CN111378602B - Bacterial strain capable of inhibiting phytophthora infestans and separation method and application thereof - Google Patents
Bacterial strain capable of inhibiting phytophthora infestans and separation method and application thereof Download PDFInfo
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Abstract
According to the invention, the Brevibacterium with the effect of inhibiting phytophthora infestans is screened out from the soil planted in the pair of test base potatoes, antagonistic bacteria WZ-502 with obvious inhibition effect is obtained, and further experiments are carried out to compare the inhibition effects of the antagonistic bacteria on the mycelium growth, zoospore release, resting spore germination and sporangium germination of the phytophthora infestans at different bacterial liquid concentrations. Experiments show that WZ-502 has remarkable inhibition effect on phytophthora infestans, and has relatively good inhibition effect on zoospore release, resting spore germination and sporangium germination of the phytophthora infestans. The strain has a practical good control effect on potato late blight.
Description
Technical field:
the invention belongs to the technical field of microorganisms, and particularly relates to a strain and a separation method and application thereof, in particular to a strain for inhibiting phytophthora infestans of potato late blight and a separation method and application thereof.
The background technology is as follows:
potatoes are the fourth most important food crop worldwide, which is produced mainly in China, russia, the United states, etc. China is one of the countries with the highest total world potato yield. For example, the total yield of potato in 2013 reaches 8892.5 ten thousand, and the total yield of potato in China reaches about 24.2% of the world.
Late blight of potato caused by phytophthora infestans (Phytophthora infestans) is the most common and economically significant potato epidemic. Phytophthora infestans can cause late blight of important crops such as potatoes, tomatoes and the like, and the late blight of potatoes is a destructive fungal disease caused by the Phytophthora infestans and can cause tuber rot and stem and leaf death of the potatoes. The disease commonly occurs in potato planting places, especially when rainy days, the damage is even more serious, and when serious, leaves wilt and the whole plant die. Late blight of potato caused by the oomycete phytophthora infestans has led to large area yield reduction of potatoes worldwide, and huge losses are caused in partial areas and even absolute yields. It is counted that the occurrence area of potato late blight in China reaches 234 ten thousand hm2 in recent years. Because of the harm of potato late blight, the disease rate of the common year is 5% -20%, the disease rate of the retransmission year is 20% -50%, and the disease rate is as high as 80% -100%. It generally results in 10% -20% of yield reduction in year, and the outbreak year can reach 50% -70% and even is out of order. The direct economic loss caused by potato late blight is several hundred million dollars each year worldwide, and about 10 million dollars in our country. Accounting for 15 percent of the total yield value of the potatoes. In addition, potato late blight can also have a serious impact on potato quality. The traditional prevention and treatment methods of potato late blight mainly comprise agricultural prevention and treatment and chemical prevention and treatment. In recent years, the traditional agricultural control method cannot fundamentally control the occurrence of diseases, so that the control effect is unstable; the long-term use of chemical pesticides can cause pathogenic bacteria to generate drug resistance, and excessive pesticide use can damage soil, aggravate the pollution of pesticides to the environment, and cause excessive toxic and harmful substances to remain in crops. Therefore, research on biological control methods is becoming more and more important, and microorganisms which do not harm crops are selected to inhibit the growth of germs by utilizing antagonism among microorganisms. If expert researches find that the EB-28 aseptic culture solution has good inhibition effect on release of zoospores of potato late blight bacteria, the research shows that the inhibition rate reaches 91.26 percent, and in a potting test, the control effect on potato late blight reaches 74.22 percent, which is obviously higher than that of an in vitro leaf blade test. The efficient antagonistic strain YR-35 of potato late blight bacteria is obtained through separation of scholars such as wave making, and the like, and experiments show that the strain has high inhibition rate on the growth of potato late blight bacteria hyphae, but can not completely and comprehensively inhibit and prevent potato late blight phytophthora infestans.
Therefore, how to provide a strain with the effect of preventing and controlling phytophthora infestans of potato so as to prevent potato late blight, the soil for planting potato is utilized to screen out bacteria with the effect of inhibiting potato late blight bacteria, and the strain is utilized to have good inhibiting effects on zoospore release, zoospore germination, sporocyst direct germination and potato late blight bacterial hypha growth of potato late blight phytophthora infestans, so that the strain with strong prevention and control potential, namely antagonistic bacteria of potato late blight phytophthora, is screened out, and the potato late blight is actually prevented and controlled in the potato planting process so as to improve the potato planting yield.
The invention comprises the following steps:
the invention provides a strain for inhibiting phytophthora infestans of potato late blight, a separation method and application thereof; the strain is prepared by preparing soil liquid from potato-planted soil with phytophthora infestans, screening to obtain the strain or antagonistic bacteria for inhibiting the phytophthora infestans, wherein the strain or antagonistic bacteria can inhibit the growth of phytophthora infestans silk, the release of zoospores, the germination of resting spores and the germination of sporangium to generate bud tubes to different degrees.
