Methods and kits for the detection of powdery mildew

A diagnostic kit and powdery mildew technology, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., can solve the problem that grapevine powdery mildew cannot be cultured in vitro, transformation scheme has not been established, and low temperature is difficult Preservation and other issues

Pending Publication Date: 2018-03-27
拜耳简易股份有限公司
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Therefore, Erysipha vine cannot be cultured in vitro, is difficult to cryopreserve, and a reliable transformation protocol has not been established, posing serious challenges for its use in laboratory experiments

Method used

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  • Methods and kits for the detection of powdery mildew

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 : Design of specific primers for powdery mildew of grape

[0074] Several powdery mildew isolates were used to perform the qPCR method. These isolates were purified and maintained on isolated grape (Vitis Vinifera cv. Cinsaut) leaves. Bioassay inoculation was performed by blowing spores from sporulated 12-14 day old leaves onto the upper surface of sterilized leaves by an air pump in a Plexiglas settling tower. Infected leaves were cultured at 22°C in a growth chamber (12 / 12h light / dark photoperiod) and transferred to fresh agar medium every 3 to 4 days. Fungal material growing on leaf surfaces was scraped into Eppendorf tubes and contaminated leaf disks were frozen at -20°C.

[0075] According to the manufacturer's instructions, according to plantII kit (Macherey-Nagel), DNA extraction from Erysiophora vine fungus isolates. DNA extracts were stored at -20°C.

[0076]Using ITS1 and ITS4 primers, PCR amplification and sequencing of the ITS region of thre...

Embodiment 2

[0089] Example 2 : In Vivo Quantification of Erysiophora viticola DNA on Grapevine Leaves

[0090] To validate the sensitivity and accuracy of the method on biological samples, qPCR analysis was applied in laboratory bioassays to total DNA extracted from leaf disks contaminated with serial dilutions of Erysipha spp. spores. Total DNA was extracted from 4 leaf discs and 100 ng of DNA per spore concentration tested at different culture periods was analyzed using qPCR analysis.

[0091] Quantities of E. viticilis target genes were determined from a plasmid DNA standard curve. The lowest concentration of 2 spores / cm was detected at cycle Ct=27,13 2 (tested immediately after spraying (d+0)), equivalent to 9.4×10 2 copies of a gene. Notably, qPCR quantification of spore concentrations from other tests showed that gene copy number increased proportionally with the number of E. viticilidis spores on the leaf surface. The highest spore concentration tested was detected at Ct = 21...

Embodiment 3

[0093] Example 3 : Evaluation of Erysiophora viticola DNA by qPCR Analysis in Field Contamination

[0094] To validate the qPCR method under field conditions, grapevine parcels were artificially contaminated on different days using laboratory prepared inoculums of different densities.

[0095] From April to August 2013, measurements were carried out at the French vineyard St Martin d’Armagnac (Gros Manseng). Planted vineyard plots with 10 plants were tested together with adjacent uninoculated 5 plant plots. By spraying different concentrations of grape powdery mildew spore solution (C1 (0.1 spores / cm 2 ), C2 (1 spore / cm 2 ) and C3 (10 spores / cm 2 )) to contaminate the leaves. Artificial inoculation with powdery mildew spores was performed on three different dates: 18 April, 7 May and 23 May. For each condition, 5 leaves were collected on d+0, d+7, d+14 and d+30 days after contamination for qPCR analysis.

[0096] For each test condition, 5 leaves were sampled and mixed...

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Abstract

The present invention relates to means, methods and kits for the specific detection of the causing agent of powdery mildew on grapes, the fungus Erysiphe necator. More specifically, the methods according to the invention are quantitative methods based on quantitative Polymerase Chain Reaction.

Description

technical field [0001] The invention relates to a method and a kit for detecting the fungus Erysiphenecator, the pathogen of powdery mildew on grapes. More specifically, the method of the present invention is a quantitative method based on quantitative polymerase chain reaction. Background technique [0002] Grapevine powdery mildew, caused by the fungus Erysiphonium vine (also known as Uncinula necator), is one of the most widespread diseases of grapevine (Vitis vinifera L.) worldwide. Erysiphe vine belongs to the class Ascomycetes and is an obligate biotrophic fungus, ie it is completely dependent on its living vine host (grapes and leaves) for growth and reproduction. Therefore, Erysipha vine cannot be cultured in vitro, is difficult to cryopreserve, and a reliable transformation protocol has not been established, posing serious challenges for its use in laboratory experiments [0003] (Spanu et al., 2012, New Phytologist 195:20-22). [0004] Despite their importance, ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6851C12Q1/06C12Q1/04
CPCC12Q1/6895C12Q1/686C12Q2545/114
Inventor P·迪布尔内S·谢拉S·瓦舍
Owner 拜耳简易股份有限公司
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