CN110713936B - Cladosporium virens and single spore separation method and identification method thereof - Google Patents
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Abstract
The invention relates to a tea leaf spot pathogen and a single spore separation method and an identification method thereof, wherein the tea leaf spot pathogen is identified as Elsinoe ampelina, is preserved in China Center for Type Culture Collection (CCTCC) in 7-15 months in 2019, and has a preservation number of M2019555. The invention provides a new Elsinoe camelllia sinensis strain Cs52 from a diseased leaf, and is identified as a pathogenic bacterium of tea leaf powdery mildew. Lays a foundation for the development of the occurrence rule and biological prevention and treatment of the disease.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a tea leaf spot pathogen and a single spore separation method and an identification method thereof.
Background
Tea white spot (Tea white scab) is one of the most serious fungal diseases in high-altitude Tea gardens, and with the further development of mountain Tea gardens, the occurrence and harm of Tea white spot become more serious. When the tea plant is infected by the tea leaf botrytis cinerea, dense brown small spots or gray white round spots can appear on leaves, stems and buds, the yield is reduced by over 10 to 50 percent, and even leaves are not harvested. The dry tea made from the infected bud leaves has the advantages of small spots on the bottom, dark liquor color, and bitter taste. In production, chemical and agricultural medicines such as carbendazim and the like are generally adopted to prevent and control the disease, and the disease development period is usually rainy season, so that the prevention effect is not good, and the technical bottleneck of tea industry development is gradually developed. Accelerating the research on pathogenic mechanism and disease epidemic rule of tea leaf spot pathogen, and having urgent need for developing ecological prevention and control measures such as disease-resistant varieties. However, the research on the disease faces a very fundamental problem-the isolation and identification of pathogenic bacteria is questionable. Therefore, the separation and identification of the tea leaf spot pathogen needs to be accelerated, further research on epidemic laws of diseases needs to be carried out, the basic premise of pathogenic bacteria of the tea leaf spot is clarified, and scientific support is provided for ecological prevention and control measures such as tea leaf spot variety breeding and the like.
Disclosure of Invention
Based on the technical problems in the background technology, the invention provides a tea leaf spot fungus and a single spore separation method and an identification method thereof.
A Cladosporium Chaxinense is identified as Elsinoe camellias sinensis Cs52, and is preserved in China center for type culture Collection 7, 15.7.2019 at the preservation address of Wuhan university school with eight paths 299 in Wuchang district (the first attached small opposite side of Wuhan university) in Wuhan City, Hubei, with the preservation number of CCTCC NO: M2019555.
In some embodiments, the polypeptide comprises 4 nucleotide sequences, wherein 4 nucleotide sequences are shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
The invention provides a method for separating single spores of Cladosporium virens, which comprises the following steps:
s1, cutting diseased tissues at the junction of diseased keys on diseased leaves;
s2, sterilizing, cleaning and airing the diseased tissue;
s3, dipping the tissue processed in the step S2 on a sterile glass slide, dripping 1 drop of sterile water, keeping the tissue moist, storing the tissue in a moisture preserving chamber at 20-24 ℃ for 5 hours, lifting the small piece, performing microscopic examination, sucking water drops of conidia by a capillary tube, enabling the water drops to flow on PDA, and distributing the water drops on the whole surface of a watch glass with the diameter of 9 cm. And (3) after the bacterial colony grows to form spores, performing single spore separation by using an agar plate surface single spore picking method to obtain the single spores of the tea white star germs.
In some embodiments, in step S2, the sterilization process employs 3% sodium hypochlorite.
In some embodiments, the PDA formulation comprises the following ingredients: 200g of potato, 20g of glucose, 20g of agar powder and distilled water are added to a constant volume of 1L.
The invention also provides an identification method of the cladosporium cucumerinum, which comprises the following steps:
1) extracting the total DNA of a sample to be detected;
2) performing PCR amplification on the total DNA;
3) carrying out agarose gel electrophoresis detection on the amplification product;
4) recovering a target fragment, cloning the target fragment onto a vector, and screening positive clones by bacterial liquid PCR;
5) sequencing the positive clones.
In some embodiments, the PCR amplification system comprises rTaq enzyme 0.25. mu.L, dNTPs 0.4. mu.L, DNA template 1. mu.L, upstream and downstream primers 2. mu.L each, ddH2O is added to 50 mu L.
In some embodiments, the reaction conditions for the PCR amplification are: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s for 35 cycles, and finally extension at 72 ℃ for 10min, and storage at 4 ℃.
The invention has the beneficial effects that: the invention provides a new Elsinoe camelllia sinensis strain Cs52 from a diseased leaf, and is identified as a pathogenic bacterium of tea leaf spot. Lays a foundation for the development of the occurrence rule and biological prevention and treatment of the disease.