The invention discloses a strain for inhibiting phytophthora infestans of potato late blight, which is preserved in China center for type culture collection, with the preservation number: cctccc No. M2019525, name of the preserved culture: brevibacterium WZ-502Brachybacterium sp.WZ-502; date of preservation, 7.5.2019, address of preservation: chinese, university of Wuhan, post code 430072.
The strain for inhibiting the phytophthora infestans of potato late blight disclosed by the invention is prepared by amplifying Brevibacterium WZ-502 of the strain through PCR, and sequencing a 16SrDNA sequence of 1424bp with primers of 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-CTACGGCTACCTTGTTACGA-3'), wherein the sequence is as follows:
GACGGGGCGGCGTGCTTACCATGCAAGTCGAACGATGACGGTGGTGCTTGCACCGCCTGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTCCACTTCGGGATAACCTCGGGAAATCGTGGCTAATACCGGATACGAGCACTCATCGCATGGTGAGTGCTGGAAAGATTTATCGGTGGGGGATGGACTCGCGGCCTATCAGTTTGTTGGTGAGGTGATGGCTCACCAAGACGATGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCCCTTTCAGTAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTTGTAGGTGGCTTGTCGCGTCTGCCGTGAAAACCCGAGGCTCAACCTCGGGCGTGCGGTGGGTACGGGCAGGCTAGAGTGTGGTAGGGGAGACTGGAACTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAAGAACACCGATGGCGAAGGCAGGTCTCTGGGCCATTACTGACACTGAGAAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCTGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGCACTGGACGGCTGCAGAGATGTGGCTTTCTTTGGACTGGTGCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTGATGGTGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCAAGCATGCTACAATGGTCGGTACAATGGGTTGCGAAACTGTGAGGTGGAGCGAATCCCAAAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCAGTGGCCCATCCTCGTGAGGGAGCTTCGAAGTGAATTCGGGT。
the phytophthora infestans of the invention preferably uses the genome DNA of the phytophthora infestans of the potato as a template and adopts ITS1 and ITS4 as primers for PCR amplification; the forward primer is ITS1, and the sequence of the forward primer is TCCGTAGGTGAACCTGCGG; the reverse primer was ITS4, which has the sequence TCCTCCGCTTATTGATATGC.
Another object of the present invention is to disclose a method for isolating a strain having an effect of inhibiting Phytophthora infestans of potato late blight, comprising the steps of,
1) Firstly, taking soil for planting different potatoes in a base, and preparing soil liquid;
2) Preparing a soil liquid, namely dissolving 8-12g of the soil in the step 1) into 90ml of sterile water, heating in a water bath at the temperature of 90-100 ℃ for 15-25min, and sequentially diluting the soil liquid into six concentration gradients by using the sterile water: 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 ；
3) Culturing, namely coating and culturing the soil liquid with each concentration in the step 2) on a pre-poured LB solid culture medium, repeating each concentration for 3-6 times, reversely culturing for 2-3d at 35-38 ℃, and screening primary bacteria;
4) Separating and purifying, namely separating and purifying the primary bacteria in the step 3), and re-screening and numbering the primary bacteria with antagonistic inhibition effect by adopting a plate counter method to obtain the Brevibacterium with the strain for inhibiting phytophthora infestans of potato late blight.
The invention relates to a strain for inhibiting phytophthora infestans of potato late blight, which is applied to inhibition of zoospores released by sporocysts of potato late epidemic phytophthora infestans.
A strain for inhibiting phytophthora infestans of potato late blight is applied to the direct germination inhibition of the sporocysts of potato late epidemic phytophthora infestans.
A strain for inhibiting Phytophthora infestans of potato late blight is applied to the germination inhibition of Phytophthora infestans of potato late blight.
A strain for inhibiting Phytophthora infestans of potato late blight is applied to the growth inhibition of potato late blight Phytophthora infestans silk.
The invention has the application of the strain for inhibiting the phytophthora infestans of the potato, and the application is that the concentration of bacterial liquid of the strain for inhibiting the phytophthora infestans of the potato is controlled to be 20-50 ml.L < -1 >.
The invention discloses a strain capable of inhibiting phytophthora infestans and application thereof, which can be applied to inhibiting and controlling harmful bacteria such as the phytophthora infestans and the phytophthora infestans, namely the antagonistic bacteria of the phytophthora infestans. The occurrence of potato late blight in large countries, which are used for potato planting and production, can cause large-area yield reduction every year, and the prevention and the control of potato late blight are also urgent. Chemical pesticide prevention and control is a main mode for preventing and controlling potato late blight at present, but a large amount of chemical pesticide can cause serious environmental pollution, food safety, human health and other problems for a long time. And the drug effect of a large amount of chemical pesticide is gradually weakened or even lost due to the drug resistance of pathogenic bacteria which is rapidly generated. Therefore, the strain for inhibiting the phytophthora infestans of the potato late blight and the application thereof are important measures for guaranteeing potato production and reducing economic loss and environmental pollution.
