Online disinfection and sterilization effect detection method for seedling substrate production

Abstract

The invention relates to a seedling substrate production online disinfection and sterilization effect detection method, and relates to the technical field of seedling substrate disinfection, the seedling substrate production online disinfection and sterilization effect detection method comprises the following steps: obtaining a continuous cropping # imgabs0 # disinfectant; the method comprises the following steps: preparing a mother solution of the disinfectant, preparing a disinfectant by using the mother solution, and uniformly spraying the disinfectant on a seedling substrate processing production line by using 1-N nozzles. The invention provides an online disinfection and sterilization effect detection method for production of a seedling substrate, which can kill pathogenic bacteria in the substrate to the maximum extent and can ensure the health and safety of tobacco seedlings; and a fluorescent quantitative PCR detection method is introduced, so that the killing effect of pathogenic bacteria is scientifically evaluated. The method has the characteristics of sensitivity, high flux, strong specificity, good repeatability, accurate quantification and the like, and provides powerful technical support for sustainable development of tobacco production.

Description

Technical Field

[0001] The invention relates to the technical field of seedling culture matrix disinfection, in particular to a method for online disinfection and sterilization effect detection of seedling culture matrix production. Background Art

[0002] The substrate is a crucial material in tobacco seedling production, directly impacting seedling quality and the severity of field diseases. According to Yang Cheng from Yunnan, over 90% of primary tobacco field infections are directly related to seedling-stage diseases. Early outbreaks of common mosaic virus (TMV) in severely affected fields are almost always directly linked to infected tobacco seedlings. Therefore, when selecting a substrate, it's important to carefully consider the tobacco seedlings' growth habits and their requirements for the substrate.

[0003] The raw materials for tobacco substrates primarily include peat, vermiculite, peat, cottonseed hulls, carbonized rice husks, corn straw, wheat straw, sugarcane bagasse, and sawdust. These materials come from a wide variety of sources, making their hygiene and safety difficult to guarantee. According to GB / T 25241.1-2010, "Technical Specifications for Intensive Tobacco Seedling Cultivation - Part 1: Floating Seedling Cultivation," the substrate production process must be rigorously disinfected and sterilized with hot steam in a closed space before use. However, in actual production, some companies, driven by cost savings, employ crude processing methods, laxly disinfecting the substrate materials, or even not at all. This results in the contamination of the substrate with numerous pathogens. This phenomenon is widespread, compromising the quality of the seedling substrate and posing a significant safety hazard to tobacco production. If farmers use substrates containing pathogens, insect eggs, pests, or weed seeds, tobacco seedlings can become invisibly infected with the virus or disease. Once these seedlings are transplanted into the field, they can easily develop tobacco diseases, particularly common mosaic virus and Fusarium oxysporum root rot, causing immeasurable economic losses to tobacco farmers. Therefore, effectively disinfecting seedling substrates is a bottleneck that substrate manufacturers urgently need to address.

[0004] Currently, common disinfection methods in China include high-temperature steam, chemical disinfection, and solar disinfection. High-temperature steam disinfection is the most effective, but requires small boiler equipment. Coal-fired boilers are strictly prohibited in many areas, making steam disinfection difficult to implement. Chemical disinfection is simple to use, but its effectiveness is poor and unsatisfactory, and there is a risk of pesticide residue. Solar disinfection requires stringent conditions, sunlight and high temperatures, and a long disinfection cycle. None of these methods can meet the requirements for in-line disinfection of seedling substrate production, making the search for new disinfection methods urgent. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for online disinfection and bactericidal effect detection in the production of seedling substrates.

[0006] The present invention provides a method for online disinfection and sterilization effect detection of seedling substrate production, which comprises the following steps:

[0007] Get disinfectant;

[0008] Prepare the mother liquor of the disinfectant, use the mother liquor to prepare the disinfectant, use 1-N nozzles to spray the disinfectant evenly on the seedling matrix processing production line; bag the disinfected matrix, seal it, and disinfect it in the bag;

[0009] The sterilization effect of the disinfectant is detected on the disinfected substrate using a fluorescent quantitative PCR method to evaluate the killing effect on pathogenic bacteria;

[0010] The disinfectant concentration of the disinfectant is ≥200ppm; the pathogenic bacteria include at least one of the black shank pathogen that causes tobacco black shank disease (referred to as black shank pathogen), the Fusarium oxysporum that causes tobacco Fusarium root rot (referred to as "Fusarium oxysporum"), and the bacterial wilt pathogen that causes tobacco bacterial wilt disease (referred to as bacterial wilt).

[0011] Furthermore, the seedling cultivation matrix includes a seedling cultivation matrix suitable for tobacco seedling cultivation, and the physical and chemical property parameters of the seedling cultivation matrix meet the requirements of the YC / T310-2009 standard.

