CN110106242B - Detection primers and detection methods for the chemotypes of the pathogenic toxin of Fusarium alfalfa root rot - Google Patents

Abstract

The invention relates to the technical field of plant inspection and quarantine, in particular to a detection primer and a detection method for the chemical type of the pathogenic toxin of Fusarium alba root rot. The present invention provides specific primers for detecting chemical types of Fusarium alfalfa root rot pathogenic toxins, the sequences of which are shown in SEQ ID NOs. 1-6. The three pairs of specific primers can be used to accurately, quickly and conveniently detect whether the pathogen of Fusarium alfalfa root rot has the potential to produce trichothecenes, zearalenone and fumonisin. Accurate, specific and sensitive detection of Fusarium alfalfa root rot pathogenic toxin chemotypes by conventional PCR technology, which effectively saves the detection time and cost, and provides the potential for toxin production of Fusarium isolated from alfalfa or alfalfa products. Assessment provides a simple, fast and accurate method.

Description

Detection primers and detection methods for the chemotypes of the pathogenic toxin of Fusarium alfalfa root rot

technical field

The invention relates to the technical field of plant inspection and quarantine, in particular to a specific primer for detecting the chemical type of the pathogenic toxin of Fusarium alfalfa root rot and a method for detecting the chemical type of the pathogenic toxin of Fusarium alfalfa root rot by using the specific primer.

Background technique

Alfalfa (Medicago sativa) is the leguminous forage with the largest planting area in the world. In recent years, the planting area of alfalfa in China has gradually increased. With the increase of alfalfa planting area and planting years, alfalfa root rot is becoming more and more serious. Alfalfa root rot can occur in various growth stages of alfalfa, and its symptoms are mostly wilting and drooping of plant tissue. In severe cases, the whole plant turns yellow and is easy to pull out from the soil. severe shrinkage. Once alfalfa root rot spreads in a large area, it will seriously reduce the yield and quality of alfalfa, and even need to be replanted.

Fusarium is one of the main pathogens that cause root rot in alfalfa. Fusarium can produce trichothecenes, zearalenone, fumonisin and other toxins, which are the cause of the disease. At the same time, it will cause harm to the nervous system, digestive system, reproductive system, etc. of humans and animals, which can lead to acute poisoning of humans and animals, and cause serious harm to the health of humans and animals. The types of toxins that can be produced by different Fusarium species will vary, and Fusarium species can be divided into different toxin chemotypes. The detection of Fusarium toxin chemotypes is of great significance for understanding the toxin-producing potential of Fusarium, further understanding the pathogenic mechanism of Fusarium and taking preventive measures in advance to prevent harm to human and animal health.

At present, the detection of Fusarium toxin chemotypes includes thin layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (high performance liquid chromatography) , HPLC), nuclear magnetic resonance spectroscopy (nuclear magnetic resonance spectroscopy, NMR) and other methods. The TLC method is simple in operation and low in cost, but it is harmful to the human body. Usually, it can only be used for qualitative detection, and cannot detect a large number of samples at the same time; ELISA detection does not require pretreatment, has high specificity, strong sensitivity, and can be quickly Detecting a large number of samples requires simple equipment and is easy to popularize. However, this method has disadvantages such as false positives, single detection objects, and the preparation and storage of antibodies. The NMR method is efficient, accurate, quantitative, and can detect target compounds without pretreatment. However, its resolution is relatively low and the instrument is relatively expensive; HPLC has the advantages of high sensitivity, rapidity and accuracy, and is suitable for the detection of trace samples, and can be combined with mass spectrometry to achieve quantitative detection. However, this method mainly uses instruments that are only equipped in large laboratories such as high-performance liquid chromatography, mass spectrometer, and LC-MS. These instruments are not only expensive, but also have high requirements for personnel operations. It involves complex processes such as toxin fermentation, purification and sample pretreatment. Therefore, it is urgent to develop a method that relies on conventional laboratory instruments, is simple and convenient to operate, has a fast detection speed, and has high sensitivity.

In recent years, research on related pathways and regulatory mechanisms of Fusarium toxin production has progressed rapidly, and the polymerase chain reaction (PCR) detection method based on related toxin-producing genes has gradually been used for the detection of Fusarium toxin chemotypes . Relatively speaking, the use of PCR technology to detect toxin-producing genes only requires the use of common laboratory instruments such as PCR instruments and electrophoresis instruments. The detection speed is fast and the operation is relatively simple.

