CN109182582B - Qualitative detection primer and detection method for fusarium oxysporum f.sp.cubense - Google Patents

Abstract

The invention discloses a primer and a detection method for qualitative detection of watermelon fusarium oxysporum, which specifically comprise primer design, primer specificity screening, sensitivity of specific primer detection and a qualitative detection method of watermelon fusarium oxysporum. The invention establishes a detection system of watermelon wilt bacteria common PCR watermelon plants and soil, can detect the watermelon wilt bacteria on the day of resisting and sensing the watermelon plants and 1-5 dpi after inoculation, and has the spore density of 5 multiplied by 10 in the soil²The detection of watermelon fusarium wilt bacteria by cfu/g verifies that PCR can be used for quickly and accurately detecting in the latent disease stage and the initial disease stage, whether the plants or soil have the fusarium wilt bacteria can be detected before the plants do not generate observable fusarium wilt disease symptoms by naked eyes, the detection method is quick, convenient and low in cost, the disease can be timely controlled before the occurrence and prevalence of the fusarium wilt disease, and the early disease detection method is established and has application potential.

Description

Qualitative detection primer and detection method for fusarium oxysporum f.sp.cubense

Technical Field

The invention relates to a qualitative detection primer and a qualitative detection method for fusarium oxysporum f.sp.cubense, and belongs to the technical field of biology.

Background

Watermelon (Citrullus lanatus (Thunb.) Matsum. etNakai) is the fifth fruit in the world, and the cultivation area, total output and per-capita consumption of watermelons in China are the first in the world. Watermelon Fusarium wilt is a worldwide fungal soil-borne disease caused by Fusarium oxysporum watermelon specialization (Fon) of Fusarium of deuteromycotina, disease residues and bacteria-bearing soil are primary infection sources of the disease, conidiospores or chlamydospores germinate to generate invasion filaments under proper conditions, the invasion filaments are subjected to branch spread among epidermal cells through wounds or invasion host epidermal cells, and a large number of conidiospores and chlamydospores are formed for re-infection. Due to the lack of specific medicaments and resistant varieties at present, the treatment difficulty of the watermelon fusarium wilt after the disease is quite large, the yield of a diseased field is generally reduced by 20-30%, the yield of a serious field can reach 50-60%, and even the watermelon fusarium wilt cannot be produced. At present, the disease is mainly prevented and controlled by breeding for disease resistance, crop rotation and stubble changing, grafting, medicament prevention and control and the like. Because the land resources are limited, the crop rotation method is not easy to implement; the grafting operation is complicated, the cost is high, and the quality of the grafting is easily influenced; chemical control is easy to cause problems of environmental pollution, pesticide resistance, pesticide residue and the like, and no chemical pesticide can effectively control the blight so far, and the blight prevention and control is one of the main problems in watermelon production. Therefore, when the plant has no disease, the qualitative detection is carried out on the watermelon blight bacteria in the watermelon and the soil, effective prevention and control measures can be taken before the conidia of the bacteria are greatly expanded and spread to breed, and the method has important significance for preventing and controlling the disease.

The traditional method for detecting the fusarium oxysporum mainly comprises tissue separation, microscopic observation, morphology and fusion group identification, but has complicated procedures and longer period. Therefore, the problem to be solved by the technical personnel in the field is to provide a qualitative detection primer and a detection method for fusarium oxysporum f.sp.

Disclosure of Invention

The invention aims to provide a qualitative detection primer and a method for simply, conveniently, quickly and accurately detecting fusarium oxysporum f.sp.cubense.

In order to achieve the purpose, the invention adopts the following technical scheme:

the primers for qualitatively detecting the fusarium oxysporum have the following sequences:

FONDX-15F：5’-GCTCGTAAGTCAGGTTCA-3’；SEQ ID NO：1；

FONDX-15R：5’-AAGCCACTCCATCACATC-3’；SEQ ID NO：2。

further, the primer screening method comprises the following steps:

(1) designing a primer: based on a database of fusarium oxysporum genome, designing and screening specific primers of fusarium oxysporum according to the difference fragments;

(2) and (3) primer specificity screening: using genomic DNA of watermelon fusarium wilt bacteria Fon race 0, Fon race1, Fon race2, tomato fusarium wilt bacteria, cucumber fusarium wilt bacteria, melon fusarium wilt bacteria, cabbage fusarium wilt bacteria, cotton verticillium wilt bacteria and tomato verticillium wilt bacteria as templates, and RNase-free ddH₂Taking O as negative control, screening specific primers of the fusarium oxysporum f.sp.cubense, and screening out the specific primers;

(3) the sensitivity of the specific primers was detected.

