CN110734922A - Detection method of Colletotrichum camelliae - Google Patents

Abstract

The invention relates to a detection method of kinds of anthracnose bacteria Colletotrichum camelliae of tea trees, which comprises the steps of extracting genome DNA of tea tree leaves, amplifying the genome DNA by using specific primers CcGAPDH-F/CcGAPDH-R and Cs18SrDNA1-F/Cs18SrDNA1-R through a fluorescent quantitative PCR method to obtain an amplified Ct value, and calculating 2^‑ΔCtValue, obtain 2^‑ΔCt(CcGAPDH) and 2^‑ΔCt(Cs18SrDNA1), and the pathogenic bacteria onset can be compared according to the ratioThe invention can detect and monitor the occurrence, expansion and prevalence of the Colletotrichum camelliae in the tea-producing area, is widely used for resistance screening of tea plant varieties and judging pathogenicity difference of different pathogenic bacteria, and particularly, the sequence of the pathogenic bacteria is adopted to be compared with the sequence of a host plant, so that the development of the pathogenic bacteria can be monitored along with the change of time.

Description

Detection method of Colletotrichum camelliae

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a detection method of kinds of tea tree anthracnose pathogenic bacteria Colletotrichum camelliae, which is applied to early diagnosis of tea tree anthracnose, monitoring and identification of pathogenic bacteria, and is particularly suitable for identification of tea tree resistant varieties and evaluation of pathogenic bacteria pathogenicity.

Background

Tea is prepared from sprout of Camellia sinensis (L.) O.Kuntze. Up to now, there are over 300 kinds of tea plant diseases that are internationally described and cause a reduction in tea yield, and the harmful parts include leaves, roots, flowers, fruits, and the like of tea plants. Because the harvesting part of the tea tree is new, the damage of the leaf diseases of the tea tree is larger than that of other crops.

More than 100 tea tree diseases recorded in China exist, in recent years, the diseases are more in an ascending trend due to the fact that fertilizer and water conditions are greatly improved and clone improved varieties are pushed in a large area, tea tree anthracnose is of main leaf diseases of tea trees, and the Colletotrichum camelliae is of dominant pathogenic bacteria causing the tea tree anthracnose and is widely distributed in various tea production areas in China.

Currently, analysis of disease resistance and pathogenicity of pathogenic bacteria in tea varieties relies on quantifying the growth of pathogenic bacteria using the size of lesions in the infected area of leaves. The disadvantages of the above methods are that they do not detect subtle changes in the pathogenic process acutely and do not quantify the growth of pathogens in the early stages of infection.

Disclosure of Invention

The invention provides genes for coding Colletotrichum camelliae GAPDH of tea tree, the nucleotide sequence is shown as SEQ ID NO.1, the length is 82bp, and the genes are named as CcGAPDH, the nucleotide sequence is searched in an international gene library, and the genes belong to 3-glyceraldehyde phosphate dehydrogenase, the invention provides specific primers for amplifying the genes for coding Colletotrichum camelliae GAPDH of tea tree, the nucleotide sequences are shown as SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequences are named as CcGAPDH-F, CcGAPDH-R, the invention provides specific primers for amplifying the SrDNA sequence cloned from the genome of tea tree Camellia sinensis, the nucleotide sequence is shown as Cs18SrDNA1, the nucleotide sequence is shown as SEQ ID NO.4, the sequence length is bp., the invention provides specific primers for amplifying the SrDNA sequence cloned from the genome of tea tree, the nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO. 866, the nucleotide sequence is shown as SEQ ID NO.5, the sequence, the SrGAPDH-8678 and the Sr18 DNA shown as 3618-8678 respectively.

The invention provides a detection method of kinds of tea tree Colletotrichum camelliae, which comprises the following steps:

, extracting genomic DNA of tea tree leaves, wherein the tea tree leaves comprise diseased leaves and non-diseased leaves;

step two, amplifying the genomic DNA obtained in the step by a fluorescent quantitative PCR method by using the primer CcGAPDH-F, CcGAPDH-R to obtain a Ct value, and calculating 2^-ΔCtA value;

step three, amplifying the genomic DNA obtained in the step by using the primer Cs18SrDNA1-F, Cs18SrDNA1-R through a fluorescent quantitative PCR method to obtain a Ct value, and calculating 2^-ΔCtA value;

step four, 2 obtained in step two^-ΔCt(CcGAPDH) value is molecular, 2 obtained in step three^-ΔCt(Cs18SrDNA1) as denominator, if the ratio is 0, the pathogen is not detected; if the ratio is more than 0, the tea plant is infected with the pathogenic bacteria; and comparing the pathogenic degree of the pathogenic bacteria and the disease resistance difference of the tea tree varieties according to the ratio.

