A selective culture medium for tobacco black shank pathogen and its preparation method and application

Abstract

The present invention relates to a tobacco black shank pathogen selective culture medium and a preparation method and application thereof. The present invention uses different types and different concentration gradients of poisonous oat culture medium plates to respectively measure the growth inhibition rates of Fusarium oxysporum, Fusarium solani and Phytophthora nicotianae, the main pathogens of root rot, on the plates, and finds that tebuconazole and imazalil have the best inhibition effect on tobacco root rot and have no effect on the growth of tobacco black shank pathogen. Therefore, the above culture medium can be used to screen and separate tobacco black shank pathogen strains. The tobacco black shank pathogen selective culture medium and screening and separation method provided by the present invention can be used to separate pathogens from tobacco black shank pathogenic plants, especially for plants with tobacco root rot complex infection, and has the characteristics of excellent separation effect, simple operation, time saving and labor saving, etc., and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial screening, and in particular relates to a selective culture medium for tobacco black shank pathogen and a preparation method and application thereof. Background Art

[0002] Tobacco is an important cash crop in China, and has made positive contributions to the increase of national and local fiscal revenue and economic development. With the large-scale cultivation of tobacco in my country, more than 60 tobacco infectious diseases have been reported in China, including tobacco virus disease, brown spot disease, target spot disease, root rot, etc. In recent years, the occurrence of tobacco Fusarium root rot in major tobacco-growing areas in my country has gradually increased, which is easy to cause complex infection with tobacco black shank disease, causing significant economic losses.

[0003] Tobacco Fusarium root rot is one of the main rhizome diseases of tobacco, usually caused by Fusarium solani and Fusarium oxysporum. Tobacco Fusarium root rot can occur from the seedling stage to the field stage of tobacco, with a disease center, and the disease mainly occurs in the field stage. The disease mainly infects tobacco rhizomes, causing root vascular bundle rot, blocking the transport of water and nutrients in the body, and thus causing tobacco plants to wither and die. The pathogen mainly overwinters in the diseased remains of tobacco plants and diseased seeds, or in the soil with hyphae and spores. It can infect alone or mixed with tobacco black shank disease, bacterial wilt, etc., causing root rot, tobacco plant death, tobacco leaf yield reduction, etc. Tobacco black shank disease is a soil-borne fungal disease caused by Phytophthora parasitica var. nicotianae. Tobacco black shank disease can occur throughout the growth period of tobacco, with relatively few cases in the seedling stage, and mainly in the field stage. The main site of disease is the rhizome of tobacco. When the base of tobacco seedlings is infected during the seedling stage, black spots will appear, and the infection will spread from bottom to top, and in severe cases, the tobacco seedlings will die. In the field stage, tobacco plants infected mainly show symptoms such as "black legs", "wearing a coat", "black plaster", "disc-shaped", and "waist rot". It mainly overwinters in the remains of diseased plants, soil and fertilizer in the form of mycelium and thick-walled spores, which are the main source of infection in the early next year. The pathogen is mainly spread through rainwater or irrigation water. Zoospores are affected by root secretions, gather and germinate on their surface, and can invade directly or from wounds to complete the initial infection. At present, tobacco sickle root rot and tobacco black shank are seriously infected in many tobacco areas in my country, making it difficult to study a single disease. In recent years, predecessors have conducted more research on the prevention and control of tobacco sickle root rot and tobacco black shank, but there are few reports on the isolation and purification of single strains of diseases in the combined infection of tobacco sickle root rot and tobacco black shank. Summary of the invention

[0004] The present invention has found through research that imazalil and tebuconazole have significant inhibitory effects on tobacco oxysporum Fusarium and tobacco solani Fusarium at a certain concentration, but have poor inhibitory effects on tobacco Phytophthora. Therefore, a tobacco black shank pathogen selective culture medium can be prepared based on these two substances, and the tobacco Phytophthora parasitica var.nicotianae that causes tobacco black shank disease can be accurately separated from tobacco Fusarium root rot and tobacco black shank disease complex infection strains by effectively inhibiting two main tobacco Fusarium. This provides a basis for subsequent disease resistance and prevention and control research on tobacco black shank disease.

[0005] The invention provides a culture medium for selectively culturing tobacco black shank pathogenic bacteria. The culture medium comprises a basic culture medium and cephalosporin, tebuconazole and / or imazalil, wherein the concentration of tebuconazole is not less than 10 mg / L, and the concentration of imazalil is not less than 5 mg / L.

[0006] Furthermore, the concentration of tebuconazole is not less than 50 mg / L, and the concentration of imazalil is not less than 20 mg / L.

[0007] Furthermore, the basic culture medium is oat culture medium.

[0008] The present invention also provides a method for isolating tobacco black shank pathogenic bacteria, which comprises culturing a sample using any of the above culture media.

[0009] Furthermore, the pathogen of tobacco black shank disease is tobacco phytophthora parasiticavar.nicotianae.