Further, the method for detecting the sensitivity of the specific primer in the step (3) is as follows: the genomic DNA of the No. 1 physiological race of the fusarium oxysporum f.sp.cubense is continuously diluted into six concentration gradients of 36.5 ng/mu L, 3650 pg/mu L, 365 pg/mu L, 36.5 pg/mu L, 3.65 pg/mu L and 0.365 pg/mu L by a 10-fold concentration gradient, and the sensitivity of the specific primer is detected.

Further, the primers for qualitatively detecting the watermelon fusarium oxysporum are used for detecting the watermelon fusarium oxysporum in watermelon plants and soil.

Further, a qualitative detection method for watermelon fusarium wilt germs comprises the following specific steps:

(1) extracting the watermelon plant genome DNA or soil genome DNA infected with pathogenic bacteria;

(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer of claim 1;

the PCR reaction system is as follows: 2 XEs Taq PCR Master Mix 12.5. mu.L, upstream and downstream primers 0.5. mu.M each, DNA template 2. mu.l (watermelon plant DNA concentration 200ng/uL, soil DNA concentration 50ng/uL), RNase-free Water ddH₂O is complemented to 25 mu L;

the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, and extension at 74 ℃ for 30s for 30 cycles; finally, extension is carried out for 10min at 72 ℃, and supplementary extension is carried out for 10 min;

and (3) carrying out agarose gel electrophoresis detection on the PCR product obtained in the step (2).

The invention has the beneficial effects that: the invention screens out 1 pair of primers for PCR qualitative detection of the fusarium oxysporum f.sp.cubense, utilizes the primers to carry out common PCR amplification on the DNA of pathogenic bacteria to be tested, and can amplify strips only in the fusarium oxysporum f.sp.cubense, which shows that the primers have strong specificity to the fusarium oxysporum f.cubense; and the concentration of the genomic DNA of the fusarium oxysporum is 34.9 pg/uL. The watermelon fusarium oxysporum PCR detection system established by the invention can detect the watermelon fusarium oxysporum on the day of resisting and sensing watermelon plants and 1-5 dpi after inoculation, and the spore density of the soil is 5 multiplied by 10²The fusarium oxysporum f.sp.citrullus can be detected in cfu/g, the PCR can be verified to be capable of rapidly and accurately detecting in the disease incubation period and the initial period, and whether the plant carries the fusarium oxysporum or not can be detected before the plant generates no observable fusarium oxysporum disease symptoms; the common PCR detection system for watermelon plants and soil is quick, convenient and low in cost, establishes a disease early detection method for timely controlling diseases before blight occurs and prevails, meets actual requirements, and has application potential.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the detection results of the specificity of the primers of the present invention;

wherein, M: 2000bp DNAmarker; 1: negative control; 2-4: physiological race1, 2, 0 of watermelon wilt pathogen; 5: tomato fusarium wilt bacteria; 6: cucumber fusarium wilt bacteria; 7: melon fusarium wilt bacteria; 8: cabbage wilt bacteria; 9: cotton wilt germs; 10: rhizoctonia solani f cotton; 11: tomato verticillium wilt;

FIG. 2 is a diagram showing the results of detecting the sensitivity of the primers of the present invention;

wherein, M: 2000bp DNAmarker; 1: negative control; 2-7: fon race1 was serially diluted in a 10-fold concentration gradient, i.e., 36.5ng/μ L, 3650pg/μ L, 365pg/μ L, 36.5pg/μ L, 3.65pg/μ L, 0.365pg/μ L;

FIG. 3 is a diagram showing the detection of pathogenic germs of 'Zaojia 8424' watermelon plant by the general PCR detection system of the present invention;

wherein, M: 2000bp DNAmarker; a: negative control; b: positive control (DNA of fusarium oxysporum race 1); c: before root dipping; 1-19: sequentially (dipping the roots, sampling on days 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 after planting);

