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Radopholus-similis control method through mediation of radopholus-similis associated bacteria

A technology of banana perforating nematode and associated bacteria, which is applied in the field of banana perforating nematode control and banana perforating nematode control, can solve the problems of difficult commercial production, easy degradation, complex components, etc., and achieves remarkable control effect and genetic stability. Good results

Active Publication Date: 2018-05-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex composition of its secondary metabolites, easy degradation in vitro, and difficulty in commercial production, its application in plant nematode control is limited.

Method used

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  • Radopholus-similis control method through mediation of radopholus-similis associated bacteria
  • Radopholus-similis control method through mediation of radopholus-similis associated bacteria
  • Radopholus-similis control method through mediation of radopholus-similis associated bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Introduction of protease pase4 gene into bananas to wear associated bacteria - Pseudomonas fluorescens

[0034] 1. Conversion method

[0035] The protease pase4 gene was introduced into the target bacteria Pseudomonas fluorescens pf36 through vector transformation and conjugation transposition respectively.

[0036] The nucleotide sequence of the protease pase4 gene is shown in SEQ ID NO:1.

[0037] The target bacterium, Pseudomonas fluorescens pf36, was screened by the inventor and was deposited in the Guangdong Microbial Culture Collection Center on November 8, 2017; the preservation number is GDMCC No: 60278.

[0038]① Vector transformation method

[0039] Using the recombinant plasmid pase4-pEASY connected with the protease pase4 gene preserved in the laboratory as a template, the following primers were used to amplify the protease pase4 gene.

[0040] Upstream primer M1 (SEQ ID NO: 2):

[0041]

[0042] The lower horizontal line is the EcoR I restr...

Embodiment 2p4

[0059] Example 2 Determination of Nematode Biological Activity of Fermentation Supernatant of p4MCS-pf36 and p4Tn5-pf36

[0060] 1. Method

[0061] Inoculate p4MCS-pf36 and p4Tn5-pf36 respectively in LB medium containing Kan (34 μg / mL), culture at 37°C and 200 rpm for 48 hours, and collect the fermentation supernatant by centrifugation. The experiments were set up as follows: p4MCS-pf36 fermentation supernatant, MCS-pf36 fermentation supernatant, p4Tn5-pf36 fermentation supernatant, Tn5-pf36 fermentation supernatant, pf36 fermentation supernatant, LB+Kan and H 2 0, after soaking for 48h, count the nematode mortality rate in each treatment solution, and every process was repeated 5 times.

[0062] 2. Results

[0063] The result of soaking nematodes with p4MCS-pf36 and p4Tn5-pf36 fermentation supernatant for 48h showed that ( figure 2 Middle A), p4MCS-pf36 and p4Tn5-pf36 treated nematodes had mortality rates of 63.9% and 42.0%, respectively, with significant differences (p0....

Embodiment 3p4

[0064] Embodiment 3p4MCS-pf36 and p4Tn5-pf36 are to the control effect of banana borer nematode and its safety determination to crops

[0065] 1. Method

[0066] The tested banana nematode is a population isolated from the rhizosphere soil of Banana braziliana, the tested banana variety is plantain (Musaparaolisiac), and the planting substrate is sandy soil: charcoal soil: organic soil = 1:1:2 (sterilized at 121°C for 30 min), pots and pots 1.8L plastic flower pots (1.5L of matrix soil). The experiments were set up as follows: p4MCS-pf36 + banana perforator (RS), p4Tn5-pf36+RS, MCS-pf36+RS, Tn5-pf36+RS, pf36+RS, LB+RS, H 2 O+RS, p4MCS-pf36, p4Tn5-pf36, pf36 and H 2 O. When the banana seedlings grow to the 5-6 leaf stage, the treatment that needs to be inoculated is based on the bacterial concentration of 10 6 Cfu / g (substrate soil) was injected into the rhizosphere soil of banana seedlings, and then inoculated with the nematode Nematode punctatus 3 days after inoculation. ...

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Abstract

The invention discloses a radopholus-similis control method through mediation of radopholus-similis associated bacteria. A method for establishing radopholus-similis biocontrol engineering bacteria mediated by the associated bacteria is provided, wherein coding genes of biological enzyme with the nematocidal activity are transformed into the radopholus-similis associated bacteria, and the engineering bacteria are obtained after establishing; then radopholus similis is controlled through the engineering bacteria, and the aim that the nematocidal protease is mediated and expressed through the associated bacteria to control the radopholus similis is achieved. The genetic-modification pseudomonas fluorescens engineering bacteria obtained with the radopholus-similis control method are good in genetic stability, remarkable in control effect of the radopholus similis and low in commercialized production difficulty; two biocontrol factors of living bacteria and thallus secondary metabolites are effectively combined, the continuous self-propagation expanding and automatic diffusion characteristics of the living bacteria and the activities when the radopholus similis is efficiently killed through metabolites are fully developed, and the radopholus-similis control method is a new method and a new path which is safe, efficient, stable and capable of sustainably controlling plant nematode parasites.

Description

technical field [0001] The present invention relates to the technical field of preventing and treating the banana nematode, and more particularly, relates to a method for preventing and controlling the banana nematode mediated by bacteria associated with the banana nematode. Background technique [0002] Banana borer nematode (Radopholus similis) is a migratory endoparasitic plant pathogenic nematode. It hosts more than 250 species of plants. It causes serious economic losses to crops in tropical and subtropical regions every year. It is one of the 10 most destructive plant pathogens in the world. One of the nematodes. [0003] At present, the control of nematodes is mainly based on chemical nematicides, and the extensive use of chemical agents has caused great harm and other negative effects on the environment, humans, animals, plants and other beneficial organisms, and has been strictly restricted in many countries and regions. . Biological control methods have the chara...

Claims

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Application Information

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IPC IPC(8): C12N15/78C12N15/57C12N1/21A01N63/00A01N63/02A01P5/00C12R1/39
CPCA01N63/00A01N63/10C12N9/50C12N15/78
Inventor 谢辉王东伟陈德强郑小玲丁善文徐春玲
Owner SOUTH CHINA AGRI UNIV
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