The antagonistic property of microorganism bacteria, namely the antagonistic bacteria of the phytophthora infestans can be utilized to inhibit the phytophthora infestans, so that the potato late blight can be prevented and treated; the researches of scholars are reported to find that 6 related antagonistic strains have obvious inhibition effects on zoospore release, telogen germination and sporangium direct germination of phytophthora potato. And WL2 strain among 6 strains shows the strongest inhibitory activity. It has also been reported that Bacillus licheniformis has different degrees of inhibition on phytophthora potato silk growth, zoospore release, telogen germination and sporangium germination to produce the germ tube, but has little inhibition on sporangium direct germination to produce the germ tube. The method provided by the invention is used for screening out the Brevibacterium with the effect of inhibiting the phytophthora infestans in the soil planted by the potato in the test base, so that a plurality of antagonistic bacteria with obvious inhibition effects such as WZ-406, WZ-502 and WZ-503 are obtained, and the better inhibition effects on the hypha growth, zoospore release, resting spore germination and sporangium germination of the phytophthora infestans under different bacterial liquid concentrations are compared through further experiments. Experiments show that WZ-502 has remarkable inhibition effect on phytophthora infestans, and has good inhibition effect on zoospore release, resting spore germination and sporangium germination of phytophthora infestans. The strain provided by the invention has a remarkable practical control effect on potato late blight.
Description of the drawings:
FIG. 1 is a phylogenetic tree of Brevibacterium WZ-502, a strain inhibiting Phytophthora infestans of potato late blight of the present invention;
FIG. 2 is an electrophoresis chart of the amplification of 16SrDNA by Brevibacterium WZ-502Taq enzyme which is a strain for inhibiting phytophthora infestans of potato late blight of the present invention;
FIG. 3 is a gram stain (1000X) of Brevibacterium WZ-502, a strain having the effect of inhibiting Phytophthora infestans of potato late blight of the present invention;
FIG. 4 is a colony morphology diagram of Brevibacterium WZ-502, a strain inhibiting Phytophthora infestans in potato late blight of the present invention;
FIG. 5 shows the effect of Brevibacterium WZ-502, which is a strain for inhibiting Phytophthora infestans in potato late blight, on inhibiting Phytophthora infestans in potato late blight (left) and blank (right);
FIG. 6 is a PLB-2 evolutionary tree diagram of phytophthora infestans isolated in the present invention;
FIG. 7 is an electrophoretogram of the PLB-2 isolated from Phytophthora infestans of the present invention; in the PLB-2 electrophoresis diagram of phytophthora potato in FIG. 7, 1 and 2 are total genome DNA,3 and Taq enzyme amplification PCR, and 4 and Pfu enzyme amplification PCR;
FIG. 8 is a sequence diagram of 16SrDNA of Brevibacterium WZ-502, which is a strain inhibiting phytophthora infestans of potato late blight of the present invention.
The specific embodiment is as follows:
the following examples further illustrate the invention, but are merely illustrative of the invention and should not be construed as limiting the invention. Variations, changes, modifications, or alterations to the methods, steps, or conditions of the present invention will be apparent to those of ordinary skill in the art without departing from the spirit and nature of the invention and are intended to be within the scope of the invention. The meanings of the phytophthora potato pathogenic bacteria and the phytophthora potato pathogenic bacteria in the following embodiments are the same, or the phytophthora potato pathogenic bacteria and the phytophthora potato pathogenic bacteria are the same; the strain capable of inhibiting the phytophthora infestans has the same meaning as the phytophthora infestans antagonistic bacteria with the inhibition effect, namely the strain capable of inhibiting the phytophthora infestans is the antagonistic bacteria of the phytophthora infestans, and the Brevibacterium WZ-502 or the called WZ-502 has the same meaning.
Example 1
According to the test, the strain of the Brevibacterium with the effect of inhibiting the phytophthora infestans is screened from the soil of the potato planted in the project test base, the inhibiting effect of the strain on the phytophthora infestans, namely the zoospore release, the resting spore germination, the sporangium direct germination and the growth of the phytophthora infestans silk of the potato is further discussed, and the strain with stronger control potential is screened from the strain.
The invention discloses a strain for inhibiting phytophthora infestans of potato late blight, which is preserved in China center for type culture collection, with the preservation number: cctccc No. M2019525, name of the preserved culture: brevibacterium WZ-502Brachybacterium sp.WZ-502; date of preservation, 7.5.2019, address of preservation: chinese, university of Wuhan, post code 430072.
