactis from-70 deg.C ultra-low temperature refrigerator, streaking on MRS solid culture medium, culturing at 30 deg.C for 36 hr, selecting single colony, inoculating to MRS liquid culture medium, and standing at 30 deg.C for culture to OD600Is approximately equal to 0.5 for standby; adding the above cultured pathogenic bacteria seed liquid into liquid low temperature 2YT agar culture medium at a ratio of 1% (v/v), mixing rapidly, and preparing 2YT solid nutrition plate; after the plate was solidified, 3 holes were punched evenly on the plate using a sterilization punch for future use.
36h and 48h sterile fermentation supernatants of JSPB14 were prepared as described in example 2, 40. mu.L of sterilized water (clear water control CK), 40. mu.L of 48h sterile fermentation supernatant (treatment 1), and 40. mu.L of 36h sterile fermentation supernatant (treatment 2) were dropped into the prepared 2YT solid plate wells containing pathogenic bacteria, the plates were placed at 30 ℃ for culture for 36h, the diameter of the antagonistic circle was investigated, and the radius of the antagonistic circle (minus the radius of the well, 3mm) was calculated. As a result, the JSPB14 secondary antibacterial substance shows stronger antagonistic activity not only on postharvest Pleurotus eryngii ulceration pathogenic bacterium lactococcus lactis subsp lactis, but also on rice bacterial blight, rice bacterial streak germ and wild rape Xanthomonas campestris wild rape pathogenic varieties, and particularly has the radius of the inhibition zone of the rice bacterial streak germ as high as 17.1mm (36h) and 18.4mm (48h), as shown in FIGS. 6 and 7.
Example 4:
preparation of JSPB14 biocontrol agent A and prevention and treatment of postharvest abalone fester
Taking a glycerol strain of the bacillus amyloliquefaciens strain JSPB14, streaking and inoculating the glycerol strain to a 2YT solid plate, and culturing for 24 hours at the temperature of 30 ℃; selecting JSPB14 single colony with sterilized toothpick, inoculating into 250mL conical flask containing 50mL2YT liquid culture medium, and culturing at 30 deg.C for 36 hr at 100r/min to obtain seed solution; inoculating the seed liquid into a Landy culture medium at a ratio of 1% (v/v) for fermentation, wherein the fermentation temperature is 26 ℃, the initial pH of the culture medium is 6.5, and the fermentation aeration is carried out at an aeration ratio of 0.5:1, wherein the aeration ratio is: the ratio of the volume of air passing through the fermentation broth per minute to the volume of the fermentation broth; the stirring speed is 75r/min, and the fermentation time is 48 h. Centrifuging the fermentation liquid for 20min at the speed of 6,000r/min, taking the sterile supernatant, adding potassium sorbate with the final concentration of 1g/L, namely JSPB14 biocontrol microbial inoculum A, then carrying out sterile filling, and storing at room temperature.
An application method of the biocontrol microbial inoculum A in preventing and treating post-harvest pleurotus eryngii ulceration disease comprises the following steps: diluting the biocontrol microbial inoculum A by 1-5 times, treating the harvested pleurotus eryngii by using a soaking mode, airing, and placing according to the conditions of a harvested goods shelf, so that a good control effect can be obtained.
The application case is as follows:
a single colony of lactococcus lactis subsp lactis SLPE1-3 is picked up and placed in a 250mL conical flask containing 50mL of MRS liquid medium, and is subjected to static culture at the temperature of 30 ℃ for 16h to serve as a pathogen inoculation body for later use. Selecting uniform pleurotus eryngii, and scratching a cross wound at the middle lower part of the pleurotus eryngii stem by using a sterilization scalpel, wherein the size of the wound is about 2cm multiplied by 2cm, and the depth of the wound is about 1 mm. A total of 3 treatments: (1) performing biocontrol treatment, namely inoculating 20 mu L of the prepared lactococcus lactis subspecies lactis inoculum to a wound of pleurotus eryngii, standing at room temperature for 30min, and then respectively soaking in 2-fold diluent of the biocontrol microbial inoculum prepared by the method for 30S, wherein the treatment number is JSPB14-S + SLPE 1-3; (2) performing contrast treatment on clear water, dripping 20 mu L of sterilized water on the wound, soaking in sterile water for 30s, and numbering as CK; (3) control treatment with pathogen SLPE1-3 alone, inoculating 20 μ L of the prepared lactococcus lactis subsp lactis inoculum to wound, and soaking in sterile water for 30s, wherein the treatment number is SLPE 1-3. And fishing out and airing the soaked pleurotus eryngii, and then placing the pleurotus eryngii in a plastic preservation box with the inoculation wound upward. Sealing the preservation box, placing the preservation box in a constant-temperature incubator at 15 ℃ for incubation for 4d, then adjusting the temperature of the constant-temperature incubator to 20 ℃, continuing incubation, and investigating the incidence rate of pleurotus eryngii at 8d and 12d after treatment respectively. As a result, as shown in FIGS. 8 and 9, the incidence of Pleurotus eryngii treated with the control inoculated with only the pathogenic bacteria was as high as 88.9% (8 th) and 97.2% (12 th); the morbidity of the pleurotus eryngii treated by the biocontrol agent is 0 (8 d) and 5.6 (12 d), and the biocontrol agent has excellent control effect.
