Method for identifying pathogenic bacteria of torreya grandis black spot

Abstract

The invention relates to an identification method of torreya grandis black spot pathogenic bacteria. The identification method of the Chinese torreya black spot pathogenic bacteria comprises the following steps: step S102, obtaining Chinese torreya infected black spot seeds; performing a tissue isolation method based on seed shells and seed coats to obtain pathogenic bacteria strains; obtaining pathogenic bacterium form identification data; step S104, obtaining rDNA-ITS, TUB and TEF gene sequence characteristics of the pathogenic bacteria, and obtaining an identification result of the pathogenic bacteria; step S106, obtaining the biological characteristics of the pathogenic bacteria based on the biological characteristics of the pathogenic bacteria; according to the identification method for the pathogenic bacteria of the torreya grandis black spot, pathogenic bacteria strains are obtained through a tissue isolation method, morphological identification is performed on the pathogenic bacteria, pathogenicity of the pathogenic bacteria is verified through a Koch's law, and rDNA-ITS, TEF and TUB gene sequence characteristics of the pathogenic bacteria are analyzed, so that the pathogenic bacteria of the torreya grandis black spot are determined; and a theoretical basis is provided for formulating a disease law and prevention and control measures of the torreya grandis black spot.

Description

Technical Field

[0001] The invention belongs to the technical field of disease identification, and particularly relates to a method for identifying the pathogen of Torreya grandis black spot disease. Background Art

[0002] The kernel of Torreya grandis is rich in high-quality fatty acids, protein and various trace elements, and has extremely high nutritional value. Especially the nut part, which has a unique flavor and is widely welcomed in the market.

[0005] Although Torreya grandis has shown great economic potential in the market, the diseases that exist in its planting and processing, especially Torreya grandis black spot disease, have become one of the main factors affecting Torreya grandis quality and yield. During the growth of Torreya grandis trees, the occurrence of diseases and pests seriously affects the appearance quality and edible value of Torreya grandis, and may even lead to a significant decrease in yield. As a common disease in Torreya grandis production, black spot disease has become one of the main factors affecting the quality of Torreya grandis, especially during the processing process, causing great damage to the appearance and taste of Torreya grandis.

[0006] During the production process of Torreya grandis, factors such as the timing of picking, the method of post-ripening treatment, and climatic conditions may affect the occurrence of diseases. Specifically, the post-ripening period of Torreya grandis (i.e., the green cattail stage) is a high-incidence period of diseases. During the post-ripening process, due to the high humidity inside and outside the Torreya grandis fruit, various pathogens are prone to breed, resulting in problems such as mold and black spots on the surface of the seed shell. In addition, due to the special components of Torreya grandis kernels and seed coats, such as tannins, Torreya grandis seeds are susceptible to infection by pathogens, further exacerbating the occurrence of Torreya grandis diseases.

[0007] At present, the occurrence of Torreya grandis black spot disease is often accompanied by the occurrence of kernel rot, hollow seeds and other phenomena, which seriously affects the appearance and quality of Torreya grandis. This not only affects the market competitiveness of Torreya grandis, but also makes growers face huge economic losses. In some areas, due to the lack of effective prevention and control measures, the occurrence of black spot disease has become a bottleneck problem in the development of Torreya grandis industry.

[0008] The typical symptoms of Torreya grandis black spot disease are black spots on the fruit surface. These spots are usually round or irregular in shape. As the disease progresses, the range of the spots gradually expands, eventually causing the entire kernel or shell to rot and deteriorate. Especially after Torreya grandis fruit has been ripened, the occurrence of black spots is more serious. The surface of the shell is often covered with a layer of white hyphae. As time goes by, the black spots gradually increase in size and even affect the quality of the kernel, causing the kernel to become soft and smelly, seriously affecting the quality of the product.

