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Two-step method for screening biocontrol bacteria against citrus Huanglongbing

A technology for citrus huanglongbing and citrus huanglongbing, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., and can solve problems such as inability to adopt antibacterial screening methods, screening, and inability to in vitro culture of pathogens.

Active Publication Date: 2020-11-13
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the technical problem that the pathogen of citrus huanglongbing cannot be cultured in vitro, and the biocontrol bacteria cannot be screened by conventional biocontrol bacteria screening methods, the present invention provides a two-step method for screening citrus huanglongbing biocontrol bacteria, which includes The following steps:

Method used

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  • Two-step method for screening biocontrol bacteria against citrus Huanglongbing
  • Two-step method for screening biocontrol bacteria against citrus Huanglongbing
  • Two-step method for screening biocontrol bacteria against citrus Huanglongbing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Isolation of cultivable endophytic bacteria for biocontrol: The healthy and diseased plants in Wenzhou tangerine citrus Huanglongbing disease were collected from the experimental farm of Yongzhou University in Hunan Province, and the healthy and diseased plants were detected by conventional real-time fluorescent quantitative PCR The method of citrus huanglongbing pathogenic bacteria is determined; get the root, stem and leaf of the healthy plant and diseased plant respectively, and use 75% (v / v) alcohol disinfection for 30 seconds respectively, and 2.5% (v / v) sodium hypochlorite surface disinfection for 1 Minutes, after fully grinding respectively, dilute 10 times and 100 times concentration with 0.85% w / w sodium chloride and smear on TSA (soybean peptone 5g / L, tryptone 15g / L, sodium chloride 5g / L, agar powder 20g / L) medium, cultured at 30°C for 48 hours, picked bacteria with different shapes and streaked and purified to obtain endophytic bacteria for biocontrol, and...

Embodiment 2

[0064] Embodiment 2 is except that following test and result are different, all the other operation steps are identical with embodiment 1.

[0065] 1. The materials used for the isolation of cultivable endophytic bacteria for biocontrol are healthy and diseased plants collected from sweet orange citrus Huanglongbing disease-infected fields in Yongshan County, Yunnan Province.

[0066] 2. Results and Analysis

[0067] 2.1 A total of 258 strains of biocontrol endophytic bacteria were isolated, and the statistical results are shown in Table 4.

[0068]The separation statistics of table 4 embodiment 2 biocontrol endophytic bacteria

[0069]

[0070] 2.2 The 10 strains with the highest scores are listed in Table 5. The HR26 strain isolated from healthy roots was assigned the highest value.

[0071] 2.3 The control effect results of citrus leaves treated with endophytic bacteria in vitro, as shown in Table 6. The corrected control effect of HR26 with the highest assigned valu...

Embodiment 3

[0079] Embodiment 3 is except that following test and result are different, all the other operation steps are identical with embodiment 1.

[0080] 1. The materials used for the isolation of cultivable endophytic bacteria for biocontrol are the healthy and diseased plants of Wocitrus huanglongbing disease-infected fields collected from the fifth group of citrus fields in Huaqiao Farm Community, Zhoucheng Town, Binchuan County, Dali, Yunnan Province.

[0081] 2. Results and Analysis

[0082] 2.1 A total of 286 strains of biocontrol endophytic bacteria were isolated, and the statistical results are shown in Table 7.

[0083] The separation statistics of table 7 embodiment 3 biocontrol endophytic bacteria

[0084]

[0085] 2.2 Table 8 shows the top 10 strains with the highest score. The HL33 strain isolated from healthy tree leaves was assigned the highest value.

[0086] Table 8 Example 3 Biocontrol Endophytic Bacteria Top 10 Strain Results

[0087]

[0088] Note: Inhi...

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Abstract

The invention relates to the technical field of biological control, in particular to a method for screening biocontrol bacteria of Candidatus Liberibacter spp. by a two-step method. According to the method disclosed by the invention, five indicator pathogenic bacteria are used for performing confrontation culture, potential biocontrol bacteria are screened, a half leaf method is adopted, the content of Candidatus Liberibacter spp. pathogenic bacteria of half leaves which are not treated is detected through real-time fluorescent quantitation PCR, after the other half leaves are treated with thepotential biocontrol bacterium liquid, the content of the Candidatus Liberibacter spp. pathogenic bacteria of the other half leaves treated with the potential biocontrol bacterium liquid is detectedthrough the real-time fluorescent quantitation PCR, the efficacy of the potential biocontrol bacteria is obtained through a calculating formula, and the potential biocontrol bacteria high in control effects are screened to be used as target Candidatus Liberibacter spp. biocontrol bacteria. The method disclosed by the invention can be used for various hosts of the Candidatus Liberibacter spp., hasthe characteristics of being high in efficiency, quick and the like, and has important significance in biological control of the Candidatus Liberibacter spp..

Description

technical field [0001] The invention relates to the technical field of plant disease biological control, in particular to the screening of citrus huanglongbing biocontrol bacteria. Background technique [0002] Citrus Huanglongbing (HLB) is a worldwide bacterial disease that has occurred in more than 40 countries. It is mainly transmitted by citrus psyllids and grafting, and almost all citrus plants can be infected with Huanglongbing. Once the citrus greening disease occurs, it will have a great impact on the quality and yield of the fruit, and will cause serious economic losses. For example, since the first discovery of citrus greening disease in Florida in 2005, the disease has caused more than $4.5 billion in losses to the state. In recent years, my country's citrus industry is also facing huge losses and severe challenges caused by Huanglongbing. [0003] Since the pathogen of citrus huanglongbing cannot be cultured in vitro, Koch's law cannot be fulfilled, but plant ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/18C12Q1/04
CPCC12Q1/04C12Q1/18C12Q1/689
Inventor 何月秋李永梅谢扎得·缪纳尔何鹏搏何鹏飞吴毅歆
Owner YUNNAN AGRICULTURAL UNIVERSITY
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