FIG. 4 is a diagram showing the detection of the common PCR detection system of the present invention for the disease-resistant watermelon plant pathogen of 'West farming 8';

wherein, M: 2000bp DNAmarker; a: negative control; b: a positive control; c: before root dipping; 1-19: sequentially (dipping the roots, sampling on days 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 after planting);

FIG. 5 is a diagram showing the detection of soil inoculated with Fusarium oxysporum by the general PCR of the present invention;

wherein, M: 2000bp DNAmarker; 1: negative control; 2-6 are DNAs with different soil disease bacteria amounts, respectively 5X 10⁵cfu/g，5×10⁴cfu/g，5×10³cfu/g，5×10²cfu/g，5×10cfu/g。

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 seedling raising

Watermelon variety is selected from watermelon wilt resistant variety 'Xinong No. 8' and susceptible variety 'Zaojia 8424'.

The seedling raising method comprises the following specific steps:

(1) cleaning the vessels (plug tray and nutrition bowl) for seedling and cultivation, sterilizing the substrates (grass peat, vermiculite and perlite), and fumigating the greenhouse with bactericide and insecticide;

(2) watermelon seeds of 'Xinong No. 8' and 'Zaojia 8424' are respectively sterilized in 1.5% NaClO solution for 20min, during which time they are agitated once;

(3) after disinfection, washing with clear water and kneading until the water becomes colorless and transparent;

(4) soaking the disinfected seeds in warm water at 50-60 ℃ for 6 hours, wrapping the seeds with gauze or a wet towel, accelerating germination in a constant temperature box at 27 ℃, and germinating after 24-30 hours;

(5) picking out the seeds with uniform germination potential for sowing: and filling a seedling raising hole disc with a substrate (peat: vermiculite: 1), watering thoroughly, sowing single seeds, covering with a mulching film, preserving heat and moisture, controlling the temperature to be 24-30 ℃ in the daytime and the temperature to be about 18 ℃ at night after seedling emergence, and preventing vain growth.

Example 2 preparation of pathogenic bacteria

The pathogenic materials are: physiological race number 0(Fon race 0, BJ), physiological race number 1(Fonrace 1, BJ), physiological race number f.sp.niveum race 2(Fonrace 2, BJ), physiological race number f.sp.niveum race, physiological race number f.sp.lysporium sp.2, b.j.solani, physiological race number f.sp.lysporium sp.lysporium sp.lyspenci (Fol), cucumis melo Fusarium oxysporum f.sp.melo (Fom), cucumis sativus Fusarium oxysporum f.sp.cu.cur (Fusarium oxysporum), Fusarium oxysporum f.sp.sp.concenum, Fusarium oxysporum f.sp.sp.sp.fusarium oxysporum (Fusarium oxysporum), Fusarium oxysporum f. Foc, Fusarium oxysporum sp.sp.sp.sp.oxysporum, Fusarium oxysporum f.sp.sp.oxysporum, Fusarium oxysporum sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.c.sp.oxysporum, Fusarium oxysporum f.sp.oxysporum f.sp.sp.oxysporum f.c.c.c.c.sp.sp.c.c.sp.sp.sp.sp.c.c.c.c.c.c.c.c.c.f.f.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.f.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.f.c.c.c.c.f.f.c.c.f.c.c.c.c.c.c.f.c.c.c.c.c.c.c.c.c.f.c.c.c.f.f.c.c.c.f.f.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.

(1) Strain preparation

Respectively picking a bacterial block with the size of about 0.2cm multiplied by 0.2cm from the original bacterial strain preservation test tube by adopting aseptic operation, placing the bacterial block on the surface of a culture medium at the lower part in a PDA culture medium test tube, inoculating, quickly sealing after the bacterial strain is inoculated, placing the bacterial block in an incubator at the temperature of 25-28 ℃ for culture, and when hyphae are basically fully distributed on the surface of the culture medium (about 5-7 days), using the bacterial block for DNA extraction and subsequent tests.

