CN107119139A

CN107119139A - The method of quick detection lily gray mold infective pathogen bacterium Botrytis cinerea

Info

Publication number: CN107119139A
Application number: CN201710446999.6A
Authority: CN
Inventors: 明军; 袁素霞; 曹雨薇; 徐雷锋; 杨盼盼
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2017-06-14
Filing date: 2017-06-14
Publication date: 2017-09-01

Abstract

The present invention relates to disease fungus technical appraisement field, it is related to the specific primer pair of PCR-based Molecular Detection gray mold pathogen Botrytis cinerea (Botrytis cinerea).Present invention solves the technical problem that being to provide specificity amplification primer to using method for identifying molecules, quick detection lily gray mold pathogen Botrytis cinerea.Not only overcome the strong shortcoming of traditional length fungal morphology qualification cycle, subjectivity, moreover, overcoming the problem of identification of ITS universal primers is not special accurate, reach that lily gray mold early stage efficiently and accurately is identified, precise and high efficiency prophylactico-therapeutic measures is taken as early as possible, the target of favourable effectively symptom management.The present invention is used for the specific primer for expanding Botrytis cinerea to being named as B.cin F/B.cin R.The primer pair of the present invention can accurately amplify the special purpose fragment of Botrytis cinerea.

Description

The method of quick detection lily gray mold infective pathogen bacterium Botrytis cinerea

Technical field

The present invention relates to Identification of The Fungal Species Causing technical field, it is related to a kind of for quick detection lily gray mold infective pathogen The specificity amplification primer pair of bacterium Botrytis cinerea.

Background technology

Lily is Liliaceae (Liliaceae) lilium (Lilium spp.) perennial herb bulbous plant.Lily be compared with Easily susceptible plant, wherein lily gray mold are to endanger one of lily more serious disease, seriously endanger lily growth and Quality, it is the most serious to blade and bud harm, with the plantation gradually expanded with long-term continuous cropping for the bulb planting area that carries disease germs Mode, disease occurrence degree is on the rise, and damaging range gradually expands, as seriously endanger lily production in important disease it One (Zhu Limei etc., 2009；2012).

Botrytis cinerea is the pathogen for causing lily gray mold, and Botrytis cinerea (Botrytis cinerea) is main cause of disease One of bacterium, the withered type hair Main Pathogenic Bacteria of leaf margin is Botrytis cinerea, and separation rate is 93.7%.Botrytis cinerea main harm is lily Blade, is started gradually to fall ill to whole blade from blade or leaf margin, circular or oval scab is formed on blade, scab is in Brown, finally makes leaf malformation withered, or even whole blade is withered, necrosis.During wet environment, scab has the mould layer of grey, after have Black sclerotium is generated.Occur yellowish-brown or bronzing bar shaped scab when stem catches an illness, basal part of stem hang contracting, fall to roll over, formed " on one side " bend gesture, blade along basal part of stem gradually upwards wither come off, bulb catch an illness can cause whole plant rot (Xu Qiong etc., 2006)。

Fungal identification mainly has traditional fungal morphology to identify and molecular biology identification.Traditional fungal morphology identification Cycle is long, and process is complicated, and strongly professional, and operating personnel's subjective consciousness is stronger.Molecular biology identification mainly uses fungi The universal primer of rDNA-ITS sequences Designs, ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 '), ITS4 (5 '- TCCTCCGCTTATTGATATGC-3 '), ITS5 (5 '-GGAAGTAAAAGTCGTAACAAGG-3 ') expanded (White et Al., 1990；Chen Fengmao, 2007), due to it is amplifiable go out most of basidiomycetes and sac fungus, specificity is strong, therefore also needs to Further it is sequenced and is compared, qualification cycle is long, somewhat expensive is sequenced, bring some difficult to germ identification, therefore exists scarce Fall into, it is necessary to improve improvement.

This patent provides the specificity amplification primer pair of rapid amplifying lily gray mold pathogen Botrytis cinerea, can be once Amplified reaction quick and precisely detects lily Botrytis cinerea pathogenic bacteria, shortens qualification cycle, reduces testing cost.