[0010] The present invention also provides the use of tebuconazole and / or imazalil in preparing reagents for separating and / or screening tobacco black shank pathogens.

[0011] Furthermore, the concentration of tebuconazole is not less than 50 mg / L, and the concentration of imazalil is not less than 20 mg / L.

[0012] Furthermore, the pathogen of tobacco black shank disease is tobacco phytophthora parasiticavar.nicotianae.

[0013] The present invention also provides a method for distinguishing tobacco Phytophthora and tobacco Fusarium, wherein a sample is inoculated into any of the above culture media for culture.

[0014] The present invention also provides the use of tebuconazole and / or imazalil in preparing reagents for selectively distinguishing and / or screening pathogens of tobacco root rot and tobacco black shank disease.

[0015] Beneficial effects: The present invention provides a culture medium for selectively culturing / screening tobacco black shank pathogens, which can effectively inhibit tobacco Fusarium, thereby accurately separating tobacco Phytophthora parasitica var.nicotianae from tobacco Fusarium root rot and tobacco black shank compound infection, with excellent separation effect and the characteristics of simple operation, time-saving and labor-saving, and low requirements for test personnel, etc., with broad application prospects, and provides a basis for subsequent research on disease resistance and prevention and control of tobacco black shank. In addition, the present invention also screens out a suitable concentration of agent for field trials, thereby effectively reducing the occurrence of field diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 The results of the plate test in Example 1 were as follows: P. nicotianae, F. solani and F. oxysporum were cultured separately on different selective culture media;

[0018] Figure 2 This is the growth situation of mycelial interruption plate with 50μg / mL tebuconazole.

[0019] Figure 3 This is the growth situation of mycelial interruption plate with 20μg / mL imazalil.

[0020] Figure 4 This is the selective culture of Fusarium solani L7, Fusarium oxysporum L88, and Phytophthora nicotianae LS4-1 on the selective culture medium containing imazalil or tebuconazole. DETAILED DESCRIPTION

[0021] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0022] Example 1: Plate test of tobacco root rot pathogen and tobacco black shank pathogen

[0023] The agents selected in this experiment include 62.5g / L fluazifop, 30% fluazifop, 40% difenoconazole, 500g / L fluazifop, 25% tebuconazole, and 20% imazalil. Five poisoned oat culture medium plates with different concentration gradients were prepared, and the colony growth inhibition rates of the main pathogens of tobacco root rot, Fusarium oxysporum and Fusarium solani, as well as the pathogen of tobacco black shank, Phytophthora nicotianae, on the plates were determined.

[0024] (1) Preparation of culture medium: The concentration of the culture medium is fixed to 90% of the standard culture medium concentration, and then the six test fungicides are diluted with sterile water to 10 times the corresponding mass concentration of the stock solution. In a sterile operating table, when the culture medium is cooled to 50-60°C, the fungicide stock solution and the culture medium are gently mixed at a ratio of 1:9 to prepare culture medium containing different mass concentrations of drugs, and poured into a culture dish to prepare a plate. After the plate is cooled, it is the drug culture medium.

[0025] (2) Determination of growth rate: The prepared bacterial cakes were inoculated in the center of the drug-containing plates with different mass concentrations, cultured at 28°C for 7 days, and the diameter of each colony was measured by the cross method. The two bacteria and each fungicide were repeated 3 times at each mass concentration. SPSS statistical software was used to analyze and obtain the inhibition rate of fungicides on the mycelial growth of tobacco root rot pathogens and tobacco black leg pathogens, as well as the toxicity regression equation, correlation coefficient, and effective inhibitory concentration (EC 50 ). The inhibition rate was calculated according to the following formula: inhibition rate (%) = (control colony growth diameter - treated colony growth diameter) / control colony growth diameter × 100%. The logarithm of the set mass concentration was used as the abscissa (x), and the inhibition rate probability value was used as the ordinate (y), and the toxicity regression equation y = a + bx was obtained.

[0026] Table 1 Determination of the toxicity of fungicides to Fusarium solani

[0027]

[0028]

[0029] Table 2 Determination of the toxicity of fungicides to Fusarium oxysporum

[0030]

[0031] It can be seen from Tables 1 and 2 that the EC values ​​of the tested fungicides against Fusarium oxysporum, Fusarium solani and Phytophthora nicotianae are 50 There were obvious differences, among which the EC values ​​of imazalil and tebuconazole of Fusarium oxysporum and Fusarium solani were 50 The lowest, the best antibacterial effect; on the contrary, imazalil and tebuconazole have the highest EC 50 The largest, and the worst inhibitory effect on tobacco blight.