The strain of the invention, namely Brevibacterium WZ-502, is amplified by PCR, the primers are 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-CTACGGCTACCTTGTTACGA-3'), 1424bp is obtained by 16SrDNA sequence sequencing, and the sequence is as follows:
GACGGGGCGGCGTGCTTACCATGCAAGTCGAACGATGACGGTGGTGCTTGCACCGCCTGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTCCACTTCGGGATAACCTCGGGAAATCGTGGCTAATACCGGATACGAGCACTCATCGCATGGTGAGTGCTGGAAAGATTTATCGGTGGGGGATGGACTCGCGGCCTATCAGTTTGTTGGTGAGGTGATGGCTCACCAAGACGATGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCCCTTTCAGTAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTTGTAGGTGGCTTGTCGCGTCTGCCGTGAAAACCCGAGGCTCAACCTCGGGCGTGCGGTGGGTACGGGCAGGCTAGAGTGTGGTAGGGGAGACTGGAACTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAAGAACACCGATGGCGAAGGCAGGTCTCTGGGCCATTACTGACACTGAGAAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCTGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGCACTGGACGGCTGCAGAGATGTGGCTTTCTTTGGACTGGTGCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTGATGGTGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCAAGCATGCTACAATGGTCGGTACAATGGGTTGCGAAACTGTGAGGTGGAGCGAATCCCAAAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCAGTGGCCCATCCTCGTGAGGGAGCTTCGAAGTGAATTCGGGT。
the invention has a 16SrDNA sequence diagram of a strain for inhibiting phytophthora infestans of potato late blight, namely Brevibacterium WZ-502,
the following is a specific preparation method and application description of the invention:
materials and methods
1. Materials and reagents
Potato late blight disease plants: the invention is collected in the project test base, in particular to a country in Yichun city of Jiangxi province of potato land;
oat medium: oat 50 g.L-1, carrot 50 g.L-1, agar powder 20 g.L-1, pH=5.8;
LB solid medium: tryptone 10 g.L-1, yeast extract 5 g.L-1, naCl 10 g.L-1, agar powder 18 g.L-1, and adjusting pH to 7.2+ -0.1 (no agar is added in the liquid culture medium); ampicillin or streptomycin: 200 mg.L-1.
2. Experimental method
(1) Isolation and screening of Phytophthora infestans
Disease standard samples are collected on a project test base of the invention, stem and leaf of late blight samples of different potato varieties are separated by adopting a tissue separation method, phytophthora infestans, namely phytophthora potato (hereinafter the same as) is inoculated on an oat culture medium, but oat and carrot are crushed firstly in the process of preparing the oat culture medium, and then filtered to obtain filtrate; 200 mg.L-1 of ampicillin was added to kill bacteria prior to inoculation. When separating mould, washing the disease leaves with water, washing with sterilized water, cutting the disease spots into small pieces with the size of 1.0cm2, soaking in 75% alcohol for 30s, taking out, and cleaning with sterile water. Placing on different oat culture medium plates, culturing at 18 deg.C for 5-6d, inoculating white plush phytophthora infestans to fresh oat culture medium (streptomycin 200 mg.L-1) with a puncher, then separating and purifying phytophthora infestans by punching and inoculating bacterial cake, and observing phytophthora infestans spore and growth form by microscopic examination, with the number of PLB-2.
(2) Morphological identification of Phytophthora infestans PLB-2
Taking out phytophthora infestans PLB-2 cultured at 18 ℃ for 7d, firstly observing colony characteristics, and then observing microscopic structures by a microscopic morphological identification method. The slide glass is manufactured by the inserting method, the cover glass is firstly inserted into the culture medium in an inclined mode by 45 degrees, then the cover glass is placed into an incubator at 18 ℃ to be cultured for 1d, and hyphae are allowed to spread on the cover glass. And performing microscopic examination, staining with a lactic acid stone carbonic acid cotton orchid staining solution, and observing the morphological structure of hyphae, sporangia and zoospores of phytophthora infestans under an optical microscope. (3) Molecular biological identification of Phytophthora infestans PLB-2
Extraction of Phytophthora infestans genomic DNA
200mg of Phytophthora infestans is firstly taken, ground into powder by liquid nitrogen and then added into a centrifuge tube with the volume of 1.5 ml. According to the rapid extraction of fungal genome DNA, the kit is used for extracting DNA by a method described by Shanghai company. 2ul of the DNA obtained by the extraction was taken and the concentration and purity of the nucleic acid in the sample were measured by a nucleic acid protein meter. The ratio of sample A260/A280 was between 1.8 and 2.0. This indicates that the DNA content of the sample is high. The DNA was subjected to agarose gel electrophoresis.
ITS-rDNAPCR amplification detection of Phytophthora infestans PLB-2
PCR amplification is performed by using phytophthora infestans genome DNA as a template and ITS1 and ITS4 as primers. The forward primer is ITS1, and the sequence of the forward primer is TCCGTAGGTGAACCTGCGG; the reverse primer was ITS4, which has the sequence TCCTCCGCTTATTGATATGC. PCR reaction system 20. Mu.L: 0.6. Mu.L ITS1, 0.6. Mu.L ITS4, 10. Mu.L 2X Taq PCR MasterMin, 1. Mu.L DNA template, 7.8. Mu.L ddH2O. Amplification procedure: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 55℃for 60s, extension at 72℃for 90s, and final extension at 72℃for 10min. After the PCR amplification is finished, agarose gel electrophoresis is carried out on the amplified product, a gel imager is used for observation, and an electrophoresis pattern is compared and analyzed, and the electrophoresis pattern is shown as a PLB-2 electrophoresis pattern of phytophthora infestans in figure 7.
Identification of Phytophthora infestans PLB-2 molecule
The phytophthora infestans PLB-2PCR product is sent to a certain biological company in Shanghai for sequencing, and the sequences are subjected to gene sequence alignment by BLAST tools of NCBI database. The evolved tree is obtained by analysis by MEGA10.0 software, and is shown in figure 6, namely the PLB-2 evolved tree diagram of phytophthora infestans.