Most of the previous studies on the analysis of Fusarium toxin-producing types have focused on the Fusarium that causes grain diseases, and there are very few studies on the chemotypes of Fusarium toxins that cause alfalfa root rot, and due to the fact that Fusarium is in the taxa of the species. It can be divided into different specialized types, variants, etc., which leads to the complication of different Fusarium toxin-producing types. Therefore, the development of an accurate and sensitive PCR detection method for the identification of the pathogenic toxins of Fusarium alfa root rot is of great significance for the analysis of the toxin production potential of Fusarium and the prevention and control of alfalfa root rot.

SUMMARY OF THE INVENTION

In order to solve the technical problems existing in the prior art, the purpose of the present invention is to provide three pairs of specific primers for detecting chemotypes of Fusarium alfalfa root rot pathogenic toxin, and these three pairs of specific primers have high specificity and sensitivity. and amplification efficiency, the three pairs of specific primers were used to further develop a PCR detection method for identifying the chemotype of the pathogenic toxin of Fusarium alfalfa root rot.

In order to achieve the above object, the technical scheme of the present invention is as follows: the present invention carries out a large amount of analysis, comparison and screening of the sequences and functions of the toxin-producing metabolic pathways and toxin-producing-related genes of Fusarium in the prior art, and it is determined that Tri5 Gene (encoding trichothecenes synthase, which catalyzes the cyclization of farnesyl pyrophosphate to synthesize trichothecenes (TND), which is the synthesis of trichothecenes (TCTs) The first step), Pks13 gene (essential gene for zearalenone (ZEN) synthesis) and Fum8 gene (encoding α-aminotransferase, which is an essential gene for fumonisins (FUM) synthesis) to detect the target. Specific primers were designed for the detection of chemotypes of Fusarium alfalfa root rot pathogenic toxin for Tri5 gene, Pks13 gene and Fum8 gene, respectively. three pairs of specific primers. Based on these three pairs of specific primers, a PCR method was developed for the detection of the chemotypes of the Fusarium alfalfa root rot pathogen toxin.

Specifically, the present invention provides specific primers for detecting chemotypes of Fusarium alfalfa root rot pathogen toxin, including the following three pairs of specific primers:

SEQ ID NO. 1: Tri5H1-F: AGTTGGGCCAAGGTTTCCAA;

SEQ ID NO. 2: Tri5H1-R: AAGGAACTGCTTGCGCTCAT;

SEQ ID NO. 3: ZEA13H1-F: AGTTGGAATACCCCGCTTGG;

SEQ ID NO. 4: ZEA13H1-R: CCACGATCCAAGACCGTTGA;

SEQ ID NO. 5: FUM8H1-F: TTGCATAGGGGTTGGTGCAA;

SEQ ID NO. 6: FUM8H1-R: GGAACAATCCAGCAAGCGTC.

A kit comprising specific primers as shown in SEQ ID NOs. 1 to 6 for detecting chemotypes of Fusarium alfalfa root rot pathogenic toxin.

To facilitate detection, the kit may also contain other reagents required for PCR detection.

Preferably, the kit contains one or more of dNTP, Mg ²⁺ , DNA polymerase, PCR reaction buffer, positive template, and ddH ₂ O.

The above-mentioned dNTP, Mg ²⁺ , DNA polymerase, and PCR reaction buffer can be individually packaged or mixed into a premix.

The present invention also provides any of the following applications of the specific primers or the kit comprising the specific primers as shown in SEQ ID NO.

(1) application in the detection of the chemotypes of the pathogenic toxin of Fusarium alfalfa root rot;

(2) The application in detecting the toxin-producing potential of Fusarium alfalfa;

(3) Application in the inspection and quarantine of plant pathogens or the prevention and control of Fusarium alfalfa root rot.

The present invention provides a method for detecting the chemical type of the pathogenic toxin of Fusarium alfalfa root rot, which is to use the specific primers shown in SEQ ID NO. or a kit containing the specific primers for detection.

Specifically, the detection method of the above-mentioned Fusarium alfalfa root rot pathogenic toxin chemotype comprises the following steps:

(1) extracting the genomic DNA of the pathogen of Fusarium alfalfa root rot to be tested;

(2) using the genomic DNA as a template, using specific primers as shown in SEQ ID NO. 1 to 6 or a kit comprising the primers to carry out PCR amplification;

(3) Determine the type of toxin-producing genes contained in the pathogen of Fusarium alfalfa root rot according to the PCR amplification product, and determine the chemotype of the pathogenic toxin of Fusarium alfalfa root rot.

