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Phyllachora dalbergiicola molecular detection primer and fast detecting method for dalbergia odorifera

A Dalbergia Dalbergia, molecular detection technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of isolating pathogenic bacteria, time-consuming, heavy workload, etc., and achieve high sensitivity Effect

Active Publication Date: 2015-09-16
HAINAN KEDA FORESTRY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional plant disease diagnosis methods need to go through the process of pathogenic bacteria isolation, cultivation, identification, etc., which is time-consuming and heavy workload, and may miss the best period of disease control
And because the nevus fungus is a kind of obligate parasitic biotrophic fungus, that is, it cannot grow on the culture medium, so the pathogenic bacteria cannot be isolated by tissue separation method, which brings great difficulties to the accurate diagnosis of the disease

Method used

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  • Phyllachora dalbergiicola molecular detection primer and fast detecting method for dalbergia odorifera
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  • Phyllachora dalbergiicola molecular detection primer and fast detecting method for dalbergia odorifera

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Detect the specific amplification of the specific primer designed by the present invention to Dalbergia nigra

[0056] 1. Preparation of Black Mole Leaf Samples

[0057] See the test samples listed in Table 1 and their collection locations. After the diseased leaf samples of Dalbergia sinensis, white grass, Pennisetum, Goose-view grass and Gangzhu black nevus were surface-sterilized with 75% alcohol, the diseased leaves were cut into 4 × 4 mm tissue blocks using sterilized scissors. Grind with liquid nitrogen. Sample numbers of black mole diseased leaves: B01~B13.

[0058] Table 1 Collection sites and molecular detection results of each tested sample

[0059]

[0060] "+" indicates that the target band of 273 bp can be detected, and "-" indicates that the target band of 273 bp cannot be detected.

[0061] 2. Preparation of Control Strain Samples

[0062] (1) Use sterilized scissors to cut the diseased and healthy junctions of typical lesions, and c...

Embodiment 2

[0072] Example 2: Sensitivity detection of primers to Dalbergia nigra

[0073] 1. DNA concentration dilution

[0074] After the concentration of the extracted Dalbergia nigra genomic DNA was determined by ultraviolet spectrophotometer, the Dalbergia nigra genomic DNA was gradually diluted down to 10ag / μL by a 10-fold order of magnitude starting from a concentration of 100ng / μL.

[0075] 2. Sensitivity detection of specific primers

[0076] The first round of PCR reaction: ITS4 / ITS5 was used as the primer to carry out the first round of PCR, and the amplification reaction system was: 2×Taq PCR MasterMix 12.5 μL, step 1) The total DNA of Dalbergia officinale leaves was 1 μL, 10 μmol / L ITS4 1 μL each of / ITS5, with ddH at the end 2 O supplemented to 25 μL, centrifuged for 15 sec and placed in a PCR instrument for amplification; PCR reaction conditions were: 94 °C pre-denaturation for 4 min; 94 °C denaturation for 30 sec, 55 °C annealing for 30 sec, 72 °C extension for 1 min, a ...

Embodiment 3

[0081] Embodiment 3: the detection of Dalbergia nigra bacteria in the diseased tissue of Dalbergia sinensis

[0082] 1. Sample Collection

[0083]A total of 23 samples of diseased and healthy tissues of Dalbergia japonica leaves were collected from the state-owned Chengmai Forest Farm in Hainan Province, and were detected by nested PCR.

[0084] 2. DNA extraction and detection

[0085] Using the plant tissue DNA extraction method described in step 2 in the summary of the invention, carry out nested PCR amplification according to the above-mentioned implementation method, PCR reaction system 25 μ L, nested PCR reaction system uses primer ITS4 / ITS5 as the first round of reaction primers, take 1 μL of the first-round PCR reaction product was used as a template, combined with Dalbergia spp. specific primers for the second-round PCR amplification, and the reaction procedure and detection method were the same as those described in the summary of the invention.

[0086] 3. Test res...

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Abstract

The invention belongs to the field of molecular detection of plant diseases, and particularly relates to a phyllachora dalbergiicola molecular detection primer and a fast detecting method for dalbergia odorifera. Nest PCR amplification, PCR product amplification analysis, agarose gel electrophoresis and result determination are carried out on a pair of designed phyllachora dalbergiicola specific primers of dalbergia odorifera. If phyllachora dalbergiicola exits in a sample, a target strip of 273 bp can be generated in sepharose gel under ultraviolet light; and if the strip does not appear, no phyllachora dalbergiicola exists. The primer is high in specificity, and the phyllachora dalbergiicola, phyllachora graminis, phyllachora phyllostachydis, and other pathogenic bacteria and endophytic fungi separated from dalbergia odorifera and pterocarpus macrocarpus can be distinguished. Moreover, the primer is high in sensitivity and can be used to detect a phyllachora dalbergiicola genome DNA with the lower limit of 100 ag / [mu]L. The detecting method is fast, and an accurate detecting result can be obtained within eight hours while a detecting result only can be obtained within at least four days or more than one week through a traditional disease diagnosis method.

Description

technical field [0001] The invention belongs to the field of molecular detection of plant diseases, and in particular relates to a molecular detection primer of Dalbergia serrata and a detection method thereof, which can be used for the early diagnosis of Dalbergia serrata and the monitoring and identification of pathogens. Background technique [0002] Dalbergia sinensis is one of the precious native tree species in the tropics in Hainan Province. It is a national second-class protected tree species. Among the 34 species of mahogany in 5 genera and 8 categories in our national standard, it is the mahogany second only to the red sandalwood. Its material is hard and heavy, strong, dry without cracking, deformation, and uniform in structure. [0003] With the increasing planting area of ​​Dalbergia japonica plantation in Hainan Province, the problem of pests and diseases has become increasingly prominent. Dalbergia mellifera disease is caused by the fungus Dalbergia melena, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2549/119
Inventor 周国英刘君昂董文统何苑嗥刘倩丽刘成锋
Owner HAINAN KEDA FORESTRY CO LTD
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