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Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora

A technology of banana fusarium wilt and soft rot bacteria, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve problems such as easy to miss farming time, labor and money consumption, time-consuming detection process, etc. , to achieve the effect of eliminating the spread of diseased seedlings and fast gene evolution rate

Active Publication Date: 2014-05-14
广东粤科植保农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in actual agricultural production, the detection, monitoring and control of important banana diseases are time-consuming, labor-intensive and money-intensive, and it is easy to miss the farming time, which does not meet the actual production requirements.

Method used

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  • Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora
  • Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora
  • Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Design and Screening of Specific Primers for Physiological Race No. 1 and No. 4 of Banana Fusarium wilt

[0066] According to the complete genome sequences of Fusarium wilt No. 1 and No. 4 physiological races published by NCBI, search for gene sequences that may be related to the pathogenicity of Fusarium wilt of Banana Fusarium wilt and have insert / deletion differences between No. 1 and No. 4 physiological races . Sequence differential genes that may be related to pathogenicity were searched on Fusarium oxysporum f.sp.cubense race1contig438 (AMGP01000438.1) and Fusarium oxysporum f.sp.cubense race4contig195 (AMGQ01000195.1). See Seq1 and Seq2 for specific sequences. At 1515-1678bp of FOC4 (seq2), FOC4 has an insert fragment of about 160bp compared with FOC1; design suitable primers for extending the sequence on both sides of this fragment. The equivalent genome differential sequence can also be obtained by the following method: design appropriate primers ac...

Embodiment 2

[0068] Example 2: Design and screening of bacterial soft rot Dickeya sp. specific primers

[0069]Primers were designed based on the gyrB gene sequences of Dickeya bacteria that can cause bacterial soft rot and other genera, including Dickeya sp.MS1 (Genbank accession number: JQ284039), Dickeya dadantii3937 (Genbank accession number: NC_014500.1), Dickeya paradisiaca Ech703 (Genbank accession number: CP001654.1), Dickeya chrysanthemi NCPPB402 (Genbank accession number: CM001974.1), Dickeya dianthicola NCPPB3534 (Genbank accession number: NZ_CM001840.1), Pectobacterium carotovorum subsp.carotovorum PC1, CP001657.1), Pectobacterium wasabiae WPP163, CP001790.1), Pectobacterium atrosepticum SCRI1043, NC_004547.2), Escherichia coli (Escherichia coli P12b, CP002291. 1), Serratia marcescens WW4, CP003959.1, Citrobacter koseri ATCC BAA-895, CP000822.1, Serratia plymuthica AS9, CP002773.1, mouse Citrobacter rodentium ICC168, NC_013716.1, Shigella sonnei53G, NC_016822.1, Cronobacter sa...

Embodiment 3

[0071] Example 3: DNA Extraction and Specific Primer Identification of Banana Fusarium wilt No. 1 and No. 4 Physiological Race Strains

[0072] The strains of physiological race 1 and 4 of Fusarium wilt were preserved in 25% glycerol as spore suspension at -80°C. Inoculate the spore suspension onto the slant of the PDA test tube (potato dextrose agar medium), culture at 25°C for 7 days, then wash the surface of the cultured bacteria with 5mL sterile water to obtain the spore suspension, then draw 1mL of the spore suspension and add it to the liquid containing 150mL PDA Culture medium in a 250mL Erlenmeyer flask at 28°C and 150rpm for 3 days, and filter mycelium with 3 layers of lens-cleaning paper.

[0073] The extraction steps of fungal DNA are as follows:

[0074] a. take the mycelium and grind it into a fine powder with liquid nitrogen;

[0075] b. Take several 2mL centrifuge tubes, add 0.5mL volume of powder to each, then add 800μL FPCB solution (purchased from Sangon Bi...

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Abstract

The invention discloses a multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora. The invention provides a group of specific primers, No.1 and No.4 physiological races of the banana wilt bacteria, and bacterial Erwinia carotovora can be simultaneously detected in a one-step PCR (Polymerase Chain Reaction) amplified reaction. When a 636bp stripe presents in the PCR product, the sample contains FOC1; when a 796bp stripe presents in the PCR product, the sample contains FOC4; when a 218bp stripe presents in the PCR product, the sample contains the bacterial Erwinia carotovora. The method is easy to operate, low in time consumption and high in detection sensitivity. The sensitivity of detecting the banana wilt bacteria mycelium can be 1 mu g, and the sensitivity of detecting the bacterial Erwinia carotovora can be 10<3>CFU / ml. The method can be applied to detection of banana disease tissues, pathogenic bacteria which are directly separated or soil and has the advantages of high accuracy and the like, diffusion and spread of the banana wilt disease and bacterial Erwinia carotovora can be avoided, and safety production of bananas is guaranteed.

Description

technical field [0001] The invention belongs to the fields of crop disease prevention and plant quarantine, and particularly relates to a multiple molecular rapid detection method for banana wilt pathogen and bacterial soft rot pathogen. Background technique [0002] Banana is the fourth largest food crop in the world after rice, wheat and corn; according to the statistics of FAO, the planting area of ​​bananas in the world has shown an increasing trend in the past 10 years. In 2007, the planting area reached 66.1575 million mu, and the output reached more than 80 million tons, while China has an area of ​​more than 4.5 million mu, ranking fifth, and an output of more than 7 million tons, ranking second. However, the development of this industry is being threatened by diseases, especially the devastating diseases of banana wilt and bacterial soft rot. The pathogen of banana wilt is Fusarium oxysporum f.sp.cubense FOC (Wang Z Z et al, 2006), which has 4 physiological races; ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/04C12Q1/686C12Q2537/143
Inventor 林壁润张景欣沈会芳蒲小明潘群英
Owner 广东粤科植保农业科技有限公司
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