CN114128725A - Bacillus subtilis KT-10 and application thereof - Google Patents
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Abstract
The invention discloses a bacillus subtilis KT-10 and application thereof in preventing and treating brown spot of kiwi fruit, wherein the bacillus subtilis KT-10 is classified and named as follows: bacillus subtilis with the Latin chemical name:Bacillus subtilis KT-10, which has been deposited in China general microbiological culture Collection center (CGMCC) on 12.11.2021 with the deposition number of CGMCC No.23792, and the deposition unit address of No. 3, Xilu No.1, Beijing, the area facing the Yangyang. The bacterial strain has a good antagonistic effect on the kiwi fruit brown spot, has a good prevention and control effect on the kiwi fruit brown spot, and has a bacteriostasis rate of 100% on the kiwi fruit brown spot pathogenic bacteria.
Description
Technical Field
The invention relates to bacillus subtilis KT-10 and application thereof, in particular to a strain KT-10 extracted from tailing soil and application thereof in preventing and treating brown spot of kiwi fruit, and belongs to the technical field of microorganisms.
Background
The fructus Actinidiae chinensis is a perennial deciduous vine plant belonging to Actinidia of Actinidiaceae. The kiwi fruit is rich in various vitamins such as vitamin A, C, E, B6, B12 and the like, is a fruit with extremely high nutritive value, is deeply loved by consumers and has wide market development prospect. China is the largest distribution area and large planting country of wild kiwi fruit resources, and main production areas are concentrated in Shaanxi, Sichuan and other provinces. The kiwi fruit is one of the special fruits in Sichuan, has large-area cultivation in places such as Chengdu, Yaan and Guangyuan, and the area of the kiwi fruit in the whole province reaches 2.97 multiplied by 104hm2The yield reaches 1.3 multiplied by 105t. In recent years, with the ever-increasing market demand of kiwi fruits, the popularization planting area is also expanding. In the rapid development process of the industry, the problem of plant diseases and insect pests of the kiwi fruits gradually becomes serious, so that the yield of the kiwi fruits is reduced, the quality of the kiwi fruits is reduced, serious economic loss is brought to fruit growers, and the kiwi fruit disease and insect pest control method becomes one of main factors restricting the development of the kiwi fruit industry. The disease of the kiwi fruits is more than 30, wherein bacterial canker, brown spot, root rot, flower rot, fruit soft rot, phytophthora root rot, leaf blight, root knot nematode, root cancer, black spot and the like are common and seriously harmful. In Sichuan areas, the problem of damage of the brown spot of the kiwi fruit is particularly prominent, the disease generally occurs in 7-8 months, the disease is rapidly transmitted, leaves can be withered and fall off early, the disease leaf rate reaches 60-100%, the photosynthesis of plants is seriously influenced, the branches and stems are withered in the later period of disease occurrence, the fruit is withered and falls off, the yield loss reaches 30-50%, and the method is one of important factors for restricting the development of Sichuan kiwi fruit industry.
Related research reports have been conducted by previous researchers on actinidia brown spot, and the causes of brown spot are classified as Mycosphaerella globiformis (Mycosphaerella sp.), Alternaria alternata (Alternaria alternata), and phoma stigmata (Phyllosticta sp.), respectively. Through identification of kiwi brown spots in Sichuan area, pathogenic bacteria of kiwi brown spot in Sichuan area are determined to be corynebacterium polystachyum (Corynespora cassiicola (Berk. & Curt.)) Wei. The corynebacterium polystachyum produces corynebacterium leaf spot mainly by infecting plant leaves, can infect various crops such as cucumbers, eggplants, hot peppers, kidney beans, tomatoes, lentils and the like, and also can infect flowers, stems and fruits of the plants, and can cause phenomena such as leaf fall, fruit fall and the like in severe cases. In autumn every year, after many Chinese gooseberry varieties in Sichuan are infected by brown spot pathogenic bacteria, large-area yield reduction is caused. Although good effect can be achieved in chemical prevention and control, the problem of pesticide residue is easy to occur in 7-9 months of the peak of the brown spot disease in the mature season of kiwi fruits, so that the search for a good biological control preparation is urgent.
