CN103725782B - Method, kit and primer for detecting pathogenic bacteria of lotus fungal rot disease carried by lotus seeds - Google Patents

Abstract

The invention discloses a method, kit and primer for detecting the pathogenic bacteria of lotus fungal rot disease carried by lotus seeds. In the invention, by utilizing the molecular biological technology, the type of the pathogenic bacteria of lotus rot disease is effectively identified and determined to be a fusarium oxysporum lotus-specialized type; a pair of primers with high specificity on the pathogenic bacteria genome of lotus rot disease is designed according to the sequencing result of the pathogenic bacteria exclusively causing the lotus rot disease; in combination with the PCR (polymerase chain reaction) technology, a fast and high-sensitivity molecular detection technology of the pathogenic bacteria of lotus rot disease is established; the technology can perform bacterium-carrying detection on the lotus seeds to realize early diagnosis and prevention of the lotus rot disease, and is of great significance to the green control of lotus disease and pollution-free planting.

Description

Detect lotus root kind and carry the method for lotus fungoid rot pathogenic bacteria, test kit and primer

Technical field

The invention belongs to the fungoid rot detection of pathogens technical field of lotus rhizome, be specifically related to a kind of lotus root kind that detects and carry the method for lotus fungoid rot pathogenic bacteria, test kit and primer.

Background technology

Lotus rhizome (containing lotus root lotus and seed lotus) belongs to nymphaeaceae plant, is a kind of perennial root herbaceous plant of extensively plantation both at home and abroad.China lotus rhizome plantation history is very long, and the root of lotus rhizome, stem, fruit are all containing abundant starch, protein and VITAMIN, and having very high economic worth, is a kind of excellent aquatic vegetable, has become one of important crops of peasant economy growth.In recent years, along with Planter industry structure adjustment, the lotus rhizome cultivated area of China has had to be increased by a relatively large margin, according to estimates, and current national lotus rhizome cultivated area about 330,000 hm ²above.

Lotus rhizome rot infects the important soil-borne disease of the one caused by Fusarium.In lotus rhizome planting process, it is the main primary source of infection of lotus rhizome rot that lotus root kind is carried disease germs, and this disease main harm lotus subterraneous stem and root, generally can cause lotus rhizome underproduction 15%-40%, reach more than 60%, even have no harvest time serious.First there is symptom at the subterraneous stem of being injured (rhizoma nelumbinis) in this disease, thereafter just disease is shown at the ground blade that sick stem bears and petiole gradually, because the pathogenetic environment singularity of corruption and blade show the hysteresis quality of disease, often cause the proper control time missing this disease, cause Severe Reduction.And at present, to be that fusarium fungus is many to lotus rhizome rot pathogenic bacterium admit scholars, but to the determination of its kind we were telling you method differs, reported just have Fusarium oxysporum (Fusarium oxysporum), pearl sickle-like bacteria (F.moniliforme), Fusarium solani (F.poae), fusarium sambucinum (F.sambucinum) and Fusarium semitectum (F.avenaceum) etc.Therefore, the classification position of lotus rhizome rot pathogenic bacteria is identified, and then whether the research that rot germ detects is carried to lotus root kind have positive effect; If can be combined with sequencing by round pcr, the phylogenetic systematics between kinds different in effective implemention fungi; Further by sequence alignment, determine the pathogenic bacterium kind monoid of lotus rhizome rot; In conjunction with sequence measurement, the specific detection primer of design lotus rhizome rot pathogenic bacteria, realizes the rapid detection of the lotus fungoid rot to lotus root kind, is the early diagnosis that can reach lotus rhizome rot with prevention object, has important actual application value.