Preferably, in the above step (2), the reaction procedure of the PCR amplification includes: pre-denaturation at 95°C for 2-5 min; denaturation at 94°C for 20-30s, annealing at 52-54°C for 30s, extension at 72°C for 30-60s, 30 ~35 cycles; 10 min extension at 72°C.

Further preferably, when using the Tri5H1-F/Tri5H1-R primer pair, the PCR amplification reaction procedure includes: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 40s, 35 Cycle; 72°C extension for 10 min.

When the ZEA13H1-F/ZEA13H1-R primer pair is used, the PCR amplification reaction procedure includes: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 30s, 35 cycles; 72°C for 30s Extend for 10min.

When using the FUM8H1-F/FUM8H1-R primer pair, the PCR amplification reaction procedure includes: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 1 min, 35 cycles; 72°C for 1 min Extend for 10min.

Preferably, in the above step (2), the 25 μL reaction system for PCR amplification is as follows: 1 μL of 10 μmol/L upstream and downstream primers, 12.5 μL of 2× PCR reaction premix, 1 μL of 100ng/μL genomic DNA, supplemented with ddH ₂ O to 25 μL.

Preferably, in the above step (3), judging the type of the toxin gene contained in the Fusarium alfalfa root rot pathogen according to the type of the PCR amplification product is judging the Fusarium alfalfa root rot pathogen according to the band type of the electrophoresis band Whether it contains trichothecenes Tri5 gene, zearalenone Pks13 gene, and fumonisin Fum8 gene.

As a preferred technical solution of the present invention, the target fragments amplified by the primer pairs of Tri5H1-F/Tri5H1-R, ZEA13H1-F/ZEA13H1-R, FUM8H1-F/FUM8H1-R are 448bp, 273bp, and 742bp, respectively. Glycogel electrophoresis is used to determine whether the pathogen of Fusarium alba root rot has the potential to produce trichothecenes, zearalenone and fumonisin according to the presence or absence and size of target fragments.

The beneficial effects of the present invention are:

The present invention provides specific primers for detecting chemical types of Fusarium alfalfa root rot pathogenic toxins, specifically three pairs of synthetic genes Tri5 with trichothecenes, zearalenone and fumonisin Gene, Pks13 gene and Fum8 gene as the target of high-efficiency specific primers, using these three pairs of specific primers can accurately, quickly and conveniently detect whether the pathogen of Fusarium alfalfa root rot has the ability to produce trichothecenes, corn The potential of zearalenone and fumonisin to achieve the accurate chemotype of Fusarium alfalfa root rot pathogenic toxin using conventional PCR technology (using high performance liquid chromatography and liquid-mass spectrometry for PCR The detection results were verified, and the two detection results were consistent), specific, and sensitive (the sensitivity of the three pairs of primers for DNA detection reached 10 pg/μL, 100 pg/μL, and 1 ng/μL, respectively).

On the basis of the above-mentioned specific primers, the present invention develops a PCR method for the detection of the chemical type of the pathogenic toxin of Fusarium alfalfa root rot. It is fast and relatively simple to operate. It can be used to quickly and conveniently detect the potential of Fusarium alfalfa root rot pathogen to produce trichothecenes, zearalenone and fumonisin, and to determine the chemotypes of the corresponding pathogenic toxins. , which greatly simplifies the detection method of the toxin-producing potential of the corresponding pathogen, saves the detection time and cost, and provides a simple, rapid and accurate method for the assessment of the toxin-producing potential of Fusarium isolated from alfalfa or alfalfa products. The method provides technical support for ensuring the safety of alfalfa feed, human and animal health and the healthy development of alfalfa industry, and has great economic and social benefits.

Description of drawings

Fig. 1 is the product electrophoretogram of PCR amplification using Tri5-F/R, ZEA13-F/R, FUM8-rp679/rp680 primers in Example 1 of the present invention, wherein, A is strain D27-1 as template, primer Tri5 - The electrophoresis image of PCR products of F/R, M is DNA marker, lane 1 is the PCR product of primer Tri5-F/R; B is the electrophoresis image of PCR product of strain N6-1 as template, primer ZEA13-F/R, M is DNA marker, lane 1 is the PCR product of primer ZEA13-F/R; C is the electrophoresis image of strain KLX2 as template, PCR product of primer FUM8-rp679/rp680, M is DNA marker, lane 1 is primer FUM8-rp679/rp680 the PCR product.

