Specific primers for detecting endogenousness of metarrhizium anisopliae in plant roots

A specific, plant root technology, applied in the field of specific primers for detecting the endophyte of Metarhizium anisopliae in plant roots, can solve the problems of unsuitable plants and cumbersome detection process, etc.

Inactive Publication Date: 2016-08-31
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, isotope or fluorescent protein labeling methods require special detection instruments and environments, and the detection process is cumbersome
The method of marking first and then detecting is obviously not suitable for the detection of plants in natural conditions, because the expected endophytic insecticidal fungus has no visible artificial markers

Method used

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  • Specific primers for detecting endogenousness of metarrhizium anisopliae in plant roots
  • Specific primers for detecting endogenousness of metarrhizium anisopliae in plant roots
  • Specific primers for detecting endogenousness of metarrhizium anisopliae in plant roots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1 distinguishes Metarhizium anisopliae from other fungi with the PCR detection of specific primer F1 / R1

[0014] There are a total of 30 experimental strains, including 12 Metarhizium anisopliae strains from different geographical locations and host sources, 3 imported Metarhizium anisopliae strains, and 5 other known fungal strains. 3 strains were identified as Metarhizium anisopliae and 7 strains were identified as other types of fungi (Table 1). Each strain was inoculated and cultured separately, the mycelia were collected, and DNA was extracted according to the instructions of the Fungi DNA Extraction Kit. Perform PCR amplification with primers F1 / R1, and the PCR conditions are: the total volume of the reaction system is 50 μL, containing 5 μL of 10×Ex Taq Buffer, 0.25 μL of 5U / μL Ex Taq, 4 μL of 2.5 mmol / L dNTPMixture, and 10 μmol / L of F1 and R1 1 μL each, 1 μL DNA template, 37.75 μL ultrapure water. The reaction program is: 94°C for 3min→94°C for 15se...

Embodiment 2

[0018] Example 2 Using F1 / R1 Primer PCR to Detect the Endophyte of Metarhizium anisopliae in Peanut Roots

[0019]Set up an experiment in which two strains of Metarhizium anisopliae inoculate peanuts. On the PDA medium, Metarhizium anisopliae strains MBJQH2-2 and Ma9 strains were respectively inoculated, and cultured at 25°C for 15 days to obtain conidia powder. Prepare the spore suspension and adjust the concentration to 1×10 7 Spores / ml. When sowing peanuts, pour 100ml of spore liquid in the seed hole. The non-inoculated control was taken as water pouring. The peanuts were planted under routine management under consistent conditions, and the peanut roots were collected on the 75th and 90th days after sowing for detection. The method steps are as follows:

[0020] 1) Wash the peanut roots, blot the surface moisture, and store them at -20°C for later use. 2) DNA extraction. 3) PCR detection. ①Synthesize primer F1 5'-GGGGTAGTCAGTATTCTGGCGGGC and primer R1 5'-GGGGTTCCACG...

Embodiment 3

[0022] Embodiment 3 detects the endogenous property of metarhizium anisopliae in corn root with F1 / R1 primer PCR

[0023] Set up an experiment in which the gpd-ben-marked Metarhizium anisopliae strain M2-2-44044 and the original strain MBJQH2-2 were inoculated in maize. On the PDA medium, Metarhizium anisopliae strains M2-2-44044 and MBJQH2-2 were respectively inoculated and cultured at 25°C for 15 days to obtain conidia powder. Prepare the spore suspension and adjust the concentration to 1×10 7 Spores / ml. On the 4th day after corn sowing, 100ml of spore liquid was poured around the base of the seedlings. The non-inoculated control was taken as water pouring. The plants were planted under routine management under the same conditions, and the corn roots were collected on the 28th day after the fungus application.

[0024] The steps of the method are as follows: 1) Wash the corn root, blot the surface moisture, and store it at -20°C for later use. 2) DNA extraction. 3) PCR...

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Abstract

The invention relates to the field of plant protection, in particular to specific primers for detecting endogenousness of metarrhizium anisopliae in plant roots. The sequences of the specific primers are F1:5'-GGGGTAGTCAGTATTCTGGCGGGC and R1:5'-GGGGTTCCACGGCGAGACCGCCAAT. The detection sensitivity reaches 1:2500.

Description

technical field [0001] The invention relates to the field of plant protection, in particular to a specific primer for detecting the endogenous nature of Metarhizium anisopliae in plant roots. Background technique [0002] Entomopathogenic fungi can infect parasitic insects and make them sick and dead. Their insecticidal function has been used in the prevention and control of agricultural, forestry, grass and other plant pests. In recent years, studies have found that entomopathogenic fungi can not only infect pests and reduce plant pests, but also enter plant persistence and promote plant development and growth. So far, at least 8 genera and species of insecticidal fungi have been proven to colonize plants, including Metarhizium anisopliae (M. robertsii), Beauveriabassiana, Paecilomyces sp., Verticillium lecanii (Lecanicillium lecanii), Isariafarinosa, Acremonium sp., Cladosporium sp., Plectosphaerella cucumerina, and Clonostachysrosea, which inhibit soil-borne diseases of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 农向群刘少芳王广君李兴佳曹广春张泽华
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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