With regard to prior art, the molecular detection primer obtaining lotus rhizome rot pathogenic bacteria needs the Identification of Species overcoming pathogenic bacterium, because the kind of the pathogenic bacterium of lotus rhizome rot also fails to determine completely, applied molecular biology technology of the present invention has carried out Identification of Species to lotus rhizome rot pathogenic bacterium, specify that the kind of lotus rhizome rot pathogenic bacteria, belong to Fusarium oxysporum lotus specialized form (F.oxysporum Schl.sp.nelumbicola (Nis.et Wat.) Booth), and design specific detection primer further according to this Fusarium oxysporum lotus specialized form, realize the Molecular Detection to lotus rhizome rot pathogenic bacteria of efficiently and accurately.At present, also not about specific detection primer design for Fusarium oxysporum lotus specialized form, and use it for and detect the method whether lotus root kind carry these lotus rhizome rot pathogenic bacterium and report or be disclosed.

Summary of the invention

An object of the present invention is to provide a kind of method that rapid detection lotus root kind carries lotus fungoid rot pathogenic bacteria, is directed to the efficient detection method of Fusarium oxysporum lotus specialized form pathogenic bacterium especially especially.

Two of object of the present invention is to provide the supporting test kit of aforesaid method and primer.

The object of the invention is to realize in the following manner:

A kind ofly detect the method that lotus root kind carries lotus fungoid rot pathogenic bacteria:

Lotus root kind is sampled, extracts sample gene group DNA as template, with

Upstream primer: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 ';

Carry out PCR reaction, if amplification obtains the band of 337bp, then carry lotus fungoid rot pathogenic bacteria in testing sample.

Contain in pcr amplification reaction system: 2.5 μ L are containing 15mM MgCl ₂10 × Trans Start Buffer; 2.5 μ L concentration are the dNTPs of 2.5mM; 1U Trans Start Taq DNA Polymerase; 2 μ L concentration are 10mM PCR upstream and downstream mix primer; 50ng template DNA; ddH ₂o supplies 25 μ L.

Pcr amplification reaction program is:

Stage 1:94 DEG C denaturation 5min; Stage 2:94 DEG C 20s, 55 DEG C of 20s, 72 DEG C of 30s, circulate 34 times altogether; Stage 3:72 DEG C extends 4min; Stage 4:4 DEG C of maintenance.

Described lotus fungoid rot pathogenic bacteria is Fusarium oxysporum lotus specialized form.

Lotus root kind described in aforesaid method comprises the lotus root kind of lotus root lotus or seed lotus.

A kind ofly detect the test kit that lotus root kind carries lotus fungoid rot pathogenic bacteria: comprise

Upstream primer: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 ';

Described lotus fungoid rot pathogenic bacteria is Fusarium oxysporum lotus specialized form.

A kind ofly detect the primer that lotus root kind carries lotus fungoid rot pathogenic bacteria:

Upstream primer: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 '.

Described lotus fungoid rot pathogenic bacteria is Fusarium oxysporum lotus specialized form.

Beneficial effect of the present invention is:

The present invention utilizes Protocols in Molecular Biology, has carried out effective qualification be defined as Fusarium oxysporum lotus specialized form to the kind of lotus rhizome rot pathogenic bacteria; The pair of primers to lotus rhizome rot germ genome high degree of specificity is designed for this exclusive sequencing result causing lotus rhizome rot pathogenic bacteria, in conjunction with round pcr, establish the molecular detection technology of a set of quick, highly sensitive lotus rhizome rot germ, this technology can detect carrying disease germs property of lotus root kind, realize early diagnosis and the prevention of lotus rhizome rot, have great meaning to the green prevention and control of lotus rhizome disease and Harmless plantation.

Accompanying drawing explanation

Fig. 1 is confession examination pathogenic bacteria and the source figure A of different sources in embodiment 1: morbidity lotus root leaf; B: morbidity lotus root block; C: isolated strains front; D: the isolated strains back side;

Fig. 2 is the pcr amplification electrophorogram of ITS sequence in embodiment 1;

Fig. 3 is different sources lotus rhizome rot germ genomic dna pcr amplification figure in embodiment 1;

Fig. 4 is different pathogenic bacterium genomic dna pcr amplification figure in embodiment 1;

Fig. 5 is Fusarium oxysporum lotus specialized form different DNA content pcr amplification figure in embodiment 1;

Fig. 6 is the Molecular Detection of lotus rhizome sample rot germ in embodiment 1.

