CN103725782A - 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 method, test kit and the primer of lotus fungoid rot pathogenic bacteria

Technical field

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

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 planting both at home and abroad.China's lotus rhizome plantation history is very long, and the root of lotus rhizome, stem, fruit all contain abundant starch, protein and VITAMIN, have very high economic worth, are a kind of good aquatic vegetables, has become one of important crops of peasant economy growth.In recent years, along with crop mix adjustment, the lotus rhizome cultivated area of China has had by a relatively large margin to be increased, according to estimates, and current national lotus rhizome cultivated area approximately 330,000 hm ²above.

Lotus rhizome rot is to infect by Fusarium a kind of important soil-borne disease causing.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%, reaches more than 60%, even total crop failure when serious.First at the subterraneous stem of being injured (rhizoma nelumbinis), there is symptom in this disease, thereafter the ground blade just bearing at sick stem gradually and petiole show disease, hysteresis quality due to the pathogenetic environment singularity of corruption and the aobvious disease of blade, often causes and misses preventing and treating optimum period of this disease, causes Severe Reduction.And at present, scholars are how fusarium fungus is admitted to lotus rhizome rot pathogenic bacterium, but to determining we were telling you method differs of its kind, reported just have Fusarium oxysporum (Fusarium oxysporum), pearl sickle-like bacteria (F.moniliforme), Fusarium solani (F.poae), fusarium sambucinum (F.sambucinum) and a Fusarium semitectum (F.avenaceum) etc.Therefore, the classification position of lotus rhizome rot pathogenic bacteria is identified, and then whether lotus root kind is carried to the research that rot germ detects have positive effect; If can combine with sequencing by round pcr, efficiently realize the phylogenetic systematics between kinds different in 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 to the lotus fungoid rot of lotus root kind, is can reach the early diagnosis of lotus rhizome rot and prevent object, has important actual application value.

With regard to prior art, the molecular detection primer that obtains lotus rhizome rot pathogenic bacteria need to overcome the Identification of Species of 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 been carried out Identification of Species to lotus rhizome rot pathogenic bacterium, the clear and definite kind of lotus rhizome rot pathogenic bacteria, belong to Fusarium oxysporum lotus specialized form (F.oxysporum Schl.sp.nelumbicola (Nis.et Wat.) Booth), and further design specific detection primer 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 the specific detection primer for the design of Fusarium oxysporum lotus specialized form, and use it for detection lotus root kind and whether carry the method report of these lotus rhizome rot pathogenic bacterium or disclosed.

Summary of the invention

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

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 is carried lotus fungoid rot pathogenic bacteria:

Lotus root kind is sampled, extract 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, in testing sample, carry lotus fungoid rot pathogenic bacteria.

In pcr amplification reaction system, contain: 2.5 μ L are containing 15mM MgCl ₂10 * Trans Start Buffer; The dNTPs that 2.5 μ L concentration are 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 ℃ of denaturation 5min; Stage 2:94 ℃ 20s, 55 ℃ of 20s, 72 ℃ of 30s, circulate 34 times altogether; Stage 3:72 ℃ is extended 4min; Stage 4:4 ℃ 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 is carried 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 is carried 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, the kind of lotus rhizome rot pathogenic bacteria has been carried out to effective evaluation and be defined as Fusarium oxysporum lotus specialized form; For this exclusive sequencing result that causes lotus rhizome rot pathogenic bacteria, design the pair of primers to lotus rhizome rot germ genome high degree of specificity, in conjunction with round pcr, set up 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, the green prevention and control of lotus rhizome disease and Harmless plantation are had to great meaning.

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 piece; C: isolated strains is positive; 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 the different DNA content pcr amplification of Fusarium oxysporum lotus specialized form figure in embodiment 1;

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

Embodiment

Below in conjunction with embodiment, be intended to further illustrate the present invention, and unrestricted the present invention.

Embodiment 1:

A, the separation that supplies examination pathogenic bacteria:

For examination pathogenic bacteria, derive from the lotus rhizome that gathers in the lotus rhizome rot morbidity cultivation field on the ground such as Changsha County, Huang Xing town and Xiangtan County in spite of illness the separated pathogenic bacteria obtaining.

Described separating step is as follows:

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

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

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

(d), a small amount of mycelia of all growth colony edges on picking lotus root piece after 5d, be inoculated on PDA substratum and be further purified cultivation, observe the cultivation proterties of bacterial strain and also carry out microscopy, tentatively to judge the purifying of isolate.The spore again isolate being produced is with under sterilizing washing, suitably after dilution (being roughly diluted to the spore that only has 30 left and right in each culture dish), after getting a certain amount of spore suspension and melting, be cooled to the nutrient agar of 45 ℃ of left and right fully to mix, be poured in culture dish, 25 ℃ of constant temperature culture, transplant Mycelium culture from the bacterium colony of the single dispersion that forms.By single bacterium colony culture by separation and purification thing tieback to healthy lotus rhizome, 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 the rot pathogenic bacteria for examination as identical.