Figure 2 is an electrophoresis diagram of PCR products amplified by using the Tri5H1-F/R primer pair in Example 4 of the present invention, wherein M is DL2000 marker, and lanes 1 to 10 are strains S49-1, D1, D14-1, D16 respectively , D18, D21-1, N5-2, KL2, KL2-1, N1-3 PCR products.

Figure 3 is an electrophoresis diagram of PCR products amplified by ZEA13H1-F/R primer pair in Example 4 of the present invention, wherein M is the DL2000 marker, and lanes 1 to 10 are strains N6-1, D25, D38, D23, PCR products of N1-3, N4-1, N9-1, N11-1, N15-2, QD11-1.

Figure 4 is an electrophoresis diagram of PCR products amplified by FUM8H1-F/R primer pair in Example 4 of the present invention, wherein M is DL2000 marker, and lanes 1 to 10 are strains KLX1, KLX2, S16, S33-1, PCR products of S45, S47-1, S47-2, D26, S39-1, KL5-3.

Figure 5 is an electrophoresis diagram of PCR products amplified by primer pairs of template Tri5H1-F/R at different concentrations in Example 5 of the present invention, wherein M is DL2000 marker, and lanes 1 to 8 are template concentrations of 100 ng/μL and 10 ng/μL, respectively , 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, 0.1pg/μL, 0pg/μL.

Figure 6 is an electrophoresis diagram of PCR products amplified by primer pairs of template ZEA13H1-F/R at different concentrations in Example 5 of the present invention, wherein M is DL2000 marker, and lanes 1 to 8 are template concentrations of 100ng/μL and 10ng/μL, respectively , 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, 0.1pg/μL, 0pg/μL.

Figure 7 is an electrophoresis diagram of PCR products amplified by primer pairs of template FUM8H1-F/R at different concentrations in Example 5 of the present invention, wherein M is the DL2000 marker, and lanes 1 to 8 are the template concentrations of 100 ng/μL and 10 ng/μL, respectively , 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, 0.1pg/μL, 0pg/μL.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Screening of specific primers for the detection of Fusarium alfalfa root rot pathogenic toxin chemotypes

1. Selection of strains for screening primers

At present, most of the reporter genes for Fusarium toxin production in NCBI and other databases are Fusarium from cereal crops, and there are no reports of Tri5, Pks13, and Fum8, which are the pathogens of Fusarium alfalfa root rot. The specific primers used to detect the chemotype of Fusarium alfalfa root rot pathogenic toxin have good versatility. The present invention aims at a variety of different sources of alfalfa root rot pathogenic Fusarium to first try to use it for the detection of isolated from grains. The screening of primers for the toxin production of Fusarium spp The primers are not universal for the detection of the virulence of Fusarium alfalfa root rot.

For the 224 strains of Fusarium isolated and purified from the alfalfa root rot plants collected in our laboratory from 2013 to 2018 (as shown in Table 1, references: Ruan Liu, Ma Zhanhong, Liu Zhenyu, Qin Feng, Wang Haiguang. Alfalfa root rot Isolation and identification of pathogens and determination of virulence of fungicides. Journal of China Agricultural University, 2016, 21(6): 56～67; Kong Qianqian, Qin Feng, Zhang Yuzhu, Ma Zhanhong, Liu Zhilong, Wang Haiguang. Mycorrhizal root of Fusarium alfa Determination of pathogenicity and toxin chemotypes of rot pathogens. Journal of China Agricultural University, 2018, 23(5): 74～85.); the latter 38 strains of Fusarium were the pathogenic bacteria isolated and purified in 2018) to sort out, according to the bacterial species , collection site, collection year, disease index, etc., 11 strains were selected for primer screening, and the strain information is shown in Table 2.