Embodiment

Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.

Embodiment 1:

The separation of A, confession examination pathogenic bacteria:

Derive from for examination pathogenic bacteria in the lotus rhizome rot morbidity cultivation field on the ground such as Changsha County, Huang Xing town and Xiangtan County gather lotus rhizome in spite of illness be separated the pathogenic bacteria obtained.

Described separating step is as follows:

(a), the described clear water of lotus rhizome is in spite of illness cleaned, be cut into about 2mm with the scissors of sterilizing ²fritter or small pieces;

B (), the alcohol being placed in 75% carry out surface sterilization, soak 3-5min again after 10s in the acid mercuric chloride aqueous solution of 0.1%, then to use in sterilized water rinsing 3 times;

(c), utilize the thieving paper of sterilizing to blot the sterilized water of lotus rhizome block remained on surface, finally move into containing agricultural streptomycin (40 μ g/ml) PDA substratum in, the constant temperature culture of 25 DEG C;

D after (), 5d, a small amount of mycelia of all growth colony edges on picking lotus root block, is inoculated on PDA substratum and is further purified cultivation, observe the cultural colony of bacterial strain and carry out microscopy, tentatively to judge the purifying of isolate.Under the spore sterilizing washing again isolate produced, (spore only having about 30 is roughly diluted in each culture dish) after suitable dilution, getting a certain amount of spore suspension is cooled to the nutrient agar of about 45 DEG C fully to mix with after thawing, be poured in culture dish, 25 DEG C of constant temperature culture, transplant Mycelium culture from the bacterium colony of the single dispersion formed.By single bacterium colony culture by separation and purification thing tieback on the lotus rhizome of health, treat that lotus rhizome shows rot classical symptom, incidence tissue is repeated to the separation of the pathogenic bacteria of aforesaid method, determining whether to obtain the pure growth identical with the pathogenic micro-organism that sets out, is then the rot pathogenic bacteria for examination as identical.

B, the cluster analysis of confession examination pathogen species: extract pathogenic bacteria DNA, ITS sequence amplimer ITS1 and ITS4 of fungi is utilized to carry out pcr amplification, reclaim by cutting glue to pcr amplification product and check order, sequence alignment is carried out again, from the attribute of DNA molecular level qualification lotus rhizome rot germ in conjunction with ncbi database.

Concrete steps are as follows:

(a), described confession examination pathogenic bacteria gene group DNA extraction:

I. get mycelium and be about 0.2g grind into powder in liquid nitrogen, then add 2%CTAB extracting solution (2%CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0,20mM EDTA pH8.0,1%PVP-40,0.2% beta-mercaptoethanol), mixing, in 65 DEG C of water-baths 1 hour, obtains mixture A;

II. described mixture A is stopped water-bath, and add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down mixing, under 12000g, centrifugal 15min, discards precipitation; Add the sodium acetate soln of the 5M of 1/3 volume, mixing, ice bath 20min; Add isopyknic chloroform/primary isoamyl alcohol (24:1) extracting twice again, obtain supernatant A;

III. in described supernatant A, add 2/3 volume isopropanol be used for precipitating DNA; Use lavation buffer solution (76% ethanol, 10mM ammonium acetate) to wash once again, dry up, then add TE damping fluid (10mM Tris-HCl, 1mM EDTA, pH7.4) dissolving, obtain solution A;

IV. in described solution A, add RNase A, make its final concentration reach 100 μ g/ml, then mix 37 DEG C of water-baths 1 hour; Use equal-volume chloroform/primary isoamyl alcohol (24:1) extracting more once, obtain supernatant liquor B;

V. add 1/2 volume 7.5M ammonium acetate, 2 times of volume dehydrated alcohols to described getting in supernatant liquor B, obtain DNA precipitation;

VI. by the DNA precipitation described in 70% washing with alcohol, dry up, add ddH ₂o dissolving DNA, obtains described genomic dna.