Example 5:
preparation of JSPB14 biocontrol microbial inoculum B and control of bacterial leaf streak of rice
Taking a glycerol strain of the bacillus amyloliquefaciens strain JSPB14, streaking and inoculating the glycerol strain to a 2YT solid plate, and culturing for 36h at the temperature of 30 ℃; selecting JSPB14 single colony with sterilized toothpick, inoculating into 1,000mL conical flask containing 200mL 2YT liquid culture medium, and culturing at 30 deg.C at 200r/min for 16h to obtain seed solution; inoculating the seed solution into Landy culture medium at a ratio of 5% (v/v) for fermentation at 31 deg.C under an initial pH of 7.2, and aerating at an aeration ratio of 2.5:1 under a stirring speed of 200r/min for 36 h. Adding a coco oil-based esterification compound with the final concentration of 8% (v/v) and sodium benzoate with the final concentration of 1g/L into the fermentation liquor, adjusting the pH value to about 4.5 by using dilute hydrochloric acid to obtain JSPB14 biocontrol microbial inoculum B, then carrying out sterile filling, and storing at room temperature.
The application method of the biocontrol microbial inoculum B in preventing and treating the bacterial leaf streak of rice comprises the following steps: diluting the biocontrol microbial inoculum B by 60-80 times, uniformly spraying at the initial stage of occurrence of bacterial leaf streak of rice, and spraying for 1 time at intervals of 7-9 days to obtain good control effect.
The application case is as follows:
in 2017, a JSPB14 biocontrol microbial inoculum B is used for a plot test for field prevention and control of rice bacterial leaf streak, the prevention and control effect on the rice bacterial leaf streak under the test dose is investigated, and 4 treatments are set in the test: clear water contrast, JSPB14 biocontrol agent B750 mL/mu, JSPB14 biocontrol agent B1000 mL/mu, 20% thiazole zinc suspending agent 100 mL/mu, 60kg of water is used for each mu, and the mixture is uniformly sprayed. The rice variety is Y Liangyou 900, and diseases are found in the investigation of 8 months and 13 days (heading and flowering period of rice). The first dose was given on day 8/15 and the second dose was given on day 8/22, and disease indices were investigated at 8d and 15d, respectively, after the second dose. The results are shown in table 1, after the second administration, the control effect of the JSPB14 biocontrol agent B at the dosage of 750 mL/mu reaches 75.9%, the control effect of the JSPB14 biocontrol agent B at the dosage of 1000 mL/mu reaches 79.1%, and the control effect is similar to that of the chemical agent 20% thiazole zinc suspending agent (82.6%); according to the investigation at 15d after the second application, the control effect of the JSPB14 biocontrol agent B at the dose of 750 mL/mu is slightly reduced (72.3%), but the control effect of the JSPB14 biocontrol agent B at the dose of 1000 mL/mu reaches 80.1%, and is basically the same as the control effect of 20% thiazole zinc suspending agent (79.8%).
Table 1 field test result of preventing and controlling rice bacterial leaf streak by JSPB14 biocontrol microbial inoculum B
The above examples illustrate that the bacillus amyloliquefaciens strain JSPB14 provided by the present invention has strong antagonistic activity against various plant pathogenic bacteria including post-harvest pleurotus eryngii ulceration and rice bacterial streak, has a good prevention and control effect on post-harvest pleurotus eryngii ulceration and rice bacterial streak, can reduce or avoid residues of chemical pesticides and agricultural antibiotics, improves food and environmental safety, and has a good market development prospect.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and all equivalent substitutions or substitutions made on the above-mentioned embodiments are included in the scope of the present invention.