(4) The strain for inhibiting the potato late blight phytophthora infestans, namely Brevibacterium WZ-502, is screened, and is called screening of phytophthora infestans antagonistic bacteria or screening of antagonistic bacteria;
firstly, soil liquid is prepared from soil obtained from a test base for planting potatoes, and the specific method is as follows: dissolving 10g of soil in 90ml of sterile water, heating in a water bath at 100deg.C for 10min, and sequentially diluting with sterile water to 10 -6 Gradient, plating method onto LB solid medium, repeating each concentration 4 times, and culturing for 2-3d under 37 deg.C. Then separating and purifying, re-screening antagonistic bacteria by adopting a flat plate counter method, re-screening three strains for inhibiting phytophthora infestans of potato and numbering three antagonistic bacteria WZ-406, WZ-502 and WZ-503.
Preparing antagonistic bacteria stock solution, selecting three antagonistic bacteria pure products WZ-406, WZ-502 and WZ-503, respectively inoculating into three bottles of LB culture medium by inoculating loop, and inoculating each bottle of LB culture mediumA bacterium is then placed at 37deg.C, 200r.min-1, 24h, OD value is detected at about 1.5, and bacterial number concentration is 3.65X10 7 CFU·ml -1 The method comprises the steps of carrying out a first treatment on the surface of the And then placed in a refrigerator at 4 c for standby.
3. The concentration of different phytophthora infestans antagonistic bacteria stock solutions is that the bacterial stock solutions (the same as below) of the strain for inhibiting the phytophthora infestans of the potato late blight have influence on the inhibition effect of the phytophthora infestans of the potato;
three antagonistic bacteria were diluted to 4 concentrations of 50 ml.l -1 、25ml·l -1 、12ml·l -1 、6ml·l -1 Bacterial solutions of different concentrations were then spread onto sterilized coagulated oat solid medium, each treatment repeated 3 times, with no bacterial stock added as a blank. 1 phytophthora infestans cake (diameter=0.8 cm) with growth vigor of about 10d was placed in the middle of each plate, and colony diameters were measured after culturing in a constant temperature incubator at 18 ℃ for about 10d, and the average value was calculated.
(1) The influence of different antagonistic bacteria stock solution concentrations on the release of zoospores from phytophthora infestans sporangium of potato late blight,
bacterial stock of antagonistic strains, i.e. having a capacity to inhibit phytophthora infestans, potato late blight was diluted to 5 concentrations: 50 ml.l -1 、25ml·l -1 、12ml·l -1 、6ml·l -1 、3ml·l -1 The sporangium brush of phytophthora infestans growing for about 10d is dropped into sterile water to prepare sporangium suspension, the sporangium suspension is diluted to 300ml, and then the sporangium suspension is split into 3 bottles of 100ml each. The total number of sporocysts was counted under a microscope to about 3.25X106. Multidot.ml-1 by the hemocytometer method. Bacterial stock solutions with different concentrations and sporangium suspensions (3 ml) were mixed in a sterilized clean and dry culture dish in equal volumes, each concentration was repeated 3 times, LB liquid medium was used as a blank control, and the culture was kept at 4℃for 3 hours in the dark to promote sporangium release of zoospores, and at the same time 50 sporangia were counted and the zoospore release rate was calculated. Zoospore release (%) = number of sporangia/total number of sporangia 100%.
(2) The influence of different antagonistic bacteria stock solution concentrations on the direct germination of phytophthora infestans sporocysts of potato late blight,
the preparation method of the sporangium suspension with 300mL is carried out again, the total number of the sporangium is 1.5X106-mL < -1 > by using a hemocytometer method, the sporangium suspension of phytophthora infestans is mixed with bacterial solutions with different concentrations of 3 strains of bacteria according to the ratio of 1:1 (3 mL: 3 mL), and the mixture is mixed with equal volume of sterile water to be used as a control. Each treatment was repeated 3 times and left to stand in the dark at 25 ℃ for 5 hours to stimulate direct germination of the sporangia. The number of the spores is counted to 50, and the average percent of sporangium germination under different bacterial liquid concentrations is calculated. Sporangium germination rate (%) = sporangium germination number/total number of spores x100%.
(3) The influence of different antagonistic bacteria stock solution concentrations on the germination of phytophthora infestans rest spores of potato late blight,
using 100ml of the sporangium suspension prepared in the above step, the solution was left for 3 hours at 4℃in the dark to promote release of zoospores, and a zoospore suspension was prepared, and the release number of zoospores was examined by microscopy and found to be about 1.0X106. Ml-1. Zoospore suspensions were mixed with 3 strains of bacteria at different concentrations 1:1 (3 mL: 3 mL) to control mixing with an equal volume of sterile water, and each treatment was repeated 3 times. Dark conditions were used for 3h at 10℃to stimulate the germination of telogen. The average percentage of telogen germination at different bacterial fluid concentrations was calculated. Resting spore germination rate = resting spore germination number/zoospore release number X100%.