[0012] Furthermore, the disinfectant is Disinfectant; the disinfectant includes A powder and B tablets.

[0013] Furthermore, the disinfectant A powder and B tablets are sprinkled into water in a ratio of 1:1 by weight to 10-20 parts by weight of water, left to stand for 30 minutes, and then stirred evenly to prepare a mother solution for use.

[0014] Furthermore, the mother liquor prepared from the disinfectant is poured into water at a weight ratio of 1:50-100, and stirred evenly to form a disinfectant.

[0015] Furthermore, the step of spraying the disinfectant on the seedling matrix processing production line to kill pathogenic bacteria in the seedling matrix includes the following process:

[0016] The disinfectant is evenly sprayed onto the surface of the substrate moving on the conveyor belt through 1-N nozzles;

[0017] Among them, the spray width of 1-N nozzles is 3 / 4-4 / 5 of the width of the conveyor belt, so that the disinfectant is sprayed on the surface of the substrate and the two are fully in contact;

[0018] Disinfectant sprayed onto the substrate surface is 900kg-1000kg per hour;

[0019] The weight of each bag of substrate spray disinfectant is 2.0kg-3.0kg;

[0020] The weight of each bag of matrix after disinfection is 15kg-20kg.

[0021] Furthermore, the packaging bag for containing the matrix is a double-layer packaging; the inner packaging bag is made of polyethylene (PE) material; the outer packaging bag is made of plastic or composite material;

[0022] The sterilized seedling medium should be directly bagged and sealed at the end of the conveyor belt; it is strictly forbidden to scatter the sterilized medium to avoid secondary contamination.

[0023] Furthermore, after the matrix is bagged and sealed, the matrix is sterilized in the bag for 5-7 days;

[0024] The substrate in the bag after disinfection is plated, sown and raised according to the conventional seedling raising method;

[0025] After disinfection, the killing rate of black leg pathogen and Fusarium oxysporum in the matrix reached more than 99.89%, and the killing rate of bacterial wilt pathogen was 78.54%.

[0026] Furthermore, the step of detecting the sterilization effect of the disinfectant using a fluorescent quantitative PCR method includes the following process:

[0027] The DNA of the pathogenic bacteria was extracted using the Ezup column soil genomic DNA extraction kit;

[0028] Performing fluorescent quantitative PCR amplification using the pathogenic primers, and then constructing a standard curve of the pathogenic bacteria;

[0029] The number of pathogens in different seedling substrate treatments is calculated using the standard curve of the pathogens to determine the online disinfection and sterilization effects of the seedling substrate production;

[0030] Wherein, the disinfectant concentration of the disinfectant is 200ppm to 400ppm.

[0031] Furthermore, when the disinfectant concentration of the disinfectant is 200 ppm, the killing effects on the black leg pathogen, the Fusarium oxysporum, and the bacterial wilt pathogen are 89.33%, 78.26%, and 46.26%, respectively;

[0032] When the disinfectant concentration of the disinfectant is 400 ppm, the killing effects on the black leg pathogen, the Fusarium oxysporum, and the bacterial wilt pathogen are 99.997%, 99.89%, and 78.54%, respectively.

[0033] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0034] The present invention provides a method for online disinfection and sterilization efficacy testing of seedling substrate production. This method not only addresses the challenges of coal-fired boilers' inability to utilize steam to disinfect substrates, the difficulty of implementing conventional solar disinfection, and the poor and unsatisfactory efficacy of chemical disinfection agents, but also maximizes the elimination of pathogenic bacteria in the substrate, ensuring disease-free, healthy, and safe tobacco seedlings during substrate seedling cultivation, achieving the goal of "a good seedling is half the harvest, and a good seedling is seven-tenths of the harvest." Furthermore, a fluorescent quantitative PCR method is introduced to analyze and evaluate the sterilization efficacy of pathogens such as black shank pathogen, Fusarium oxysporum, and bacterial wilt pathogens. This method is rapid, sensitive, high-throughput, highly specific, highly automated, reproducible, and accurately quantified, providing strong technical support for the sustainable development of tobacco production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0037] Figure 1 A schematic flow chart of a method for online disinfection and bactericidal effect detection of seedling substrate production provided by an embodiment of the present invention.

[0038] Figure 2 In the embodiment of the present invention, 1-N nozzles spray disinfectant on the seedling substrate on the production line for disinfection.

[0039] Figure 3 The figure shows the electrophoresis test results of DNA extracted from matrix pathogens in the examples of the present invention.

[0040] Figure 4 This is the standard curve of Psoralea corylifolia constructed after fluorescent quantitative PCR in the examples of the present invention.