Leading reference：

1) diagnosis of the pretty lily gray molds of Zhu Limei, Hou Jianwen, Luo Fengxia, Jiang Qian and its isolating and purifying for pathogen [J] Jinling School of Science and Technology journals, 2009,25 (3)：59-63.

2) 8, Zhu Limei, Luo Fengxia, Li Mingfeng, Yin Ying Detroits etc. lily cultivar reflects to the disease resistance of lily gray mold Fixed [J] Jiangsu's agriculture science, 2012,40 (5)：81-83.

3) Xu Qiong, Xu Bingliang, Wang Fang bird watching leaf blight symptom types and pathogen identification [J] plant protection, 2006,32 (5)：61-64.

4) White T J, Bruns T, Lee S.Analysis of phylogenetic relationships by amplification and direct sequencing of ribosomal RNA genes[C].PCR Protoco is A Gudie to Methos and Applications, New York：Academic, 1990,15-22.

5) Chen Feng maos of fungies ITS region sequences structures and its application [J] forestry science and technology exploitations, 2007, (2)

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of quick detection lily gray mold infective pathogen bacterium Botrytis cinerea Specific primer pair, the molecular method of Rapid identification.

This programme inventive technique scheme is as follows：

1st, CTAB methods extract fungal DNA

2nd, for the PCR amplifications for the specific primer pair for detecting lily gray mold pathogen

The specific primer for designing Botrytis cinerea according to specific sequence enters performing PCR amplification to B.cin F/B.cin R, Rapid amplifying goes out the specific purpose band of Botrytis cinerea.

Brief description of the drawings

Fig. 1 is the testing result figure that to ITS1, ITS4 a variety of fungies are entered with performing PCR amplification using universal primer.M in Fig. 1 Swimming lane is Marker I, and clip size is 600bp, 500bp, 400bp, and 1 swimming lane is that universal primer expands Botrytis cinerea pathogen The PCR primer of increasing, 2 swimming lanes are the PCR primer that universal primer is expanded to tree peony grape spore pathogen, and 3 swimming lanes are universal primer pair The PCR primer of Fusarium oxysporum amplification, 4 swimming lanes are the PCR primer that universal primer is expanded to penicillium commune, and 5 swimming lanes are general The PCR primer of primer pair Phytophthora cactorum amplification, 6 swimming lanes are the PCR primer that universal primer is expanded to water, are used as negative control (negative control)。

Fig. 2 is the testing result that to B.cin F/B.cin R a variety of fungies are entered with performing PCR amplification using specific primer Figure.M swimming lanes are Marker I in Fig. 2, and clip size is 600bp, 500bp, 400bp, and 1 swimming lane is primer pair B.cin F/ B.cin R are to the pcr amplification product of Botrytis cinerea pathogen, and fragment length is 742bp, and 2 swimming lanes are primer pair B.cin F/ B.cin R are to the pcr amplification product of tree peony grape spore pathogen, and 3 swimming lanes are primer pair B.cin F/B.cin R to sharp spore reaping hook The pcr amplification product of bacterium, 4 swimming lanes are primer pair B.cin F/B.cin R to the pcr amplification product of penicillium commune, 5 swimming lanes For pcr amplification products of the primer pair B.cin F/B.cin R to Phytophthora cactorum, 6 swimming lanes are primer pair B.cin F/B.cin R to water The PCR primer of amplification, is used as negative control.

Fig. 3 is the aim sequence that primer pair B.cinF/B.cinR is expanded to Botrytis cinerea PCR, and length is 742bp.