(b), described ITS1/ITS4 primer:

Described primer I TS1 is as follows:

ITS1 primer sequence: 5 '-TCCGTAGGTGAACCTGCGG-3 ';

Described primer I TS4 is as follows:

ITS4 primer sequence: 5 '-TCCTCCGCTTATTGATATGC-3 ';

Described ITS1/ITS4 primer choosing is synthesized by Shanghai Sheng Gong company Shanghai combining unit;

Described pcr amplification, utilizes described in ITS1 and ITS4 primer pair and carries out containing in the reaction system (25 μ L) of full genome screening amplified reaction for examination pathogenic bacteria: 2.5 μ L are containing 15mM MgCl ₂10 × Buffer; 2.5 μ L concentration are the dNTPs of 2.5mM; 1U Taq archaeal dna polymerase; Concentration is primer I TS1 and the ITS4 1 μ L respectively of 10mM; 50ng template DNA; ddH ₂o supplies 25 μ L; Taq archaeal dna polymerase and reaction buffer are purchased from TaKaRa company.DNTPs is purchased from Beijing Quanshijin Biotechnology Co., Ltd;

Pcr amplification reaction program is: stage 1:95 DEG C denaturation 3min; Stage 2:95 DEG C 30s, 60 DEG C of 30s, 72 DEG C of 2min, circulate 30 times altogether; Stage 3:72 DEG C extends 7min; Stage 4:4 DEG C of maintenance.Wherein PCR instrument is the Veriti96well Thermal Cycler purchased from Applied Biosystems company;

The detection of pcr amplification and product: get amplified production and add 3 μ l sample-loading buffers (6 × Loading Buffer) in 15 μ l PCR primer, clicks and enters in the sepharose of 1% after pipettor mixing.100V electrophoresis, the time is 40min.

The recovery of (c), described PCR primer

Adopt QIAquick Gel Extraction Kits (Wei Er biotech firm) to reclaim test kit, concrete step is as follows successively:

1) use the agarose after aseptic operation cutter cutting electrophoresis, agarose is little as far as possible, and then move in 2ml centrifuge tube, this operation steps is carried out under ultra violet lamp;

2) add binding buffer II 400ul, at temperature 50-60 DEG C, water-bath is about 10min, after mixed solution shakes mixing gently, moves in adsorption column;

3) leaving standstill of 2min is carried out at normal temperatures, then centrifugal 1min under rotating speed 5000rpm;

4) add 500wash solution, under rotating speed is 8000rpm, centrifugal 1min, outwells liquid;

5) repeating step 4;

6) the centrifugal 30s of 10000rpm;

7) adsorption column moves on in the centrifuge tube of new 2ml, adds Elution buffer30ul, leaves standstill 2min, centrifugal 1min under rotating speed 10000rpm under normal temperature;

8) discard adsorption column, the product getting 5ul carries out electrophoresis detection, and remaining is stored in-20 DEG C.

(d), described pcr amplification product cloning and characterization

Check order by being sent to three rich polygala root companies to the PCR primer after reclaiming, the DNA sequence dna of acquisition also carries out sequence alignment in ncbi database, identifies the kind of lotus rhizome rot pathogenic bacteria from DNA molecular level.

The numerator detection mark of C, acquisition lotus rhizome rot germ, and described mark is verified: for supplying the ITS sequence sequencing result of examination pathogenic bacteria and designing Auele Specific Primer; In checking material, verify described primer, obtain the molecule marker stablizing, accurately detect lotus rhizome rot germ.

Described checking material comprises:

For examination lotus rhizome rot isolated strains as following table:

Described checking material comprises: Trichoderma (Trichoderma harzianum), anthrax bacillus (Bacillus anthraci), fusarium solani (F.solani), Alternariaspp (Alternaria), Phytophthora capsici (Phytophthora capsici) and Powdery Mildew (powdery mildew)

Step is as follows:

With described checking material genomic dna for template, carry out pcr amplification reaction, obtain pcr amplification product;

The extracting method of described checking material genomic dna is see step B;

Described primer is synthesized by Shanghai Sheng Gong company Shanghai combining unit;