The invention also provides a detection kit for tea tree Colletotrichum camelliae, which comprises the following specific components of a kit 1 and a kit 2, wherein the kit comprises primers CcGAPDH-F and CcGAPDH-R and quantitative PCR reaction mixed liquor, and the primers Cs18SrDNA1-F and Cs18SrDNA1-R and quantitative PCR reaction mixed liquor.

The detection method provided by the invention is used for detecting and monitoring the occurrence, expansion and prevalence of the tea-producing region Colletotrichum camelliae, is widely used for resistance screening of tea plant varieties, and judging pathogenicity difference of different pathogenic bacteria, particularly the sequence comparison of the pathogenic bacteria and the sequence of a host plant is adopted, and the development of the pathogenic bacteria can be monitored along with the change of time.

Drawings

FIG. 1 is a schematic diagram of the electrophoresis of CcGAPDH and Cs18SrDNA1 for the amplification of Colletotrichum tea camelliae and Camellia sinensis respectively;

FIG. 2 is a schematic diagram showing the amplification curves of CcGAPDH and Cs18SrDNA1 for respectively amplifying Colletotrichum tea camelliae and tea tree Camellia sinensis;

FIG. 3 is a schematic diagram showing the electrophoretic detection of CcGAPDH-specific amplified Colletotrichum camelliae;

FIG. 4 is a schematic diagram showing the onset symptoms and quantitative PCR detection of L J43 inoculated by Colletotrichum camelliae;

FIG. 5 is a schematic diagram showing the onset symptoms and quantitative PCR detection of tea anthracnose bacterium Colletotrichum camelliae inoculated with LJ43 at different times;

FIG. 6 is a schematic diagram showing the quantitative PCR detection of different varieties of tea tree inoculated with Colletotrichum camelliae;

FIG. 7 is a schematic diagram of quantitative PCR detection of different strains of Colletotrichum camelliae inoculated with LJ43 tea tree.

Detailed Description

Through the amplification ratio of CcGAPDH and Cs18SrDNA1, can be widely applied to detecting and monitoring the generation and growth of Colletotrichum Colletotrichum, resistance screening of tea tree varieties and tea charcoalThe pathogenicity of different strains of the Scutellaria melilotrichum camelliae. Inoculating tea tree Longjing No. 43 (LJ43) with tea anthracnose bacterium Colletotrichum calameliae spores, performing moisture-retention closed culture for 3 days, collecting spores and leaves, extracting genome DNA, amplifying the genome DNA by using a primer CcGAPDH-F/CcGAPDH-R and a primer Cs18SrDNA1-F/Cs18SrDNA1-R respectively through a fluorescent quantitative PCR method to obtain an amplified Ct value, and calculating 2^-ΔCtValue, obtain 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA 1). If the ratio is 0, the pathogenic bacteria are not detected; if the ratio is more than 0, the tea plant is infected with the pathogenic bacteria; based on the ratio, the degree of the pathogenic bacteria can be compared. Inoculating Colletotrichum gallinae spore to tea tree Longjing No. 43 (LJ43) and tea 108(ZC108) in tea tree variety, extracting genome DNA, and performing quantitative PCR analysis on disease resistance difference of tea tree variety to be detected. Inoculating different strains of tea anthracnose bacterium Colletotrichum camelliae to tea tree Longjing No. 43 (LJ43), extracting genome DNA, and performing quantitative PCR analysis on pathogenicity difference of different strains of tea anthracnose bacterium Colletotrichum camelliae to be detected.

Example 1: primer design and quality detection of CcGAPDH sequence and Cs18SrDNA1 sequence

Primers CcGAPDH-F and CcGAPDH-R designed for amplifying a Colletotrichum camelliae CcGAPDH gene fragment (82bp) and primers Cs18SrDNA1-F and Cs18SrDNA1-R for amplifying a tea plant Cs18SrDNA1 gene fragment (167bp) are screened from an international nucleotide database.

CcGAPDH-F and CcGAPDH-R, Cs18SrDNA1-F and Cs18SrDNA1-R are used as primers, and tea anthracnose bacterium and tea plant genome DNA are used as templates, and CcGAPDH and Cs18SrDNA1 genes are respectively amplified to obtain an amplification band of a single shown in figure 1 and an amplification curve of a single shown in figure 2.

