JP2012147699A - Method for quickly detecting calonectria ilicicola in soil by soil dna extraction-pcr method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly detect soilborne Calonectria ilicicola.SOLUTION: A soil sample is collected from the soil of a field from the surface to the depth of 5 cm, and the collected soil is dried at 60°C by using a ventilation dryer. The whole soil DNAs are extracted from about 0.5 g of the dried soil sample by using a soil DNA extraction kit. The extracted DNAs are PCR-amplified by using a primer set peculiar to a marker gene (a β-tubulin gene) of Calonectria ilicicola with the extracted whole soil DNAs as templates. The amplified product is electrophoresed with agarose gel, and the Calonectria ilicicola marker gene is detected by a DNA band of the electrophoresed gel. Presence of the Calonectria ilicicola is detected by the detected DNA band, and the fungus density is semi-quantitated from the density of the band.

Description

  The present invention is a technique for rapidly detecting soil-borne soybean black root rot fungi, and specifically detects and semi-quantitatively amplifies soybean black root rot fungus marker genes contained in total DNA extracted from soil. Technology.

  In recent years, soybean black root rot has occurred frequently in various parts of Japan. The reason for this is not clear, but it is often caused by soybean crops. If soybean black root rot occurs, soybean growth is strongly inhibited and the yield is greatly reduced. Effective coping techniques for soybean black root rot have not been developed, and if soybean black root rot frequently occurs, soybean cultivation must be stopped. Soybean black root rot is soil-infectious, so if you can know the presence of pathogenic bacteria in the soil before planting soybeans, you can deal with soil disinfection and crop changes. There was no such technique (Patent Document 1, Patent Document 2, Patent Document 3).

JP 2005-269966 A JP 2006-151898 A JP2007-202529

It was difficult to detect soybean black root rot fungi from soil samples, and quantitative analysis was impossible. In order to detect soybean black root rot fungus, it is necessary to detect soybean black root rot fungus quickly using a soil sample.

Extraction of total DNA from soil sample, PCR amplification using primers targeting marker genes specific to soybean black root rot fungus, electrophoresis of amplified products on agarose gel to detect soybean black root rot marker marker gene To do.

(Soil DNA extraction method)
Dry the soil sample at 60 ° C. and weigh about 0.5 g. Soil DNA Isolation
Kit, MO Bio) to extract total DNA in the soil.

(Soil sample collection method)
Take several soil samples from one field. Collect at least 4 points per area (10 ares). The depth is between 5 cm from the surface, and about 500 g is collected. Collected soil samples should be refrigerated until analysis.

(Pretreatment method of soil sample)
A soil sample (raw soil) is dried by ventilation at 60 ° C for 24 hours. A dry soil sample is pulverized using a mortar.

(Soil DNA extraction method)
Weigh about 0.5 g of dry soil sample. Total DNA in the soil is extracted from the soil sample using a soil DNA isolation kit (MO Bio).

(Create primers that amplify the marker gene)
In order to detect soybean black root rot fungi in soil, it is necessary to PCR-amplify only the genes possessed by soybean black root rot fungi. Among the genes possessed by soybean black root rot fungus, a primer set was designed to amplify Histone H3 and β-tubulin gene regions as shown in the sequence listing.
Primer set 1 is a combination of SEQ ID NO: 1 and SEQ ID NO: 2, and primer set 2 is a combination of SEQ ID NO: 2 and SEQ ID NO: 3.

(PCR amplification and detection method)
PCR amplification was performed on two soybean black root rot fungi using Histone H3 and a total of three primer sets designed in the β-tubulin gene region. Thereafter, the PCR amplification product was electrophoresed using an agarose gel and stained with ethidium bromide. The stained electrophoresis gel was irradiated with ultraviolet rays using a transilluminator, and the band was detected.

The conventional soybean black root rot detection method takes time and labor, and cannot be detected before soybean cultivation. By using this method, not only soybean black root rot fungus in soil can be detected in a short time, but also semi-quantitative determination of the fungus fungus is possible, so that the possibility of soybean black root rot occurring before soybean cultivation can be determined.

Detection of soybean black root rot fungus by three kinds of primer sets Detection limit of primer set 1 (β-tubulin)

Collect the soil that will be planted with soybeans in advance. By using this detection method, we investigate the presence and density of soybean black root rot fungi in the soil, and obtain information on the risk of soybean black root rot occurrence to take measures such as soil disinfection and crop change.

(Primer verification)
In any strain, the only primer sets in which gene amplification was stably observed were the two primer sets 1 and 2 designed in the β-tubulin region. Therefore, as a result of examining the detection limits of these three primer sets shown in FIG. 1, the primer set 1 (β-tubulin) detected up to 5 pg / μl per reaction solution as shown in FIG. It was possible. In addition, the primer set 1 demonstrated the specificity of the primer set 1 because no gene amplification was observed in the pesticidal, white silkworm, and mycorrhizal fungi.

Claims (2)

A method for rapidly detecting soybean black root rot fungi in soil by extracting soil DNA and specifically PCR-amplifying only the marker gene (β-tubulin) of soybean black root rot fungus. A primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 shown in the Sequence Listing, which amplifies only the marker gene (β-tubulin) of soybean black root rot of claim 1.