CN117106677A - Bacillus saxifrage Ws-1 for antagonizing kiwi canker pathogenic bacteria and application thereof - Google Patents
Bacillus saxifrage Ws-1 for antagonizing kiwi canker pathogenic bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus safoci Ws-1 for antagonizing kiwi canker pathogenic bacteria and application thereof, which are excellent antagonistic strains of pseudomonas syringae kiwi pathogenic varieties，Preservation number: CGMCC No.27263, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing. The method for preventing and treating kiwi fruit canker by using the strain Ws-1 comprises the following steps: inoculating the strain Ws-1 to an LB liquid culture medium for culturing, obtaining fermentation seed liquid, then performing expansion culture, and finally preparing a diluent to spray plants. The strain is planted from healthy kiwi fruitsEndophytic bacteria separated from the strain has strong antagonism on pseudomonas syringae and kiwi fruit pathogenic varieties which are pathogenic bacteria of kiwi fruit bacterial canker, can obviously inhibit the growth of the pathogenic bacteria, can effectively prevent and treat kiwi fruit canker, and provides a new microbial germplasm resource for the biological prevention and treatment of kiwi fruit canker.
Description
Technical Field
The invention belongs to the technical field of biological control of plant protection, and particularly relates to bacillus saxifrage Ws-1 capable of effectively antagonizing pathogenic varieties of pseudomonas syringae and kiwi fruits and a method for controlling kiwi fruit canker by using the strain Ws-1.
Background
Kiwi fruitActinidiaLindl) is a perennial vine belonging to the genus actinidia of the family actinidiaceae, known separately as wild peach, sheep peach, kiwifruit (Kiwifruit). The fruits contain rich dietary fibers, vitamins, mineral elements, amino acids and bioactive substances, especially the Vc content is high (90-420 mg in 100 g fresh pulp), and the fruit is a reputation of 'king' of fruits; the kiwi fruit can be eaten fresh, can be processed into jam, fruit juice and the like, has important medical care value, and has wide market prospect.
In recent years, the kiwi fruit industry has been rapidly developed in more than 30 countries worldwide, the total cultivation area is about 20 ten thousand hectares, the yield is up to 240 ten thousand tons, wherein the cultivation area and yield of Chinese kiwi fruit are the largest, respectively account for 53% and 38% of the world share, in Italy, and in New Zealand, the third. Along with the annual increase of the planting area and the gradual expansion of the production scale of the kiwi fruits, the problem of kiwi fruit diseases is also increasingly highlighted. Of these, the most typical is bacterial canker of kiwifruit, which is a disease produced by a pathogenic variant of Pseudomonas syringae kiwifruitPseudomonas syringae pv. actinidiae) The main trunk, branches, tendrils, leaves and the like of the kiwi fruit are mainly damaged, and the kiwi fruit has the characteristics of rapid transmission, strong pathogenicity, high control difficulty and the like, and is extremely easy to cause large-area death of the tree body in a short period.
At present, the measures for controlling the kiwi canker mainly comprise excellent resistant variety breeding, chemical control and biological control besides scientific and standard agricultural management, wherein the biological control has the characteristics of environmental friendliness, human and animal safety, sustainability, strong specificity for pathogenic bacteria and the like, meets the requirements of people on green foods and is favored by a plurality of scholars. Shao Baolin et al (2015) obtain bacillus subtilis B2 from kiwi fruit rhizosphere soil, and both pot culture and field experiments show a certain effect of preventing and treating canker; cui Ligong et al (2017) obtain bacteria (JDG 6, JDG16, JDG 23) from gynostemma pentaphylla rhizosphere soil, which can obviously reduce the occurrence of kiwi fruit canker; kim et al (2019) are separated into 3 actinomycetes from rhizosphere and leaf circumference of kiwi fruits, and have remarkable control effect on canker of kiwi fruits; wicaksono et al (2018) isolated from the medicinal plant M ā nuka to 3 endophytes inhibiting the canker Psa, can reduce the occurrence of canker in the Chinese goosebeery and the like to a certain extent. Although some achievements are achieved in the study on the biological control of kiwi fruit canker, serious disadvantages still exist: at present, most biocontrol strains are derived from soil, kiwi fruit rhizosphere and leaf circumference, and are extremely easily influenced by environmental factors (climate, temperature and humidity, soil property and the like) in the application process, so that the biocontrol effect is unstable and not ideal. Since the endophyte has specificity to the choice of host plant, and is not easy to be interfered by external environment factors. Therefore, the research and development of the kiwi fruit indigenous endophytic bacteria as the canker biocontrol strain thereof has wide application prospect.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a kiwi fruit indigenous endophytic bacterium which is an antagonistic strain of a pseudomonas syringae kiwi fruit pathogenic variety which is a kiwi fruit canker pathogenic bacterium, and the kiwi fruit canker can be effectively prevented and treated by using the strain; meanwhile, the invention also provides a method for biologically preventing and treating kiwi fruit canker by using the strain.