[0041] Figure 5 This is the standard curve of Fusarium oxysporum constructed after fluorescent quantitative PCR in the examples of the present invention.

[0042] Figure 6 This is the standard curve of Ralstonia solanacearum constructed after fluorescent quantitative PCR in the examples of the present invention.

[0043] Figure 7 These are the results of fluorescent quantitative PCR amplification of DNA samples of black leg pathogen in different treated matrices in the examples of the present invention.

[0044] Figure 8 These are the results of fluorescent quantitative PCR amplification of DNA samples of Fusarium oxysporum root rot pathogens in different treated matrices in the examples of the present invention.

[0045] Figure 9 These are the results of fluorescent quantitative PCR amplification of the bacterial wilt pathogen DNA samples in different treated matrices in the examples of the present invention. DETAILED DESCRIPTION

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0047] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0048] The present invention provides a method for detecting the online disinfection and sterilization effect of seedling substrate production. Figure 1 As shown, the method for online disinfection and sterilization effect detection of seedling substrate production includes the following steps:

[0049] Get disinfectant;

[0050] Prepare the mother liquor of the disinfectant, use the mother liquor to prepare the disinfectant, use 1-N nozzles to spray the disinfectant evenly on the seedling matrix processing production line; bag the disinfected matrix, seal it, and disinfect it in the bag;

[0051] The sterilization effect of the disinfectant is detected on the disinfected substrate using a fluorescent quantitative PCR method to evaluate the killing effect on pathogenic bacteria;

[0052] The disinfectant concentration of the disinfectant is ≥200ppm; the pathogenic bacteria include at least one of the black shank pathogen that causes tobacco black shank disease (referred to as black shank pathogen), the Fusarium oxysporum that causes tobacco Fusarium root rot (referred to as "Fusarium oxysporum"), and the bacterial wilt pathogen that causes tobacco bacterial wilt disease (referred to as bacterial wilt).

[0053] The embodiments of the present invention provide a method for online disinfection and sterilization effectiveness testing of seedling substrate production. This method not only addresses the difficulties of using steam from coal-fired boilers to disinfect the substrate, as well as the poor and unsatisfactory disinfection effects of chemical agents, but also maximizes the elimination of pathogenic bacteria in the substrate, ensuring that tobacco seedlings remain disease-free, healthy, and safe during the substrate seedling cultivation period, achieving the goal of "a good seedling is half the harvest, and a good seedling is seven-tenths of the harvest." Simultaneously, a fluorescent quantitative PCR detection method is introduced to analyze and evaluate the sterilization efficacy of pathogens such as black shank pathogen, Fusarium oxysporum, and bacterial wilt pathogens. This method is rapid, sensitive, high-throughput, highly specific, highly automated, reproducible, and accurately quantified, providing strong technical support for the sustainable development of tobacco production.

[0054] The present invention uses real-time fluorescence quantitative PCR (RTFQPCR) for detection. Fluorescence quantitative PCR combines fluorescence resonance energy transfer with fluorescently labeled probes on the basis of conventional PCR, cleverly integrating nucleic acid amplification, hybridization, spectral analysis, and real-time detection technologies. It has the characteristics of rapidity, sensitivity, high throughput, strong specificity, high degree of automation, good repeatability, and accurate quantification. Therefore, this study first uses a disinfectant to kill pathogens in the seedling culture medium, and then uses fluorescence quantitative PCR technology for detection, ultimately providing a theoretical basis for the disinfectant to kill harmful pathogens in the seedling culture medium.

[0055] In some specific embodiments, the seedling cultivation matrix includes a seedling cultivation matrix suitable for tobacco seedling cultivation, and the physical and chemical property parameters of the seedling cultivation matrix meet the requirements of the YC / T310-2009 standard.

[0056] In some specific embodiments, the disinfectant is Disinfectant; the disinfectant includes A powder and B tablets.

[0057] In some specific embodiments, the disinfectant A powder and B tablets are sprinkled into water at a ratio of 1:1 by weight to 15 parts by weight of water, left to stand for 30 minutes, and then stirred to form a mother solution for use.

[0058] In some specific embodiments, the mother liquor prepared from the disinfectant is poured into water at a weight ratio of 1:70 and stirred evenly to form a disinfectant.

[0059] In some specific embodiments, the step of spraying the disinfectant on the seedling substrate processing production line to kill pathogenic bacteria in the seedling substrate includes the following process:

[0060] The disinfectant is evenly sprayed onto the substrate surface moving on the conveyor belt through 1-N nozzles (such as Figure 2 shown);

[0061] Among them, the spray width of 1-N nozzles is 3 / 4-4 / 5 of the width of the conveyor belt, so that the disinfectant is sprayed on the surface of the substrate and the two are fully in contact;

[0062] Disinfectant sprayed onto the substrate surface is 900kg-1000kg per hour;

[0063] The weight of each bag of substrate spray disinfectant is 2.0kg-3.0kg;

[0064] The weight of each bag of matrix after disinfection is 15kg-20kg.