[0009] The occurrence of Torreya grandis black spot disease not only affects the appearance of the fruit, but may also reduce the quality of the Torreya grandis kernel. Since Torreya grandis kernels contain a large amount of fat and protein, these nutrients may be degraded by pathogens after being infected by the disease, causing the kernels to become soft and rotten, moldy, or hollow, further reducing their edible and commercial value. Black spot disease also makes the frying and processing of Torreya grandis more complicated, increasing production costs and processing difficulties.

[0010] Although Torreya grandis black spot has become one of the main diseases affecting Torreya grandis quality, there is still a lack of systematic research on this disease. Most of the research on Torreya grandis diseases at home and abroad focuses on cultivation management and optimization of cultivation environment, while the research on the specific pathogens, pathogenesis and prevention and control measures of Torreya grandis black spot is relatively weak, especially in terms of pathogen identification and pathogenic mechanism, and existing research has not yet given a clear answer.

[0011] In the study of Torreya grandis diseases, the pathogens of black spot disease have not received enough attention. Most of the existing literature has not systematically identified and classified the pathogens of Torreya grandis black spot disease, which has led to blindness and uncertainty in disease prevention and control. Although some studies have shown that Torreya grandis black spot disease may be related to infection by microorganisms such as mold and bacteria, the specific types of pathogens and their pathogenic mechanisms are still unclear. In addition, the existing prevention and control technologies are mostly at the level of simple pesticide spraying and physical control, lacking precise prevention and control measures based on the characteristics of pathogens.

[0012] Based on the above background, the present application provides a method for identifying the pathogen of Torreya grandis black spot disease, which is used to quickly separate and identify the pathogen. Summary of the invention

[0013] The purpose of the present invention is to provide a method for identifying the pathogen of Torreya grandis black spot disease with a simple structure and reasonable design in order to solve the above problems.

[0014] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0015] A method for identifying the pathogen of Torreya grandis black spot disease, the method comprising the following steps:

[0016] Step S102, obtaining diseased black-spotted Torreya grandis seeds;

[0017] The pathogen strains were obtained by tissue separation method based on seed coat and seed coating;

[0018] Obtain pathogen morphological identification data;

[0019] Step S104, obtaining rDNA-ITS, TUB and TEF gene sequence characteristics of the pathogen, and obtaining the identification result of the pathogen;

[0020] Step S106, obtaining the biological characteristics of the pathogen based on the biological characteristics of the pathogen;

[0021] The biological characteristics include suitable temperature, suitable pH and suitable carbon and nitrogen sources.

[0022] As a further optimization scheme of the present invention, a pathogenic bacteria strain is obtained, the seed shell and seed coat are separated at a preset frequency, and after activation, a 5 mm diameter bacterial cake is taken from the edge of the colony and inoculated into the center of a plate with different treatment culture media, one plate per dish, and 3 dishes are repeated for each treatment. The plates are cultured in a constant temperature incubator at 25°C for 2 days, and the colony diameter is measured by the cross method to obtain the morphological characteristics of the colonies. The experimental data are subjected to a one-way analysis of variance, and a significance test for the difference is performed.

[0023] As a further optimization scheme of the present invention, the separation frequencies of seed husk and seed coating are 76.92% and 60% respectively.

[0024] As a further optimization scheme of the present invention, a pathogen strain is obtained, and the pathogen is separated and purified by a tissue separation method. The seed shell is surface disinfected with 75% ethanol and rinsed with sterile water three times. Then, a seed shell and seed coat tissue block of about 5×5 mm in size is cut from the black spot, placed on a PDA culture medium, cultured at 25°C, and purified for a preset number of times to obtain a pathogen numbered XFHK3.

[0025] As a further optimization scheme of the present invention, the purified strain was inoculated on PDA medium and cultured at 25° C. The color, morphology, and surface characteristics of the colonies were observed and recorded, and the color and size of the conidia were recorded to obtain the morphological data of the XFHK3 pathogen.

[0026] As a further optimization scheme of the present invention, an oat branch crushed grain culture medium is obtained, and the isolated strain is cultured in the oat branch crushed grain culture medium at 25° C. with alternating light and dark conditions, and spore production is induced for the isolated strain. Then, conidiophores are picked and crushed on a glass slide to collect morphological data of the spores.