The inventor collects healthy branches and leaves of Miliang No. 1 and red sun kiwi fruits at a kiwi fruit plantation sampling point in Hunan Xiang West area, puts the kiwi fruits into an ice box to be brought back to a laboratory, separates and screens 1 endophytic bacterial strain inhibiting the growth of kiwi fruit canker pathogenic bacteria from the sample, namely antagonistic strain of pseudomonas syringae kiwi fruit pathogenic varieties, and is named as bacillus safari Ws-1 after identificationBacillus safensis Ws-1), and deposited with deposit number: CGMCC No.27263, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: beijing city, chaoyang area, north Chenxi lu 1, 3 ChineseThe institute of microbiology.
The isolation and screening steps of the strain Ws-1 comprise:
(1) The plantation frequently suffering from kiwi fruit canker in the western Hunan province is taken as a sampling point, the branches and leaves of healthy 'Miliang No. 1' and 'hongyang' kiwi fruit plants are collected and put into an ice box to be brought back to a laboratory for standby. Surface sterilizing the branch and leaf sample, shearing to proper size, adding into sterilized grinding bowl, adding 5-10 mL sterile water, and grinding. Standing for 5-10 min, sucking upper liquid, and sequentially performing 10 -1 Multiple of 10 -2 Multiple of 10 -3 Dilution with multiple gradients; dilutions of 0.1. 0.1 mL were applied to LB medium solid plates, each dilution was repeated 3 times, and the culture was inverted at 28 ℃.
(2) Single colony grows on the plate to be coated, colonies with different forms are picked, and the colonies are respectively inoculated on LB solid culture medium for culture by adopting a four-area streak method; repeating the purification for 3 generations, transferring the culture to an LB solid culture medium inclined surface test tube for culture, and then preserving at 4 ℃.
(3) Inoculating the pathogenic bacteria of the kiwi canker of the pseudomonas syringae and the kiwi fruits to an LB liquid culture medium for culturing 24 h, and sucking the bacterial liquid and coating the bacterial liquid on an LB solid culture medium plate for later use. And (3) selecting single colony points of the separated endophytic bacteria on the plates, inoculating 3-4 sites on each plate, culturing at the constant temperature of 28 ℃, observing the antibacterial condition, and generating a antibacterial zone, namely the antagonistic strain of the pseudomonas syringae kiwi fruit pathogenic variety.
(4) Fermenting and culturing the primary screened antagonistic strain, centrifuging, absorbing 200 mL fermentation liquor, transferring into oxford cup coated with pseudomonas syringae kiwi fruit pathogenic variant plate, observing the size of bacteriostasis zone around oxford cup, and screening strain with strong antagonistic effect. The species were identified based on morphology and 16S rRNA gene sequence analysis.
The endophytic bacteria with excellent effect of antagonizing the pseudomonas syringae and the kiwi fruit pathogenic varieties is the biocontrol strain of the kiwi fruit canker, can be used for biological control of the kiwi fruit canker, and comprises the following steps:
(1) Inoculating antagonistic strains of the pseudomonas syringae kiwi fruit pathogenic varieties into an LB liquid culture medium for culture to obtain fermentation seed liquid;
(2) Inoculating the fermentation seed liquid to an LB liquid culture medium for expansion culture to obtain a fermentation culture liquid.
(3) Preparing a fermentation culture solution into a dilution solution with a certain concentration (comprising thalli, and the bacterial concentration is more than or equal to 1.2X10) 3 cfu/mL, namely the total bacterial colony number of each milliliter is more than or equal to 1200), aiming at the kiwi fruit plants, the effective control of kiwi fruit canker can be realized.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a novel kiwi canker biocontrol strain Ws-1, which is an indigenous endophyte strain of kiwi, namely an antagonistic strain of a pathogenic variety of kiwi, namely pseudomonas syringae, which is identified and named as bacillus safoci Ws-1%Bacillus safensis Ws-1）。
The strain is endophytic bacteria obtained by separating and purifying branches of the acquired kiwi fruit plants, has a strong inhibition effect on the pathogenic bacteria of the kiwi fruit bacterial canker, namely pseudomonas syringae and kiwi fruit, can obviously inhibit the growth and propagation of the pathogenic bacteria of the kiwi fruit bacterial canker, can effectively prevent and treat the kiwi fruit canker, and provides a new microbial germplasm resource for the biological prevention and treatment of the kiwi fruit canker.