[0065] In some specific embodiments, the packaging bag for containing the matrix is a double-layer packaging; the inner packaging bag is made of polyethylene (PE) material; and the outer packaging bag is made of plastic or a composite material;

[0066] The sterilized seedling medium should be directly bagged and sealed at the end of the conveyor belt; it is strictly forbidden to scatter the sterilized medium to avoid secondary contamination.

[0067] In some specific embodiments, after the matrix is sealed in a bag, the matrix is sterilized in the bag for 5-7 days;

[0068] The substrate in the bag after disinfection is plated, sown and raised according to the conventional seedling raising method;

[0069] After disinfection, the killing rate of black leg pathogen and Fusarium oxysporum in the matrix reached more than 99.89%, and the killing rate of bacterial wilt pathogen was 78.54%.

[0070] In some specific embodiments, the step of detecting the sterilization effect of the disinfectant using a fluorescent quantitative PCR method includes the following process:

[0071] The DNA of the pathogenic bacteria was extracted using the Ezup column-type soil genomic DNA extraction kit;

[0072] Performing fluorescent quantitative PCR amplification using the pathogenic primers, and then constructing a standard curve of the pathogenic bacteria;

[0073] The number of pathogens in different seedling substrate treatments is calculated using the standard curve of the pathogens to determine the online disinfection and sterilization effects of the seedling substrate production;

[0074] Wherein, the disinfectant concentration of the disinfectant is 200ppm to 400ppm.

[0075] In some specific embodiments, when the disinfectant concentration of the disinfectant is 200 ppm, the killing effects on the black leg pathogen, the Fusarium oxysporum, and the bacterial wilt pathogen are 89.33%, 78.26%, and 46.26%, respectively;

[0076] When the disinfectant concentration of the disinfectant is 400 ppm, the killing effects on the black leg pathogen, the Fusarium oxysporum, and the bacterial wilt pathogen are 99.997%, 99.89%, and 78.54%, respectively.

[0077] It should be noted that in the online disinfection and bactericidal effect detection method for seedling matrix production provided by the embodiment of the present invention, the raw materials involved, unless otherwise specified or restricted, can all be commercially available products; at the same time, the process steps and parameters involved can be carried out in accordance with the existing preparation process or by using existing equipment such as "a disinfection spray equipment for seedling matrix production line (ZL20222 1843756.9)" and "a disinfection spray device for seedling matrix production line (ZL2022 21843761.X)" according to conventional operation methods, and the present invention document will not elaborate on them one by one.

[0078] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. If there are no corresponding national standards, then the methods are carried out in accordance with general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0079] Example

[0080] This example provides a method for online disinfection and bactericidal effect detection of seedling substrate production, comprising the following steps:

[0081] 1 Materials and Methods

[0082] 1.1 Test materials

[0083] 1.1.1 Test seedling culture medium

[0084] The technical parameters and indicators of the seedling medium meet the requirements of the YC / T310-2009 standard and are provided by Hubei Luhu Tianhui Agricultural Technology Co., Ltd.

[0085] 1.1.2 For testing disinfectant

[0086] Disinfectant (hereinafter referred to as disinfectant), including powder A and tablet B. Supplied by Zhengzhou Huinong Pest Control Co., Ltd.

[0087] 1.1.3 Test pathogens

[0088] Tobacco black shank pathogen (Phytophthoranicotianae) bacterial solution (abbreviated as black shank pathogen), concentration 1×10 5 cfu / ml, provided by the tobacco leaf quality evaluation and maintenance technology research team of the Tobacco Agriculture Research Laboratory, Zhengzhou Tobacco Research Institute, China National Tobacco Corporation (abbreviated as: provided by Zhengzhou Tobacco Research Institute);

[0089] Tobacco Fusarium oxysporum root rot fungus (Fusarium oxysporum) bacterial solution (abbreviated as Fusarium oxysporum), concentration:

[0090] 2.6×10 7 cfu / ml, provided by the Plant Protection Research Laboratory, Tobacco Research Institute, Henan Academy of Agricultural Sciences (abbreviated as: provided by Henan Academy of Agricultural Sciences).

[0091] Tobacco bacterial wilt pathogen (Ralstonia solanacearum) bacterial solution (referred to as bacterial wilt pathogen), concentration: 4.4×10 9 cfu / ml, provided by the Agronomic Research and Development Center of Guizhou Tobacco Research Institute (abbreviated as: provided by Guizhou Tobacco Research Institute).