Described FO1_FR primer is as follows:

Upstream primer is: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer is: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 ';

Described FO1_FR primer carries out containing in pcr amplification reaction system (25 μ L): 2.5 μ L are containing 15mMMgCl ₂10 × Trans Start Buffer; 2.5 μ L concentration are the dNTPs of 2.5mM; 1U Trans Start Taq DNA Polymerase; 2 μ L concentration are 10mM PCR upstream and downstream mix primer; 50ng template DNA; ddH ₂o supplies 25 μ L; Trans Start Taq DNA Polymerase is purchased from Beijing Quanshijin Biotechnology Co., Ltd, and dNTPs is purchased from TaKaRa company;

Pcr amplification reaction program is: stage 1:94 DEG C denaturation 5min; Stage 2:94 DEG C 20s, 55 DEG C of 20s, 72 DEG C of 30s, circulate 34 times altogether; Stage 3:72 DEG C extends 4min; Stage 4:4 DEG C of maintenance; Wherein PCR instrument is the Veriti96well Thermal Cycler of Applied Biosystems company;

The result in the present embodiment be analyzed as follows:

1, for the separation of examination pathogenic bacteria

In steps A, by gathering lotus rhizome in spite of illness and blade to the ground rot generation lotus root Tanaka such as Changsha County, Xiangyin County and Xiangtan County, utilize plate isolation method, picking list bacterium colony obtains 60 pathogen strain bacterium, as shown in Figure 1.Fig. 1 is confession examination pathogenic bacteria and the source (A: morbidity lotus root leaf of different sources; B: morbidity lotus root block; C: isolated strains front; D: the isolated strains back side).

2, for the cluster analysis of examination pathogen species

In step B-b, with the representational sickle-like bacteria bacterial strain screened for DNA profiling, with ITS1 and ITS4 for primer carries out pcr amplification.

Analytical results shows, representative bacterial strain all obtains electrophoretic band by pcr amplification, and the theoretical value of primer conforms to the length of the DNA of this bacterial strain, this result illustrates that all strains testeds all obtain the ITS sequence of rDNA by pcr amplification, as shown in Figure 2.Fig. 2 is the pcr amplification electrophorogram (M:marker of ITS sequence; Swimming lane 1 ~ 18: be separated representative bacterial strain).

In step B-c, cut glue reclaim described target fragment, the three rich polygala root companies that are sent to check order, the DNA sequence dna of acquisition, and carry out sequence alignment by ncbi database.

Analytical results shows, all Fusarium oxysporum is belonged to for examination pathogenic bacteria from the qualification of DNA molecular level, and reach 100% with the homology of Fusarium oxysporum lotus specialized form sequence D Q002550.1, therefore we can judge that lotus rhizome rot pathogenic bacteria is that Fusarium oxysporum belongs to lotus specialized form.

3, for the specificity verification of examination primer

Utilize the sequencing result design Auele Specific Primer FO1_FR described in step B-c, germ genomic dna is separated to described different sources lotus rhizome rot and carries out pcr amplification, analytical results display (as shown in Figure 3), 12 strain different sources checking pathogenic bacterias all can amplify the target fragment of a 337bp specifically.

Fig. 3 is different sources lotus rhizome rot pathogenic bacteria gene group DNA pcr amplification figure (M1:600bp DNA ladder; M2:2kb DNA ladder; Swimming lane 1-12: different sources lotus rhizome rot germ strain DNA; Swimming lane 13: negative blank)

The different bacterial strain for test card in step B-c is utilized to continue to verify Auele Specific Primer FO1_FR, result shows, only energy specific amplification 337bp fragment in Fusarium oxysporum lotus specialized form bacterial strain, and other strains tested and blank bacterium can not amplify target fragment (as shown in Figure 4).

