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Molecular diagnosis method for gray mold

A technique for botrytis cinerea and botrytis cinerea, which is applied in the field of detection and identification of botrytis cinerea pathogens, can solve problems such as long time period and difficult control, and achieves good early diagnosis, rapid method and good specificity.

Inactive Publication Date: 2006-03-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It is difficult to judge with the naked eye at the initial stage of the onset of gray mold in fields and protected areas. When it is visible to the naked eye, the disease will develop rapidly and be difficult to control. , humidity and experience judgment, give guidance to vegetable farmers, misjudgment is inevitable
[0008] The routine research in the laboratory is to isolate and cultivate pure pathogens, and identify the species under the microscope. This method requires a long period of time, and the operator needs to have rich experience in morphological identification.

Method used

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  • Molecular diagnosis method for gray mold
  • Molecular diagnosis method for gray mold
  • Molecular diagnosis method for gray mold

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, specificity test

[0049] Weigh 0.03g each of 9 plant tissues including hm1, hm2, hm3, hm4, bf1, bf2, sm1, sm2, and LJYM, add 200μL of extraction buffer respectively, grind thoroughly, then add 300μL of extraction buffer, and mix well , in a water bath at 65°C for 10 min; add an equal volume of chloroform: isoamyl alcohol (24:1), mix well, and place on ice for 10 min; centrifuge at 12000 rpm / min for 10 min; take the supernatant, add 0.6 times the volume of isopropanol, mix Mix well, place at -20°C for 30min, centrifuge at 12000rpm / min for 10min, discard the supernatant; wash the precipitate twice with pre-cooled 70% ethanol and dry; add 50μL ddH 2 O dissolved.

[0050] Add 2 μL of template, 5 μL of 10×PCR buffer, 2.5 μL of dNTP (2.0 mmol / L), 0.04 μL of Taq enzyme (5U / μL), and 0.25 μL each of forward and reverse primers (20 μmol / L) into 9 reaction tubes , plus ddH 2 O to a total volume of 50 μL.

[0051] PCR amplification was carried out according to th...

Embodiment 2

[0053] Embodiment 2 sensitivity test

[0054] According to the ratio shown in Table 1, healthy tomato leaves were mixed with in vitro cultured Botrytis cinerea mycelium, and about 0.03 g of plant tissue mixed with a certain amount of Botrytis cinerea mycelium was weighed, and the template DNA was extracted in the same way as in Example 1, Carry out PCR amplification, the result is as follows figure 2 shown. The results showed that No. 2, No. 3 and No. 4 could amplify clear and bright bands, No. 1 and No. 5 had no amplification.

[0055] label

[0056] After the No. 4 template was diluted by 2 times, 5 times, 10 times, 20 times and 50 times, PCR amplification was carried out in the same way as in Example 1. The result is as image 3 shown. It can be seen that positive amplification can be obtained after dilution of 2 times, 5 times, 10 times, 20 times and 50 times, but the amplification efficiency also decreases. When tested under ultraviolet transmission, 20-fo...

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Abstract

A molecular diagnosing method for botrytis of tomato includes such steps as designing a pair of PCR primers specific to the pathogen of tomato botrytis, using them for PCR amplifying of the specimen to be tested, and judging if the tomato plant is suffered from botrytis. Its advantages are high speed, high sensitivity and high specificity.

Description

technical field [0001] The invention relates to the field of plant disease and insect pest control, in particular to the detection and identification of the gray mold pathogen. Background technique [0002] Botrytis cinerea is a very harmful disease in the vegetable area in northern my country. Grapes, tomatoes, peppers, cucumbers, kidney beans and other vegetables cultivated in protective facilities such as plastic greenhouses, greenhouses, and small sheds often occur and become popular, and in severe cases, the yield can be reduced by 100%. 20% to 30% or more. In the southern vegetable area of ​​my country, this disease rarely occurred in the past, but in recent years, due to the development of protected cultivation, Botrytis cinerea has also begun to occur and has a tendency to increase year by year. [0003] Botrytis mainly damages fruits, but can also damage leaves and stems. Fruit damage generally starts from the remaining petals, receptacles, etc., and moist, gray-br...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/447
Inventor 马雅军王娜
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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