[0027] As a further optimization scheme of the present invention, healthy and clean Torreya grandis seeds are collected, surface disinfected with 75% ethanol, and then rinsed with sterile water. The treated seeds are placed in a culture dish covered with moisturizing sterile filter paper, and a bacterial cake is punched from the cultured colony with a hole puncher. The bacterial cake is attached to the middle of the Torreya grandis seed shell, and a PDA culture block is used as a control. Each replicate has 9 seeds and each treatment has 3 replicates. The seeds are continuously observed for disease infection and the symptoms of the disease are recorded. Finally, the diseased tissue is isolated again from the seeds inoculated with pathogens to obtain the pathogenicity data of the isolated bacteria.

[0028] As a further optimization scheme of the present invention, the purified strain is inoculated on PDA medium for culture, the mycelium is collected, and the DNA of the isolated and purified pathogenic strain is extracted using the Ezup column fungal genomic DNA extraction kit. First, primers ITS1 / ITS4 are selected to amplify the ITS gene, and the pathogenic bacteria species are preliminarily determined after comparison. Primers Bt2a / Bt2b and EF1-688F / EF1-986R are further selected to perform PCR amplification on the TUB and TEF genes of the strain, and sequencing results are obtained and homology comparison analysis is performed. The sequences are compared and the multiple genes are spliced ​​after shearing to construct a phylogenetic tree.

[0029] As a further optimization scheme of the present invention, universal primers ITS1 / ITS4 are obtained to perform PCR amplification on the selected representative strain XFHK3. After sequence determination, the obtained sequences are aligned, and multiple sequence alignment is performed based on the amplified strain-specific conserved sequence molecular sequences to construct a molecular phylogenetic tree to complete the identification of the pathogen XFHK3.

[0030] The beneficial effects of the present invention are:

[0031] The present invention obtains the pathogenic bacteria strain by tissue separation method, performs morphological identification on the pathogen, verifies the pathogenicity of the pathogen by Koch's postulates, and analyzes the rDNA-ITS, TEF and TUB gene sequence characteristics, in order to clarify the pathogenic bacteria of Torreya grandis black spot disease and provide a theoretical basis for the pathogenesis of Torreya grandis black spot disease and the formulation of prevention and control measures.

[0032] The specific primers for the pathogen of Torreya grandis black spot disease provided by the present invention provide a feasible solution for the rapid and accurate identification of the pathogen; the specific primers are used to confirm the colony, so that the pathogen can be quickly identified.

[0033] The invention separates, cultures, screens and identifies the black spot disease caused by Lasiodiplodia theobromae.

[0034] The disease symptom characteristics and specific symptoms of Torreya grandis black spot disease disclosed in the present invention provide an important basis for accurately identifying the disease and successfully isolating the pathogen; by collecting seeds with typical disease symptoms for separation, the target pathogen can be accurately isolated.

[0035] The separation and identification method of the present invention is not only rapid but also simple and easy to implement, and those skilled in the art can easily master the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the process structure of a method for identifying the pathogen of Torreya grandis black spot disease of the present invention;

[0037] Figure 2A schematic diagram of symptoms of Torreya grandis black spot disease in order to invent a method for identifying the pathogen of Torreya grandis black spot disease;

[0038] Figure 3 To invent a method for identifying the pathogen of Torreya grandis black spot disease, including morphological and molecular biological identification of Torreya grandis black spot disease;

[0039] Figure 4 A schematic diagram of the pathogenicity determination of Torreya grandis black spot disease in order to invent a method for identifying the pathogen of Torreya grandis black spot disease;

[0040] Figure 5 A schematic diagram of the effect of temperature on the mycelial growth of XFHK3 in a method for identifying the pathogen of Torreya grandis black spot disease;

[0041] Figure 6 A schematic diagram of the effect of pH on the mycelial growth of Torreya grandis black spot disease XFHK3 in order to invent a method for identifying the pathogen of Torreya grandis black spot disease;