[0032] Table 3 Toxicity determination of fungicides against tobacco phytophthora

[0033]

[0034]

[0035] Table 4 Inhibitory effects of six fungicides on the colony diameters of Fusarium solani and Phytophthora nicotianae

[0036]

[0037]

[0038] Table 5 Average inhibition rate of six fungicides on the colony diameter of Fusarium solani and Phytophthora nicotianae

[0039]

[0040]

[0041]

[0042] As shown in Table 3, Table 4 and Table 5, the six fungicides tested had different degrees of inhibition on the mycelial growth of Fusarium oxysporum and Phytophthora nicotianae, and the inhibition rate increased with the increase of the mass concentration of the fungicide. Among them, the inhibition effects of imazalil and tebuconazole on Fusarium oxysporum and Fusarium solani were the best when the mass concentrations were 20 μg / mL and 50 μg / mL, respectively, while the inhibition effect on Phytophthora nicotianae was poor. As shown in Table 5, the inhibition rates of the six fungicides tested on Fusarium oxysporum and Fusarium solani were different degrees of inhibition, among which the inhibition rates of 20 μg / mL imazalil and 50 μg / mL tebuconazole on Fusarium oxysporum and Fusarium solani were both above 75%, while the inhibition rates on Phytophthora nicotianae were all below 1.5%. In summary, 20 μg / mL imazalil and 50 μg / mL tebuconazole had better inhibitory effects on Fusarium oxysporum and Fusarium solani, but had poor inhibitory effects on Phytophthora nicotianae. Based on this, effective doses of imazalil and tebuconazole can be prepared into a selective culture medium for the isolation and screening of Phytophthora nicotianae.

[0043] Experimental Example 2 Mycelium interruption test of tobacco root rot pathogen and tobacco black shank pathogen

[0044] In this experiment, tobacco oxysporum Fusarium L88, tobacco solani Fusarium L7 and tobacco Phytophthora nicotianae LS4-1 were selected and purified respectively, and then made into bacterial cakes and inoculated into the center of OA culture medium. When they grew to 2 / 3 of the plate, the mycelium of the three strains was divided into two groups (L88 and LS4-1, L7 and LS4-1). The common weight was taken for full interruption and then evenly coated on two new poisonous OA culture media with different mass concentrations (20μg / mL imazalil, 50μg / mL tebuconazole). Each group was repeated three times and cultured at 28℃ in the dark. When the mycelium grew out of the culture medium surface visible to the naked eye, the grown mycelium was picked into the new OA culture medium. Six newly grown interrupted mycelium were randomly picked from each selective culture medium and cultured in the dark at 28℃.

[0045] like Figure 4 As shown, the results showed that the proportion of mycelium in the two groups (L88 and LS4-1, L7 and LS4-1) after being broken in the two selective culture media was more than 90%, indicating that the two selective culture media have a good effect in inhibiting tobacco Fusarium without affecting tobacco Phytophthora.

[0046] Experimental Example 3: Application of selective culture medium in the separation of tobacco root rot and tobacco black shank combined infection

[0047] In Fangyuan Village, Fangyuan Town, Guiyang County, Chenzhou City, Hunan Province, 20 diseased plants with tobacco root rot and tobacco black shank were collected, and two selective culture media (20μg / mL imazalil, 50μg / mL tebuconazole), mixed culture media and OA culture media were used for conventional tissue separation. The results are shown in Table 6. It can be seen that the separation success rate of Phytophthora is high in the case of complex infection using selective culture media, which can reach 80-90%, and when 20μg / mL imazalil and 50μg / mL tebuconazole are mixed and added to the selective culture media at the same time, the separation effect can be further improved, and the separation success rate reaches 100%, which plays a good synergistic effect. The separation effect is significantly better than that of OA culture media.

[0048] Table 6 Selection of culture media for separation applications in combined infection situations

[0049]

[0050] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A culture medium for selectively isolating the pathogen of tobacco black shank disease, Phytophthora parasiticavar.nicotianae, characterized in that: The culture medium consists of a basic culture medium, tebuconazole and imazalil, wherein the concentration of tebuconazole is 50 mg / L and the concentration of imazalil is 20 mg / L.

2. The culture medium according to claim 1, characterized in that The basic culture medium is oatmeal culture medium.

3. A method for isolating the tobacco black shank pathogen Phytophthora parasitica var.nicotianae, characterized in that: The sample is cultured using the culture medium according to any one of claims 1-2.

4. Use of tebuconazole and imazalil in the preparation, separation and / or screening of products for the pathogenic bacteria of tobacco black shank disease Phytophthoraparasitica var.nicotianae, characterized in that: The concentration of tebuconazole is 50 mg / L, and the concentration of imazalil is 20 mg / L.

5. The use of tebuconazole and imazalil in the preparation of a reagent for selectively distinguishing tobacco root rot and tobacco black shank pathogens, characterized in that: The pathogens of tobacco root rot are Fusarium solani and Fusarium oxysporum, the pathogen of tobacco black shank is Phytophthora parasitica var. nicotianae, the concentration of tebuconazole is 50 mg / L, and the concentration of imazalil is 20 mg / L.

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