Biological prevention and control mainly means that biological pesticides such as antagonistic bacteria, metabolites of the antagonistic bacteria, plant source extracts and the like are used for preventing and controlling diseases. Various studies have shown that the use of beneficial microbial agents can be reduced or avoided. For example: the invention patent with the publication number of CN101438664A discloses a biological comprehensive prevention and control method for diseases of fruits, vegetables and economic crops. By applying the biological antagonistic bacteria agent containing bacillus subtilis BS2-5, BS6 and BS8-3 bacteria and the biochemical yellow rot bacterial manure in a compounding way, the pathogenic bacteria on the liquid surface of the plant and around the root can be inhibited and killed by utilizing the beneficial microbial flora. And the invention patent with the publication number of CN112280714A discloses pseudomonas aeruginosa 8-7 and application thereof. The pseudomonas aeruginosa 8-7 related to the patent has broad-spectrum antibacterial activity and good antagonistic action on various pathogenic bacteria, particularly the pathogenic bacteria of fungi imperfecti have strong antibacterial action on the pathogenic bacteria of the leaf spot of the corynespora hevea and the pleonaspora minitans, the antibacterial rates are 79.33% and 79.67% respectively, and in addition, the pseudomonas aeruginosa also has great biocontrol potential in the aspect of preventing and controlling the plectalopsis multocida and the pleonadospora minitans.
Although the existing microbial agent with better broad-spectrum antibacterial activity can have good antagonistic action on various pathogenic bacteria, the specificity is poor, so that when the specific pathogenic bacteria are prevented and treated, chemical agents are applied or combined with other strains, or the antibacterial effect is limited. Because few research reports are specially directed at the biological control of the brown spot of the kiwi fruit at present, the research and development of the biocontrol strain applied to the control of the brown spot of the kiwi fruit are of great significance.
Disclosure of Invention
The invention aims to provide bacillus subtilis KT-10 and application thereof in preventing and controlling kiwi brown spot, the bacillus subtilis has a good antagonistic effect on kiwi brown spot, the prevention and control effect is good, and the bacteriostatic rate of the bacillus subtilis on kiwi brown spot pathogenic bacteria (Corynespora cassiicola) reaches 100%.
The invention is realized by the following technical scheme: the application of bacillus subtilis KT-10 in preventing and treating brown spot of kiwi fruits is characterized in that the classification and the designation of the bacillus subtilis KT-10 are as follows: bacillus subtilis with the Latin chemical name: bacillus subtilis KT-10 has been preserved in China general microbiological culture Collection center (CGMCC) at 11.12.2021, with the preservation number of CGMCC No.23792 and the preservation unit address of No. 3 Hospital No.1 of Xilu, North Cheng, the Korean area, Beijing.
The microbial inoculum, powder or fermentation liquor of the bacillus subtilis KT-10 is used for preparing a biocontrol preparation for the brown spot of the kiwi fruit.
The other technical scheme of the invention is the bacillus subtilis KT-10.
The 16S rDNA sequence of the bacillus subtilis KT-10 is shown in SEQ ID NO. 1.
The bacillus subtilis KT-10 is obtained by separating and purifying waste ore soil bacteria.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention solves the problems of pesticide residue and environmental pollution caused by long-term application of chemical pesticides and unsatisfactory bacteriostatic effect of a broad-spectrum biocontrol agent in the existing prevention and control of the kiwi fruit brown spot, and provides the bacillus subtilis KT-10 with better antagonistic action on the kiwi fruit brown spot pathogenic bacteria.
Drawings
FIG. 1 is a primary screening chart of antagonistic confrontation culture method
FIG. 2 is a plate diagram of antagonistic bacteria effect evaluation
FIG. 3 is a BOX-A1R cluster analysis chart of antagonistic bacteria
FIG. 4 is a phylogenetic tree of 13 antagonistic strains 16S rRNA.