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

Concrete steps are as follows:

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

I. get the about 0.2g of mycelium 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), mix, in 65 ℃ of water-baths 1 hour, obtain mixture A;

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

III. in described supernatant A, add 2/3 volume Virahol to be used for precipitating DNA; Use again lavation buffer solution (76% ethanol, 10mM ammonium acetate) washing once, 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 ℃ of water-baths 1 hour; Use again equal-volume chloroform/primary isoamyl alcohol (24:1) extracting once, obtain supernatant liquor B;

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

VI. the DNA precipitation with 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 synthetic by the synthetic portion in Shanghai Sheng Gong company Shanghai;

Described pcr amplification, utilizes described in ITS1 and ITS4 primer pair for examination pathogenic bacteria and carries out containing in the reaction system (25 μ L) of full genescreen amplified reaction: 2.5 μ L are containing 15mM MgCl ₂10 * Buffer; The dNTPs that 2.5 μ L concentration are 2.5mM; 1U Taq archaeal dna polymerase; Concentration is primer I TS1 and the ITS4 difference 1 μ L 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 ℃ of denaturation 3min; Stage 2:95 ℃ 30s, 60 ℃ of 30s, 72 ℃ of 2min, circulate 30 times altogether; Stage 3:72 ℃ is extended 7min; Stage 4:4 ℃ 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 products, click and enter after pipettor mixes in 1% sepharose.100V electrophoresis, the time is 40min.

(c), the recovery of described PCR product

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 as far as possible little, then moves in 2ml centrifuge tube, and this operation steps is carried out under ultra violet lamp;

2) add binding buffer II 400ul, the about 10min of water-bath at temperature 50-60 ℃, after mixed solution shakes gently and mixes, moves in adsorption column;

3) carry out at normal temperatures the standing of 2min, then centrifugal 1min under rotating speed 5000rpm;

4) adding 500wash solution, is centrifugal 1min under 8000rpm at rotating speed, 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, standing 2min under normal temperature, centrifugal 1min under rotating speed 10000rpm;

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

(d), described pcr amplification product order-checking and analysis

By the PCR product to after reclaiming, be sent to San Bo polygala root company and check order, the DNA sequence dna of acquisition also carries out sequence alignment in ncbi database, from DNA molecular level, identifies the kind of lotus rhizome rot pathogenic bacteria.

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 that can stablize, 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:

The described checking material genomic dna of take is template, carries out pcr amplification reaction, obtains pcr amplification product;

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

Described primer is synthetic by the synthetic portion in Shanghai Sheng Gong company Shanghai;

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; The dNTPs that 2.5 μ L concentration are 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 ℃ of denaturation 5min; Stage 2:94 ℃ 20s, 55 ℃ of 20s, 72 ℃ of 30s, circulate 34 times altogether; Stage 3:72 ℃ is extended 4min; Stage 4:4 ℃ 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 that tries pathogenic bacteria

In steps A, by the ground rot generation lotus root Tanakas such as Changsha County, Xiangyin County and Xiangtan County are gathered to lotus rhizome and blade in spite of illness, utilize plate isolation method, picking list bacterium colony has obtained 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 piece; C: isolated strains is positive; D: the isolated strains back side).

2, for the cluster analysis of examination pathogen species

In step B-b, the representational sickle-like bacteria bacterial strain screening of take is DNA profiling, take ITS1 and ITS4 to carry out pcr amplification as primer.

Analytical results shows, representative bacterial strain all can obtain electrophoretic band by pcr amplification, and the length of the theoretical value of primer and the DNA of this bacterial strain conforms to, all strains testeds of this presentation of results have all obtained 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: separated representative bacterial strain).

In step B-c, described target fragment is cut to glue and reclaim, be sent to San Bo polygala root company and check order, the DNA sequence dna of acquisition, and carry out sequence alignment by ncbi database.

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

3, for the specificity of examination primer, verify

Utilize the sequencing result design Auele Specific Primer FO1_FR described in step B-c, the separated germ genomic dna of described different sources lotus rhizome rot is carried out to pcr amplification, analytical results shows (as shown in Figure 3), and 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)

Utilize the different bacterial strains for test card in step B-c to continue Auele Specific Primer FO1_FR to verify, result shows, only can specific amplification in Fusarium oxysporum lotus specialized form bacterial strain 337bp fragment, 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 entrained Fusarium oxysporum lotus rhizome specialized form of lotus rhizome and other pathogenic bacteria.

4, the sensitivity for examination primer detects

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

Fig. 5 is the different DNA content pcr amplification of Fusarium oxysporum lotus specialized form 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 piece, extract DNA and carry out pcr amplification, in morbidity lotus root piece, all can amplify the specific fragment of 337bp, and can not increase (as shown in Figure 6) in healthy lotus root piece.The Molecular Detection that this cover technology can be carried for the early stage rot germ of lotus rhizome is described, 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 piece-1; Swimming lane 2: healthy lotus root piece-2; Swimming lane 3: morbidity lotus root piece-1; Swimming lane 4: morbidity lotus root piece-2).

Claims (7)

1. detect lotus root kind and carry a method for lotus fungoid rot pathogenic bacteria, it is characterized in that:

Lotus root kind is sampled, extract 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, in testing sample, carry lotus fungoid rot pathogenic bacteria.

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

In pcr amplification reaction system, contain: 2.5 μ L are containing 15mM MgCl ₂10 * Trans Start Buffer; The dNTPs that 2.5 μ L concentration are 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 ℃ of denaturation 5min; Stage 2:94 ℃ 20s, 55 ℃ of 20s, 72 ℃ of 30s, circulate 34 times altogether; Stage 3:72 ℃ is extended 4min; Stage 4:4 ℃ 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 lotus root kind and carry a test kit for 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 lotus root kind and carry a primer for 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.

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