[0092] 1.1.4 Instruments

[0093] Sartorius BSA224S-CW electronic analytical balance (Germany), Guohua Electric HH-2 digital constant temperature water bath, Eppendorf MiniSpinPlus micro high-speed centrifuge (Germany), DYY-6C electrophoresis apparatus (Beijing Liuyi Instrument Factory), Bio-Rad BIO-RADGelDoeXR gel imaging analysis system (USA), Thermo Fisher NanoDrop One UV spectrophotometer (USA), Miou MINIP-2500 microplate mini centrifuge (Hangzhou), and LightCycler 480II real-time fluorescence quantitative PCR instrument (Roche, USA).

[0094] 1.1.5 Reagents

[0095] Ezup column-type soil DNA extraction kit, Sangon Biotech (Shanghai) Co., Ltd.

[0096] 1.2 Test methods

[0097] This experiment was conducted in the laboratory of the Tobacco Agriculture Research Laboratory of the Zhengzhou Tobacco Research Institute of China National Tobacco Corporation (hereinafter referred to as the Zhengzhou Tobacco Research Institute Laboratory). A total of three treatments were set up, each with three replicates. The specific treatment settings are shown in Table 1 below.

[0098] Table 1 Treatment design for continuous cropping Wuyou disinfectant matrix disinfection test

[0099]

[0100] 1.2.1 Matrix pathogen inoculation

[0101] The concentrations of the black leg pathogen, Fusarium oxysporum and bacterial wilt pathogen were diluted to 1×10 5 cfu / ml, 2.6×10 5 cfu / ml, 4.4×10 5 cfu / ml, reserve for use.

[0102] Weigh 150g of seedling medium and put it in a small basin. Then inoculate 150ml of the mixture of three pathogens into 150g of the medium. When inoculating, use a small spray bottle to evenly spray the bacterial solution into the medium. Stir while spraying to ensure that the bacterial solution and the medium are fully mixed. Let it stand for 30min-60min and then disinfect it with disinfectant.

[0103] 1.2.2 Disinfection of disinfectant matrix

[0104] The disinfectant concentrations used in this test were 200ppm and 400ppm, respectively. Clean water was used as a control. The specific treatment settings are shown in Table 1. The disinfectant preparation method is as follows:

[0105] 1.2.2.1 Preparation of disinfectant stock solution

[0106] The ratio of disinfectant to water is 1:10. Take 5g of powder A and tablet B respectively, and sprinkle them slowly in a "Z-shape" into 100ml of water. Cover and let it stand for 30 minutes to prepare the mother liquor. Stir well and set aside.

[0107] 1.2.2.2 Preparation of disinfectant

[0108] Measure 200ul and 400ul of the mother solution prepared in 1.2.2.1 respectively, add them to 100ml of clean water, and stir well to make 200ppm and 400ppm disinfectants.

[0109] 1.2.2.3 Disinfection of Disinfectant Matrix

[0110] Weigh 3 portions of the inoculated substrate (prepared in 1.2.1), each weighing 50 g. Measure 10 ml each of 200 ppm and 400 ppm disinfectant and clean water, add them to a small spray bottle, and spray the 3 portions of substrate with clean water, 200 ppm, and 400 ppm disinfectant, respectively, for disinfection.

[0111] 1.2.3 Extraction of matrix pathogen DNA

[0112] The Ezup column-type soil genomic DNA extraction kit was used to extract pathogen DNA. The extraction method is as follows:

[0113] Weigh 300mg of matrix, add 400μl of 65℃ preheated Buffer SCL, shake to mix, and place in a 65℃ water bath for 5 minutes. Centrifuge at 12000rpm at room temperature for 3 minutes. Pipette the supernatant into a clean 1.5ml centrifuge tube. Then add an equal volume of Buffer SP, invert to mix, and place on ice for 10 minutes. Centrifuge at 12000rpm at room temperature for 3 minutes. Pipette the supernatant into a clean 1.5ml centrifuge tube. Add 200μl of chloroform, mix thoroughly, and centrifuge at 12000rpm for 5 minutes. Pipette the upper aqueous phase into a clean 1.5ml centrifuge tube. Add 1.5 times the volume of Buffer SB, mix thoroughly, and then pipette it all into the adsorption column and let it stand at room temperature for 2 minutes. Centrifuge at 12000rpm for 30 seconds and discard the waste liquid in the collection tube. Return the adsorption column to the collection tube, add 700 μl of WashSolution, and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid from the collection tube. Return the adsorption column to the collection tube, add 300 μl of WashSolution, and centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube. Return the adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 minutes. Remove the adsorption column and place it in a new 1.5 ml centrifuge tube. Add 50 μl of TEBuffer to the center of the adsorption membrane, let it stand for 3 minutes, and centrifuge at 12,000 rpm for 2 minutes. Store the resulting DNA solution at -20°C or use it directly in subsequent experiments.

