Kit for detecting phytophthora root rot of medicago sativa and detection method thereof

Abstract

The invention discloses a kit for detecting phytophthora root rot of alfalfa and a detection method thereof, and relates to the technical field of biology. The detection kit and the detection method provided by the invention can specifically detect whether an alfalfa sample to be detected is infected with phytophthora root rot fungi or not, can identify whether the alfalfa phytophthora root rot fungi exist or not from a small amount of alfalfa samples, and have the characteristic of high sensitivity; besides, the PCR primer combination for detecting the phytophthora medicaginis also has the characteristics of short time consumption and simplicity and convenience in operation, and the detection can be completed in a relatively short time under laboratory conditions; the detection method provided by the invention is helpful for an inspector of phytophthora rot of medicago to quickly identify whether the medicago seeds are infected with phytophthora rot of medicago, so that medicago can better serve animal husbandry production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a kit for detecting alfalfa Phytophthora root rot pathogen and a detection method thereof. Background Art

[0002] Alfalfa (Medicago sativa L.) is known as the "king of forage grass" in the world for its high quality, high yield, high feeding value (crude protein content 21%), good palatability and wide range of uses. This versatile plant can be grown in various soils and climate conditions, so the planting area in more than 80 countries has reached 35 million hectares. As a high-value crop, due to its well-developed root system and the ability to reduce soil erosion, it can also be used to restore pastures. Therefore, alfalfa has important economic and ecological value.

[0003] As the area of ​​cultivation increases, alfalfa diseases are becoming more serious, resulting in a significant decline in alfalfa production worldwide. Root rot was first reported in 1989. Root rot is mainly caused by fungi, oomycetes and nematodes. The infected tissue changes color to form brown (black) and necrotic areas, which may cause plant death. Several plant pathogenic fungi and oomycetes can cause root rot. These pathogens include Fusarium oxysporum and Fusarium equisetum, Rhizoctonia solani, Pythium malathione and other fungi.

[0004] Alfalfa root rot is one of the main diseases that causes alfalfa yield loss. Susceptible alfalfa varieties can cause a yield loss of nearly 68%. The pathogen is Phytophthora medicaginis. Once alfalfa is infected, the damage is very serious. Quarantine is the most economical and effective way to prevent the colonization and spread of the pathogen. Accurate, fast and effective detection methods are the technical basis for achieving effective quarantine.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a kit for detecting Phytophthora alfalfa root rot pathogen and a detection method thereof. The kit and the detection method of the present invention can accurately, quickly and effectively detect Phytophthora alfalfa root rot pathogen.

[0007] In a first aspect, the present invention provides a primer set for detecting Phytophthora alfalfa root rot, which comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0008] In a second aspect, the present invention provides the use of the above primer set in detecting Phytophthora alfalfa root rot pathogen.

[0009] In a third aspect, the present invention provides the use of the above primer set in preparing a kit for detecting Phytophthora alfalfa root rot pathogen.

[0010] In a fourth aspect, the present invention provides a kit for detecting Phytophthora alfalfa root rot pathogen, comprising the above-mentioned primer set.

[0011] In a fifth aspect, the present invention also provides the use of the above-mentioned kit in detecting alfalfa Phytophthora root rot pathogen.

[0012] In a sixth aspect, the present invention also provides a method for detecting Phytophthora alfalfa root rot pathogen, comprising:

[0013] Using the DNA of the sample to be tested as an amplification template, the above primer set is used to establish a PCR reaction system and perform PCR amplification;

[0014] The amplified product is detected. If the target band is amplified, it is determined that the sample to be tested is infected with alfalfa phytophthora root rot bacteria; otherwise, it is determined that the sample to be tested is not infected with alfalfa phytophthora root rot bacteria.

