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A method for rapidly diagnosing poplar black spot caused by Erwinia poplar

A technology for rapid diagnosis and black spot disease, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of no obvious effect in the prevention and control of P. chinensis, and achieve easy identification and detection Fast, highly specific results

Active Publication Date: 2019-01-11
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, many conventional fungicides have been ineffective against P. poplaris, and therefore, more effective rapid diagnostic methods are needed to detect this pathogen before the onset of symptoms
At present, there is no report on the molecular detection of Dispora poplaris

Method used

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  • A method for rapidly diagnosing poplar black spot caused by Erwinia poplar
  • A method for rapidly diagnosing poplar black spot caused by Erwinia poplar
  • A method for rapidly diagnosing poplar black spot caused by Erwinia poplar

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The primers of LAMP are designed based on the rDNA ITS sequences (Genbank accession no.KU508806 and Genbank accession no.KM246324) of the multi-sprouting tube specialization and single-sprouting tube specialization of Pseudomonas porphylla published by Genbank. Simultaneously download the ITS sequences of other pathogenic bacteria of the genus Pseudomonas from the Genbank database (Pseudomonas apple-EU520097, Pseudomonas rosea-AY904059, and Pseudomonas apple-JN587494), and use BioEditV7.2.0 for sequence comparison analysis (Alzohairy, 2011) ). The primer design software PrimerExplorer V4 (http: ∥primerexplorer.jp / e / ) was used to design LAMP primers (Eiken Chemical Co., Ltd., Tokyo, Japan) according to the design principles of LAMP primers. A total of six primers were designed, including two outer primers (F3 / B3) and two inner primers (FIP / BIP), which can recognize six different regions of target DNA, and two additional loop primers (FL / BL) , Can accelerate the reaction ...

Embodiment 2

[0043] 1. Extraction of fungal materials and genomic DNA

[0044] The 20 tested strains, sources and quantities used for the test are shown in Table 2. Among them, there are 6 multi-sprouting and single-sprouting specific strains of Pseudomonas spp. There are 1 strains of Diospora vulgaris and 1 Diospora rosea, and 6 strains belonging to other fungal groups, namely Phytophthora sojae, Colletotrichumtruncatum, Magnaporthe oryzae, and Fusarium solani Bacteria (Fusarium solani), Verticillium dahlima (Verticillium dahlima) and Chestnut blight (Cryphonectria parasitica). The test strains were transferred to potato dextrose agar medium (PDA) (add 200 grams of potato, 2% glucose and 2% agar per liter), and cultivated in the dark at 25°C. Cut 10 (2mm×2mm) mycelium blocks from the edge of the colony and transfer them to potato dextrose liquid medium (PDB) for shaking culture for 3-5 days, filter and collect the hyphae, freeze, drain and grind into mycelium powder, -20℃ spare. Then DNea...

Embodiment 3

[0053] Example 3 The traditional PCR detection method of Populus

[0054] For traditional PCR, primer F3 / B3 is used to amplify a specific region of rDNA ITS sequence. PCR reaction system: 1.0μL template DNA, 12.5μL PrimerSTAR Max Premix (2×, Takara, R045A), each 0.5μL F3 / B3 (10μM), add deionized water to the total system to 25μL. Thermal cycle program: 98°C, 4 minutes; 31 cycles (98°C, 10 seconds; 56°C, 5 seconds; 72°C, 5 seconds); 72°C, 5 minutes. The PCR reaction product was verified by 1.5% agarose gel electrophoresis (120v, 30 minutes), stained with ethidium bromide, and then photographed and recorded under a UV gel imager. DNAladder Marker II (Tiangen, MD102, China) is used as a size reference.

[0055] A total of 20 strains were used to evaluate the specificity of the LAMP technology, including 6 strains each of the multi-sprouting tube-specialized and single-sprouting tube-specialized strains of Phyllanthus spp. and 1 each There are 6 strains of other fungal groups, namel...

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Abstract

The invention discloses a method for rapidly diagnosing poplar black spot caused by Erwinia poplar, which extracts genomic DNA of poplar black spot and carries out LAMP reaction. The color of the solution was directly identified by naked eyes. The blue color was positive and the purple color was negative. Positive solution was further confirmed by gel electrophoresis, and the product showed a ladder-like band, which was caused by poplar black spot caused by Dictyotrichum poplar. The diagnostic method established by the present application is a detection technique with high reaction speed, strong specificity, simple equipment, and easy identification and identification of reaction results. Isothermal amplification and naked eye detection of HNB dye make LAMP analysis suitable for the detection and monitoring of Erwinia poplar under natural conditions, and has broad application prospects.

Description

Technical field [0001] The invention belongs to the technical field of poplar black spot diagnosis, and specifically relates to a method for quickly diagnosing poplar black spot caused by Marssonina brunnea. Background technique [0002] Poplar has become a model species for forest genomics research due to its unique biological characteristics. It is a major plantation tree species and has high economic and environmental value. At present, the planting area of ​​poplar plantations in my country has exceeded 7 million hectares, ranking first in the world. However, the single stand structure of poplar plantations is likely to lead to large-scale outbreaks of pests and diseases, loss of its ecological, economic and social value, resulting in huge loss. Poplar black spot is one of its important diseases. It is caused by three pathogenic fungi (Marrssonina populi) in the genus Marrssonina (Marrssonina) of the subphylum Coelospora. Marrssonina brunnea (Marrssonina brunnea); White popl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/173
Inventor 熊琴郑新月张琳琳张晨钱雨琳
Owner NANJING FORESTRY UNIV