[0042] Figure 7 A schematic diagram showing the effect of carbon sources on the mycelial growth of XFHK3 in an identification method for the pathogen of Torreya grandis black spot disease;

[0043] Figure 8 Schematic diagram of the effect of nitrogen source on the mycelial growth of XFHK3 in the invention of a method for identifying the pathogen of Torreya grandis black spot disease. DETAILED DESCRIPTION

[0044] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0045] References Figure 1 The flowchart of the identification method shown in the figure shows a method for identifying the pathogen of Torreya grandis black spot disease, which method comprises the following steps:

[0046] Step S102, obtaining diseased black-spotted Torreya grandis seeds;

[0047] The pathogen strains were obtained by tissue separation method based on seed coat and seed coating;

[0048] Obtain pathogen morphological identification data;

[0049] Step S104, obtaining rDNA-ITS, TUB and TEF gene sequence characteristics of the pathogen, and obtaining the identification result of the pathogen;

[0050] Step S106, obtaining the biological characteristics of the pathogen based on the biological characteristics of the pathogen;

[0051] The biological characteristics include suitable temperature, suitable pH and suitable carbon and nitrogen sources.

[0052] In this embodiment, specific reference is made to Figures 2 to 8 As shown in the schematic diagram, the biological characteristics of the pathogen of Torreya grandis black spot disease were studied. After the representative strain XFHK3 was activated, a 5 mm diameter bacterial cake was taken from the edge of the colony and inoculated into the center of the plates with different treatment culture media, one piece per dish, and 3 dishes were repeated for each treatment. The plates were cultured in a constant temperature incubator at 25°C for 2 days, and the colony diameter was measured by the cross method, and the morphological characteristics of the colonies were observed. SPSS software was used to perform one-way analysis of variance on the experimental data, and Duncan was used to perform significance test for differences.

[0053] Furthermore, the disease symptoms of Torreya grandis black spot disease were investigated and disease samples were collected, the symptoms were recorded, and the disease samples were collected and further separated and identified;

[0054] Isolation and purification of the pathogen of Torreya grandis black spot disease: The pathogen was isolated and purified by tissue separation method. The Torreya grandis black spot seeds were sterilized with 75% ethanol for 30 seconds, rinsed with sterile water 3 times, placed on sterile filter paper to dry, and then the seed shell was crushed with a sterilized nut clamp. A tissue block of about 5×5mm was cut from the seed shell and seed coat black spot with a sterile blade. The inside of the seed shell and the outside of the seed coat were transferred to PDA medium for constant temperature culture at 25℃. The morphology of the isolated colonies was observed after 5 days. After the fungus grew, it was purified and numbered XFHK3;

[0055] Further, morphological identification of pathogens: the purified strains were inoculated on PDA medium and cultured at 25°C, the color, morphology, surface characteristics of the colonies were observed and recorded, and the color and size of the conidia were recorded as the basis for morphological identification;

[0056] In addition, an oat branch granule medium (100 mL oat medium, 10 g Torreya grandis branch granule) can also be prepared. The oat branch granule medium is used for alternating light and dark culture at 25°C to induce spore production in the isolated strain, and then the conidiophores are selected and crushed on a glass slide to observe the morphological characteristics of the spores for morphological identification;

[0057] Furthermore, Koch's postulates were used to verify the isolated pathogens: healthy and clean Torreya grandis seeds were collected, and the surface was disinfected with 75% ethanol, and then rinsed with sterile water, and the treated seeds were placed in a culture dish covered with moisturizing sterile filter paper. A bacterial cake (about 5 mm in diameter) was punched out from the cultured colony with a hole puncher, and the bacterial cake was attached to the middle of the Torreya grandis seed shell. PDA culture blocks were used as controls, with 9 seeds per replicate and 3 replicates for each treatment. Continuously observe whether the seeds are susceptible to the disease and the symptoms of the disease and keep records. Finally, the diseased tissues were isolated again from the seeds inoculated with pathogens to further verify Koch's postulates and complete the identification of the pathogenicity of the isolated bacteria;

