CN104846094A - Quick detection method for a variety of pathogen molecules of strawberry root rot diseases and application of quick detection method - Google Patents

Abstract

The present invention discloses a rapid detection method and application of various pathogenic bacteria molecules of strawberry root rot. The method is to perform PCR reaction on pathogenic bacteria DNA with universal and specific primers for pathogenic fungi. When the PCR product presents a single band of about 500bp, the PCR product is sequenced. , the sequencing results were compared with the NCBI database, and it was clear that the disease-like tissues contained different pathogenic bacteria that caused strawberry root rot. The method is simple to operate, less time-consuming and high-throughput. The qualitative detection of root disease samples of strawberry nursery seedlings by this method can effectively avoid the spread and occurrence of strawberry root rot, and ensure the construction and safe production of strawberry disease-free seedling production bases in my country.

Description

Molecular rapid detection method and application of various pathogens of strawberry root rot

technical field

The invention belongs to the technical field of fruit tree disease prevention and plant quarantine, and relates to a molecular rapid detection method and application of various pathogenic bacteria of strawberry root rot.

Background technique

Strawberry root rot is a general term for a large class of diseases caused by soil-borne pathogenic fungi. More than 20 species of root rot pathogens have been reported in major strawberry producing areas around the world, and it is a soil-borne disease that is difficult to control. Common pathogenic bacteria are: the main causes of strawberry black root rot are Fusarium spp., F.oxysporum, Rhizoctonia solani, Pythium sp. Pestalotiopsis sp.; the pathogen causing root rot of strawberry red stem is Phytophthora fragariae C.J.Hickman. The typical symptom of strawberry root rot is that when the diseased root is cut or the outer skin of the root is peeled off, dark red diseased tissue can be seen, which is in sharp contrast to the healthy part; The incidence rate of root rot is 20% to 30%, reaching 50% to 80% in some areas, and the mortality rate of affected plants is 100%. In recent years, strawberry black root rot and red central column root rot are the most harmful and most influential among strawberry diseases, seriously affecting the yield and benefit of strawberry, and becoming the bottleneck of strawberry planting development.

The pathogenic fungus causing strawberry root rot has obvious characteristics of latent infection. Latent infection of root rot pathogens on strawberries is a common phenomenon, and strawberry root rot will occur when environmental conditions are suitable for the onset. Strawberry production generally uses asexual seedlings for propagation, and the mobility of seedlings between regions is also very frequent. If there is latent infection of root rot pathogens on transplanted seedlings, it will often cause long-distance spread of pathogens across regions, resulting in regional outbreaks of root rot and bringing huge economic losses to strawberry production. Therefore, the aseptic seedlings propagated in the nursery base without root rot can avoid the occurrence of strawberry root rot from the root. However, with the expansion of the planting area and the wider spread of the disease, this method is very difficult. Therefore, it is very important to establish an effective seedling detection technology, which can effectively avoid the spread and occurrence of strawberry root rot. However, because the infected seedlings do not have obvious symptoms, and some inspection methods currently developed are not easy to be popularized and implemented because they are cumbersome and time-consuming, they still bring great difficulties to the current inspection of root rot pathogens. The etiology of strawberry root rot is complex. The premise of rational use of chemical control methods is to find out the pathogenic microorganisms of strawberry root rot in the area, and prescribe the right medicine to get twice the result with half the effort. At the same time, the residual problems caused by chemical control and the damage to the ecological environment cannot be ignored. The traditional method of isolation, culture and identification of pathogenic bacteria relies on the rich experience of professionals and systematic theoretical knowledge of pathogenic bacteria classification, which will delay the best time for prevention and control. At present, there is no effective method to control strawberry root rot. It is mainly to take comprehensive control measures with agricultural control as the main and chemical control as the supplement. Therefore, the prevention of strawberry root rot should be given priority to, and the quarantine inspection of the disease should be strengthened. In order to prevent the spread of strawberry root rot from infected areas to non-infested areas and ensure the safe production of strawberries in China, it is necessary to establish a set of stable, simple, fast and sensitive molecular detection methods for quarantine and detection of strawberry seedlings. The PCR detection method has the advantages of high sensitivity, high precision, small sampling volume, and short detection time. Qualitative detection of strawberry seedlings through this technology is of great significance to ensure the construction and safe production of strawberry disease-free seedling production bases in my country, and to block the introduction of dangerous foreign strawberry seedlings into my country.

Contents of the invention

The purpose of the present invention is to overcome the existing defects in the prior art, and to provide a rapid detection method for pathogenic bacteria of strawberry root rot disease. The method is simple and easy to operate, and can be directly applied to production practice for high-sensitivity and rapid detection of infected plant tissues, ensuring healthy sterile seedlings for production, and having very important significance for early warning of pathogenic bacteria molecules.

