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Application of Acidovorax avenae subsp.avenae phage and biocontrol agent

A technology of brown streak bacteria and biocontrol agents, applied in the direction of virus/bacteriophage, phage, application, etc.

Active Publication Date: 2018-12-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a large number of studies by relevant scientific researchers at home and abroad have shown that external stimuli such as ultraviolet light irradiation, mitomycin C and other antibiotics, H 2 o 2 , nalidixic acid, salt ions or other inducers that can cause DNA damage can induce phage lysogeny / lysis cycle switching, but it is still unclear which external stimulus can induce which phage cycle switching

Method used

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  • Application of Acidovorax avenae subsp.avenae phage and biocontrol agent
  • Application of Acidovorax avenae subsp.avenae phage and biocontrol agent
  • Application of Acidovorax avenae subsp.avenae phage and biocontrol agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The bacteriophage AP1 of the rice bacterial brown streak pathogen, the preservation number is CCTCC M 2014599, was obtained by the applicant in the previous experiment. For related information, please refer to the Chinese invention patent with the publication number CN104630154A.

[0022] Rice bacterial blight (Xanthomonas oryzae subsp.oryzae, Xoo for short), of which 6 strains were isolated from Liaoning (L1-L6), 4 strains were isolated from Zhejiang (Z1-Z4), and 12 strains were isolated from Guangdong Province. Diseased plants (G8~G16, C2, C4, C8).

[0023] Pick a single colony of fresh bacterial blight of rice, place it in 5ml of NA liquid medium, and cultivate it in a constant temperature shaker with a rotation speed of 200rpm at 30°C until the bacterial concentration is about 10 8 cfu / ml; take 1ml of the bacterial solution to a new 5ml NA medium, add 6μl concentration of 10 9 pfu / ml of AP1 phage ( figure 1 : 0-11 respectively represent 10-fold gradient dilution o...

Embodiment 2

[0025] Mix 5ml of NA semi-solid medium (cooled to about 50°C) with 1ml of rice bacterial blight suspension (10 8 cfu / ml) were mixed evenly, spread on the NA solid medium to make the creep of Xoo bacteria, and set aside after solidification.

[0026] 2 μl of the lysate with different concentrations prepared in Example 1 was dropped onto the bacteria plate of Xoo bacteria, dried in the air, and incubated in a constant temperature incubator at 30° C. for 24 hours.

[0027] Judgment on the successful induction of the lysogenic / lytic cycle transition of the rice bacterial blight prophage: 22 strains were isolated from rice bacterial blight in Liaoning, Zhejiang and Guangdong, and the lysates produced transparent phages after co-cultivation spot( figure 2 ).

Embodiment 3

[0029] Specific primers were designed by using the conserved gene of exogenous phage AP1 to rule out the judgment of AP1 self-amplification and reproduction: bands could be detected in AP1, but no bands could be detected in the induced release phage. The specific primers for the AP1 conserved gene, the nucleotide sequences of which are:

[0030] Upstream primer F1: 5'-ATAGGATCCATGAAGTTCTACGCCCCCACCG-3',

[0031] Downstream primer R1: 5'-TGTCAGTCGACTCAGCCGTTCACGTCTTCGAAG-3'.

[0032] The sequence fragment obtained by using F1 and R1 to amplify AP1 is shown in SEQ ID No.1, and the fragment length is 395bp.

[0033] PCR detection system:

[0034] 2×T5 Super PCR mix enzyme

10μL

10μmol / L upstream and downstream primers

1μL each

template

1μL

Sterilized double distilled water

7μL

total

20 μL

[0035]PCR reaction conditions: pre-denaturation at 98°C for 10min; denaturation at 98°C for 30S, annealing at 60°C for 20S, exte...

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Abstract

The invention discloses application of Acidovorax avenae subsp.avenae phage and a biocontrol agent. Researches find that the Acidovorax avenae subsp.avenae phage AP1 (with the preservation number of CCTCC M 2014599) isolated from Acidovorax avenae subsp.avenae has the function of inducing transformation of Xanthomonas oryzae subsp.oryzae prophage into a lytic cycle from a lysogenic cycle, after the prophage enters the lysis cycle, the prophage may proliferate in a large amount and then is released from bacterial cells after cleavage of Xanthomonas oryzae pv. oryzae so as to affect the growth and quantity of the Xanthomonas oryzae pv. oryzae, namely the AP1 indirectly kills the Xanthomonas oryzae pv. oryzae. Therefore, the Acidovorax avenae subsp.avenae phage AP1 can be used to control ricebacterial leaf blight caused by the Xanthomonas oryzae pv. oryzae, and the AP1 is isolated from the bacteria in a rice field, does not cause toxic effects on the environment or animals, and is safe and environmentally friendly.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a rice bacterial brown streak pathogen phage and a biocontrol preparation. Background technique [0002] Rice is the main food crop and economic crop in my country, and is the staple food of one-third of the human beings in the world. Rice bacterial brown streak pathogen (Acidovorax avenae subsp. avenae) is a bacterium of the genus Pseudomonas, which mainly overwinters on rice seeds and straw, and invades and causes damage through stomata and wounds of rice plants. The main symptoms caused by the pathogen are brown stripes on the leaf sheath along the leaf veins on the rice seedlings, which can cause the heart and leaves to die in severe cases; early ear deformities and increased false fruit rate will also appear on rice seedlings. [0003] The applicant disclosed a rice bacterial brown streak bacteriophage and its application in the Chinese invention patent with ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01G13/00A01C1/00
CPCA01C1/00A01G13/00C12N7/00C12N2795/00011
Inventor 邱文杨敏李斌张阳索拉博密·欧莱覃·欧衮叶咪陈杰刘梦菊
Owner ZHEJIANG UNIV
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