[0058] Molecular biological identification of pathogens: The purified strains were inoculated on PDA medium and the mycelium was collected. The DNA of the isolated and purified pathogenic strain was extracted using the Ezup column fungal genomic DNA extraction kit. First, primers ITS1 / ITS4 (5'-TCCGTAGGTGAACCTGCGG-3', 5'-TCCTCCGCTTATTGATATGC-3') were selected to amplify the ITS gene. After comparison, the species of the pathogen was preliminarily determined. Primers Bt2a / Bt2b (5'-GGTAACCAAATCGGTGCTGCTTTC-3', 5'-ACCCTCAGTGTAGTGACCCTTGGC-3') and EF1-688F / EF1-986R (5'-CGGTCACTTGATCTACAAGTGC-3', 5'-TACTTGAAGGAACCCTTACC-3') were further selected to perform PCR amplification of the TUB and TEF genes of the strain. The sequencing results were compared with the NCBI database for homology analysis, and the sequences were downloaded from GenBank. The obtained sequences were aligned using MEGAX software, and after cutting, multiple genes were spliced ​​to construct a phylogenetic tree.

[0059] Pathogenicity test: In the early stage, large areas of dense white hyphae appeared on Torreya grandis seeds, which spread and spread out. The hyphae gradually turned black, and the hyphae bulged vigorously. The color of the seed shell began to darken, forming irregular black spots. Black bulges appeared at random points on the surface of the seed shell. With the development of the disease, the area of ​​the lesions gradually increased, and the hyphae penetrated the seed shell and attached to the seed coat. At 15 days, half of the seeds and the inner seed coat turned black. At 25 days, the hyphae of some seeds were extremely vigorous, and the inner seed kernel rotted.

[0060] Morphological characteristics of pathogenic bacteria: The pathogenic bacteria XFHK3 is cultured on PDA plates. The colonies are initially white and fluffy, with extremely vigorous aerial hyphae and a dense texture. The color of the colony center gradually deepens to black. The conidia are elliptical or ovate. The initial conidia are transparent, then the color changes to dark brown, and finally the mature conidia have a septum in the center. The spore size is about 24.31μm×13.29μm;

[0061] The selected representative strain XFHK3 was amplified and sequenced using universal primers ITS1 / ITS4. After sequencing, the obtained sequence was compared by BLAST and found that XFHK3 and Lasiodiplodia theobromae had a 100% homology. At the same time, multiple sequence alignments were performed based on the molecular sequences of the amplified specific conservative sequences of each strain, and a molecular phylogenetic tree was constructed. It was found that XFHK3 and Lasiodiplodia theobromae were clustered in one branch, which further established the identification of the pathogen XFHK3;

[0062] Further, the effects of temperature and pH on mycelial growth:

[0063] The pathogen was cultured at 25°C for 3 days. A 5-mm-diameter sterile punch was used to collect bacterial cakes from the edge of the colony and inoculated on PDA medium under the following different conditions: 5 temperature gradients were set at 15°C, 20°C, 25°C, 30°C, and 35°C; 1 mol·L -1 NaOH and 1 mol·L -1 HCl mother solution, the pH of PDA medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, a total of 10 gradients, each treatment condition was set to 3 replicates, and cultured at 25°C in the dark for 2 days. The colony diameter was measured using the cross method, and the morphological characteristics of the colonies were observed;

[0064] Effects of carbon and nitrogen sources on mycelial growth:

[0065] A 5 mm bacterial cake was inoculated into the center of Cazpek medium containing different carbon and nitrogen sources. The different carbon source treatments were to replace the sucrose in the Cazpek medium with fructose, lactose, galactose, maltose, sorbitol, xylose, sucrose, glucose, soluble starch, and mannitol respectively; the different nitrogen source treatments were to replace the sodium nitrate in the Cazpek medium with potassium nitrate, ammonium sulfate, ammonium chloride, ammonium nitrate, alanine, beef extract powder, glycine, peptone, trypsin, and yeast extract powder respectively. Each treatment was repeated 3 times and cultured at 25°C for 2 days. The colony diameter was measured using the cross method.