CcGAPDH-F and CcGAPDH-R are used as primers to amplify different pathogenic bacteria of tea tree leaf, and the primers can specifically amplify different strains of Colletotrichum camelliae and Colletotrichum camelliae (figures 3, 1-6).

TABLE 1

Serial number	Primer name	Primer sequences
				1	CcGAPDH-F	Shown as SEQ ID NO.2
2	CcGAPDH-R	Shown as SEQ ID NO.3
			3	Cs18SrDNA1-F	Shown as SEQ ID NO.5
4	Cs18SrDNA1-R	Shown as SEQ ID NO.6

Example 2: pathogenic bacteria infecting tea tree

Inoculating the pathogenic bacteria in PDA culture medium, placing in incubator (22 + -2 deg.C, 12h light and 12h dark), and culturing for 10 d. Collecting spores, washing, and treating with ddH₂O dilution to 1 x 10⁶Inoculating clean tea tree leaves with 6-8 drops, cutting wound with blade before inoculation, and culturing all leaves at high humidity (>80%) of the cells were stored in sealed glass covers and placed in an incubator with strict control of the light and temperature (25. + -. 2 ℃,12h light and 12h dark). After inoculation, lesion size was measured. After infection for different time, collecting leaves and pathogenic bacteria, and freezing at-80 deg.CAnd (4) storing for DNA detection.

Example 3 extraction of Colletrichum camelliae and tea Tree genomic DNA

The samples were frozen in liquid nitrogen and homogenized with a tissue disruptor (Qiagen, Hilden, Germany) at 30 times/sec for 2X 30 seconds. mu.L of DNA extraction buffer (200mM Tris-HCl, pH 7.5; 250mM NaCl; 25mM EDTA; 0.5% SDS) was added and shaken again in the tissue disruptor at 30 times/sec for 10 seconds. The metal is bathed for 60min at 65 ℃, and shaken up every 5-10min in the middle. The EP tube was taken out, cooled slightly, an equal volume of chloroform-isoamyl alcohol (24:1) was added to the tube in a hood, the mixture was turned upside down and mixed, and centrifuged at 12000rpm for 15min at room temperature. The supernatant (about 450. mu.L) was aspirated, mixed with an equal volume of chloroform-isoamyl alcohol (24:1), and centrifuged at 12000rpm for 15 min. Sucking supernatant fluid 300 μ L, adding 2.5 volume of anhydrous ethanol (-precooling at 20 deg.C), and precipitating at 20 deg.C for 10 min. Centrifuge at 12000rpm for 15min, leave a precipitate, and wash twice with 70% ethanol. Blow-drying at 55 deg.C in metal bath, and blowing with 30 μ LddH₂And dissolving the O. Storing at-20 deg.C. At least three replicates per DNA sample.

Example 4: fluorescent quantitative PCR method for detecting target gene

About 30ng of the DNA template extracted in example 3 was mixed with 0.4mM primer CcGAPDH-F, CcGAPDH-R and SYBRPremix Ex Taq II (RNaseH Plus) (2 XConc.) in a total of 25. mu.L. The Ct value obtained with 12.5. mu.L LSYBR, 1.5. mu.L forward and reverse primers, 9. mu.L ddH2O and 2. mu.L template DNA.

About 30ng of the DNA template extracted in example 3 was mixed with 0.4mM primer Cs18SrDNA1-F, Cs18SrDNA1-R and SYBR Premix Ex Taq II (RNaseH Plus) (2 XConc.) in a total of 25. mu.L. The Ct value obtained with 12.5. mu.L SYBR, 1.5. mu.L forward and reverse primers, 9. mu.L ddH2O and 2. mu.L template DNA in the reaction mixture.

qPCR was performed using Applied Biosystems7500Sequence Detection System (Applied Biosystems7500Sequence Detection System, USA) from ABI, USA, and 3 repetitions were performed, with the PCR program 95 ℃ for 1min, 95 ℃ for 15s,60 ℃ for 34s followed by fluorescence Detection for 40 cycles, each primer pair contained non-templated controls in each run, Applied Biosystems from ABI, USAems7500 software and microsoft office Excel software, the results were analyzed. Calculate respective 2^-ΔCtValue, step 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA 1). And judging whether the disease occurs or not and the severity of the disease according to the ratio.