Drawings
FIG. 1 is a schematic representation of the field symptom phenotype of the bacterium California;
FIG. 2 is a schematic diagram showing the colony growth of Cladosporium virens;
FIG. 3 is a morphological schematic of T1 and T2 inoculants;
FIG. 4 is a phylogenetic tree diagram of T1;
FIG. 5 is a phylogenetic tree diagram of T2;
FIG. 6 is a schematic representation of the pathogenicity determination of the T1 and T2 pathogens.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention can be embodied in many different forms than those herein described and one skilled in the art can make similar modifications without departing from the spirit of the invention and it is therefore not limited to the specific embodiments disclosed below.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplification of description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
A Cladosporium theophrasti identified as Elsinoe camelllia sinensis, which is preserved in China center for type culture Collection in 7 months and 15 days in 2019, and the preservation number is CCTCC NO: M2019555.
In some embodiments, the polypeptide comprises 4 nucleotide sequences, wherein 4 nucleotide sequences are shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
In order to facilitate a further understanding of the present invention, the technical solutions of the present invention will now be described in detail with reference to preferred embodiments.
Example 1: single spore isolation of tea-white star germ
1. A single spore isolation method of tea leaf spot fungus comprises the following steps:
s1, collecting diseased leaves, washing with sterile water, and cutting diseased tissues into small pieces of 2mm multiplied by 2mm from the boundary of diseased positions;
s2, dipping the small piece of the diseased tissue on a sterile glass slide, dripping 1 drop of sterile water, keeping the small piece of the diseased tissue in a humidity keeping chamber for 5 hours at the temperature of 20-24 ℃, lifting the small piece, performing microscopic examination, and sucking water drops of conidia by a capillary tube. (ii) a
And S3, enabling water drops to flow on the PDA, distributing the water drops on the whole surface of a surface dish with the diameter of 9cm, placing the surface dish in an incubator for dark culture at 24 ℃ for 6d, and carrying out monospore separation by using a monospore picking method on the surface of an agar flat plate after a bacterial colony grows to form spores.
2. Morphological observation of Cladosporium virens
And (3) dyeing observation: the strain separated from the single spore is cultured to a stable stage in a constant temperature box at 25 ℃ by using a PDA plate culture medium, the morphological characteristics of the colony are recorded, and the morphological characteristics of the microspore are observed under an optical or electronic microscope. Soaking the diseased plaque in 98% absolute ethyl alcohol, boiling until the leaf color is clear, placing on a glass slide, dripping a drop of dye solution (20 ml of carbolic acid, 20ml of lactic acid, 40ml of glycerol, 20ml of distilled water and 50mg of cotton blue), covering a cover glass, slightly pressing to enable the specimen to be tiled for 5min, and performing microscopic examination.
And (3) observing by an electron microscope: separating single spore from strain, dipping in small amount of spore, fixing, dewatering, replacing, drying, preparing sample, spraying gold film, and observing germ spore form under Philips XL-30 scanning electron microscope.
3. Morphological characteristics of Cladosporium Chachiensis
The tea leaf spot is generated on tender shoots, tender leaves, petioles, tender stems and buds of a tea garden with the altitude of more than 400m, and the highest incidence is generated on the tender leaves. The primary stage of the tea leaf spot is a pinhead-shaped brown spot, the edge of the disease spot is provided with a yellow halo when the disease spot is illuminated, the center of the disease spot is sunken, the periphery of the disease spot is raised, the disease spot outwards expands around the primary spot, the number of the disease leaf spots reaches dozens, the diameter of the disease spot is not more than 2mm, and the disease spots are mutually fused to form a large irregular spot, so that the leaf is distorted, deformed or malformed, see a and b in fig. 1. There are also lesions on the tender stem and bud, but the number of lesions is small, usually scattered, see c and d in fig. 1.
358 parts of tea leaf pathogens of the tea leaf powdery mildew from different tea gardens are separated, purified and cultured, wherein most of the tea leaf pathogens comprise common tea plant disease pathogens such as anthracnose, alternaria leaf spot and the like, and endophytic fungi such as phomopsis and the like, and the content of the endophytic fungi is 35.9-64.71%. And the 150 strains obtained by separation were classified into two types of T1 type and T2 type according to colony morphology characteristics. Wherein the growth speed of the T1-HNGZ colony is 0.09cm/d, the initial stage is yellow brown, the later stage is red brown, the colony is in a shape close to a circular felt, the edge is provided with folds, the boundary is smooth and clear, the texture is compact and hard, the center position is provided with a hill-shaped bulge, the conidium pile is pink and sticky, the edge of the later stage colony is thin, white hypha is sometimes generated on the surface, and yellow pigment and other substances are secreted to ensure that the culture medium is yellow, which is shown in a and b in figure 2. The growth rate of the T2-HNBJ colony is 0.67cm/d, the early stage is gray brown, the later stage is black brown, the colony is fluffy and round, the edge is radial, and the center of the colony is provided with small granular spots, see c and d in figure 2. According to the survey, as shown in the table 1, T1 type bacteria and T2 type bacteria can be separated and obtained in 4 tea areas with different geographical positions and altitudes. Wherein, the proportion of T1 type is 5.24% -12.82%, the proportion of T2 type is 23.53% -51.28%, the proportion of T2 type is obviously larger than that of T1 type, and the higher the altitude is, the higher the proportion of T2 type separation is.