Another object of the present invention is to provide the application of the rapid molecular detection method for pathogenic bacteria of strawberry root rot.

Its specific technical plan is:

A molecular rapid detection method for multiple pathogens of strawberry root rot, comprising the following steps:

Step 1. Isolation of pathogenic bacteria

Use the tissue separation method to separate the pathogenic bacteria from the root of strawberry, cut the diseased tissue at the junction of the diseased and healthy tissues into 4-5 mm small pieces, soak them in 75% ethanol for 5 seconds, then move the diseased tissues into 0.1% mercuric chloride solution and soak them for 3- After 5 minutes, the diseased tissue was transferred to sterile water and rinsed 3 times, and finally the tissue was moved to PDA medium, and the culture dish was placed in a 25°C incubator for 3-5 days, and the colony diameter was 1 cm, and the DNA extraction experiment was performed;

Step 2. A small amount of pathogen DNA extraction

1) Use a sterilized toothpick to scrape a small amount of mycelium of pathogenic bacteria on the PDA plate, and put it into a 2.0ml centrifuge tube;

2) Add 500μl Lysis buffer, 1 crushed bead, and grind for 60-90s with a multi-sample tissue grinder;

3) Stand at room temperature for 10 minutes;

4) Centrifuge at room temperature for 10 minutes at 13200 rpm, take 400 μl of supernatant and transfer to a new centrifuge tube;

5) Add 800 μl of absolute ethanol, mix by inversion; stand at -20°C for 10 minutes to precipitate DNA;

6) 13200rpm, centrifuge at 4°C for 5min;

7) Wash the precipitate with 75% ethanol, dry it in the air, and dissolve it with 50 μl ddH ₂ O. The supernatant is the extracted DNA solution, which is used as a downstream PCR template;

Step 3. Design universal primers for pathogenic fungi

Upstream primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; Downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3';

Step 4. PCR amplification

The PCR reaction system is 25 μL, the reaction solution is: 10×buffer 2.5 μL, dNTP 1 μL, primers ITS1 and ITS4 1 μL each, Taq enzyme 0.5 μL, DNA template 3 μL, ddH2O 16 μL, PCR amplification program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C 45s, annealing at 53°C for 45s, extension at 72°C for 1min, and 35 cycles; total extension at 72°C for 10min; detect PCR results on 1% agar gel, when the PCR product shows a band of about 500bp, that is, the sample contains strawberry root Rot pathogens, randomly selected samples of CY2, JN3, JM2, and LW2 PCR products were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing;

Step 5. Sequence analysis

Sequencing results and NCBI database

http: ∥ blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM＝blastn&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome sequence alignment, to obtain clear strawberry root rot pathogen species.

The molecular quick detection method of strawberry root rot pathogenic bacteria of the present invention is applied in the quarantine of strawberry seedlings, the early diagnosis of strawberry root rot in the field, and the monitoring and identification of pathogens.

The beneficial effects of the present invention are:

The invention designs universal specific primers according to the genome sequence of the pathogen causing strawberry root rot. Through the qualitative detection of strawberry seedlings by this technology, the construction and safe production of strawberry disease-free seedling production bases in my country can be realized, and the purpose of blocking the introduction of dangerous and diseased strawberries from abroad at the same time. The method is simple to operate, less time-consuming and high-throughput. The qualitative detection of root disease samples of strawberry nursery seedlings by this method can effectively avoid the spread and occurrence of strawberry root rot, and ensure the construction and safe production of strawberry disease-free seedling production bases in my country.

Description of drawings

Figure 1 is the PCR electrophoresis image of strawberry root disease-like tissues in the same area, wherein, lane M is DNA Marker 2000; lanes 1-9 are PCR products of strawberry root tissue collected from different regions corresponding to the following table; lane ck- is a negative control Water was used as template for PCR products.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

1. Isolation of pathogenic bacteria

Pathogens from strawberry roots were isolated by tissue separation method. Cut out a small piece of 4-5mm diseased tissue at the junction of diseased and healthy tissue, soak it in 75% ethanol for 5 seconds, then move the diseased tissue into 0.1% mercuric chloride solution and soak it for 3-5 minutes, then transfer the diseased tissue to sterile water for rinsing 3 times, finally move the tissue to the PDA medium, place the petri dish in a 25°C incubator and culture for 3-5d, and the colony diameter is 1cm to carry out the DNA extraction experiment.