Table 1 Information on 224 isolated and purified Fusarium alfalfa root rot pathogenic strains

Table 2 Information of tested Fusarium strains for primer screening

strain number Collection location Year of collection strain species KD3 The sixth branch of the third sub-district of Nandagang, Huanghua City, Hebei Province 2015 Fusarium oxysporum N19 South Campus of Hebei North University, Xuanhua District, Zhangjiakou City, Hebei Province 2015 Fusarium solani N1-2 South Campus of Hebei North University, Xuanhua District, Zhangjiakou City, Hebei Province 2015 Fusarium redolens N6-1 South Campus of Hebei North University, Xuanhua District, Zhangjiakou City, Hebei Province 2015 Fusarium equiseti KLX1 The sixth branch of the third sub-district of Nandagang, Huanghua City, Hebei Province 2015 Fusarium commune KLX2 The sixth branch of the third sub-district of Nandagang, Huanghua City, Hebei Province 2015 Fusarium proliferatum S49-1 The third sub-district of Nandagang, Huanghua City, Hebei Province 2014 Fusarium incarnatum QD3-2 The third sub-district of Nandagang, Huanghua City, Hebei Province 2015 Fusarium oxysporum N9-2 South Campus of Hebei North University, Xuanhua District, Zhangjiakou City, Hebei Province 2015 Fusarium acuminatum B3-5-1 South Campus of Hebei North University, Xuanhua District, Zhangjiakou City, Hebei Province 2016 Fusarium tricinctum D27-1 Dongzhuangzi Village, Yangerzhuang Town, Huanghua City, Hebei Province 2014 Fusarium equiseti

2. Extraction of genomic DNA from Fusarium alfalfa root rot pathogen

The 11 bacterial strains in the separated and purified table 2 were activated and cultivated, and the fresh mycelium at the edge of the colony was punched with a sterile hole punch of 6 mm in diameter, and it was inoculated on a potato dextrose agar medium (PDA) plate (90 mm in diameter). ), placed in a mold incubator at 25 °C for 5 d. Extraction of Fusarium alfalfa root rot by modified CTAB method (Guo L.D., Hyde K.D., Liew E.C.Y. Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. New Phytologist, 2000, 147(3): 617～630) Pathogenic genomic DNA, the specific steps are as follows:

(1) Use a sterilized toothpick to scrape the fresh mycelium from the edge of the strain cultured for 5 days, put it into a 2.0 mL shaking tube containing 0.4 g of quartz sand, add 1 mL of CTAB extract at 65°C, and shake on a vortex shaker for 2 times, 6m/s each time, 40s.

(2) 65°C water bath for 30min, during this period, gently invert and mix 2-3 times to ensure full lysis.

(3) Centrifuge at 12000rpm for 10min, take the supernatant into a new 2.0mL sterile centrifuge tube, add an equal volume of chloroform/isoamyl alcohol (24:1), invert and mix, and then centrifuge at 12000rpm for 10min.

(4) Aspirate the supernatant into a new 2.0mL sterile centrifuge tube, add an equal volume of chloroform/isoamyl alcohol (24:1), invert and mix, and centrifuge at 12,000rpm for 10min (this step can be repeated until the aqueous phase and organic until no protein appears between the phases).

(5) Pipet the supernatant into a new 1.5mL sterile centrifuge tube, add 2/3 volume of isopropanol to the supernatant, gently invert and mix, a white fibrous precipitate can be seen, and let stand at -20°C for 4h .

(6) Centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash the precipitate with 70% ethanol and anhydrous ethanol, and centrifuge at 10,000 rpm for 5 min.

(7) Centrifuge briefly, remove excess liquid with a pipette tip, open the lid of the centrifuge tube and dry in an oven at 37°C for 10 min until the DNA precipitate becomes translucent.

(8) 50-100 μL ddH ₂ O was added to dissolve the DNA precipitate, and the DNA was digested and dissolved in a water bath at 37° C. for 1 h.

3. Obtainment of toxin-related genes Tri5, Pks13, and Fum8 gene fragments

By consulting relevant literature at home and abroad, the primers used for the detection of Fusarium toxin production-related genes were obtained, and the genomic DNAs of the 11 strains extracted in the above 2 were used as templates for PCR amplification. After screening, Tri5, Pks13, Primers for Fum8 gene fragments. PCR amplifications were first carried out using the methods reported in the literature, and the amplified products were detected by agarose gel electrophoresis. If there was no target band, the PCR system and procedures were improved, and PCR amplification was performed again and agarose gel electrophoresis was carried out. Detection, when there is no target band after multiple improvements, it is considered that the primer cannot amplify the target band. The product of a single clear target band was sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing. After many attempts, one pair of primers for each of the Tri5, Pks13, and Fum8 genes that can relatively stably amplify the target band was finally obtained. The primer sequences are shown in Table 3.

Table 3 Screened primers that can amplify the target bands of toxin-related genes Tri5, Pks13 and Fum8

The references are:

Nicholson, P., Simpson, D.R., Wilson, A.H., Chandler, E., Thomsett, M. Detection and differentiation of trichothecene and enniatin-producing Fusarium species on small-grain cereals. European Journal of Plant Pathology, 2004, 110(5- 6): 503～514.