The strain is a kiwi endophytic bacterium, can mutually and symbiotically with plant tissues, has small interference by external environmental factors, stable performance, convenient spraying and remarkable kiwi ulcer prevention and treatment effect, provides an environment-friendly, simple and effective method for kiwi ulcer prevention and treatment, and is beneficial to environmental protection and organic kiwi production.
Detailed Description
The method for preventing and treating the kiwi fruit canker by using the kiwi fruit canker biocontrol strain Ws-1 and the application strain Ws-1 is further specifically described by combining the specific embodiments.
2021, 3 months, collecting branches and leaves of unaffected Miliang No. 1 and red sun kiwi fruits by taking kiwi fruit plantation in Hunan Xiangxi area as sampling point, and collecting N26 degrees 36'30E114 degrees 07'34The sheet sample was placed in an ice bin and brought back to the laboratory for use. Sequentially soaking with 75% ethanol for 1 min, 1% sodium hypochlorite for 5 min, and 75% ethanol for 1 min, respectively sterilizing the surfaces of the branches and leaves, shearing to proper size, placing into sterilized grinding bowl, adding 5-10 mL sterile water, and grinding. Standing for 5-10 min, sucking upper liquid, and sequentially performing 10 -1 Multiple of 10 -2 Multiple of 10 -3 And (5) performing multiple gradient dilution. Respectively coating 0.1-mL gradient dilutions on LB culture medium plates, repeating each dilution for 3 times, inversely culturing in a constant temperature incubator at 28 ℃ for 1-2 d, and growing single colony on the plates to be coated; selecting colonies with different forms, and respectively inoculating the colonies on an LB solid medium for culture by adopting a four-area streak method; repeatedly purifying for 3 generations, transferring LB solid culture medium, performing slant culture, and storing in a refrigerator at 4deg.C.
Culturing pathogenic bacteria of kiwi canker, pseudomonas syringae and kiwi fruit pathogenic variety overnight, taking 50 mu L of bacterial liquid, and coating on LB culture medium for standby. And (3) selecting single colony points of the separated endophytic bacteria on the plates, inoculating 3-4 sites on each plate, culturing at the constant temperature of 28 ℃, observing the antibacterial condition after 2-3 d, and generating an antibacterial zone, namely the antagonistic strain of the pseudomonas syringae kiwi fruit pathogenic variety.
Inoculating the strain with strong antibacterial activity into 5 mL LB liquid medium, shake culturing at 28deg.C and 180 r/min for 24 h to obtain seed liquid, then taking 100 μL, inoculating into 50 mL LB liquid medium, culturing at 28deg.C and 180 r/min for 48 h, transferring the fermentation liquid into sterile centrifuge tube, centrifuging at 4deg.C and 10000 r/min for 10 min, centrifuging, and filtering with 0.22 μm microporous membrane to obtain supernatant. Meanwhile, the pathogenic bacteria pseudomonas syringae and kiwi fruit pathogenic varieties are cultured overnight, and 50 mu L of bacterial liquid is coated on an LB culture medium for standby.
Placing oxford cup on a plate coated with pathogenic bacteria, transferring 200 μl of supernatant into oxford cup, repeating each strain for 3 times, culturing at 28deg.C at constant temperature, and observing antibacterial condition after 2-3 d to obtain a good strain with antibacterial circle diameter up to 1.9 times of colony diameter.