(2) Preparation of spore suspension of No. 1 physiological race (Fonrace 1, BJ) of Fusarium oxysporum f.sp.citrulli

Adopting aseptic operation, picking a bacterium block with the size of 0.5cm multiplied by 0.5cm of watermelon wilt bacterium, vibrating the bacterium block into a PL culture medium, quickly burning and plugging a bottle mouth and a rubber plug on an alcohol lamp, culturing in a shaker at 25-28 ℃ at the rotating speed of 125rpm/min, and obtaining bacterium liquid for later use after 5-7 days.

Preparation of PL Medium: 200g of potato and 20g of lactose, peeling and cleaning the potato, and cutting into 1cm³Adding 1000ml distilled water into the left and right small blocks, decocting with slow fire for 40min until the potato blocks become soft, and taking care to prevent overflow. After filtering with double-layer gauze, the cup for boiling is washed with a small amount of distilled water, poured on the gauze and filtered. Then taking down gauze, squeezing the water in the potato into a beaker as much as possible, supplementing distilled water to 300ml, adding weighed lactose, stirring uniformly, subpackaging in 4 triangular flasks (60ml), and autoclaving at 121 ℃ for 20min for later use.

Sterilizing the ware at 121 deg.C for 20min, and oven drying. Filtering the bacterial liquid with two layers of gauze into a 1000mL beaker, centrifuging at 6000rpm for 15min, pouring out the supernatant, dissolving the spore precipitate with deionized water, pouring into the other beaker, and adding a proper amount of distilled water. Counting with a blood counting chamber, adjusting spore concentration to 5 × 10⁶cfu/mL, i.e., diluted to 1.25 spores per cell of the hemocytometer (observed under a 10X 10 microscope).

EXAMPLE 3 inoculation with Fusarium oxysporum

The method comprises the steps of inoculating No. 8 watermelon fusarium wilt bacteria No. 1 physiological microspecies to West farming No. 8 and Zaojia 8424 seedlings formed after 8-10 days of sowing (namely when cotyledons are spread and flat) in example 1 by adopting a root soaking method, wherein the spore concentration of the fusarium wilt bacteria is 5 multiplied by 10⁶cfu/mL, soaking the root for 15min, and shaking for 2-3 times to prevent spore sedimentation.

And (4) planting the seedlings after root soaking into a nutrition pot, and taking the seedlings inoculated with clear water as a control. Inoculating 60 watermelon varieties, culturing in an air-conditioning greenhouse at 26-30 ℃ after inoculation, and sampling rhizomes of 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d and 19d before root soaking and after inoculation respectively, and storing at-20 ℃ for later use.

Extracting the genome DNA of the pathogenic tissue artificially inoculated with fusarium oxysporum by adopting a CTAB method, carrying out PCR detection on the genome DNA of the artificially inoculated watermelon sample, and analyzing by combining with the disease index.

Disease index ∑ (number of disease-grade plants × representative value) × 100/total number of surveys × representative value of the most serious grade of disease.

The plants are rated as 0-5, disease-resistant at levels 2 and below 2, and susceptible at levels above 3.

Level 0: no symptoms;

level 1: in the middle of sunny day, the cotyledon wilts or partial cotyledon and true leaves will wither slightly, and the fruit can recover at night;

and 2, stage: 1 true leaf wilting or heavier leaf wilting;

and 3, level: leaf and more than 60% of true leaves will wither, hindering development;

4, level: the whole plant withers, more than 60 percent of the plant withers, and heart leaves survive;

and 5, stage: the whole plant wilted severely and died.

Example 4 extraction of genomic DNA of Fusarium oxysporum

Extracting genome DNA of watermelon fusarium wilt bacteria No. 0 physiological race, watermelon fusarium wilt bacteria No. 1 physiological race, watermelon fusarium wilt bacteria No. 2 physiological race, tomato fusarium wilt bacteria, melon fusarium wilt bacteria, cucumber fusarium wilt bacteria, cabbage fusarium wilt bacteria, cotton verticillium wilt bacteria and tomato verticillium wilt bacteria.

Extracting pathogen genome DNA, selecting an Ezup column type genome DNA extraction kit and purchasing from Beijing.