FIG. 5 is an oxford cup experiment of antagonistic strain KT-10.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is to be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention claimed, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The brown spot of kiwi fruit is relatively serious in Sichuan area, and the pathogenic bacteria is mainly corynespora polyspora (C. The infection is strong, the host is wide, the propagation way is wide, the variation is easy, and the quick and effective prevention and control measures are lacked to bring serious influence on agricultural production. Corynebacterium polystachyum (c. casilicola) is the earliest and broadest host range species found in corynebacterium (Corynespora). Currently, there are 160 pathogenic species and 42 saprophytic species of the corynebacterium (Corynespora) fungus that have been reported. The corynebacterium polystachyum mainly infects plant leaves to generate corynebacterium leaf spot (also called brown spot), and can also infect flowers, fruits and stems of plants when the corynebacterium polystachyum is serious. At present, in the control process of plant diseases, biological control is regarded by people as a substitute for chemical pesticides due to the advantages of no pollution to the environment, safety, no residue in products, strong specificity to pathogenic bacteria, no drug resistance, high efficiency and the like. However, the existing biocontrol microbial resources for biological control of actinidia brown spot by corynespora polystachya are few, and although the existing patents CN101438664A and CN112280714A disclose flora and strains with broad-spectrum antibacterial activity, the existing patents do not have specific advantages for the actinidia brown spot pathogenic bacteria, so that the invention needs to find an advantageous antagonistic strain specially used for controlling the actinidia brown spot, and makes a contribution to biological control of actinidia brown spot by utilizing microbial antagonism.
The following examples are provided to illustrate specific embodiments of the present invention, and it is understood that the scope of the present invention is not limited to the following examples. The antagonistic strains KT-10 or KT-10 described in the examples below are both Bacillus subtilis KT-10.
Example 1: separation, purification and identification of antagonistic strain KT-10
(1) Separation of bacteria from tailings
Tailing soil is collected from Panzhihuazhujiabao mining area waste mining area (N26 o37 '2', E101 o45 '60') in 9 months in 2014, three sampling areas are selected by utilizing random distribution, soil layer samples of 0-30cm are collected by quincunx distribution, the soil layer samples are mixed and then are divided by a quartering method, soil samples of about 50Kg are collected in each area, the soil samples are filled into sterile self-sealing bags, and the sterile self-sealing bags are placed in an ice box at 4 ℃ and transported back to a laboratory.
The method is characterized in that a dilution plate coating method is adopted to separate and purify bacteria from Panzhihuazhujiabao waste ore soil, and 136 bacterial strains with different bacterial colony characteristics are mainly selected according to the size, color, shape and the like of the bacterial colony morphology when a single bacterial colony is selected. In the purification process, the shape of the thallus is observed by using a microscope, wherein most of the bacteria are gram-positive bacteria, and the shape of the thallus is almost rod-shaped.
(2) Screening for antagonistic bacteria
Separating by using a dilution coating method to obtain tailing soil bacteria, selecting single colonies with different forms for multiple microscopic examination and streaking to obtain a pure culture of the tailing bacteria, and storing the pure culture in a beef extract inclined plane (-20 ℃) and 30% glycerol (-80 ℃). Screening antagonistic bacteria on the PDA flat plate by adopting a confronting culture method. Dividing the plate into four equal parts, dividing the plate into 2-3cm long straight lines, punching 5mm of main corynespora ACC10 cakes in the center of the plate by using a puncher, taking the pathogenic bacteria cake only inoculated in the center as a control CK, culturing at 28 ℃ for 5d, observing the colony shape of pathogenic bacteria, and defining that the ore soil bacteria obviously inhibit the pathogenic bacteria colony as antagonistic bacteria.