Embodiment

1st, fungal DNA is extracted using CTAB methods

(1) separation of pathogen, purifying, Liquid Culture

Using infected leaves, 75% alcohol-pickled concussion 15s, mercuric chloride processing 12min, with aseptic water washing 3 times, every time one Minute, conventional organization partition method takes the strong intersection tissue of blade disease to be separated, and is placed in PDA culture medium center, 25 DEG C, dark Culture.After 5 days, with oese by fungus colony edge mycelia picking PDA culture medium, 25 DEG C, dark culturing.Repeat previous step Suddenly, until growing single kind of form bacterium colony in PDA culture medium.In ultra-clean work, the colony edge bacterium of picking culture 3-4 days Silk is put into potato dextrose broth.Fluid nutrient medium is put into shaking table and cultivated, 25 DEG C of temperature, rotating speed 180r/ Min, concussion and cultivate 3-4d.Bacterium solution, then the mycelia stayed in aseptic water washing on gauze are filtered with sterile gauze, to wash away mycelia On culture medium, collect mycelia.The moisture on mycelia is blotted with sterile gauze, liquid nitrogen grinding is standby into powder.

(2) CTAB extracts DNA

1. the hypha powder for taking 0.2g liquid nitrogen grindings good, add 650 μ L 2%CTAB Extraction buffers (20mmol/LEDTA, 1.4mol/LNaCI, 100mmol/LTris-HCl) it is transferred in 1.5mL centrifuge tubes, 65 DEG C, water-bath 1h (periods after rapid mixing Concussion is turned upside down every 15min once).

2. taken out after water-bath terminates and be cooled to room temperature, 12000rpm centrifuges 10min.

3. supernatant is taken in another centrifuge tube after centrifuging, and isometric chloroform-isoamyl alcohol (24: 1) is added, after mixing 12000rpm, centrifuges 10min.

4. previous step is repeated.

5. supernatant is drawn, isometric absolute ethyl alcohol (- 20 DEG C of precoolings) is added, -20 DEG C of standings after mixing precipitate 30- 60min。

6. 12000rpm, centrifuges 10min, abandons supernatant, and the precipitation being centrifuged out adds 75% ethanol 1mL and washed, 6000rpm, 5min is centrifuged, is repeated 2 times.

7. 15-30min is placed in room temperature or superclean bench, dries DNA, add 40-80 μ L ddH₂O dissolving DNAs, Saved backup in -20 DEG C.

2nd, enter performing PCR amplification to the fungi not belonged to together to ITS1/ITS4 using universal primer, then carry out Ago-Gel Electrophoresis detection, as shown in figure 1, it can be seen that not belonging to fungi together amplifies band from inspection result.

3rd, present embodiment is used for the species-specific primer of quick detection Botrytis cinerea to being named as B.cin F/B.cin R, enters performing PCR amplification to the fungi not belonged to together using primer pair B.cin F/B.cin R, then enters row agarose gel electrophoresis Detection, testing result amplify an about 742bp obvious spy as shown in Fig. 2 can be clearly seen that from testing result Different in nature band, the specific purpose band sequence is as shown in Figure 3.

Claims

1. quick detection lily gray mold infective pathogen bacterium --- the method for Botrytis cinerea, it is characterised in that：For detecting grey Portugal The PCR amplification system of the specific primer pair of grape spore is 20 μ L：The μ of 2 μ L, B.cin F of masterplate DNA, 0.5 μ L, B.cin R 0.5 L, 2 × Taq PCR Master Mix 10 μ L, ddH₂O 7μL。

2. quick detection lily gray mold infective pathogen bacterium --- the method for Botrytis cinerea according to claim 1, its The pcr amplification reaction program for being characterised by the specific primer pair for detecting Botrytis cinerea is：95℃ 3min；95 DEG C of 30s, 55.7 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃ 10min.

3. quick detection lily gray mold infective pathogen bacterium --- the method for Botrytis cinerea according to claim 1, its It is characterised by for the specific primer for detecting Botrytis cinerea to being named as B.cin F/B.cin R, forward primer B.cin F's Base sequence is 5 '-CCCGAGCTAATTTATGTTTTGC-3 ', reverse primer B.cin R base sequence for 5 '- GAGCCGAAGAAAGAGTAATG-3 ', purpose fragment length is 742bp.