The specific operation is as follows:

(1) according to the body standard

PW Solution +12mL isopropanol

Wash Solution +22.5mL absolute ethanol

(2) Grinding 20mg of hypha into powder by using liquid nitrogen, putting the powder into a 1.5mL centrifuge tube, adding Buffer Digestion (200 mu L), beta-mercaptoethanol (2 mu L) and protease K (20 mu L), shaking and uniformly mixing, and carrying out water bath at 56 ℃ for 1h until the cells are completely lysed. The mixture was inverted every 10min during the water bath.

(3) BufferPF (100. mu.L) was added, mixed well by inversion, and left to stand at-20 ℃ for 5 min.

(4) Centrifuging at room temperature at 10000rpm for 5min, and transferring the supernatant to a new 1.5mL centrifuge tube.

(5) BufferBD (200. mu.L) was added and mixed well. Adding 200. mu.L of absolute ethyl alcohol and mixing evenly.

(6) And (3) putting the adsorption column back into the collection tube, transferring the solution and the semitransparent fibrous suspended matters into the adsorption column by using a liquid transfer device, standing for 2min, centrifuging at 10000rpm at room temperature for 1min, and pouring waste liquid in the collection tube.

(7) The adsorption column was returned to the collection tube, PW Solution (500. mu.L) was added thereto at 10000rpm for 30s, and the supernatant was discarded.

(8) The adsorption column was returned to the collection tube, and Wash Solution (500. mu.L) was added thereto at 10000rpm for 30s, and the supernatant was discarded.

(9) Centrifugation at 12000rpm for 2min at room temperature left a Wash Solution.

(10) The adsorption column was removed, and the column was put into a new 1.5mL centrifuge tube, TE (50. mu.L) was added thereto, the mixture was allowed to stand for 3min, centrifuged at 12000rpm for 2min at room temperature, and the DNA solution was collected and stored at-20 ℃.

Example 5 plant Total genomic DNA extraction

Extracting genome DNA of plant tissues sampled before plant root soaking and 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d and 19d after inoculation, and adopting a CTAB method, wherein the specific steps are as follows:

(1) the 2% CTAB extract was preheated in a water bath at 65 ℃.

(2) Weighing the test material (about 300mg), placing the test material in a mortar, adding a liquid nitrogen mill, and quickly grinding the test material into powder; transfer to 2mL centrifuge tube.

(3) Adding 700 μ L of 2% CTAB extraction liquid, and mixing by turning upside down; water bath at 65 ℃ for 1h, and gently shaking and mixing the mixture once every 10min to ensure that the sample is cracked fully.

(4) Taking out from the water bath, cooling for 2min, adding 800 μ L phenol chloroform extract (balance phenol: chloroform: isoamyl alcohol 25: 24: 1), and shaking vigorously to obtain emulsion at 12000rpm/min for 15 min.

(5) The supernatant was collected at 800. mu.L, added to a new 2mL centrifuge tube, and 800. mu.L of chloroform extract (chloroform: isoamyl alcohol: 24: 1) was added thereto, and the mixture was shaken at 12000rpm for 15min, and the process was repeated once.

(6) Taking 800 μ L of the supernatant, transferring into a new 1.5mL centrifuge tube, adding 0.7 times of isopropanol pre-cooled at 20 ℃, shaking up, and precipitating at-20 ℃ for 2 h.

(7) Precooling at 4 ℃ by using a high-speed centrifuge, putting a centrifuge tube containing flocculent DNA into the centrifuge at 4 ℃, 12000rpm/min for 5min, and discarding the supernatant.

(8) Adding 70% ethanol 200 μ L to precipitate, shaking at 12000rpm/min for 5min, discarding supernatant, repeating for 2-3 times, and naturally air drying the precipitate.

(9) The precipitate was dissolved by addition of 200 μ LTE. Adding 1.5 μ L RNase, and storing at-20 deg.C in water bath at 37 deg.C for 1 h.