4. The invention relates to the form and molecular identification of antagonistic bacteria WZ-502 which is a strain for inhibiting phytophthora infestans of potato late blight
The gram of the antagonistic bacteria of the present invention or the gram of the strain having the effect of inhibiting phytophthora infestans of potato is shown in fig. 3, and the specific identification method comprises: brevibacterium WZ-502 was isolated and purified by multiple streaking plates, the morphological structure of each strain was observed, and then observed with a microscope after gram staining. And the genus is identified by referring to the Bojie's bacteria identification manual. Molecular identification of the best antagonistic bacteria WZ-502 is as above; the primers for the amplification detection of 16SrDNA of the best antagonistic bacteria WZ-502 of Phytophthora infestans are 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-CTACGGCTACCTTGTTACGA-3'). 16SrDNAPCR product sequencing.
Results and analysis
Identification of phytophthora infestans and antagonistic bacteria thereof or morphological identification of phytophthora infestans and antagonistic bacteria thereof,
the phytophthora infestans PLB-2 sample is cultured in an oat culture medium by using a tissue separation method, the separated phytophthora infestans PLB-2 is pure white on the oat culture medium, and has the same form as the prior art, even hypha distribution, white filiform shape and circular colony. The slide is manufactured by an inserting sheet method for microscopic examination, the slide is dyed by lactic acid stone carbonic acid cotton blue dyeing liquid, and the slide is observed under an optical microscope, so that hyphae are thin, have no partition and branches, most hyphae are in a multi-branch form, the top ends of the hyphae are expanded and are attached to sporangia, and the sporangia are elliptical.
Three strains of WZ-406, WZ-502 and WA-503 obtained by screening by the method of the invention have better antagonistic bacteria, and the colony morphological structure of the Brevibacterium WZ-502 is shown in figure 4. Other antagonistic bacteria such as WZ-406 and WA-503 have colony morphology similar to that of the WZ-502 strain; the WZ-502 colony is observed to be yellow, the surface water content is high, the colony is smooth, the viscosity is high, and the edge of the colony is flat. As shown in fig. 3; subsequent observation with an optical microscope after gram staining revealed that the WZ-502 strain was gram-violet, indicating that this antagonistic bacterium was a gram-positive bacterium; WZ-502 is a short rod-like structure.
Molecular identification of Phytophthora infestans PLB-2
Extracting genome DNA from the phytophthora infestans PLB-2 after successful separation and purification, and carrying out ITS-rDNA PCR amplification on the extracted DNA. Then agarose gel electrophoresis is carried out, and the electrophoresis result is shown in figure 7; the molecular weight of ITS-rDNA is 546bp, the homology with Phytophthora infestans reaches 100% through sequencing result and BLAST comparison, which is shown as a PLB-2 evolutionary tree diagram of potato phytophthora infestans in figure 6, and the separated PLB-2 of potato phytophthora infestans is identified as Phytophthora infestans by combining morphological result analysis.
The invention has the identification of antagonistic bacteria WZ-502 16SrDNA of the strain for inhibiting the phytophthora infestans of potato late blight,
the strain with the function of inhibiting the phytophthora infestans of potato late blight, namely antagonistic bacteria WZ-502, is subjected to PCR amplification, and the sequence of 16SrDNA is sequenced to obtain 1424bp. As shown in FIG. 1, by BLAST comparison, the homology of WZ-502 with Brevibacterium paracoccus Brachybacterium paraconglomeratum strain LMG 19861 (NR 022502) reaches 100%, and a phylogenetic tree was constructed based on 13 strains by using NJ method to confirm classification attribution of the WZ-502 strain, and the result shows that the WZ-502 strain is classified as Brevibacterium. The WZ-502 strain is in the same branch as Brevibacterium paracoagulation NR022502, the homology is 99%, and the Renske (2003) suggests that the sequence similarity is between 95% and 99%, and can be identified as the same genus. WZ-502 was identified as Brevibacterium sp.
Influence of different antagonistic bacteria stock solution concentrations on phytophthora infestans inhibition effect
Under the same conditions and different concentrations of antagonistic bacterial solutions, the growth of phytophthora infestans was inhibited to different extents, as shown in table 1 and fig. 5. Wherein the inhibition effect of WZ-406 is not obvious, while WZ-502 shows good inhibition effect, when the concentration is 50 ml.L-1, the mould growth diameter is as small as 1.1+/-0.10 acm, almost no growth is seen, the difference is very obvious, when the concentration is 6 ml.L-1, the diameter of phytophthora infestans is 5.1+/-0.10 acm, and the difference is obvious compared with the control.
TABLE 1 Effect of different antagonistic bacterial fluid concentrations on Phytophthora infestans growth Tab.1 inhibit ionofantronisic bacteria growth of mycelial ofp infstans
FIG. 5 shows the inhibitory effect of WZ-502 on phytophthora infestans and a blank, and FIG. 5 shows the inhibitory effect of WZ-502 on the left side and the blank on the right side.