The primary screening of antagonistic action is carried out on 136 separated and purified ore soil bacteria, and the result shows that 18 ore soil bacteria have obvious dissolving action on pathogenic bacteria bacterial plaque to form a boundary line (figure 1) parallel to the lineation of the antagonistic bacteria, the pathogenic bacteria colony is completely black, and the whole pathogenic bacteria single colony is obviously smaller than that of a control panel CK pathogenic bacteria single colony, which indicates that the 18 strains have antagonistic action on the kiwi fruit brown spot pathogenic bacteria.
(3) Evaluation of Effect against bacteria
The antagonistic effect of the screened antagonistic bacteria was evaluated by two plate antagonisms (FIG. 2) and determination of the inhibitory Rate (RE). The confrontation culture method I: equivalentlyquartered on PDA plates, the centre was inoculated with a cake of M.polyspora (ACC10) (5mm) and the opposite area was inoculated with 2-3cm of bacteria, in triplicate per strain. After culturing in an incubator at 28 ℃ for 5d, measuring the diameter r of the pathogenic bacteria with obvious bacteriostatic effect and the reference diameter ckr, and calculating the growth inhibition rate c (c is ckr-r/ckr) of the pathogenic bacteria, wherein if the growth inhibition rate c is larger, the antagonistic effect is better; the second opposing culture method: the center of the plate was inoculated with 2-3cm of bacteria, and a cake (5mm) of M.polyspora (ACC10) was spotted at 1/2 from the center, and each strain was replicated three times. After 5 days of incubation in an incubator at 28 ℃, the antagonistic band R between the two corynespora polygama ACCs 10 and the diameter RCK of the control plate were measured, i.e.: the larger R compared to RCK, the stronger the antagonistic effect.
The antagonistic effect of antagonistic bacteria on the monilinia fructicola is evaluated by adopting two antagonistic culture methods, and the result shows that 18 strains of bacteria have obvious antagonistic effect on the corynespora polyspora ACC 10. The result of the detection by the culture method I (figure 2a) shows that the growth inhibition rate of 18 strains of bacteria is in the range of 14.71-51.43%, which indicates that the growth inhibition rates of the 18 strains of antagonistic bacteria are different, wherein the growth inhibition rate of KT-58 is the maximum. The results of the second counter-culture method (figure 2b) show that the R antagonistic zone of the 18 strains of bacteria to pathogenic bacteria, namely, the corynespora dorsalis ACC10, ranges from 0.95 cm to 2.9cm, wherein the antagonistic zone of KT-113 is the largest, and the antagonistic zone of KT-52 is the smallest.
The results of the determination of the bacteriostatic Rate (RE) show that 18 antagonistic bacteria have different bacteriostatic rates on 20 pathogenic bacteria of the kiwi fruit brown spot, namely corynespora polyspora, and the bacteriostatic rate range is 45-100% (Table 1). Wherein, the bacteriostatic rate of KT-115 and KT-10 is up to 100 percent; the bacteriostatic rates of KT-71 and KT-60 are 90 percent; the bacteriostatic rates of KT-83 and KT-102 are 85 percent and the bacteriostatic rate of KT-97 is 80 percent; the bacteriostasis rate of other 11 antagonistic bacteria to 20 Actinidia polygama molds is lower than 80%, and the bacteriostasis rate of KT-121 is the lowest and is 45%.
The method for measuring the antibacterial Rate (RE) comprises the following steps: randomly selecting 20 kiwi fruit pathogenic bacteria corynespora polyspora from different collection places in Sichuan area, determining the bacteriostatic Rate (RE) of antagonistic bacteria on the kiwi fruit corynespora polyspora by using a confronting culture method, observing and counting the bacteriostatic rate of the antagonistic bacteria on the 20 kiwi fruit pathogenic bacteria corynespora polyspora after culturing at the same temperature for the same time, wherein the larger RE is, the better antagonistic effect on the kiwi fruit brown spot is.