Fig. 4 is different pathogenic bacterium genomic dna pcr amplification figure (swimming lanes 1: Fusarium oxysporum lotus specialized form; Swimming lane 2: Trichoderma; Swimming lane 3: anthrax bacillus; Swimming lane 4: Alternariaspp; Swimming lane 5: fusarium solani; Swimming lane 6: Phytophthora capsici; Swimming lane 7: Powdery Mildew)

The above results shows, primers F O1_FR designed in the present invention has specificity, can distinguish the Fusarium oxysporum lotus rhizome specialized form entrained by lotus rhizome and other pathogenic bacteria.

4, for the sensitivity technique of examination primer

In step B-c, dilute for examination lotus rhizome rot germ genomic dna, carry out pcr amplification with the DNA being diluted to the concentration gradient of 20ng, 2ng, 200pg, 20pg, 2pg and 1pg for template respectively, utilize above-mentioned set up detection system to detect the sensitivity of FO1_FR.Result display (Fig. 5): Auele Specific Primer FO1_FR energy stable detection is to the genomic dna of 1pg.

Fig. 5 is Fusarium oxysporum lotus specialized form different DNA content pcr amplification figure (M1:600bp marker; M2:2kb marker; Swimming lane 1:20ng; Swimming lane 2:2ng; Swimming lane 3:200pg; Swimming lane 4:20pg; Swimming lane 5:2pg; Swimming lane 6:1pg; Swimming lane 7:0pg)

5, the detection of pathogenic bacteria in sample

In step B-c, from the lotus rhizome of field, choose morbidity lotus root block, extract DNA and carry out pcr amplification, all can amplify the specific fragment of 337bp in morbidity lotus root block, and can not increase (as shown in Figure 6) in healthy lotus root block.Illustrate that this cover technology can be used in the Molecular Detection that the early stage rot germ of lotus rhizome carries, to reach the effect of early diagnosis, early prevention.

Fig. 6 is Molecular Detection (the M1:600bp marker of lotus rhizome sample rot germ; M2:2kb marker; Swimming lane 1: healthy lotus root block-1; Swimming lane 2: healthy lotus root block-2; Swimming lane 3: morbidity lotus root block-1; Swimming lane 4: morbidity lotus root block-2).

Claims (7)

1. detect the method that lotus root kind carries lotus fungoid rot pathogenic bacteria, it is characterized in that:

Lotus root kind is sampled, extracts sample gene group DNA as template, with

Upstream primer: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 ';

Carry out PCR reaction, if amplification obtains the band of 337bp, then carry lotus fungoid rot pathogenic bacteria in testing sample.

2. method according to claim 1, is characterized in that,

Contain in pcr amplification reaction system: 2.5 μ L are containing 15mM MgCl ₂10 × Trans Start Buffer; 2.5 μ L concentration are the dNTPs of 2.5mM; 1U Trans Start Taq DNA Polymerase; 2 μ L concentration are 10mM PCR upstream and downstream mix primer; 50ng template DNA; DdH2O supplies 25 μ L.

3. method according to claim 1 and 2, is characterized in that,

Pcr amplification reaction program is:

Stage 1:94 DEG C denaturation 5min; Stage 2:94 DEG C 20s, 55 DEG C of 20s, 72 DEG C of 30s, circulate 34 times altogether; Stage 3:72 DEG C extends 4min; Stage 4:4 DEG C of maintenance.

4. method according to claim 1, is characterized in that, described lotus fungoid rot pathogenic bacteria is Fusarium oxysporum lotus specialized form.

5. method according to claim 1, is characterized in that, lotus root kind comprises the lotus root kind of lotus root lotus or seed lotus.

6. detect the test kit that lotus root kind carries lotus fungoid rot pathogenic bacteria, it is characterized in that: comprise

Upstream primer: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 ';

Described lotus fungoid rot pathogenic bacteria is Fusarium oxysporum lotus specialized form.

7. detect the primer that lotus root kind carries lotus fungoid rot pathogenic bacteria, it is characterized in that:

Upstream primer: 5 '-AGGACCCCTAAACTCTGTTTCTAT-3 ';

Downstream primer: 5 '-CAGTTGCGAGGGTTTTACTACTAC-3 '.

Described lotus fungoid rot pathogenic bacteria is Fusarium oxysporum lotus specialized form.