EXAMPLE 6 inoculation of Fusarium oxysporum in soil and Total genomic DNA extraction

Soil inoculation of blight bacteria: adding 5X 10 per 100g soil⁶cfu/mL (10mL) conidia suspension formulated as 5X 10⁵cfu/g of soil. Continuously diluting the suspension with concentration gradient of 10 times for 4 times, respectively mixing with soil to obtain 5 × 10 suspension⁵cfu/g，5×10⁴cfu/g，5×10³cfu/g，5×10²cfu/g，5×10¹cfu/g of inoculation soil, respectively planting 50 early-good 8424 watermelon plants and 50 west-nong No. 8 watermelon plants in each inoculation treatment soil, culturing the inoculated watermelon plants at the temperature of 26-30 ℃ in an air-conditioning greenhouse, and counting the diseased plant rate of the watermelon plants.

Extraction of total DNA of Soil genome Using the method of extraction with FastDNA Spin Kit for Soil Kit:

(1) 500 mg of soil was added to the Lysing Matrix E Tube. Due to the movement of the FastPrep instrument, a large amount of aggregation was seen within the tube. The same Lysing Matrix should not exceed 7/8 for the volume of the tube. The tube leaves space, improving the chance of better homogenization. Adding 978. mu.l of Sodium Phosphate Buffer and 122. mu.l of MT Buffer;

(2) a speed of 6.0 for 40 seconds of mixing in a FastPrep instrument;

(3)14000XG, centrifuging and homogenizing Matrix E Tubes for 5-10 minutes; the supernatant was transferred to a 2ml clean tube; adding 250 μ l PPS reagent and mixing, shaking the tube 10 times;

(4)14000XG is centrifuged for 5 minutes, and the supernatant is transferred to a clean 15-mm riser pipe; the supernatant was added to 1ml Binding Matrix Suspension (the Binding Matrix was resuspended uniformly before use).

(5) The DNA is allowed to bind to the matrix by placing in a spinner or manually reversing for 2 minutes. The tube was left to stand for 3 minutes to allow the silica matrix to settle.

(6) Carefully remove 500. mu.L of the supernatant to avoid encountering a sedimenting Binding Matrix; discarding the supernatant; resuspending the Binding Matrix in the remaining liquid; approximately 600. mu.l of the mixture was transferred to a SPINTMFilter, 14000XG and centrifuged for 1 minute; the Catch Tube was emptied and the remaining supernatant was added to the SPINTM Filter and centrifuged;

(7) add 500. mu.L SEWS-M to SPINTM Filter, 14000XG and centrifuge for 1 min; pouring off the flow-through and replacing the SPINTM Filter in the Catch Tube; centrifuging at 14000XG for 2 minutes, and airing the rest SEWS-M in the SPINTM Filter;

(8) moving the SPINTM Filter to a new catcher tube, and air-drying for 5 minutes at room temperature;

(9) adding 50-100 μ L DES for light resuspension, gently stirring the filter membrane, and flicking with a gun head or vortex/finger to obtain high-efficiency DNA elution silica matrix; the DNA was centrifuged for 1min at 14000XG and the eluted DNA was transferred to a Catch Tube.

Example 7 screening of specific primers

(1) Primer design

Based on the existing database (https:// www.ncbi.nlm.nih.gov/genome/genomes/707) of Fusarium oxysporum genome, specific primers suitable for Fusarium oxysporum were screened out according to the differential fragment design.

(2) Primer specificity screening

Uses the genomic DNA of watermelon fusarium wilt (race 0, race1, race 2), tomato fusarium wilt, melon fusarium wilt, cucumber fusarium wilt, cabbage fusarium wilt, cotton verticillium wilt and tomato verticillium wilt as template, and RNase-free ddH₂O is used as negative control, and watermelon withering is carried outScreening a germ specific primer, namely screening out a specific primer FONDX-15F/FONDX-15R;

the primer sequences are as follows:

FONDX-15F：GCTCGTAAGTCAGGTTCA；SEQ ID NO：1；

FONDX-15R：AAGCCACTCCATCACATC；SEQ ID NO：2。

the total PCR reaction is 25. mu.L, wherein 2 XEs TaqPCR MasterMix 12.5. mu.L, upstream and downstream primers are 0.5. mu.M each, DNA template is 2. mu.L, RNase-free Water ddH₂Make up to 25. mu.L of O.