Example 5: statistical analysis

Analysis of variance (ANOVA) was performed on lesion size and qPCR data using SPSS 18 software (IBM, USA) and LSD test. Reported values are expressed as means ± standard deviation (means ± standard deviation). All treatments were independently repeated 3 times with a P value <0.05 as a significant difference.

Example 6: quantitative PCR detection of L J43 pathogenicity of Colletrichum camelliae

Inoculating LJ43 leaf with pathogenic bacteria, keeping humidity in sterile water for 3 days, observing lesion size, collecting leaf, extracting two groups of leaf genome DNA, amplifying the two groups of leaf genome DNA by CcGAPDH-F and CcGAPDH-R, and calculating 2 according to Ct value obtained by amplification^-ΔCtThe two groups of leaf genome DNAs were amplified using the Cs18SrDNA1-F and Cs18SrDNA1-R, and Ct values obtained by the amplification were calculated 2^-ΔCtValue, obtain 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA 1).

FIG. 4CK (sterile Water group) 2^-ΔCt(CcGAPDH) and 2^-ΔCtThe ratio of (Cs18SrDNA1) was about 0, indicating that no infestation by this pathogen was detected. In FIG. 4, 2 of the samples 3 days after infection of LJ43 with Colletotrichum camelliae^-ΔCt(CcGAPDH) and 2^-ΔCtThe ratio of (Cs18SrDNA1) was about 0.0012. Compared with CK (sterile water group), the difference is significant, which indicates that Colletrichum camelliae in the case has infected LJ43 and shows pathogenicity.

Example 7: quantitative PCR detection of growth amount and growth tendency of Colletrichum camelliae on LJ43 variety

Inoculating LJ43 leaf to pathogenic bacteria, culturing in a sealed and humidified environment for 6 days, observing lesion size every day, collecting leaf tissue, extracting genome DNA, amplifying the genome DNA by using CcGAPDH-F and CcGAPDH-R, and amplifying Ct obtained by amplificationValue, calculation 2^-ΔCtThe Ct value obtained by amplifying the genomic DNA using the Cs18SrDNA1-F and Cs18SrDNA1-R was calculated as 2^-ΔCtValue, obtain 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA 1).

In FIG. 5, the upper panel shows the development of lesions after different days of inoculation; the lower diagram is according to 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA1) was plotted to obtain the growth of Colletotrichum camelliae at different times when the tea plant LJ43 was infected. As can be seen, with increasing time, the lesion was observed to spread on day 3, and then the lesion gradually increased; by using the detection method of the invention, pathogenic bacteria can be detected on the 1 st day, and the growth amount of the pathogenic bacteria is gradually increased along with the increase of time.

Example 8: quantitative PCR detection of disease occurrence of Colletotrichum camelliae on different varieties of tea trees

Selecting two national-grade varieties of Longjing 43 and Zhongcha 108 of tea tree, respectively inoculating Colletochumcamelliae, culturing in a closed environment for different times while keeping moisture, extracting genome DNA, amplifying the genome DNA by using the CcGAPDH-F and CcGAPDH-R, and calculating 2 according to Ct value obtained by amplification^-ΔCtThe Ct value obtained by amplifying the genomic DNA using the Cs18SrDNA1-F and Cs18SrDNA1-R was calculated as 2^-ΔCtValue, obtain 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA 1). The disease incidence of Colletotrichum camellliae on different varieties can be obtained by comparing the values.

FIG. 6 the results show that the tea plant LJ43 was 2 days after 6 days of Colletotrichum camelliae inoculation^-ΔCt(CcGAPDH) and 2^-ΔCtThe ratio of (Cs18SrDNA1) was as high as 0.18, whereas in tea tree ZC108, the ratio was close to 0. The detection result of the growth amount of the Colletotrichum camelliae on different varieties shows that the resistance of the tea variety ZC108 to the bacteria is higher than that of LJ 43. The method provided by the invention can be used for screening the resistance difference of different varieties of tea trees to the Colletotrichum camelliae.

Example 9: quantitative PCR detection of the disease condition of different Colletotrichum camelliae strains on tea tree LJ43

Selecting different strains of Colletrichum camelliae, taking LJ43 as a test variety, respectively inoculating pathogenic strains ZJ1A8, LS _19 and ZJ1A5, and performing closed moisture-preserving culture to extract pathogenic leaf genome DNA of three pathogenic bacteria; amplifying the genomic DNA by using the primer CcGAPDH-F, CcGAPDH-R, obtaining Ct value by amplification, and calculating^2-ΔCtThe Ct value is calculated by amplifying the genomic DNA by using the primer Cs18SrDNA1-F, Cs18SrDNA1-R and obtaining the Ct value by amplification^2-ΔCtA value; obtained 2^-ΔCt(CcGAPDH) and 2^-ΔCt(Cs18SrDNA 1).