(4) Molecular characterization of antagonistic bacteria
Screening out strains with antagonistic effect, extracting DNA by GUTC (guanidine isothiocyanate), and storing at-20 deg.C. Performing cluster analysis on antagonistic bacteria, and performing BOX-PCR (BOX-polymerase chain reaction) by using total DNA as a template and a BOX-A1R primer (5'-CTACGGCA AGG CGACGCTGACG-3'); the amplification procedure was pre-denaturation at 95 ℃ for 2min, extension at 94 ℃ for 1min, annealing at 52 ℃ for 1min, extension at 65 ℃ for 8min, and final extension at 65 ℃ for 16 min. The PCR product was electrophoresed with 2% agarose gel at 80v for 2.5 h. After the fingerprints of the BOX-PCR gel electrophoresis chart are counted by 0 and 1, a phylogenetic tree is constructed by BOXA1R-PCR fingerprint by software NTSYSpc 2.2(USA), and then clustering analysis is carried out on antagonistic bacteria.
Selecting a representative strain, taking total DNA as a template, and selecting primers 27F and 1492R to amplify 16S rRNA; PCR amplification system 25 μ L: 2 XPCR Mix 12.5. mu.L; 27F and 1492R (10. mu. mol) 0.15. mu.L; 1. mu.L of template DNA (50 ng/ml); adding double distilled water to 25 μ L; and (3) amplification procedure: 30 cycles, annealing temperature 55 ℃. And (4) detecting the amplification product by 1% gel electrophoresis, and sending the amplification product to Shanghai Ministry of Industrial and genetic engineering for sequencing. The sequencing results were subjected to BLASTn analysis in GeneBank to determine homologous sequences. A phylogenetic tree was constructed on the 16S rRNA of the antagonistic bacteria using the Neighbor-joining method (Neighbor-joining) of MEGA5.0 software.
The 18 antagonistic bacteria from the tailings soil were divided into two major groups I and II at a similarity level of 74% by BOX-A1R cluster analysis (fig. 3), indicating diversity of antagonistic strains in the soil. Wherein, the group I comprises 13 strains, and the group II only comprises 5 strains. Class I is clustered into 5 subclasses, while class II is clustered into 2 subclasses. The similarity of strains KT10, KT97 and KT99 is 100%, and the similarity of KT58 and KT71, KT113 and KT115, KT60 and KT65 is similar at the 100% level respectively.
According to a BOX-A1R clustering dendrogram, 13 strains are selected to represent antagonistic strains at a similarity level of 100% for 16S rRNA sequencing analysis, and the similarity of the 16S rRNA genes of the 13 strains of antagonistic bacteria and the 16S rRNA of Bacillus (Bacillus) is as high as 99% -100% (Table 1). Therefore, the 13 antagonistic bacteria were preliminarily determined to be bacillus. The phylogenetic tree (FIG. 4) was constructed by using the Neighbor Joining method (Neighbor-Joining) of the software MEGA6.0 for 13 representative antagonistic strains and 25 standard strains of Bacillus (Bacillus) 16S rRNA, and revealed that 13 strains were located in 3 branches and belong to different species. Wherein KT53, KT60, KT103, KT52, KT71, KT64, KT83, KT10 and KT113 are positioned on the same branch with Bacillus subtilis and Bacillus tequilensis, which indicates that the homology is high and the genetic distance is close, but the specific identification as the seed level cannot be accurately achieved; the strain KT70 is in the same branch with Bacillus licheniformis, so that KT70 belongs to Bacillus licheniformis; KT121, KT102 and KT72 are in the same branch as Bacillus pumilus, and the 3 antagonistic strains are close to the genetic distance of the Bacillus pumilus.