[0114] The integrity of the DNA was detected by 1% agarose gel electrophoresis, and its content was measured by ultraviolet spectrophotometer. The obtained DNA solution was stored at -20°C.

[0115] 1.2.4 Establishment of standard curve and LNA quantitative PCR detection of pathogens in different treatments

[0116] A 1.0ml suspension of Psoralea corylifolia, Fusarium oxysporum, and Ralstonia solanacearum was pipetted and centrifuged. Pathogen DNA was extracted using the Ezup column-based soil genomic DNA extraction kit to obtain a 50µl DNA solution. The concentrations were measured using a UV spectrophotometer. The DNA concentrations for Psoralea corylifolia, Fusarium oxysporum, and Ralstonia solanacearum were 1.3ng / µl, 30.2ng / µl, and 1.7ng / µl, respectively. The DNAs from each of the three bacterial suspensions were then diluted 5-fold in a gradient. 1µl of each diluted concentration gradient was used as a quantitative PCR template. Fluorescence quantitative PCR amplification was performed using primers for each of the three pathogens, and a standard curve was constructed.

[0117] At the same time, nine samples were extracted from the three treatments (Table 1), each replicated three times, for a total of nine samples. LNA fluorescent quantitative PCR was then performed on each of these nine samples using three primer pairs specific for each of the three pathogens. The total fluorescent quantitative PCR reaction system consisted of 25 μl of 2× fluorescent quantitative PCR reaction mixture, 12.5 μl of 10 μl forward primer, 2 μl of reverse primer, and 1 μl of the matrix pathogen DNA solution to be tested. Amplification conditions were as follows:

[0118] The quantitative PCR amplification conditions for black leg pathogen were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, 50 cycles; extension at 72°C for 10 min;

[0119] The quantitative PCR amplification conditions for Fusarium oxysporum were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, 50 cycles; extension at 72°C for 10 min;

[0120] Quantitative PCR amplification conditions for Ralstonia solanacearum: The quantitative PCR program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 20 s, for 60 cycles;

[0121] Among them, the LNA primers for black shank pathogen used in this experiment are shown in Table 2-1, and the black shank pathogen primers were provided by Zhengzhou Tobacco Research Institute; the LNA primers for Fusarium oxysporum used in this experiment are shown in Table 2-2, and were provided by the Tobacco Research Institute of Henan Academy of Agricultural Sciences; the LNA primers for bacterial wilt used in this experiment are shown in Table 2-3, and were provided by Guizhou Tobacco Research Institute.

[0122] Table 2-1: LNA primers of Psoralea corylifolia used in this experiment

[0123]

[0124] Table 2-2: Fusarium oxysporum LNA primers used in this experiment

[0125]

[0126] Table 2-3: LNA primers for bacterial wilt used in this experiment

[0127]

[0128] 3 Results and Analysis

[0129] 3.1 Matrix pathogen DNA extraction and detection results

[0130] Through the analysis of DNA extraction and detection results of different matrix pathogens, it can be seen that the DNA extracted by the Ezup kit is very complete, the main DNA band is above 10kb, and it presents a clear DNA band ( Figure 3 ), among which, the three repeat DNA concentrations of A200-YLQ treatment were 24.2ng / ul, 22.8ng / ul, and 17.6ng / ul, respectively; the three repeat DNA concentrations of A400-YLQ treatment were 9.5ng / ul, 48.1ng / ul, and 19.8ng / ul, respectively; the three repeat DNA concentrations of CK0-YLQ treatment were 15.6ng / ul, 17.4ng / ul, and 18.2ng / ul, respectively. The DNA extraction effects all met the test requirements and can be used for subsequent tests; the DNA electrophoresis test results of matrix pathogen extraction are as follows Figure 4 、 Figure 5 、 Figure 6 As shown, Figure 4-Figure 6 Middle: M:DL15000DNAMaker; 1.A200-YLQ-1; 2.A200-YLQ-2; 3.A200-YLQ-3; 4.A400-YLQ-1; 5.A400-YLQ-2; 6.A400-YLQ-3; 7.A800-YLQ-1; 8.A800-YLQ-2; 9.A800-YLQ-3; 10.CK0-YLQ-1; 11.CK0-YLQ-2; 12.CK0-YLQ-3; Table 3 shows the DNA extraction concentrations of pathogenic bacteria in different treatment matrices.

