Separated paddy bacterial blight bacterial phage and application thereof

A technology of rice bacterial blight and bacteriophage, which is applied in the direction of virus/phage, application, and microbial-based methods, can solve problems such as lack of phage resources, different species, and no guiding significance for the control of rice bacterial blight, and achieve good results. Preventive effect, disease prevention, wide application effect

Active Publication Date: 2017-07-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The phage resources that have been reported in the world to prevent and control rice bacterial blight are scarce, and only South Korea’s Chae J C et al. have made related reports, but the spe

Method used

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  • Separated paddy bacterial blight bacterial phage and application thereof
  • Separated paddy bacterial blight bacterial phage and application thereof
  • Separated paddy bacterial blight bacterial phage and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Screening and purification of phage

[0035] 1. Collection of soil samples

[0036] The soil samples in the present invention were collected from Wuxi City, Jiangsu Province, and were soil in farmland (Table 1). Use five-point sampling method to collect, that is, take at least 5 points from each sampling point, first shovel the topsoil of 1 to 2 cm, and then take the soil with a depth of 5 to 10 cm, put it in a bag, and store in a refrigerator at 4°C .

[0037] 2. Isolation of bacteriophages against rice bacterial blight in soil samples

[0038] Pre-amplified Xanthomonas oryzae PXO99A(OD 600 :0.2~0.6) (Table 1), take 5mL of each of the 10 bacterial suspensions and mix them in an Erlenmeyer flask, weigh 5g of soil sample in the Erlenmeyer flask, and place it on a shaker at 28°C, 190r / min Incubate overnight. The soil suspension shaken overnight was centrifuged to remove soil particles and bacterial cells, and the supernatant was taken, and filtered and sterilized with a steril...

Embodiment 2

[0047] Preparation and Electron Microscopic Observation of High Concentration Phage

[0048] 1. Preparation of phage

[0049] Add the bacteriophage Xoo_sp15 to the pre-prepared PXO99A bacterial suspension (OD: 0.3), incubate overnight at 28℃, 190r / min shaker, centrifuge the co-culture at 12000rpm for 5min, take the supernatant, and use 0.22μm nitrocellulose Sterilize by membrane filtration to obtain a phage suspension, which is to be further concentrated.

[0050] 2. Phage counting method (titer)

[0051] Dilute the obtained phage sample in a 10-fold ratio, take 100 μl of the sample with a certain dilution ratio, and spread the double-layer plate, and calculate the number of plaques by taking an appropriate ratio. Dilution count as figure 2 .

[0052] 3. Concentration of phage

[0053] Prepare 30ml Xoo_sp15 phage suspension (10 10 PFU / ml), the obtained phage suspension was subjected to ultracentrifugation, 110000g, centrifuged for 2h, the supernatant was discarded, the phage was suspe...

Embodiment 3

[0058] Effect of Bacteriophage in Medium on the Growth of Xanthomonas oryzae Blight

[0059] Preparation of Xoo_sp15 phage suspension (10 10 PFU / ml): Prepare two sets of test tubes, put 5ml of NB liquid medium in each test tube, transfer the prepared Xanthomonas oryzae pv. oryzae bacteria PXO99A into the test tube, culture it in a shaker at 28°C, 190r / min. Turbidity (OD: 0.6~0.8). No phage was added to the first set of test tubes, and 300 μl of Xoo_sp15 phage suspension was added to the second set of test tubes. The two groups of test tubes were placed in a shaker at 28°C and 190r / min for 12 hours, and the OD value was measured every 3 hours to observe the difference in the growth of Xanthomonas oryzae PXO99A after different treatments.

[0060] The results show that when there is no phage added, PXO99A grows normally without any inhibition. When the bacteriophage Xoo_sp15 suspension was added, the growth state of PXO99A was inhibited to a certain extent, the growth was relativel...

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PUM

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Abstract

The invention discloses a separated paddy bacterial blight bacterial phage and an application thereof. The phage disclosed by the invention is preserved at China Center for Type Culture Collection with the preservation No. CCTCC NO: M2015727. The separated phage disclosed by the invention is a virulent phage separated from natural soil, the phage Xoo_sp15 is utilized to prevent and control plant diseases caused by xanthomonas and the preventing and control effect is excellent. The phage is expected to be highly purified and developed into a biopesticide for avoiding the propagation of plant xanthomonas and the growth and propagation of xanthomonas in plants and can be used for effectively preventing and treating the plant diseases caused by xanthomonas.

Description

Technical field [0001] The invention belongs to the field of biological prevention and control, and specifically relates to a phage isolate capable of specifically lysing Xanthomonasoryzae pv.oryzae strains and its application as a biological pesticide in the prevention and control of plant diseases. [0002] technical background [0003] Bacteriophage (phage) is a virus that grows and reproduces in cells. As early as 1896, Hankin discovered through research that there is a substance that can pass through a magnetic filter in the river water of the Ganges and Yamuna in India. It has obvious antibacterial effect on cholera. Subsequently, Edward Twort and Felixd Herele independently discovered a substance in 1915 and 1917 respectively. They found that this substance could specifically lyse host bacteria and form plaques on the double-layer plate, so they named it phage ( He Mizhi. Isolation and biological characterization of E. coli K88 and K99 spectral phages. 2012). Since biologi...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/00A01P1/00C12R1/92
CPCA01N63/00C12N7/00
Inventor 彭东海孙明刘锦董朝霞
Owner HUAZHONG AGRI UNIV
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