The influence of different antagonistic bacteria stock solution concentrations on zoospores released by phytophthora infestans sporangia,
under the action of bacterial stock solutions with the same conditions and different concentrations, the inhibition effect of three antagonistic bacteria on zoosporium release of phytophthora infestans is shown in the following table 2, WZ-502 shows the highest inhibition effect under the concentration of the bacterial stock solution with the concentration of 50 ml.L-1, the zoosporium release rate is only 15.38%, the zoosporium release rate of a control group reaches 46.15%, obvious inhibition effect is shown, and the difference is very obvious; WZ-503 was second to the worst inhibition by WZ-406. WZ-502 also maintained zoospore release at about 26% at a bacterial stock concentration of 12 ml.L-1, a significant difference compared to 46.85% for the control group.
TABLE 2 antagonism of the effect of bacterial fluid concentration on zoospore release from Phytophthora infestans
Tab.2Concentration grade effection ofantagonistic bacteriaon release of zoospores from sporangia ofP.infestans
Description: the effect of different antagonistic bacterial stock concentrations on direct germination of phytophthora infestans sporangia.
The inhibition effect of different antagonistic bacteria stock solutions on the direct germination of sporangium is shown in table 3, and the results show that different antagonistic bacteria stock solutions have a certain inhibition effect on the direct germination of sporangium. As the concentration of antagonistic bacteria stock solution increases, the sporangium germination rate gradually decreases, and the relative inhibition rate correspondingly increases. When the concentration of WZ-502 is 50 ml.L-1, the relative inhibition rate of direct germination of sporangia is highest and is 13.11%, and the germination rate of the sporangia is remarkably different from that of a control. When the WZ-502 concentration is 12 ml.L-1, the relative inhibition rate of direct sporangium germination to direct sporangium germination is highest, and is 26.18%, and the sporangium germination rate is significantly different from that of a control.
TABLE 3 effects of different antagonistic bacterial liquid concentrations on direct germination of Phytophthora infestans sporangia
Tab.3Concentration grade effection ofantagonistic bacteriaon ongermination of sporangia ofP.infestans
The influence of different antagonistic bacteria stock solution concentrations on the germination of phytophthora infestans rest spores is illustrated.
Under the action of different antagonistic bacteria liquid concentrations, the inhibition effect of the three antagonistic bacteria on the germination of the phytophthora infestans is shown in table 4, and it can be seen that when the antagonistic bacteria liquid concentration is large, the inhibition effect on the germination of the phytophthora infestans is obvious, and when the concentration is 50 ml.L-1 and 25 ml.L-1, the relative inhibition rate on the germination of the phytophthora infestans is high. Wherein W Z-502 has remarkable inhibition effect, is remarkably different, and has remarkable effect after WZ-503. As the concentration of antagonistic bacterial liquid is continuously reduced, the relative inhibition rate of the telogen germination is gradually reduced, but the difference is obvious compared with the control. The results show that the concentration of different antagonistic bacteria liquids has great influence on the germination of telogen spores, the obvious difference is obvious, and the strain WZ-502 has theoretical value on the prevention and treatment of potato late blight.
TABLE 4 influence of different antagonistic bacterial liquid concentrations on the germination of P.aphanidermatum
Tab.4Concentration grade effection ofantagonistic bacteriaon ongermination ofzoospores ofP.infestans
The strain which is separated by the method and used for inhibiting the potato late blight phytophthora infestans, namely the Brevibacterium WZ-502 has an inhibiting effect on the potato late blight phytophthora infestans, and the strains have different effects, namely the strains are preserved in China center for type culture collection, and the preservation number is: CCTCC NO: M2019525, brevibacterium WZ-502 has the most remarkable effect.
Conclusion and discussion
As a large country for planting and producing potatoes, the occurrence of potato late blight can cause large-area yield reduction every year, and research on prevention and control of potato late blight is also urgent. Chemical pesticide prevention and control is a main mode for preventing and controlling potato late blight, but a large amount of chemical pesticide can cause serious environmental pollution, food safety, human health and other problems for a long time. And the drug effect of a large amount of chemical pesticide is gradually weakened or even lost due to the drug resistance of pathogenic bacteria which is rapidly generated. Therefore, the development of effective, safe and environment-friendly biopesticide is an important measure for guaranteeing potato production and reducing economic loss and environmental pollution. In recent years, biological control of plant diseases has been widely accepted in agricultural production by virtue of its advantages of no pollution, no residue, no ecological toxicity, good safety, and the like. Among them, inhibition of phytophthora infestans and thus control of potato late blight by antagonistic microorganisms is also receiving increasing attention. According to researches, the antagonistic strains have obvious inhibition effects on release of zoospores of phytophthora infestans, germination of telogen and direct germination of sporangium. If the strain WL2 shows the strongest inhibitory activity, the scholars find that bacillus licheniformis can also inhibit the growth of phytophthora infestans filaments, the release of zoospores, the germination of resting spores and the germination of sporangium to generate bud tubes to different degrees; but the inhibition effect on the direct germination of sporangia to produce the bud tube is not great. The antibacterial effect of the strains Sy11, M15 and A5295 on inhibiting phytophthora infestans is studied by using a counter culture method, a perforation method and an in vitro tissue section method. The Sy11 strain has the greatest effect of preventing and treating potato late blight. Zegeye et al found that Trichoderma was more effective in controlling potato late blight than P.fluorescens. The research of Lamsal and the like shows that the inhibition rate of 7 rhizosphere bacteria to phytophthora infestans is above 60%, wherein the inhibition effect of AB15 strain is strongest. And researches by scholars also find that endophytic bacterial strains 265ZY1, 265ZY3 and 265XY6 have stronger antagonistic capability on Fusarium solani, and the antibacterial rate is close to 75%. Through morphological observation and 16S rDNA gene sequence analysis, 265ZY1 was identified as Bacillus cereus (Bacill us vallismortis), 265ZY3 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and 265XY6 was identified as Bacillus methylotrophicus (Bacillus methylotrophicus).