Claims (10)
1. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) JSPB14, which is characterized in that the preservation number of the strain is CGMCC No. 14934.
2. A fermentation product of the Bacillus amyloliquefaciens JSPB14 of claim 1.
3. A biocontrol microbial inoculum comprising bacillus amyloliquefaciens JSPB14 and/or bacillus amyloliquefaciens JSPB14 fermentation products of claim 1.
4. A method for preparing the biocontrol microbial inoculum as described in claim 3, comprising the steps of:
(1) activation culture: taking a glycerol strain of the bacillus amyloliquefaciens strain JSPB14, streaking and inoculating the glycerol strain to a fresh 2YT solid plate, and culturing for 24-36 h at the temperature of 30 ℃;
(2) preparing a seed solution: selecting a single colony to inoculate in a 2YT liquid culture medium, and carrying out shake culture at 30 ℃ for 16-36 h, wherein the oscillation frequency is 100-200 r/min;
(3) preparing biocontrol bacterium liquid: inoculating the seed liquid obtained in the step (2) into a Landy culture medium for fermentation according to the ratio of 1-5% (v/v), wherein the fermentation temperature is 26-31 ℃, the initial pH of the culture medium is 6.5-7.2, the fermentation aeration is carried out according to the aeration ratio of 0.5: 1-2.5: 1, the stirring speed is 75-200 r/min, and the fermentation time is 36-48 h;
(4) preparing a biocontrol microbial inoculum A: and (4) centrifuging the biocontrol bacterium liquid fermented in the step (3), taking the sterile supernatant, adding potassium sorbate, then performing sterile filling, and storing at room temperature.
5. The method according to claim 4, wherein the potassium sorbate is added to the solution in step (4) to a final concentration of 1 g/L.
6. A method for preparing the biocontrol microbial inoculum as described in claim 3, comprising the steps of:
(1) activation culture: taking a glycerol strain of the bacillus amyloliquefaciens strain JSPB14, streaking and inoculating the glycerol strain to a fresh 2YT solid plate, and culturing for 24-36 h at the temperature of 30 ℃;
(2) preparing a seed solution: selecting a single colony to inoculate in a 2YT liquid culture medium, and carrying out shake culture at 30 ℃ for 16-36 h, wherein the oscillation frequency is 100-200 r/min;
(3) preparing biocontrol bacterium liquid: inoculating the seed liquid obtained in the step (2) into a Landy culture medium for fermentation according to the ratio of 1-5% (v/v), wherein the fermentation temperature is 26-31 ℃, the initial pH of the culture medium is 6.5-7.2, the fermentation aeration is carried out according to the aeration ratio of 0.5: 1-2.5: 1, the stirring speed is 75-200 r/min, and the fermentation time is 36-48 h;
(4) preparing a biocontrol microbial inoculum B: adding the coco oil-based esterification compound and sodium benzoate into the fermentation liquor prepared in the step (3), adjusting the pH value to about 4.5, then carrying out sterile filling, and storing at room temperature.
7. The method of claim 6, wherein the coco esterification complex of step (4) has a final concentration of 8% (v/v) and sodium benzoate has a final concentration of 1 g/L.
8. The application of the bacillus amyloliquefaciens JSPB14 or the fermentation product thereof or the biocontrol microbial inoculum as defined in any one of claims 1-3 in preventing and treating post-harvest pleurotus eryngii ulceration and rice bacterial leaf streak.
9. The application method of the biocontrol microbial inoculum A prepared by the method of claim 4 or 5 in preventing and treating the ulceration disease of the harvested pleurotus eryngii, wherein the biocontrol microbial inoculum A is diluted by 1-5 times, the harvested pleurotus eryngii is treated in a soaking mode, and the pleurotus eryngii is placed under the condition of a harvested goods shelf after being dried in the air.
10. The application method of the biocontrol bacterial agent B prepared by the method of claim 6 or 7 in preventing and treating the bacterial leaf streak of rice is characterized in that the biocontrol bacterial agent B is diluted by 60-80 times, is uniformly sprayed at the initial occurrence stage of the bacterial leaf streak of rice, and is sprayed for 1 time at intervals of 7-9 days.
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