Antagonistic effect and classification identification of Table 118 strains of bacteria on Monilinia fructicola
Example 2: effect verification of antagonistic strain KT-10 on prevention and treatment of kiwi fruit brown spot
(1) Analysis of toxin production time of antagonistic bacteria
Shaking the screened bacteria with better antagonistic effect by using an LB liquid culture medium, carrying out suction filtration by using a disposable bacteria filter to obtain sterile fresh filtrate, collecting the filtrate once every 8h, and carrying out an oxford cup experiment: two sterilized oxford cups were placed vertically on a corynespora spinosa ACC10 cake plate 3cm from the center with sterile forceps, 200 μ L of the collected filtrate at different time periods was added to each oxford cup, and the cells were cultured in an incubator at 28 ℃ for 2d to record the antagonistic time, so as to preliminarily analyze the time for the antagonistic bacteria to produce toxins.
6 strains of tailing soil bacteria (KT-10, KT-60, KT-71, KT-83, KT-113 and KT-115) with better antagonistic effect are selected according to the antagonistic effect evaluation to analyze the antagonistic toxin production time (Table 2). Wherein, two strains of KT-10 and KT-115 antagonistic bacteria begin to generate antagonistic toxins after being cultured for 32 hours, and the antagonistic toxins generated are increased after 40 hours; KT-60 and KT-83 are cultured for 32h to generate toxin, KT-60 is cultured for 48h to stop generating toxin, and KT-83 is cultured for 48h to still generate toxin; KT-71 is cultured for 40h and then starts to produce toxin, and the toxin is not increased in 48 h; KT-113 cultured for 48h and no bacteriostatic circle and toxin were observed in the negative group.
TABLE 2 analysis of the time of antagonistic bacteria to produce antagonistic toxins
Note: -: no bacteriostatic zone is generated; +: generating a bacteriostatic circle; ++: increase of zone of inhibition
(2) Evaluation of prevention and control effect of antagonistic bacteria on brown spot of potted kiwi fruit
Transplanting 2-year-old kiwi fruit grafted red-sun kiwi fruit seedlings to a greenhouse (25 ℃), and carrying out prevention and control effect evaluation after the seedlings adapt to greenhouse culture conditions and leaves are completely unfolded. Evaluating the prevention and control effect of the brown spot of the potted kiwi fruit according to the optimal time for antagonistic toxin production by antagonistic bacteria and the strain with obvious inhibition zone effect in an Oxford cup experiment: obtaining a corynespora polybotrya ACC10 bacterial cake (5cm), inoculating the bacterial cake on red-yang kiwi fruit leaves, inoculating 5 healthy leaves to each strain, and inoculating 5 bacterial cakes to each leaf; spraying the strain with the strongest antagonistic effect for 40h after inoculating the strain cake, fermenting for 40h, taking the same nutrient solution without antagonistic bacteria as a negative control CK, and repeating the experimental design and the control CK for 3 red-yang kiwi fruit saplings respectively; and (5 d), 10d, 15d, 20d, 25d and 30d are respectively measured to determine the diameter of the brown spot disease spot, and the diameter is compared with a negative control CK to determine the disease attack speed of the brown spot disease, if the development speed is obviously slower than that of the negative control CK, the antagonistic bacteria have the field prevention and control effect.
According to the analysis result of the antagonistic toxin production time of antagonistic bacteria, KT-10 with the strongest antagonistic effect is selected to evaluate the prevention and control effect of KT-10 on the field kiwi fruit brown spot ACC10, and the result shows that KT-10 has a strong inhibition effect on the spreading speed of kiwi fruit brown spot disease spots and can effectively prevent and control the enlargement of kiwi fruit brown spot disease spots (figure 5). After inoculation of the pathogenic bacteria cake, the brown spot on the kiwi fruit leaves begins to expand 10 days after the KT-10 fermentation liquid is sprayed, and the control CK begins to expand 5 days after inoculation of the bacteria cake. With the time of inoculating the pathogenic bacteria cake, the expansion speed of the KT10 sprayed scab is very slow, the average size of the scab is only 7.2mm when the disease is inoculated to 30d, the diameter of the scab is only 1.44 times of that of the cake, the brown spot scab of CK is rapidly expanded, the scab is expanded to 15.3mm when the disease is inoculated to 30d, and the diameter of the scab is already expanded to 3 times of the original diameter. The tailing bacterium KT-10 has a good prevention and control effect on the kiwi fruit brown spot.
