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Detection kit for Sclerotinia sclerotiorum strain drug-resistance and detection method of Sclerotinia sclerotiorum strain drug-resistance

A technology of sclerotinia and drug resistance, applied in the field of rapid detection of drug resistance of plant pathogenic fungi, can solve the problems of heavy workload, expensive equipment, difficult to promote, etc., and achieve the effect of low requirements, intuitive results and high accuracy

Inactive Publication Date: 2012-05-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Apparently, the traditional method for measuring the sensitivity of mycelia to chemicals usually takes a few weeks, the workload is heavy, the number of test samples is limited, and strict aseptic procedures are required, so it is difficult to detect the existence of drug-resistant strains in the early stage. The judgment of whether it grows on the culture medium is susceptible to the subjective influence of the researcher; the detection method based on PCR technology, especially high-throughput real-time quantitative PCR technology, requires expensive instruments, requires high technical requirements for operators, and is difficult to implement locally. Promoted by agricultural departments such as cities and counties

Method used

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  • Detection kit for Sclerotinia sclerotiorum strain drug-resistance and detection method of Sclerotinia sclerotiorum strain drug-resistance
  • Detection kit for Sclerotinia sclerotiorum strain drug-resistance and detection method of Sclerotinia sclerotiorum strain drug-resistance
  • Detection kit for Sclerotinia sclerotiorum strain drug-resistance and detection method of Sclerotinia sclerotiorum strain drug-resistance

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Embodiment 1

[0034] The screening of embodiment 1 sclerotinia drug resistance monitoring medicament concentration

[0035] In order to study the screening index of suitable resistant strains, a screening study was carried out on the detected concentration.

[0036] 1.PDA / bromophenol blue basic medium: (see "Invention Summary" for preparation)

[0037] 2. Carbendazim resistance screening medium: PDA / bromophenol blue basic medium is used as the basic medium, and the additional carbendazim is 1mg / L, 2mg / L, 3mg / L, 4mg / L, 5mg / L respectively , 6mg / L and 10mg / L;

[0038] 3. Sclerotinia resistance screening medium: take PDA / bromophenol blue basic medium as the basic medium, and add Sclerotinia at 1mg / L, 2mg / L, 3mg / L, 4mg / L, 5mg / L respectively , 6mg / L and 10mg / L.

[0039] 4. Randomly select 50 copies of Sclerotinia sclerotia collected from Hunan Province, Hubei Province, Anhui Province, Qinghai Province and Jiangsu Province on the stalks of rapeseed disease, after drying, slice with scalpel free...

Embodiment 2

[0044] The preparation of embodiment 2 kit culture medium

[0045] 1. PDA / bromophenol blue growth medium: boil 200g of peeled potatoes, 20g of glucose and a small amount of distilled water for about 15min, filter and discard the potato residue, collect the filtrate, add distilled water to 1000ml, and press 0.1g / 1000ml after the volume Bromophenol blue was added to the proportion of the medium, fully dissolved, and then autoclaved at 121°C for 20 minutes for later use;

[0046] 2. Drug resistance screening medium 1: use PDA / bromophenol blue medium as the basic medium, add carbendazim 5mg / L;

[0047] 3. Drug resistance screening medium 2: take PDA / bromophenol blue medium as the basic medium, add 5 mg / L of sclerotia net;

[0048] 4. Under aseptic conditions, add 500 mg / L of ceftriaxone sodium before the above three kinds of culture medium are solidified, and pack them into plastic spiral tubes with an inner diameter of 22 mm, an outer diameter of 25 mm and a height of 96 mm, and...

Embodiment 3

[0052] The culture property test of embodiment 3 sclerotinia on PDA / bromophenol blue growth medium

[0053] The S. sclerotiorum strain Ep-1PNA367 (Huiquan Liu et al., 2009) was cultured in PDA and activated continuously at 20°C for 2-3 times; the mycelium pieces of the activated S. sclerotiorum were inoculated in the PDA / bromophenol blue medium (See "Contents of the Invention" for preparation), cultured at 20°C for 15 days, and recorded the colony shape and color every day. see results figure 1 , Sclerotinia colony grows and produces oxalic acid, the pH value of medium is reduced to 3-4 (Marciano et al., 1983), and bromophenol blue meets acid and changes color, and the PDA medium that contains bromophenol blue changes from blue After 4 days, due to the degradation of oxalic acid, the pH value of the medium rose and gradually returned to blue.

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Abstract

The invention belongs to the field of rapid plant pathogenic fungi drug-resistance detection technologies, and specifically relates to a fast, simple and practical detection kit of plant pathogenic fungi Sclerotinia sclerotiorum strain drug-resistance. The kit is suitable for fast detection and analysis for the drug-resistance of Sclerotinia sclerotiorum to field common fungicides such as Carbendazim or Dimethachlon. According to the invention, Sclerotinia sclerotiorum sclerotium collected from the field is taken as a material, inhibition effect on sclerotium germination by a medicine is taken as an index, and color change of a culture medium is taken as a basis of the detection method. The drug-resistance of Sclerotinia sclerotiorum can be detected timely after the harvest of preseason rape. Massive and convenient detections are carried out in four days. The drug-resistance strain is identified according to color change. The result is intuitional and obvious with the accuracy being 100%. It is of practical value for taking effective measures to control Sclerotinia sclerotiorum in next year. In addition, combined with a method based on the PCR technology, drug-resistance strains with different resistance mechanisms might be obtained and strain materials are provided for the research of Sclerotinia sclerotiorum resistance mechanisms.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of drug resistance of plant pathogenic fungi, and in particular relates to a rapid, simple and practical detection technology for the drug resistance of plant pathogenic fungus Sclerotiorum sclerotiorum strains, which is suitable for the rapid resistance of sclerotiorum to field fungicides. sex detection and analysis. Background technique [0002] Sclerotinia sclerotiorum (Lib.) de Bary] is widely distributed all over the world and can infect more than 540 kinds of plants in 65 families, including major oil crops such as rapeseed, soybean, sunflower, lettuce, Chinese cabbage, celery, carrot, etc. Important vegetable crop, causes Sclerotinia sclerotiorum (Boland and Hall, 1994; Bolton et al., 2006). Sclerotinia sclerotiorum is the primary disease in rapeseed production in my country, especially in the vast rapeseed growing areas in the Yangtze River Basin. In general, the incidence rate o...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 姜道宏张霞付艳苹程家森谢甲涛
Owner HUAZHONG AGRI UNIV
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