[0131] Table 3 DNA extraction concentration of pathogenic bacteria in different treatment matrices

[0132]

[0133] 3.2 Establishment of standard curve

[0134] DNA was extracted from 1.0 ml of the three pathogenic bacteria solution to obtain 50 μl of DNA solution, which was then diluted 5-fold. 1 μl of the solution was used as a quantitative PCR template. Fluorescence quantitative PCR amplification was performed using the three pathogenic bacteria primers, and a standard curve was constructed. The results are shown in the table. Figure 4 (Standard curve of black leg pathogen constructed after fluorescence quantitative PCR), Figure 5 (Standard curve of Fusarium oxysporum constructed after fluorescence quantitative PCR), Figure 6 (Standard curve of Ralstonia solanacearum constructed after fluorescence quantitative PCR).

[0135] 3.3 Fluorescence quantitative PCR amplification results

[0136] Pathogen DNA was extracted from 0.30g of differently treated substrates to obtain 50ul DNA solution. 1ul was used as a quantitative PCR template and fluorescent quantitative PCR amplification was performed using three pathogen primers. The number of pathogens in the substrate to be tested can be calculated based on the standard curve. The amplification results show that Figure 7 (Results of fluorescence quantitative PCR amplification of DNA samples of black leg pathogen in different treatment matrices) Figure 8 (Results of fluorescence quantitative PCR amplification of DNA samples of Fusarium oxysporum root rot pathogen in different treatment matrices), Figure 9 (Fluorescence quantitative PCR amplification results of bacterial wilt pathogen DNA samples in different treatment matrices) The fluorescence intensity of the matrix samples was strong, indicating that all treated pathogen DNAs produced obvious amplification products, and there were differences in the pathogens of different treatments.

[0137] 3.4 Results of fluorescence quantitative PCR detection of the killing effect of disinfectants on pathogens in the matrix

[0138] Standard curves for three pathogens were constructed using fluorescent quantitative PCR. The Cp values of pathogen DNA from different treatments were substituted into the linear equations of the standard curves to calculate the number of pathogens in each matrix treatment (Table 4). Analysis revealed that, overall, the disinfectant concentrations of 200 ppm and 400 ppm all had varying degrees of effectiveness against blackleg pathogens, Fusarium oxysporum, and bacterial wilt. The high concentration (400 ppm) was more effective than the low concentration (200 ppm). Furthermore, the disinfectant was more effective against fungi than against bacteria. The 200 ppm and 400 ppm concentrations of disinfectant killed 89.33% and 99.997% of blackleg pathogens, respectively; 78.26% and 99.89% of Fusarium oxysporum, respectively; and 46.26% and 78.54% of bacterial wilt, respectively. Overall, the effectiveness was good.

[0139] Table 4 Comparison of the killing effects of different disinfectant concentrations on different pathogens in the matrix

[0140]

[0141] 4 Conclusion

[0142] The seedling matrix is an important seedling material in the seedling raising process. If the matrix is not thoroughly disinfected, is toxic or contaminated, it will cause the seedling growth to weaken, the tobacco seedlings to be unhealthy, and the tobacco seedlings to be infected. Once these tobacco seedlings are transplanted into the field, it will cause a concentrated outbreak of tobacco diseases, especially TMV, Fusarium oxysporum root rot, bacterial wilt, etc., which will cause immeasurable economic losses to tobacco production. Therefore, whether the seedling matrix is thoroughly disinfected has become one of the restrictive factors affecting the sustainable development of tobacco production. The present invention sprays a disinfectant on the matrix production line, allowing the disinfected matrix to continue to play a disinfecting role in the packaging bag, effectively killing the pathogens in the matrix for a long time, and using fluorescent quantitative PCR to detect the killing effect. The results show that:

[0143] 1. The disinfectant has a significant killing effect on blackleg pathogens, Fusarium oxysporum, and bacterial wilt. Furthermore, the killing effect on pathogenic fungi is significantly higher than that on bacteria. The killing effect on fungi is greater than 75.00%, with a maximum of 99.997%. The killing effect on bacteria is greater than 45.00%, with a maximum of 78.54%.

[0144] 2. The disinfectant's effectiveness against pathogens exhibits a gradient effect. Within the test range, the sterilization effect gradually increases with increasing concentration. At a concentration of 200 ppm, the disinfectant achieved kill rates of 89.33% against blackleg pathogens, 78.26% against Fusarium oxysporum, and 46.26% against Ralstonia solanacearum. At a concentration of 400 ppm, the kill rates were 99.997%, 99.89%, and 78.54%, respectively. Overall, the disinfectant demonstrated excellent effectiveness against tobacco pathogens.

[0145] 3. In view of the advantages of online matrix disinfection technology with disinfectants, which is fast, simple, safe, labor-saving and efficient, it can kill pathogenic bacteria in the matrix to the maximum extent and ensure the health and safety of tobacco seedlings.

[0146] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as an inflexible limitation of the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values within the range. In addition, whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range.