[0066] SPSS software was used to perform one-way analysis of variance on the experimental data, and Duncan test was used for significant difference test.

[0067] Field symptom investigation and disease sample collection: After picking mature and cracked Torreya grandis, there are different degrees of black spots on the surface of the seeds after the cattails are removed, and they gradually expand during the ripening process. In the post-harvest ripening warehouse, Torreya grandis seeds with black spots are investigated and collected, and the incidence of black spot disease is about 56%. Torreya grandis black spot disease mainly harms Torreya grandis seeds after the cattails are removed. The affected area of ​​​​Susceptible Torreya grandis black spot disease varies in size. In the early stage of the disease, there are fine white hyphae on the surface of the seeds, and slightly raised black balls appear. There are many and uneven distribution. Microscopic observation shows that they are aggregates of spores and hyphae; in the later stage of the disease, the hyphae expand along the surface of the seed shell into irregular large black spots, and the spots are smoothly attached to the seed shell; the black spots can penetrate the seed shell, and black hyphae are attached to the inside of the shell and the seed coat. Some black spot disease seeds are prone to soft rot of the kernel in the later stage, and hollow seeds appear.

[0068] Pathogenicity determination: The isolated strain XFHK3 was inoculated on clean Torreya grandis seeds. After 10 days of inoculation, the inoculated seeds showed symptoms similar to those during after-ripening, while the uninoculated control seeds were still symptom-free. The symptoms of inoculated Torreya grandis seeds were as follows: fine white hyphae appeared on the surface of the seeds, spreading and spreading, the color of the hyphae gradually turned black, the hyphae were vigorously raised, the color of the seed shell began to darken, and irregular black spots were formed. Black raised balls appeared at irregular points on the surface of the seed shell. With the development of the disease, the area of ​​the lesions gradually increased, and the hyphae penetrated the seed shell and attached to the seed coat. At 15 days, half of the seeds and the internal seed coat were black. At 25 days, the hyphae of some seeds were extremely vigorous, and the internal seed kernels rotted.

[0069] Morphological characteristics of pathogenic bacteria: The pathogenic bacteria XFHK3 is cultured on PDA plates. The colonies are initially white and fluffy, with extremely vigorous aerial hyphae and a dense texture. The color of the colony center gradually deepens to black. Conidia are elliptical or ovate. The initial spores are transparent, then the color changes to dark brown, and finally the mature conidia have a septum in the center. The spore size is about 24.31μm×13.29μm.

[0070] Molecular identification of pathogens: The randomly selected representative strain XFHK3 was amplified and sequenced using universal primers ITS1 / ITS4. After sequencing, the obtained sequences were compared by BLAST and found that XFHK3 and Lasiodiplodia theobromae had a 100% homology. At the same time, multiple sequence alignments were performed based on the amplified molecular sequences of the specific conservative sequences of each strain, and a molecular phylogenetic tree was constructed. It was found that XFHK3 and Lasiodiplodia theobromae were clustered in one branch, which further established the identification of the pathogen XFHK3.

[0071] Further, the effect of temperature on mycelial growth: Figure 5It can be seen that too high or too low temperature is not conducive to the growth of mycelium XFHK3, the pathogen of Torreya grandis black spot disease, and mycelium can grow in the range of 15-35℃. There is a significant effect on the colony diameter between different temperature treatments. The optimal growth temperature of mycelium of XFHK3 is 30℃, and the colony diameters are 11.30cm and 11.10cm respectively; the second most suitable mycelium growth temperature for XFHK3 is 25℃ and 35℃, and the colony diameters are 8.18cm and 7.67cm respectively; XFHK3 grows slowly at 15℃, and the colony diameter is 2.69cm. The suitable growth range of mycelium of XFHK3, the pathogen of Torreya grandis black spot disease, is 20℃-35℃, and the optimal temperature is 30℃.