[0015] The present invention has the following beneficial effects:

[0016] (1) The present invention designs a PCR primer set with a specific amplification effect on Phytophthora alfalfa root rot fungus based on the characteristics of high conservation of gene sequences within fungal species and variability between genera and species. The primer set can distinguish Phytophthora alfalfa root rot fungus from other common pathogens such as Rhizoctonia solani, and has the characteristics of high accuracy;

[0017] (2) The detection kit and the detection method of the present invention can identify whether there is Phytophthora alfalfa root rot from a small amount of alfalfa samples, and have the characteristics of high sensitivity;

[0018] (3) The detection of Phytophthora alfalfa root rot pathogen using the PCR primer combination of the present invention is also time-saving and easy to operate. The detection can be completed in a short time under laboratory conditions. The detection method of the present invention helps Phytophthora alfalfa root rot pathogen inspectors to quickly identify whether alfalfa seeds are infected with Phytophthora alfalfa root rot pathogens, so that alfalfa can better serve the livestock production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1The PCR identification results of whether 14 alfalfa seed varieties contain alfalfa Phytophthora root rot pathogen;

[0021] Figure 2 for Figure 1 Sequencing and comparison of PCR products of 20 seeds of Zhongmu 6 and Zhonglan 1;

[0022] Figure 3 The PCR identification results of the sensitivity of the kit for identifying the alfalfa Phytophthora root rot pathogen in different numbers of seeds of Zhonglan No. 1, Adina, MF4010, and Zhongmu No. 1;

[0023] Figure 4 The specific identification results of PCR primers for detecting Phytophthora alfalfa root rot pathogen;

[0024] Figure 5 The PCR results of existing primers used to identify whether 14 alfalfa seed varieties contain alfalfa root rot pathogen Phytophthora alfalfa. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] In order to quickly, accurately and efficiently detect the colonization of Phytophthora alfalfa root rot in alfalfa, the present invention designed a pair of PCR primer sets for Phytophthora alfalfa root rot based on the characteristics of high conservation of gene sequences within fungal species and variability between genera and species. The primer sets are:

[0027] MD5-F (5′-3′): CAATAAACTTGGCCTCCCTTCCTT-SEQ ID NO.1;

[0028] MD5-R(5′-3′): GAACAGACACCCTACTTCGCACC-SEQ ID NO. 2.

[0029] Based on the above primer set, the present invention can provide a detection kit for Phytophthora alfalfa root rot pathogen, which comprises the above primer pair and other conventional reagents for PCR amplification, including PCR amplification reagents, DNA templates and ddH2O.

[0030] Among them, the PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and other commonly used auxiliary reagents.

[0031] In addition to the PCR amplification reagent provided by the present invention, the PCR reaction system can also use commercial products of the same type, and PCR amplification can be performed by adding a specified amount of DNA template and primers before amplification. The premise is that the positive control sample must amplify the correct band, and the negative control sample has no amplified band.

[0032] By amplifying the DNA of the samples infected or not infected with Phytophthora alfalfa root rot, it was found that the target band could be amplified from the samples only in the presence of Phytophthora alfalfa root rot.

[0033] Since the above-mentioned detection primers or kit can be used to identify whether an alfalfa sample contains Phytophthora alfalfa root rot bacteria, the present invention also provides a method for detecting whether an alfalfa sample is infected with Phytophthora alfalfa root rot bacteria. The detection sample can be either the mycelium of Phytophthora alfalfa root rot bacteria or the roots, leaves, seeds, seedlings and soil samples of susceptible alfalfa, so that the detection of Phytophthora alfalfa root rot bacteria in various scenarios can be realized.

[0034] Taking alfalfa seeds as an example, the method for detecting whether alfalfa seeds contain alfalfa phytophthora root rot bacteria includes the following steps:

[0035] (1) Extracting genomic DNA from alfalfa seeds to be tested;

[0036] (2) using the DNA obtained in step (1) as a template and the above primer pair to perform a PCR amplification reaction;

[0037] (3) taking the PCR amplification product and detecting it by agarose gel electrophoresis;

[0038] (4) judging whether the alfalfa seeds to be tested are infected with Phytophthora alfalfa root rot fungi according to whether characteristic bands appear in the electrophoresis bands: if bands appear in the electrophoresis results, the alfalfa seeds are infected with Phytophthora alfalfa root rot fungi; if no bands appear, the alfalfa seeds are not infected with Phytophthora alfalfa root rot fungi.