TABLE 1 statistics table for separation and purification of pathogenic bacteria of tea aventurine in different producing areas
4. Morphological Observation of isolated strains
Representative T1 type bacteria were inoculated to the leaf of tea tree to be tested and observed after 10 days for cotton blue staining, resulting in loose hyphae in the middle of the lesion as shown in a and b in FIG. 3. Wherein the ascomycetes is (6.0-18.5) μm × (12.0-28.5) μm furthermore, as shown in c in FIG. 2, sporangia appeared in T1 colonies after 7 days of culture on PDA medium. Microscopic examination of T1 bacteria at mature stage shows that there are many conidia on the colony surface, which are ellipse and translucent, and the size is (1.5-5.0) μm x (1.0-2.5) μm, see d, e and f in FIG. 3, and compared with the existing research report, the T1 strain has the same shape as the Elsinoe sp. After the T2 type representative bacteria are cultured for 10 days, the microscopic observation surface of the T2 type representative bacteria has a dark brown sphere with the diameter of 90-180 um, 1-3 orifices with the diameter of 17-33 um, conidia of the T2 type representative bacteria are irregular round colorless unicellular, and the conidia have the size of (3.4-5.0) Mumx (2.2-3.1) Mum, and are shown in g, h and i in a picture 3. The morphology of the T2 type strain is consistent with the characteristics of tea leaf-point mould and stem-point mould. In view of this, based on the multigenic molecular identification of T1-HNGZ and T2-HNBJ, representative strain T1 (T1-HNGZ-1) was identified as Elsinoe camellias sinensis, and representative strain T2 (T2-HNBJ-1) was identified as Phoma sp.CPT.
Example 2: identification method of Cladosporium virens
1. An identification method of thebainsiella comprises the following steps:
1) the method for extracting the total DNA of the sample to be detected specifically comprises the following steps: taking 0.4g of hyphae of the strain cultured for 7d, extracting total DNA of the fungus by using a fungus genome DNA extraction kit, and respectively amplifying to fungus genome spacer sequences ITS, 18SrRNA, RPB2 and LSU by using the extracted DNA as a template, wherein the sequences of primers are shown in Table 2.
TABLE 2 PCR pathogen detection primers
2) For the total DNA, carrying out PCR amplification, specifically comprising the following steps: PCR amplification System: rTaq enzyme 0.25. mu.L, dNTPs 0.4. mu.L, DNA template 1. mu.L, upstream and downstream primers (10. mu. mol/L) each 2. mu.L, ddH2O make up to 50. mu.L. The reaction condition is pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ 30SAnnealing at 55 ℃ for 30 DEG CSExtension at 72 ℃ of 90SFor 35 cycles, the final extension at 72 ℃ for 10min, and storage at 4 ℃.
3) The amplification products were detected by electrophoresis on a 1.2% agarose gel.
4) Recovering a target fragment, cloning the target fragment onto a vector, and screening positive clones by bacterial liquid PCR, wherein the method comprises the following specific steps: the target fragment is recovered according to the instructions of the gel recovery kit, cloned to a pMD19-T vector, and positive clones are screened by a bacterial liquid PCR method.
5) Sequencing the positive clones, and specifically, the steps are as follows: sequencing was performed by the firm of the Committee Biotechnology (Shanghai) corporation. Vector information is removed from the sequencing result, comparison search is carried out through BLAST of NCBI, homologous comparison data are downloaded, and system tree estimation is carried out on each sequence by utilizing Maximum Likelihood (ML) and Bayesian Inference (BI). In the CIPRES portal, default parameters of ML are used. Bayesian inference analysis is implemented in Beast V2.4.3.