According to the invention, the Brevibacterium with the effect of inhibiting phytophthora infestans is screened out from the soil planted in the pair of test base potatoes, antagonistic bacteria WZ-502 with obvious inhibition effect is obtained, and further experiments are carried out to compare the inhibition effects of the antagonistic bacteria on the mycelium growth, zoospore release, resting spore germination and sporangium germination of the phytophthora infestans at different bacterial liquid concentrations. Experiments show that WZ-502 has remarkable inhibition effect on phytophthora infestans, and has relatively good inhibition effect on zoospore release, resting spore germination and sporangium germination of the phytophthora infestans. The strain has a practical good control effect on potato late blight.
<110> Yichun college
<120> strain for inhibiting phytophthora infestans of potato late blight, and separation method and application thereof
<210> 1
<211> 1424
<212> DNA
<213> Artificial sequence (Brachybacterium sp.)
<400> 1
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Claims (6)
1. A strain for inhibiting phytophthora infestans of potato late blight, which is preserved in China center for type culture collection, with a preservation number: cctccc No. M2019525, name of the preserved culture: brevibacterium WZ-502Brachybacterium sp. WZ-502; date of preservation, 7.5.2019, address of preservation: chinese, university of Wuhan, post code 430072.
2. The use of a strain having a capacity to inhibit phytophthora infestans in potato as claimed in claim 1 for inhibiting the release of zoospores from the sporangia of phytophthora infestans.
3. The use of a strain having the effect of inhibiting phytophthora infestans of potato in inhibiting direct germination of the sporocysts of phytophthora infestans of potato according to claim 1.
4. The use of a strain having the effect of inhibiting phytophthora infestans in potato in inhibiting the germination of phytophthora infestans in potato in accordance with claim 1.
5. The use of a strain having a capacity of inhibiting phytophthora infestans in potato as claimed in claim 1 for inhibiting the growth of phytophthora infestans filaments.
6. The use according to any one of claims 2 to 5, wherein the concentration of the bacterial liquid of the strain for inhibiting phytophthora infestans in potato is controlled to be 20-50 ml.L -1 。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005090553A1 (en) * | 2004-03-24 | 2005-09-29 | Korea Research Institute Of Chemical Technology | Bacillus subtilis strain having antagonistic activity for controling plant diseases |
| CN107630064A (en) * | 2017-11-01 | 2018-01-26 | 甘肃省农业科学院生物技术研究所 | A kind of phytophthora infestans antagonistic effect culture medium and its preparation method and application |
| CN108570435A (en) * | 2018-05-14 | 2018-09-25 | 清华大学 | The short shape bacillus of one plant of Nie Shi and its application |
-
2020
- 2020-02-27 CN CN202010121962.8A patent/CN111378602B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005090553A1 (en) * | 2004-03-24 | 2005-09-29 | Korea Research Institute Of Chemical Technology | Bacillus subtilis strain having antagonistic activity for controling plant diseases |
| CN1934241A (en) * | 2004-03-24 | 2007-03-21 | 韩国化学研究院 | Bacillus subtilis strain having antagonistic activity for controling plant diseases |
| CN107630064A (en) * | 2017-11-01 | 2018-01-26 | 甘肃省农业科学院生物技术研究所 | A kind of phytophthora infestans antagonistic effect culture medium and its preparation method and application |
| CN108570435A (en) * | 2018-05-14 | 2018-09-25 | 清华大学 | The short shape bacillus of one plant of Nie Shi and its application |
Non-Patent Citations (5)
| Title |
|---|
| 拮抗细菌W-7的鉴定及其对致病疫霉的抑制作用;万安琪等;《河南农业科学》;20170215;第46卷(第02期);55-59,82 * |
| 拮抗菌YQ5对致病疫霉的抑制作用及其抑菌稳定性研究;吴艳清等;《中国植保导刊》;20181025(第10期);7-14 * |
| 致病疫霉拮抗细菌WL2鉴定及其发酵液稳定性分析;王游游等;《河北农业大学学报》;20170731;第40卷(第4期);90-95 * |
| 致病疫霉拮抗细菌筛选及J-28菌株鉴定;李颖等;《中国植保导刊》;20160125(第01期);10-15,19 * |
| 马铃薯致病疫霉及其拮抗菌的筛选与鉴定;刘紫英等;《浙江农业学报》;20200531;第32卷(第5期);840-848 * |
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