[0147] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for online disinfection and bactericidal effect detection of seedling substrate production, characterized in that: The method for detecting the online disinfection and sterilization effect of the seedling culture matrix comprises the following steps: Get disinfectant; Prepare the mother liquor of the disinfectant, use the mother liquor to prepare the disinfectant, use 1-N nozzles to spray the disinfectant evenly on the seedling matrix processing production line; bag the disinfected matrix, seal it, and disinfect it in the bag; The sterilization effect of the disinfectant is detected on the disinfected substrate using a fluorescent quantitative PCR method to evaluate the killing effect on pathogenic bacteria; The disinfectant concentration of the disinfectant is ≥200 ppm; the pathogenic bacteria include at least one of the black shank pathogen that causes tobacco black shank disease, the Fusarium oxysporum that causes tobacco root rot, and the bacterial wilt pathogen that causes tobacco bacterial wilt disease.

2. The method for detecting the online disinfection and sterilization effect of the seedling culture medium according to claim 1, wherein: The seedling cultivation matrix includes a seedling cultivation matrix suitable for tobacco seedling cultivation, and the physical and chemical property parameters of the seedling cultivation matrix meet the requirements of the YC / T310-2009 standard.

3. The method for detecting online sterilization and bactericidal effect of seedling culture substrate production according to claim 1, wherein: The disinfectant is continuous cropping Disinfectant; the disinfectant includes A powder and B tablets.

4. The method for detecting the online disinfection and sterilization effect of the seedling culture medium according to claim 3, wherein: The disinfectant A powder and B tablets are sprinkled into water in a ratio of 1:1 by weight to 10-20 parts by weight of water, left to stand for 30 minutes, and then stirred to prepare a mother solution for use.

5. The method for detecting online disinfection and sterilization effect of seedling culture matrix production according to claim 4, wherein: The mother liquor prepared from the disinfectant is poured into water at a ratio of 1:50-100 by weight, and stirred evenly to form a disinfectant solution.

6. The method for detecting online disinfection and sterilization effect of seedling culture matrix production according to claim 5, wherein: The step of spraying the disinfectant on the seedling matrix processing production line to kill the pathogenic bacteria in the seedling matrix comprises the following process: The disinfectant is evenly sprayed onto the surface of the substrate moving on the conveyor belt through 1-N nozzles; Among them, the spray width of 1-N nozzles is 3 / 4-4 / 5 of the width of the conveyor belt, so that the disinfectant is sprayed on the surface of the substrate and the two are fully in contact; Disinfectant sprayed onto the substrate surface is 900kg-1000kg per hour; The weight of each bag of substrate spray disinfectant is 2.0kg-3.0kg; The weight of each bag of matrix after disinfection is 15kg-20kg.

7. The method for detecting online disinfection and sterilization effect of seedling culture matrix production according to claim 6, wherein: The packaging bag for containing the matrix is a double-layer packaging; the inner packaging bag is made of polyethylene (PE) material; the outer packaging bag is made of plastic or composite material; The sterilized seedling medium should be directly bagged and sealed at the end of the conveyor belt; it is strictly forbidden to scatter the sterilized medium to avoid secondary contamination.

8. The method for detecting online disinfection and sterilization effect of seedling culture substrate production according to claim 7, wherein: After the matrix is bagged and sealed, the matrix is sterilized in the bag for 5-7 days; The substrate in the bag after disinfection is plated, sown and raised according to the conventional seedling raising method; After disinfection, the killing rate of black leg pathogen and Fusarium oxysporum in the matrix reached more than 99.89%, and the killing rate of bacterial wilt pathogen was 78.54%.

9. The method for detecting online disinfection and sterilization effect of seedling culture medium production according to claim 1, wherein The steps of detecting the sterilization effect of the disinfectant using the fluorescent quantitative PCR method include the following process: The DNA of the pathogenic bacteria was extracted using the Ezup column-type soil genomic DNA extraction kit; Performing fluorescent quantitative PCR amplification using the pathogenic primers, and then constructing a standard curve of the pathogenic bacteria; The number of pathogens in different seedling substrate treatments is calculated using the standard curve of the pathogens to determine the online disinfection and sterilization effects of the seedling substrate production; Wherein, the disinfectant concentration of the disinfectant is 200ppm to 400ppm.

10. The method for online disinfection and bactericidal effect detection of seedling substrate production according to claim 9, characterized in that: When the disinfectant concentration is 200 ppm, the killing effects on the black leg pathogen, the Fusarium oxysporum, and the bacterial wilt pathogen are 89.33%, 78.26%, and 46.26%, respectively. When the disinfectant concentration of the disinfectant is 400 ppm, the killing effects on the black leg pathogen, the Fusarium oxysporum, and the bacterial wilt pathogen are 99.997%, 99.89%, and 78.54%, respectively.

Images