[0039] In the above detection method, the reaction system of PCR amplification is 25 μL: 12.5 μL of PCR amplification reagent, 1.0 μL of upstream primer, 1.0 μL of downstream primer, 1 μL of template DNA, 9.5 μL of ddH2O; wherein the concentration of DNA template is <600 ng / μL; the concentration of upstream primer and downstream primer is 10 μM.

[0040] The reaction program of PCR amplification was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 30 s, annealing at 56°C for 10 s, extension at 72°C for 5 s, 35 cycles, and extension at 72°C for 1 min.

[0041] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0042] Example 1

[0043] This embodiment is a kit for detecting Phytophthora alfalfa root rot, and its components include: a PCR amplification primer pair, 2×Phanta Flash Master Mix (Dye Plus) (purchased from Novozymes), a DNA template and ddH2O.

[0044] Among them, the PCR amplification primer pair is:

[0045] MD5-F (5′-3′): CAATAAACTTGGCCTCCCTTCCTT-SEQ ID NO.1;

[0046] MD5-R(5′-3′): GAACAGACACCCTACTTCGCACC-SEQ ID NO. 2.

[0047] Example 2

[0048] This embodiment is a method for detecting whether alfalfa seeds are infected with alfalfa Phytophthora root rot bacteria, which uses the detection kit in Example 1 for detection, and the specific steps are as follows:

[0049] S1. Collect alfalfa seeds of different varieties and extract genomic DNA for later use.

[0050] S2. Using the PCR amplification primer pair in Example 1, perform PCR detection on the seed genomic DNA.

[0051] The total volume of the PCR detection system was 25 μL, including: 12.5 μL of 2×Phanta Flash Master Mix (Dye Plus), 1 μL of upstream primer, 1 μL of downstream primer; 1 μL of DNA template (concentration less than 600 ng / μL); 9.5 μL of ddH2O.

[0052] The amplification conditions were as follows: (1) pre-denaturation at 98°C for 30 s; (2) denaturation at 98°C for 30 s; annealing at 56°C for 10 s; (4) extension at 72°C for 5 s; (5) steps (2) to (4) were cycled 35 times; and (6) extension at 72°C for 1 min.

[0053] S3. Use 1.5% agarose gel for detection. Weigh 1.5g agarose, add 100mL of prepared 1×TAE, heat in a microwave oven for about 3min until the agarose is completely dissolved, add 5μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 50min, run the gel for 25min at 130V in a vertical plate electrophoresis instrument, and then observe the results under a gel imager. The above steps were repeated three times to ensure the credibility of the experiment.

[0054] Test results such as Figure 1 As shown in Figure A and Figure B, 1-14 represent genomic DNA of 20 seeds and leaves of different alfalfa varieties, respectively; CK(+) represents Phytophthora alfalfa root rot pathogen (positive control); CK(-) is water (negative control); M is a marker; the names of alfalfa varieties represented by 1-14 are shown in Table 1. All genomic DNAs were extracted three times independently and PCR assays were performed respectively. The gel images are random images of three biological replicates.

[0055] Table 1 Information on the 14 participating alfalfa varieties

[0056]

[0057] from Figure 1 It can be seen that no bands were detected in the seeds of alfalfa varieties No. 2 and No. 8, while bands were detected in the other 12 varieties.

[0058] right Figure 1 The PCR products with detected bands were sequenced and analyzed, specifically, the PCR products of 20 seeds of Zhonglan No. 1 and Zhongmu No. 1 were sequenced and compared. The results are as follows Figure 2 As shown in the figure, it can be seen that the sequencing results are highly similar to the template sequence.