(3) Kiwi brown spot field simulation evaluation
And selecting orchards with flat terrain, uniform fertility, consistent growth vigor of kiwi fruit trees and 8-year tree age for carrying out tests. Experiment design 3 treatments (each treating 3 kiwi trees), fermentation broth (10) of bacillus subtilis KT-109cells/mL) was diluted 400-fold and the fermented medium was used as a control, 3 replicates per treatment. The field plot is arranged in random blocks, each treatment is separated by two plants,and a row of kiwi fruits is reserved in the middle as a protection row. Spraying the fermentation liquor at the early stage of the disease occurrence of the brown spot of the kiwi fruit, and continuously applying the medicine for 3 times at intervals of 7 days. Disease indices were investigated before, 7d after the first and 7d after the last application of the inoculum. During investigation, 3 treated trees are taken from each cell, five points of each tree are sampled, 20 leaves are investigated at each point, the disease condition of the leaf spot of the kiwi fruit is investigated, and the disease index and the control effect are calculated according to the disease condition.
The calculation method comprises the following steps:
in the formula: CK 0-pre-drug disease index for placebo;
CK 1-disease index after drug administration in placebo zone;
PT 0-pre-dose disease index in the drug treatment area;
PT 1-disease index after drug treatment area administration;
according to statistics of field efficacy test results (table 3), the bacillus subtilis KT-10 fermentation broth shows a remarkable prevention and treatment effect on kiwi brown spot. After the KT10 fermentation inoculum is applied for the first time, the disease index of the kiwi fruit brown spot is slightly increased, the relative prevention effect is 78.48%, and the control effect is obvious compared with the control. After the KT10 fermentation inoculum is applied for the 3 rd time, although the disease index is increased, the prevention and treatment effect is 83.89 percent, and the prevention and treatment effect is obvious compared with a control strain. The result proves that the bacillus subtilis KT10 fermentation inoculant has good prevention and control effect on the kiwi brown spot.
TABLE 3 field control of Actinidia deliciosa cern brown spot by Bacillus subtilis KT10 microbial inoculum
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
EQUENCE LISTING
<110> institute of science of natural resources of Sichuan province
<120> Bacillus subtilis KT-10 and application thereof
<130> 12 months of 2021 and 08 days
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1454
<212> DNA
<213> Artificial sequence
<400> 1
tatttgcgtc gtgctataat gcagtcgagc ggacagatgg gagcttgctc cctgatgtta 60
gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120
aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa aaggtggctt 180
cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aatggctcac 240
caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360
gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga 420
acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag ccacggctaa 480
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540
taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 600
ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660
gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720
tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840
ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt 1020
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200
gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat 1260
ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc agccgccgaa 1440
gggaacaggg agtt 1454
Claims (5)
1. An application of bacillus subtilis KT-10 in preventing and treating kiwi brown spot is characterized in that: the classification and the designation of the bacillus subtilis KT-10 are as follows: bacillus subtilis with the Latin chemical name:Bacillus subtilis KT-10, which has been deposited in China general microbiological culture Collection center (CGMCC) on 12.11.2021 with the deposition number of CGMCC No.23792, and the deposition unit address of No. 3, Xilu No.1, Beijing, the area facing the Yangyang.
2. Use according to claim 1, characterized in that: the microbial inoculum, powder or fermentation liquor of the bacillus subtilis KT-10 is used for preparing a biocontrol preparation for the brown spot of the kiwi fruit.
3. A bacillus subtilis KT-10 according to any one of claims 1 or 2.
4. Bacillus subtilis KT-10 according to claim 3, wherein: the 16S rDNA sequence of the bacillus subtilis KT-10 is shown in SEQ ID NO. 1.
5. Bacillus subtilis KT-10 according to claim 3, wherein: the bacillus subtilis KT-10 is obtained by separating and purifying waste ore soil bacteria.
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