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Method for identifying pathotype of verticillium dahlia, kit and contained nucleic acid and primer pair

A technology of Verticillium dahliae and primer pairs is applied in the field of identifying pathogenic types of fungal strains and kits, and can solve the problems of farmland yield of severe cotton diseases, large differences in pathogenic types, and farmland yield of mild cotton diseases.

Active Publication Date: 2014-02-12
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the pathogenic types of different Verticillium dahliae strains are very different. For example, some strains with high pathogenicity will cause serious diseases of cotton after infecting cotton and cause a decline in farmland yield, while some strains with low pathogenicity The strains infected cotton only cause mild diseases of cotton and have little impact on farmland yield

Method used

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  • Method for identifying pathotype of verticillium dahlia, kit and contained nucleic acid and primer pair
  • Method for identifying pathotype of verticillium dahlia, kit and contained nucleic acid and primer pair
  • Method for identifying pathotype of verticillium dahlia, kit and contained nucleic acid and primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This example is used to illustrate the preparation of samples to be tested. Specifically, genomic DNA was prepared from 80 different Verticillium dahliae strains (numbered VDG1 to VDG80).

[0023] Cut the stem of the cotton plant about 10-35cm above the ground and cut it into 1mm 3 Small pieces of about the size are inoculated in PDA medium (containing 200 g / L of potato, 20 g / L of glucose, and 20 g / L of agar) containing antibiotics (ampicillin, rifampicin and streptomycin, all at a concentration of 100 μg / ml) Above, cultivated at 25°C for 7 days, and identified the colonies of Verticillium dahliae according to the standard colony morphology of Verticillium dahliae. The identified colonies of Verticillium dahliae were taken as samples of Verticillium dahliae strains.

[0024] According to the above method, 80 different Verticillium dahliae bacterial strains were obtained from 80 different cotton plants, and the above-mentioned 80 different Verticillium dahliae bacteria...

Embodiment 2

[0026] This example is used to illustrate the detection results of the pathogenic types of the above 80 different Verticillium dahliae strains.

[0027] Refer to literature (Zhu Heqin, Feng Zili, Li Zhifang, Zhao Lihong, Shi Yongqiang. Identification of Verticillium wilt resistance of cotton varieties (lines) by quantitative dipping in vermiculite sandy bottomless paper bowl. China Cotton, 2010, 37(12) : 15-17) in the vermiculite sandy soil bottomless paper pot quantitatively dipped in bacterial solution method, the 80 different Verticillium dahliae strains described in Example 1 were carried out to identify the pathogenic type of cotton seedlings. The level of the pathogenic type was characterized by the disease index of the 80 different Verticillium dahliae strains after inoculating healthy cotton plants.

[0028] Specifically, Verticillium dahliae was cultured in Chapic medium (2g / L NaNO 3 , K of 1g / L 2 HPO 4 , 0.5g / L KCl, 0.5g / L MgSO 4 , 0.01g / L FeSO 4 , 30g / L sucrose...

Embodiment 3

[0034] The whole genome sequence of Verticillium dahliae strains (numbered VDG1 and VDG2) was determined according to the method specified in the instruction manual (Solexa technology) of Genome Analyzer of Illumina Company.

[0035] In this example, the whole genome sequences of the above two different Verticillium dahliae strains were obtained. Through sequence comparison, it was identified that the whole genome sequence of VDG1 does not contain the sequence shown in SEQ ID NO: 1; while the whole genome sequence of VDG2 The genome sequence contains the sequence shown in SEQ ID NO:1.

[0036] By the same method as above, 61 of the above 80 different Verticillium dahliae strains (respectively VDG3, VDG63, VDG61, VDG18, VDG35, VDG32, VDG73, VDG31, VDG33, VDG26, VDG55, VDG23, VDG57, VDG36, VDG11, VDG67, VDG47, VDG5, VDG29, VDG59, VDG49, VDG44, VDG21, VDG48, VDG8, VDG50, VDG25, VDG43, VDG37, VDG46, VDG16, VDG4, VDG4, VDG38, VDG2 VDG45, VDG12, VDG76, VDG27, VDG10, VDG41, VDG64, V...

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Abstract

The invention provides nucleic acid which is provided with a sequence as shown in SEQ ID NO:1. The invention further provides a primer pair for amplifying the nucleic acid and a method for identifying a pathotype of a fungi strain. The fungi strain is verticillium dahlia. The method comprises the following steps: (1) preparing a sample to be tested, containing whole genome DNA (deoxyribonucleic acid) of the verticillium dahlia; (2) taking the sample to be tested, containing whole genome DNA obtained from the step (1) as a template and carrying out PCR (polymerase chain reaction) amplification by the primer pair; detecting if nucleic acid as shown in SEQ ID NO:4 is existent in an amplified product; (3) judging the pathotype of the verticillium dahlia according to a detection result in the step (2), and when nucleic acid as shown in SEQ ID NO:4 is inexistent in the amplified product, indicating that the pathotype of the verticillium dahlia belongs to a high pathotype; when nucleic acid as shown in SEQ ID NO:4 is existent in the amplified product, indicating that the pathotype of the verticillium dahlia does not belong to the high pathotype.

Description

technical field [0001] The invention relates to the field of crop disease prevention and control, in particular to a nucleic acid, a pair of primers, a method for identifying pathogenic types of fungal strains and a kit. Background technique [0002] Verticillium dahliae is a plant pathogenic fungus that can cause diseases such as verticillium wilt in plants (especially cotton). However, the pathogenic types of different Verticillium dahliae strains are very different. For example, some strains with high pathogenicity will cause serious diseases of cotton after infecting cotton and cause a decline in farmland yield, while some strains with low pathogenicity The strains infected cotton only caused mild diseases of cotton and had little impact on farmland yield. In addition, the pathogenic type of Verticillium dahliae also shows a certain degree of latency, that is, the highly pathogenic strains infected in the current year may not cause disease, but will cause serious diseas...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12R1/645
Inventor 戴小枫李蕾陈捷胤马雪峰孔志强包郁明肖红利桂月晶徐明
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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