PCR reaction procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, and extension at 74 ℃ for 30s for 30 cycles; finally, extension is carried out for 10min at 72 ℃ and for 10min in a supplementary manner.

PCR products of 5. mu.L were separately subjected to 2% agarose gel electrophoresis for 30min (90V), and the amplification results were detected by a gel imager.

As a result, only the genomic DNA of Fusarium oxysporum (Fonrace 0, Fon race1, Fon race 2) can amplify 1 specific band of 556bp respectively, and the results are shown in FIG. 1. The result shows that the primer FONDX-15F/FONDX-15R has specificity and can distinguish the fusarium oxysporum f.sp.citrulli from other pathogenic bacteria.

Example 8 detection of primer sensitivity

Genomic DNA of a test watermelon fusarium wilt strain Fon race1 is serially diluted into six concentration gradients of 36.5 ng/mu L, 3650 pg/mu L, 365 pg/mu L, 36.5 pg/mu L, 3.65 pg/mu L and 0.365 pg/mu L by a 10-fold concentration gradient, and the diluted samples are respectively used as templates, and RNase-free ddH is used as a template₂And O is used as a negative control, and PCR amplification is carried out. As shown in FIG. 2, the minimum detection concentration of the primers FONDX-15F/FONDX-15R to the test strain genome Fonrace1 was 365pg/μ L.

Example 9 detection of Fusarium oxysporum of Mikan and Western melons in diseased plants

And performing PCR amplification by using the genome DNA of plant tissues 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d and 19d before root soaking and after inoculation of the extracted plants as a template, and detecting the fusarium oxysporum f.sp.sp.citrullus in the plants with diseases.

PCR reactionThe total amount should be 25 μ L, wherein 2 × Es Taq PCR MasterMix 12.5 μ L, upstream and downstream primers each 0.5 μ M, DNA template 2 μ L (200ng/uL), RNase-free Water ddH₂Make up to 25. mu.L of O.

PCR reaction procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, and extension at 74 ℃ for 30s for 30 cycles; finally, extension is carried out for 10min at 72 ℃ and for 10min in a supplementary manner.

PCR products of 5. mu.L were separately subjected to 2% agarose gel electrophoresis for 30min (90V), and the amplification results were detected by a gel imager.

The detection result of 'Zaojia 8424' is shown in FIG. 3, the 'Zaojia 8424' has no specific amplification before root dipping and negative control, 1dpi has weak bands, then until 7dpi, the brightness is gradually enhanced, the brightness of 9-15dpi does not obviously change, the brightness of the 17dpi and 19dpi samples is slightly reduced, and the detection result is probably related to that most of the plants are wilted and died after 15 dpi. After 'Zaojia 8424' is dipped in roots, 1-5 dpi of plants have no disease symptoms, and the watermelon disease index is 0; when the watermelon plant is at 7dpi, the watermelon plant begins to show slight wilting symptom, and the disease index of the watermelon plant is 38.33 percent; the disease index of a watermelon plant is continuously increased from 7dpi to 15dpi, and 17dpi reaches 95 percent; almost all watermelon plants of 19dpi withered and die (see table 1); the detection system can be used for qualitatively detecting pathogenic bacteria before and after the sick watermelon plants show diseases.

The detection result of the disease-resistant variety 'West farming 8' is shown in figure 4, the 'West farming 8' has no strip before root dipping and no negative control, specific strips can be amplified at 1-19dpi after inoculation, the strips are brighter on the 7 th day, watermelon plants have no disease at 0-13 dpi, slight wilting and wilting diseases begin to appear at 15dpi, the disease index is 5.00, and the disease index is 17.20 (see table 1); the detection system can be used for qualitative detection of pathogenic bacteria before and after disease of the disease-resistant watermelon plant.