[0072] Further, the effect of pH on mycelial growth: Figure 6 It can be seen that the pathogen of Torreya grandis black spot disease XFHK3 can grow at pH = 3-10, and the mycelium grows best at pH = 5, with an average colony diameter of 11.30 cm. XFHK3 inhibits its growth in an overacidic environment of pH = 3, with a colony diameter of 2.60 cm; it has a significant inhibitory effect on mycelium growth under alkaline conditions of pH = 10-12, with a colony diameter of 2.21 cm-0.70 cm. The above results show that the pathogen of Torreya grandis black spot disease can grow faster in an environment of pH = 4-9, and is more suitable for growth in a slightly acidic environment. pH = 5 is the most suitable environment for mycelium growth.

[0073] Further, the effects of carbon and nitrogen sources on mycelial growth: Figure 7 and Figure 8 It can be seen that the pathogen of Torreya grandis black spot disease XFHK3 can utilize a variety of carbon sources and nitrogen sources. The best carbon source for XFHK3 is sucrose, with the largest colony diameter of 7.31cm, followed by galactose, with a colony diameter of 6.73cm. It grows slower on xylose, with a colony diameter of 2.12cm. The results show that the best carbon source for Torreya grandis black spot disease XFHK3 is sucrose. ( Figure 7 (in Chinese, NC, no carbon source; Lac, lactose; Gal, galactose; Mal, maltose; Sor, sorbitol; Xyl, xylose; Suc, sucrose; Glu, glucose; Sol, soluble starch; Fru, fructose; Man, mannitol).

[0074] The best nitrogen source for XFHK3 was tryptone, with the largest colony diameter of 8.40 cm, significantly higher than other treatments; followed by ammonium chloride, alanine, beef extract powder and yeast extract, with colony diameters ranging from 7.74 cm to 7.77 cm. The results showed that the best nitrogen source for XFHK3, the pathogen of Torreya grandis black spot, was tryptone. ( Figure 8In the formula, NN, no nitrogen source; (NH4)2SO4, ammonium sulfate; NH4Cl, ammonium chloride; NaNO3, sodium nitrate; Ala, alanine; Beef, beef extract; KNO3, potassium nitrate; Gly, glycine; Pep, peptone; Try, tryptone; Yea, yeast extract).

[0075] Torreya grandis black spot disease is common and often occurs during the post-harvest ripening process. There is no research on this disease at home and abroad. This study used morphological, molecular biological identification and pathogenicity identification methods, and used the method of multi-gene splicing comparison to determine that the pathogen causing Torreya grandis black spot disease is Lasiodiplodia theobromae. Studies have shown that Lasiodiplodia theobromae is an important plant pathogen that can cause diseases in a variety of tropical and subtropical plants, and can harm flowers, oil crops, grain crops, fruit trees, including tea trees, mulberry trees, rubber trees, kiwis, mangoes, etc. Lasiodiplodia theobromae can cause chestnut black rot, with hyphae attached to the surface of the kernels and a large number of hyphae. The kernels are severely affected, and the affected tissues become soft, rotten, and sour and smelly. The symptoms are similar to those of Torreya grandis black spot disease in this study.

[0076] The results of this experimental study showed that the suitable growth temperature of XFHK3 (Lasiodiplodia theobromae) mycelium is 25℃-30℃, and the optimal growth temperature is 30℃; the pH suitable for mycelium growth is between 4-9, and pH=5 is optimal for mycelium growth; the optimal carbon source is sucrose, and the optimal nitrogen source is trypsin.

[0077] This application identifies the pathogen of Torreya grandis black spot disease and studies its biological characteristics, clarifies that the pathogen that causes Torreya grandis black spot disease is Lasiodiplodia theobromae, clarifies the relationship between pathogen growth and environmental conditions, and provides an important basis for explaining the occurrence and development of Torreya grandis black spot disease.