[0059] Experimental Example 1

[0060] This experiment is a sensitivity detection test of the kit in Example 1, and is as follows:

[0061] S1. The genomic DNA of 5, 10, 20 and 40 alfalfa seeds detected to contain or not contain alfalfa Phytophthora root rot pathogen (Zhonglan No. 1, Zhongmu No. 1, MF4010 and Adina) in Example 2 was re-extracted for PCR detection.

[0062] The total volume of the PCR detection system is 25 μL, including: 2×Phanta Flash Master Mix (Dye Plus) 12.5 μL, upstream primer 1 μL, downstream primer 1 μL; DNA template: 1 μL; ddH2O 9.5 μL.

[0063] Among them, in the DNA template: the concentration of 1 seed is 153ng / μL; the concentration of 10 seeds is 191ng / μL; the concentration of 20 seeds is 305ng / μL; the concentration of 40 seeds is 387ng / μL; the concentration of 60 seeds is 443ng / μL.

[0064] The amplification conditions were as follows: (1) pre-denaturation at 98°C for 30 s; (2) denaturation at 98°C for 30 s; annealing at 56°C for 10 s; (4) extension at 72°C for 5 s; (5) steps (2) to (4) were cycled 35 times; and (6) extension at 72°C for 1 min.

[0065] S2. Use 1.5% agarose gel for detection, weigh 1.5g agarose, add 100mL of prepared 1×TAE, heat in a microwave oven for about 3min until the agarose is completely dissolved, add 5μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 50min, run the gel for 25min at 130V in a vertical plate electrophoresis instrument, and then observe the results under a gel imager. The above steps were repeated three times to ensure the credibility of the experiment.

[0066] Test results such as Figure 3 As shown in Figures A, B, C and D, No. 1-4 represent the PCR identification results of genomic DNA extracted from 40, 20, 10 and 1 seeds of alfalfa seeds, respectively, Zhonglan No. 1, Adina, MF4010 and Zhongmu No. 1; CK(+) represents Phytophthora alfalfa root rot (positive control); CK(-) is water (negative control); M is a marker. All genomic DNA was extracted three times independently and PCR assayed respectively. The gel image is a random image from three biological replicates.

[0067] Figure 3 It can be seen that the genomic DNA of 40, 20, 10, and 5 seeds of Zhonglan No. 1 and Adina alfalfa varieties can detect bands, and the bands become darker in sequence. The genomic DNA of 40, 20, 10, and 5 seeds of MF4010 and Zhongmu No. 1 alfalfa varieties cannot detect bands. It can be concluded that even if one alfalfa seed is used as a sample, the detection primer of the present invention can detect whether the sample to be tested is infected with Phytophthora alfalfa root rot, which also proves that the kit of the present invention has high sensitivity.

[0068] Experimental Example 2

[0069] This experiment is a specificity verification test of the kit in Example 1, and is as follows:

[0070] S1. Collect the bacterial liquid of Rhizoctonia solani, streak it on the culture medium with corresponding resistance, and culture it upside down in a 37℃ incubator until obvious monoclonal colonies appear.

[0071] Pick a single colony, then place it in 20 μL triton-x100 and stir it. Mark the corresponding bacteria and the streaked area on the plate, and boil the EP tube containing 20 μL triton-x100 at 100℃ for 2 minutes.

[0072] S2. Using the PCR amplification primer pair in Example 1, perform PCR detection on the genomic DNA of Rhizoctonia solani.

[0073] The total volume of the PCR detection system was 25 μL, including 12.5 μL of 2×Phanta Flash Master Mix (Dye Plus), 1 μL of boiled bacterial supernatant, 1 μL of upstream primer (concentration of 10 μM), 1 μL of downstream primer (concentration of 10 μM), and 8.5 μL of ddH2O.