TABLE 1 index of disease of watermelon plants inoculated with Fusarium oxysporum

Example 10 detection of pathogenic bacteria in soil

Inoculating the spores of No. 1 physiological race of the watermelon fusarium wilt pathogen with a certain concentration gradient to soil, extracting soil DNA, and performing PCR amplification by using a primer FONDX-15F/FONDX-15R. The primers can be from 5X 10⁵-5×10²Specific bands are amplified in cfu/g soil, the result is shown in figure 5, and the detection sensitivity of the primer to fusarium oxysporum spores in soil is 5 multiplied by 10²cfu/g. In the soil treatment of different germ amounts, disease-resistant varieties of watermelons all show the characteristics of late onset of disease and low plant incidence of plant diseases; as the quantity of germs decreases, the disease processes of resistant and susceptible varieties are gradually slowed down, and the disease incidence rate of plants is gradually reduced. 5X 10⁵cfu/g and 5X 10⁴Watermelon hosts treated by cfu/g soil have similar performances, disease symptoms of the anti-susceptible hosts and the susceptible hosts appear on 8 th and 5 th days after inoculation respectively, the disease symptoms reach the maximum on 20 th day, and the plant disease incidence rate of the plants is finally 8% and 100%; watermelon as host of 5X 10³cfu/g and 5X 10²In the treatment of cfu/g soil, the disease is more than 5 multiplied by 10⁴cfu/g～5×10⁵The cfu/g treatment performance is light, and the host disease resistance and host infection incidence rate is also small; the disease condition of 50cfu/g is present 12 days after inoculation, the disease condition is the slowest, the disease incidence rate of the final resistant and susceptible host plants is 2 percent and 32 percent, and the results are shown in table 2.

TABLE 2 watermelon incidence in soil of different germ contents (%)

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> university of agriculture in Hebei

<120> qualitative detection primer and detection method for watermelon fusarium wilt

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence

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aagccactcc atcacatc 18

Claims (5)

1. The primer for qualitatively detecting the fusarium oxysporum f.sp.cubense is characterized by comprising the following primer sequences:

FONDX-15F：5’-GCTCGTAAGTCAGGTTCA-3’；SEQ ID NO：1；

FONDX-15R：5’-AAGCCACTCCATCACATC-3’；SEQ ID NO：2。

2. a screening method of primers for qualitatively detecting fusarium oxysporum f.sp.cubense is characterized by comprising the following specific steps:

(1) designing a primer: based on a database of fusarium oxysporum genome, designing and screening specific primers of fusarium oxysporum according to the difference fragments;

(2) and (3) primer specificity screening: using genomic DNA of watermelon fusarium wilt bacteria Fon race 0, Fon race1, Fon race2, tomato fusarium wilt bacteria, cucumber fusarium wilt bacteria, melon fusarium wilt bacteria, cabbage fusarium wilt bacteria, cotton verticillium wilt bacteria and tomato verticillium wilt bacteria as templates, and RNase-free ddH₂Screening specific primers of fusarium oxysporum f.sp.cubense by taking O as a negative control, and screening out the specific primers of claim 1;

(3) the sensitivity of the specific primers was detected.

3. The screening method of primers for qualitatively detecting Fusarium oxysporum F sp niveum as claimed in claim 2, wherein the method for detecting sensitivity of specific primers in step (3) is as follows: the genome DNA of the No. 1 physiological race of the fusarium oxysporum f.sp.cubense is continuously diluted into six concentration gradients of 36.5 ng/mu L, 3.65 ng/mu L, 365 pg/mu L, 36.5 pg/mu L, 3.65 pg/mu L and 0.365 pg/mu L by a 10-fold concentration gradient, and the sensitivity of the specific primer is detected.

4. The use of the primer for qualitative detection of Fusarium oxysporum F.sp.sp.citrulli of claim 1 in the detection of Fusarium oxysporum F.sp.citrulli in plants and soil.

5. A qualitative detection method for watermelon fusarium wilt germs is characterized by comprising the following specific steps:

(1) extracting the watermelon plant genome DNA or soil genome DNA infected with pathogenic bacteria;

(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer of claim 1;

the PCR reaction system is as follows: 2 XEs Taq PCR Master Mix 12.5. mu.L, upstream and downstream primers 0.5. mu.M each, DNA template 2. mu.l, RNase-free Water ddH₂O is complemented to 25 mu L;

the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, and extension at 74 ℃ for 30s for 30 cycles; finally, extension is carried out for 10min at 72 ℃, and supplementary extension is carried out for 10 min;

(3) and (3) carrying out agarose gel electrophoresis detection on the PCR product obtained in the step (2).

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