After the strain is cultured in LB culture medium for 24 h, the colony is white, irregular in shape, irregular in edge and flat,smooth, glossy, and sticky. Extracting and separating total DNA of the bacterial strain by using a bacterial genome DNA extraction kit, and carrying out PCR amplification on 16S rRNA gene segments of the separated bacterial strain by using a bacterial universal primer PA/PB (PA: 5'-AGAGTTTGATCCTGGCTCA G-3'; PB: 5'-TTAAGGTGATCCA GCCGCA-3'), wherein the reaction system is as follows: taq PCR Mix (2×) 25 μl; 1 mu L of each forward primer and reverse primer; 2 mu L of template; sterile water was filled to 50 μl. The PCR procedure was: pre-denaturation at 95℃for 2.5 min; denaturation at 95℃for 15 s, annealing at 55℃for 30 s, elongation at 72℃for 1 min,30 cycles; extending for 10 min at 72 ℃. The PCR amplified products were subjected to agarose gel electrophoresis and then sent to the biological engineering (Shanghai) Co., ltd for sequencing. Submitting the detected sequence to GenBank (http:// www.ncbi.nlm.nih.gov) database, searching homologous sequences by Blast tool software, comparing, constructing a phylogenetic tree by using Mega7.0 Neighbor Joining (NJ), and identifying and naming the strain as bacillus safoci Ws-1 according to phylogenetic relation analysisBacillus safensis Ws-1), and deposited with deposit number: CGMCC No.27263, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The biocontrol strain for kiwi fruit canker is used for biological control of kiwi fruit canker, and comprises the following specific implementation steps:
inoculating the strain Ws-1 into 100 mL LB liquid medium, and carrying out shake culture at 28 ℃ and 180 r/min for 24 h to obtain fermentation seed liquid; inoculating the fermentation seed liquid into 1L LB liquid medium at 1% inoculum size, shake culturing at 28deg.C and 180 r/min, and culturing whenOD 600 And harvesting the fermentation culture solution when the fermentation culture solution reaches 1.2.
The fermentation culture solution is prepared into a diluent with a certain concentration (containing thalli, the bacterial concentration is 1250 cfu/mL, namely, the total bacterial colony number per milliliter is 1250), which is taken as a treatment group 1,1.6 percent of a 800-time diluent of thiazolone, which is taken as a treatment group 2, and a 72 percent of a 800-time diluent of agricultural streptomycin, which is taken as a treatment group 3, and sterile clear water is taken as a control group (treatment group 4). And (3) field efficacy test: 4 red sun kiwi fruit gardens with 3-5 a tree ages are selected, 4 cells are randomly divided in each orchard for 4 treatments, 50 kiwi fruits are treated each time, 4 orchards are repeated, and 200 kiwi fruits are investigated each group. And in the key period from fruit picking in autumn to defoliation, spraying the whole kiwi fruit plant with each corresponding medicament for 3 times at intervals of 10 d. And counting the percentage of the trunk, yellow brown or rust red ulcer disease plants in each treatment group in the high-incidence period (2-3 months) of the kiwi ulcers in the spring, taking the percentage as the disease rate of the ulcer disease rate, and calculating the field disease prevention effects of different treatments according to the following formula.
Disease preventing effect= (control morbidity-treatment morbidity)/control morbidity x 100%
Through statistics, the average inhibition rate of the field efficacy test of the strain Ws-1 group (treatment group 1) reaches 85.4%, the average inhibition rate of the field efficacy test of the 1.6% thiazolone 800-time diluent group (treatment group 2) is 79.2%, and the average inhibition rate of the field efficacy test of the 72% agricultural streptomycin 800-time diluent group (treatment group 3) is 69.8%.
Further field control experiments prove that: when the concentration of the fermentation culture solution of the strain Wt-1 reaches 2000 cfu/mL, the control effect of the kiwi canker is greatly enhanced, the average inhibition rate can reach 90 percent, and the average inhibition rate is far higher than that of a 1.6 percent thiazolone 800-time diluent and a 72 percent agricultural streptomycin 800-time diluent. Therefore, the Steiner Li Bake Hold's bacteria Ws-1 has very ideal control effect on kiwi fruit canker, and is an excellent kiwi fruit canker biocontrol strain.
Claims (2)
1. A bacillus safoci Ws-1 for antagonizing pathogenic bacteria of kiwi canker belongs to bacillus safoci @Bacillus safensis) The strain has excellent antagonistic effect on the pathogenic bacteria pseudomonas syringae and kiwi fruit pathogenic varieties of kiwi fruit canker, is a biological control strain for kiwi fruit canker, and has the preservation number: CGMCC No.27263, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
2. A method for preventing and treating kiwi fruit canker by utilizing bacillus subtilis Ws-1 is characterized by comprising the following steps:
(1) Inoculating the strain Ws-1 into an LB liquid culture medium for culture to obtain fermentation seed liquid;
(2) Inoculating the fermentation seed liquid into an LB liquid culture medium for expansion culture to obtain a fermentation culture liquid;
(3) Preparing a dilution liquid with a certain concentration (the concentration of the thallus is more than or equal to 1.2X10) 3 cfu/mL), aiming at kiwi fruit plants.
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