[0078] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0079] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for identifying the pathogen of Torreya grandis black spot disease, characterized in that: The method comprises the following steps, Step S102, obtaining diseased black-spotted Torreya grandis seeds; The pathogen strains were obtained by tissue separation method based on seed coat and seed coating; Obtain pathogen morphological identification data; Step S104, obtaining rDNA-ITS, TUB and TEF gene sequence characteristics of the pathogen, and obtaining the identification result of the pathogen; Step S106, obtaining the biological characteristics of the pathogen based on the biological characteristics of the pathogen; The biological characteristics include suitable temperature, suitable pH and suitable carbon and nitrogen sources.

2. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 1, characterized in that: The seed coat and seed shell were separated by tissue separation to obtain the pathogenic bacteria strain. After activation, a 5mm diameter bacterial cake was taken from the edge of the colony and inoculated into the center of the culture medium plates with different treatments, one piece per dish, and 3 dishes were repeated for each treatment. The plates were cultured in a constant temperature incubator at 25℃ for 2 days. The colony diameter was measured by the cross method to obtain the morphological characteristics of the colonies. The experimental data were subjected to one-way analysis of variance and a significance test was performed.

3. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 2, characterized in that: The separation frequencies of this strain in seed shell and seed coating were 76.92% and 60% respectively.

4. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 2, characterized in that: The pathogen strain was obtained, and the pathogen was isolated and purified by tissue separation method. The seed shell was surface disinfected with 75% ethanol and rinsed with sterile water for 3 times. Then, the seed shell and seed coating tissue blocks of about 5×5 mm in size were cut at the black spots, placed on PDA culture medium, cultured at 25°C, and purified for a preset number of times to obtain the pathogen numbered XFHK3.

5. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 4, characterized in that: The purified strain was inoculated on PDA medium and cultured at 25°C. The color, morphology, and surface characteristics of the colonies were observed and recorded, and the color and size of the conidia were recorded to obtain the morphological data of the XFHK3 pathogen.

6. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 5, characterized in that: Obtain an oat branch crushed grain culture medium, culture it in the oat branch crushed grain culture medium at 25° C. with alternating light and dark, induce spore production of the isolated strain, and then pick out conidiophores and crush them on a glass slide to collect morphological data of the spores.

7. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 1, characterized in that: Healthy and clean Torreya grandis seeds were collected, surface disinfected with 75% ethanol, and then rinsed with sterile water. The treated seeds were placed in a culture dish covered with moisturizing sterile filter paper, and a bacterial cake was punched from the cultured colony with a hole puncher. The bacterial cake was attached to the middle of the Torreya grandis seed shell, and PDA culture blocks were used as controls. Each replicate had 9 seeds and 3 replicates for each treatment. The seeds were continuously observed for disease infection and the symptoms of the disease were recorded. Finally, the diseased tissues were isolated again from the seeds inoculated with pathogens to obtain the pathogenicity data of the isolated bacteria.

8. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 1, characterized in that: The purified strain was inoculated on PDA medium for culture, and the mycelium was collected. The DNA of the isolated and purified pathogenic strain was extracted using the Ezup column fungal genomic DNA extraction kit. Primers ITS1 / ITS4 were first selected to amplify the ITS gene, and the pathogenic bacteria species were preliminarily determined after comparison. Primers Bt2a / Bt2b and EF1-688F / EF1-986R were further selected to perform PCR amplification on the TUB and TEF genes of the strain, and sequencing results were obtained and homology comparison analysis was performed. The sequences were obtained by comparison, and the multiple genes were spliced ​​after shearing to construct a phylogenetic tree.

9. The method for identifying the pathogen of Torreya grandis black spot disease according to claim 8, characterized in that: The universal primers ITS1 / ITS4 were used to perform PCR amplification on the selected representative strain XFHK3. After sequencing, the obtained sequences were aligned, and multiple sequence alignments were performed based on the amplified strain-specific conserved sequence molecular sequences to construct a molecular phylogenetic tree to complete the identification of the pathogen XFHK3.

Images