The pathogenic conditions of different strains of Colletotrichum camelliae on the strain LJ43 can be obtained by comparing the values. FIG. 7 shows that the pathogenicity of Colletrichum camelliae strain ZJ1A8 is higher than LS _19 and ZJ1A5, which indicates that the method provided by the invention can be used for evaluating the pathogenicity difference of different strains of Colletrichum camelliae.

SEQUENCE LISTING

<110> Jilin university

<120> detection methods of Colletotrichum camelliae

<130>2019.10.28

<160>6

<170>PatentIn version 3.5

<210>1

<211>82

<212>DNA

<213> Artificial Synthesis

<400>1

cccgcatctg gtagacaaga aggctatctt gacttgatgc caattaaaac catgggctgg 60

gacggaggga cacatgctat ca 82

<210>2

<211>20

<212>DNA

<213> Artificial Synthesis

<400>2

cccgcatctg gtagacaaga 20

<210>3

<211>20

<212>DNA

<213> Artificial Synthesis

<400>3

tgatagcatg tgtccctccg 20

<210>4

<211>167

<212>DNA

<213> Artificial Synthesis

<400>4

tgaaagacga acaactgcga aagcatttgc caaggatgtt ctcattaatc aagaacgaaa 60

gttgggggct cgaagacgat cagataccgt cctagtctca accataaacg atgccgacca 120

gggatcagcg gatgttactt ttaggactcc gctggcacct tatgaga 167

<210>5

<211>20

<212>DNA

<213> Artificial Synthesis

<400>5

tgaaagacga acaactgcga 20

<210>6

<211>19

<212>DNA

<213> Artificial Synthesis

<400>6

tctcataagg tgccagcgg 19

Claims (6)

1, genes for coding Colletotrichum camelliae GAPDH of tea tree, which is characterized in that the nucleotide sequence is shown as SEQ ID NO.1, the length is 82bp, and the gene is named as CcGAPDH.

The specific primers for amplifying the gene coding the Colletotrichum camelliae GAPDH as claimed in claim 1 are , and the nucleotide sequence is shown as SEQ ID NO.2 and SEQ ID NO.3 and is named as CcGAPDH-F, CcGAPDH-R.

3, kinds of 18SrDNA sequences cloned from the genome of tea tree Camellia sinensis, named Cs18SrDNA1, the nucleotide sequence is shown as SEQ ID NO.4, and the sequence length is 167 bp.

specific primers for amplifying the 18SrDNA sequence cloned from the genome of Camellia sinensis as claimed in claim 3, wherein the primers are represented by SEQ ID NO.5 and SEQ ID NO.6 and named Cs18SrDNA1-F and Cs18SrDNA1-R, respectively.

5, detection method of Colletotrichum camelliae of tea tree, which is characterized by comprising the following steps:

, extracting genomic DNA of tea tree leaves, wherein the tea tree leaves comprise diseased leaves and non-diseased leaves;

step two, amplifying the genomic DNA obtained in the step by a fluorescent quantitative PCR method by using the primer CcGAPDH-F, CcGAPDH-R to obtain a Ct value, and calculating 2^-ΔCtA value;

step three, amplifying the genomic DNA obtained in the step by using the primer Cs18SrDNA1-F, Cs18SrDNA1-R through a fluorescent quantitative PCR method to obtain a Ct value, and calculating 2^-ΔCtA value;

step four, 2 obtained in step two^-ΔCt(CcGAPDH) value is molecular, 2 obtained in step three^-ΔCt(Cs18SrDNA1) as denominator, if the ratio is 0, the pathogen is not detected; if the ratio is more than 0, the tea plant is infected with the pathogenic bacteria; and comparing the pathogenic degree of the pathogenic bacteria and the disease resistance difference of the tea tree varieties according to the ratio.

The detection kit for varieties of tea tree anthracnose bacteria Colletotrichum camelliae comprises the following specific components of a kit 1 and a kit 2, wherein the kit comprises primers CcGAPDH-F and CcGAPDH-R and quantitative PCR reaction mixed liquor, and the primers Cs18SrDNA1-F and Cs18SrDNA1-R and quantitative PCR reaction mixed liquor.

Images