[0074] The amplification conditions were as follows: (1) pre-denaturation at 98°C for 30 s; (2) denaturation at 98°C for 30 s; annealing at 56°C for 10 s; (4) extension at 72°C for 5 s; (5) steps (2) to (4) were cycled 35 times; and (6) extension at 72°C for 1 min.

[0075] S3. Use 1.5% agarose gel for detection. Weigh 1.5g agarose, add 100mL of prepared 1×TAE, heat in a microwave oven for about 3min until the agarose is completely dissolved, add 5μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 50min, run the gel for 25min at 130V in a vertical plate electrophoresis instrument, and then observe the results under a gel imager. The above steps were repeated three times to ensure the credibility of the experiment.

[0076] Test results such as Figure 4 As shown in the figure, 1 represents Phytophthora alfalfa root rot pathogen; 2-4 represent Rhizoctonia solani; CK(+) represents positive control (Phytophthora alfalfa root rot pathogen); CK(-) represents negative control (water); M is a marker. All genomic DNA was extracted three times independently and PCR assays were performed respectively. The gel image is a random image from three biological replicates.

[0077] from Figure 4 It can be seen that there are no bands in lanes 2-4, indicating that the detection primers of the present invention can only specifically identify samples containing alfalfa Phytophthora root rot bacteria.

[0078] Experimental Example 3

[0079] In this experiment, primers were designed based on the gene sequence of alfalfa Phytophthora root rot pathogen in an existing authorized patent (CN102051415A). Two sets of PCR tests were performed using DNA amplified from 14 alfalfa seed varieties as templates, and the results were compared with the PCR identification results using the special primers designed in the present invention to further illustrate the sensitivity of the detection primers of the present invention.

[0080] Test results such as Figure 5 As shown: Figures 1-4 in A, B, C and D represent the PCR identification results of genomic DNA extracted from 40, 20, 10 and 1 seeds of alfalfa seeds of Zhonglan No. 1, Adina, MF4010 and Zhongmu No. 1, respectively; CK(+) represents Phytophthora alfalfa root rot (positive control); CK(-) is water (negative control); M is a marker. All genomic DNA was extracted three times independently and PCR assayed respectively. The gel image is a random image of three biological replicates.

[0081] from Figure 5 It can be seen that the published primers for detecting Phytophthora root rot cannot detect all alfalfa seeds infected with Phytophthora root rot.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer set for detecting Phytophthora alfalfa root rot pathogen, characterized in that: It comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

2.

2. Use of the primer set as claimed in claim 1 in detecting alfalfa Phytophthora root rot pathogen.

3. Use of the primer set as claimed in claim 1 in preparing a kit for detecting Phytophthora alfalfa root rot pathogen.

4. A kit for detecting Phytophthora alfalfa root rot pathogen, characterized in that: Comprising the primer set of claim 1.

5. The kit according to claim 4, characterized in that The kit also includes PCR amplification reagents, DNA templates and ddH2O.

6. The kit according to claim 5, characterized in that The PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer.

7. Use of the kit as described in any one of claims 4 to 6 in detecting Phytophthora alfalfa root rot pathogen.

8. A method for detecting Phytophthora alfalfa root rot pathogen, characterized in that: include: Using the DNA of the sample to be tested as an amplification template, using the primer set described in claim 1, establishing a PCR reaction system, and performing PCR amplification; The amplified product is detected. If the target band is amplified, it is determined that the sample to be tested is infected with alfalfa phytophthora root rot bacteria; otherwise, it is determined that the sample to be tested is not infected with alfalfa phytophthora root rot bacteria.

9. The method according to claim 8, characterized in that The reaction system is 25 μL: 12.5 μL of PCR amplification reagent, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template DNA, and 9.5 μL of ddH2O; The concentrations of the upstream primer and the downstream primer are: 10 μM; the concentration of the template DNA is <600 ng / μL.

10. The method according to claim 8, characterized in that The reaction program of PCR amplification was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 30 s, annealing at 56°C for 10 s, extension at 72°C for 5 s, 35 cycles, and extension at 72°C for 1 min.

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