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Method for systematically creating watermelon fusarium wilt strains with different pathogenicity and accurate identification method for resistance to watermelon fusarium wilt

A watermelon wilt pathogen and pathogenicity technology, applied in the field of bioengineering, can solve the problems of long breeding cycle, unclear genetic background of watermelon materials and pathogens, repeatability of disease resistance identification, pesticide residues, etc., to overcome differences in genetic background, Accelerating the breeding process for disease resistance and the effect of accelerating the breeding process

Inactive Publication Date: 2017-11-21
河南省农业科学院园艺研究所 +1
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Problems solved by technology

[0004] It is very difficult to prevent and control watermelon Fusarium wilt. People have taken various measures to carry out research on the prevention and control of watermelon Fusarium wilt, such as: crop rotation, grafting, chemical control and disease-resistant breeding, etc., but there are certain limitations
Because pathogenic bacteria can survive in the soil for more than 6 years, crop rotation and intercropping are limited; grafting seedlings consumes more manpower, material and financial resources, takes longer to grow seedlings and will affect the quality of watermelons, and at the same time cannot solve the problem of seeds carrying pathogens, and watermelon grafting The incidence of root rot and stem rot will increase significantly; the use of chemicals will cause pesticide residues, environmental pollution, and poisonous effects on humans and livestock
[0005] Disease resistance breeding is one of the most effective and safest ways to control Fusarium wilt. Issues such as maturity and repeatability of disease resistance identification lead to too long breeding cycle, limitations of variety resistance, and relatively high R&D costs and prices, thus limiting its large-scale promotion and application

Method used

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  • Method for systematically creating watermelon fusarium wilt strains with different pathogenicity and accurate identification method for resistance to watermelon fusarium wilt
  • Method for systematically creating watermelon fusarium wilt strains with different pathogenicity and accurate identification method for resistance to watermelon fusarium wilt
  • Method for systematically creating watermelon fusarium wilt strains with different pathogenicity and accurate identification method for resistance to watermelon fusarium wilt

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the acquisition of watermelon fusarium wilt transformant

[0062] ① The watermelon Fusarium oxysporum FON1 inoculated for 7 days (preserved by the Watermelon and Melon Research Laboratory of the Horticultural Institute of Henan Academy of Agricultural Sciences, collected by Liang Shen; collected time: 2015.5; collected place: Kaifeng, Henan) was used for PDA culture plate, and 15 mL of no Bacteria water to prepare conidia suspension, after filtering through 2-3 layers of sterile lens-cleaning paper, the concentration is 1×10 6 Conidia / ml conidia suspension for genetic transformation of Fusarium;

[0063] ② Inoculate Agrobacterium AGL1 containing the pATMT1 plasmid (purchased from Shanghai Weidi Biotechnology Co., Ltd.) into LB liquid medium, culture overnight at 28°C, 200rpm, and wait until the OD 600 When the temperature reaches 0.5-0.6, draw 200 μl of bacterial liquid and inoculate it into 5 ml of AIM liquid medium containing 200 μg / ml acetosyringone, s...

Embodiment 2

[0069] Embodiment 2: the establishment of different pathogenicity watermelon wilt pathogen reference standard bacterial strains

[0070] ① Take the PDA plate of Fusarium wilt of watermelon cultured for 10 days, add 20ml of sterile water, filter through sterile lens tissue, collect the conidia of Fusarium wilt, and prepare the concentration of 1x10 5 Conidia / ml spore suspension for inoculation experiments;

[0071] ② Soak watermelon seeds in 2% sodium hypochlorite solution for 5 minutes, wash them with water, sow them in a sterilized substrate, and grow them in an artificial climate box at 25°C, 12h, 16°C, 12h, until they grow to two leaves and one heart vaccination;

[0072] ③ Take watermelon seedlings with two leaves and one heart, remove the root matrix and rinse, cut off part of the fibrous roots, dip the roots in the conidia suspension for 30 minutes, and replant them on the plug trays. Each strain is inoculated with 30 watermelon seedlings, 22- Under the temperature of ...

Embodiment 3

[0084] Embodiment 3: Identification of resistance of different watermelon varieties

[0085] ① Cultivate watermelon Fusarium wilt bacteria FON1, FON1-1, FON1-2, FON1-3,

[0086] FON1-4 and FON1-5 strains were inoculated into 4 disease-resistant varieties (Zhengkang No. 1, Zhengkang No. 2, Zhengkang No. 3, and Zhengkang No. 6) for disease resistance identification, pathogenicity analysis, and calculation Disease index, according to the watermelon variety disease incidence index and disease resistance grading standard, determine the material disease resistance and specificity of disease resistance. Its disease resistance classification standard is:

[0087] High resistance: less than 20%;

[0088] Medium resistance: 21%-50%;

[0089] Light resistance: 51%-80%;

[0090] Infection: 80%-100%,

[0091] ② Accurate identification of watermelon disease resistance According to the resistance grading standard, the following results were obtained. The disease resistance of 4 resistant ...

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Abstract

The invention discloses a method for systematically creating watermelon fusarium wilt strains with different pathogenicity and an accurate identification method for resistance to watermelon fusarium wilt. The method for systematically creating the watermelon fusarium wilt strains with different pathogenicity comprises the following steps: respectively creating the strains with different pathogenicity through the technical means of establishing a fusarium wilt mutant library for 4 physiological races of the watermelon fusarium wilt, obtaining genetic transformation mutants of the fusarium wilt, identifying pathogenicity strength of the strains, screening mutant strains with different pathogenicity and the like; and the accurate identification method for the resistance to the fusarium wilt comprises the following steps: carrying out qualitative disease resistance determination on the same material or variety by using the obtained strains with different pathogenicity of the same physiological race, and further being capable of determining resistance strength quantitative disease resistance for a certain physiological race of the same material. The method disclosed by the invention used for identifying the resistance to the fusarium wilt of the watermelon material or variety is simple in steps, produces visual results, and has strong consistency and repeatability; and the method further can be used for other pathogens causing vascular bundle diseases and accelerates resistance identification and disease resistance breeding process of different fusarium wilt host materials and varieties.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for systematically creating strains of watermelon Fusarium wilt with different pathogenicities and an accurate identification method for watermelon resistance to Fusarium wilt. Background technique [0002] Watermelon is one of the top ten fruits in the world, known as the "king of summer fruits". According to FAO statistics, in 2013, the global watermelon harvested area was about 3.48 million hectares, ranking seventh among fruits, with an output of about 109.27 million tons. Rank fruit first. However, due to the watermelon wilt disease caused by Fusarium oxysporum, the watermelon yield has been greatly reduced, and this disease has become one of the most serious diseases that endanger the watermelon industry. Especially in recent years, watermelon planting has been developing in the direction of facilities, bases, and specialization. The phenomenon of heavy cro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15A01G7/06C12R1/77
CPCA01G7/06C12N1/14
Inventor 梁慎尚泓泉徐小利古勤生邓云赵卫星常高正李晓慧程丹丹高宁宁康利允王彬
Owner 河南省农业科学院园艺研究所
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