2. A small amount of pathogen DNA extraction

1) Use a sterilized toothpick to scrape a small amount of mycelium of pathogenic bacteria on the PDA plate, and put it into a 2.0ml centrifuge tube;

2) Add 500μl Lysis buffer, 1 crushed bead, and grind for 60-90s with a multi-sample tissue grinder;

3) Stand at room temperature for 10 minutes;

4) Centrifuge at room temperature for 10 minutes at 13200 rpm, take 400 μl of supernatant and transfer to a new centrifuge tube;

5) Add 800 μl of absolute ethanol, mix by inversion; stand at -20°C for 10 minutes to precipitate DNA.

6) 13200rpm, centrifuge at 4°C for 5min;

7) The precipitate was washed with 75% ethanol, dried in the air and dissolved in 50 μl ddH ₂ O. The supernatant was the extracted DNA solution, which could be used as a downstream PCR template.

3. Design universal primers for pathogenic fungi

Upstream primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (step 3 can be performed simultaneously with steps 1 and 2).

4.PCR amplification

The PCR reaction system is 25 μL, and the reaction solution is: 10×buffer 2.5 μL, dNTP 1 μL, primers (ITS1 and ITS4) 1 μL, Taq enzyme 0.5 μL, DNA template 3 μL, ddH ₂ O 16 μL. PCR amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 53°C for 45 s, extension at 72°C for 1 min, and 35 cycles; total extension at 72°C for 10 min. The PCR result is detected on 1% agar gel, and when the PCR product presents a band of about 500bp, that is, the sample contains the pathogenic bacteria of strawberry root rot. Randomly selected samples CY2, JN3, JM2, and LW2 PCR products were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.

5. Sequence Analysis

Sequencing results and NCBI database

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) sequence alignment, the clear strawberry root rot pathogen species was obtained.

The experimental results are shown in Figure 1 and Table 1.

Table 1: Information table of strawberry samples collected in different regions

Note: + represents a positive result, and a false positive result occurred in No. 4 sample.

CY2: 99% homologous identity with Pestalotiopsis sp (FJ175373.1), as shown in SEQ ID NO:1.

JN3: 99% homologous to Alternaria alternata (JF796073.1), as shown in SEQ ID NO:2.

JM2: 99% homologous to Colletotrichum gloeosporioides (KM357285.1), as shown in SEQ ID NO:3.

LW2: 99% homologous to Fusarium solani (JN786598.1), as shown in SEQ ID NO:4.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

Claims (1)

1. the multiple nosophyte numerator method for quick of Strawberry Root Rot evil, comprises the following steps:

The separation of step 1. pathogenic bacteria

The pathogenic bacteria of tissue isolation to strawberry root is adopted to be separated, cut the fritter of the diseased tissues 4-5mm of the strong intersection of disease, be placed in 75% ethanol and soak 5s, then diseased tissues is moved in 0.1% mercuric chloride solution and soak 3-5min, again diseased tissues to be transferred in sterilized water rinsing 3 times, finally moved on to by tissue on PDA substratum, placed by culture dish in 25 DEG C of incubators and cultivate 3-5d, namely colony diameter 1cm carries out DNA extraction experiment;

Step 2. pathogenic bacteria DNA extracts on a small quantity

1) with a small amount of pathogenic bacteria mycelia on sterilizing toothpick scraping PDA flat board, load in the centrifuge tube of 2.0ml;

2) 500 μ l Lysis buffer are added, 1 broken pearl, Multi-example tissue grinder grinding 60-90s;

3) room temperature leaves standstill 10min;

4) 13200rpm, the centrifugal 10min of room temperature, get supernatant liquor 400 μ l and proceed in new centrifuge tube;

5) add 800 μ l dehydrated alcohols, put upside down mixing;-20 DEG C of standing 10min precipitate DNA;

6) 13200rpm, 4 DEG C of centrifugal 5min;

7) precipitation uses 75% washing with alcohol, with 50 μ l ddH after air drying ₂o dissolves, and supernatant is the DNA solution of extraction, for downstream PCR template;

Step 3. designs pathogenic fungi universal primer

Upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 '; Downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ';

Step 4.PCR increases

PCR reaction system is 25 μ L, and reaction solution is: each 1 μ L of 10 × buffer2.5 μ L, dNTP1 μ L, primer I TS1 and ITS4, Taq enzyme 0.5 μ L, DNA profiling 3 μ L, ddH2O 16 μ L, pcr amplification program: 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 53 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations; 72 DEG C of total elongation 10min; The agar gel of 1% detects PCR result, when PCR primer presents 500bp band, namely contains Strawberry Root Rot pathogenic bacteria in sample, random choose sample CY2, JN3, JM2, LW2PCR product is sent to the order-checking of raw work biotechnology Shanghai limited-liability company;

Step 5. sequential analysis

Sequencing result and ncbi database

Http:// blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn & PAGE_TYPE=BlastSearch & LINK_LOC=blasthome sequence alignment, obtains clear and definite Strawberry Root Rot Species of Pathogens.