2. Molecular characterization of isolated strains
Extracting T1-HNGZ of T1 type bacteria and T2-HNBJ genome DNA of T2 type bacteria, cloning and sequencing ITS, 18S rRNA, RBP2 and LSU regions of rDNA, downloading ITS, 18S rRNA, RBP2 and LSU region sequences through NCBI nucleic acid database homologous comparison, and constructing a phylogenetic tree with corresponding sequences obtained by sequencing. The sequencing results were submitted to NCBI database, accession numbers of T1-HNGZ were MK312256, MK312257, MK814964 and MK793793, respectively. The phylogenetic tree results show that T1-HNGZ has the closest relationship with Elsinoe hederae, Elsinoe leucogens and Elsinoe theae, see FIG. 4, the accession numbers of ITS, RBP2, 18S rRNA and LSU region sequences amplified by T2-HNBJ are MK814959-MK814963 in sequence, and the phylogenetic tree results show the closest relationship with Phoma sp, Phoma herbarum and Didymellia bellidis, see FIG. 5. Thus, the T1-HNGZ strain is initially identified as Elsinoe sp, and the T2-HNBJ strain is identified as Phoma sp.
Example 3: pathogen isolation pathogenicity assay
The pathogenicity of tea tree Mingfeng is determined.
The inoculation method with wounds comprises the following steps: shearing newly-grown branches of 2-year-old test tea trees potted in a greenhouse, cleaning the newly-grown branches with sterile water, drying the newly-grown branches, symmetrically manufacturing 5 inoculation wounds with the same depth and size on the left side and the right side of a leaf of a 2 nd flat leaf on the upper portion by using an inoculation needle, respectively placing a test strain bacterium disc with the diameter of 5mm and cultured for 14d and a sterile PDA culture medium control disc on a puncture part, soaking the puncture part with sterile water, covering absorbent cotton for moisturizing, and repeating inoculation with wounds for 10 times.
A wound-free inoculation method: the test strain cultured for 14d was prepared at 2.0X 105And (4) mL of conidium suspension, soaking the collected branches in the suspension for 5h, taking out, and soaking the branches in sterile water for 5h to serve as a control. Respectively placing the tea trees and branches inoculated with wounds and without wounds in an illumination incubator for culture (constant temperature of 26 ℃, 95% humidity, light and dark alternation of 12/12 hours), removing the inoculum after 48 hours, continuing to culture, observing the disease condition every day, after the inoculated part is diseased, separating pathogenic bacteria again, observing the forms of bacterial colonies and conidia and comparing with the test strains. The woundless inoculation was repeated 10 times.
3 representative strains of T1 type and T2 type were selected and inoculated onto leaves of young leaves of tea trees to be tested for culture observation. The study found that after the T1-HNGZ strain is inoculated and cultured for 4 days, the leaves are needle-shaped and light brown small spots, yellow halos are arranged on the edges, after the T1-HNGZ strain is cultured for 6 days, the diseased spots are red brown, the center is slightly concave, the periphery is provided with brown bulges, and the diameter is about 0.2-1.5mm, as shown in a and b in figure 6. Especially, the symptoms consistent with the initial stage of the development of the tea white spot reported by field observation appear at the 4 th d of non-wound inoculation. After inoculating T2-HNBJ strain and culturing for 2d, the leaves are 4-8mm brown patches, and after 6d, irregular black brown patches grow, the diameter is about 1.0-1.5cm, and the color of the edge is darker, as shown in c and d in figure 6. The character is different from the symptoms of the tea leaf spot reported by field observation, and no symptoms appear after non-invasive inoculation. Therefore, T1-HNGZ is identified as the pathogenic bacterium of the tea leaf spot.
The features of the above-described embodiments may be combined in any combination, and for the sake of brevity, all possible combinations of features in the above-described embodiments will not be described in detail, but rather, unless there is a conflict between such combinations, the scope of the present disclosure should be considered to be within the scope of the present disclosure.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Claims (4)
1. A Cladosporium theophrasini identified as Elsinoe camelllia sinensis, is preserved in China center for type culture Collection in 7 months and 15 days in 2019, and has a preservation number of CCTCC NO: M2019555.
2. The method for identifying cladosporium cucumerinum according to claim 1, comprising the steps of:
1) extracting the total DNA of a sample to be detected;
2) performing PCR amplification on the total DNA;
3) carrying out agarose gel electrophoresis detection on the amplification product;
4) recovering a target fragment, cloning the target fragment onto a vector, and screening a positive clone body by bacterial liquid PCR;
5) sequencing the positive clones.
3. The method of claim 2, wherein the PCR amplification system comprises rTaq enzyme 0.25. mu.L, dNTPs 0.4. mu.L, DNA template 1. mu.L, upstream and downstream primers 2. mu.L, ddH2O is added to 50 mu L.
4. The method of claim 2, wherein the PCR amplification is performed under the following conditions: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s for 35 cycles, final extension at 72 ℃ for 10min, and storage at 4 ℃.
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