Priyanka, S.R., Venkataramana, M., Balakrishna, K., Murali, H.S., Batra, H.V. Development and evaluation of a multiplex PCR assay for simultaneous detection of major mycotoxigenic fungi from cereals. Journal of Food Science and Technology, 2015, 52(1) :486～492.

Zhang, L.P., Wang, J.S., Zhang, C.L., Wang, Q.M. Analysis of potentialfumonisin-producing Fusarium species in corn products from three main maize-producing areas in eastern China. Journal of the Science of Food and Agriculture, 2013, 93(3) :693～701.

Taking the genomic DNA of strain D27-1 as a template, and using Tri5-F/R as a primer, the sequencing result (as shown in SEQ ID NO. The sequencing result of strain D27-1 has the highest sequence homology with the sequence annotated as Fusarium sp.FIESC_29tri5gene for trichodiene synthase, strain ITEM10392 (GenBank accession number: LN995586.1), and the Ident value is 99%. Taking the genomic DNA of strain N6-1 as a template and ZEA13-F/R as a primer, the sequencing results (as shown in SEQ ID NO.14) were compared with the existing sequences in the NCBI nucleic acid sequence database for BLAST analysis, and the results showed that the strains The sequencing result of N6-1 has the highest sequence homology with the annotated Gibberellazeae PKS gene cluster, complete sequence; and polyketide synthase (PKS13) (GenBank accession number: DQ019316.1), with an Ident value of 90%; N6-1 is annotated as Fusariumpseudograminearum CS3096 PKS13 (PKS13) (GenBank accession number: XM_009259982.1) has a high sequence homology with an Ident value of 89%; the genomic DNA of strain KLX2 was used as the template, FUM8-rp679/rp680 was used as the primer, and the sequencing results (such as SEQ ID No. 15) was compared with the existing sequences in the NCBI nucleic acid sequence database, and the results showed that the sequencing results of strain KLX2 were annotated as Fusarium fujikuroi strain HKM 41 fumonisinbiosynthetic alpha-oxoamine synthase (FUM8) gene (GenBank accession number) : KF415157.1) had the highest sequence homology, and the Ident value was 100%.

4. Design and screening of specific primers for the detection of chemotypes of Fusarium alfalfa root rot pathogen toxins

Through a large number of amplification experiments, it was found that the stability of Tri5-F/R, ZEA13-F/R, FUM8-rp679/rp680 primers was poor, and under the same conditions, the target band could not be guaranteed to be amplified every time, that is, the repeatability was poor. , and the clarity of the bands is poor, which cannot meet the requirements for primers and PCR amplification for the chemotype detection of Fusarium alfalfa root rot pathogenic toxins. Tri5-F/R, ZEA13-F/R, FUM8-rp679/rp680 The electropherograms of primers used for PCR amplification of strains strains D27-1, N6-1 and KLX2 are shown in Figure 1 .

In order to make the detection of Fusarium alfalfa root rot pathogenic toxin chemotypes have higher specificity, sensitivity and accuracy, the present invention is based on the amplified trichothecenes Tri5 gene fragment of the primers in the above table 3, The sequences of the zearalenone Pks13 gene fragment and the fumonisin Fum8 gene fragment were designed to amplify and detect specific primers for the Tri5 gene, the Pks13 gene and the Fum8 gene, respectively.

After a large number of screening and comparison experiments, the present invention finds that good detection effect of Tri5, Pks13 and Fum8 genes can be achieved not only by satisfying common primer design principles or by using primers designed by primer design software. On the basis of satisfying general design principles, the present invention has carried out researches on the binding force between primers and target sequences, the structure between primers or between primers and target sequences, GC content, Tm value, primer length, amplified fragment length, etc. After a lot of manual optimization design and screening, three pairs of specific primers with excellent performance in all aspects were finally obtained as shown in Table 4.

Table 4 Specific primers for the detection of Fusarium alfalfa root rot pathogenic toxin chemotypes

Example 2 Determination of PCR amplification reaction conditions for detection of Fusarium alfalfa root rot pathogenic toxin chemotypes

In order to achieve rapid and efficient target band amplification and detection, the PCR amplification reaction conditions of the specific primer pairs Tri5H1-F/R, ZEA13H1-F/R and FUM8H1-F/R were optimized and screened respectively. The best annealing temperature and the shortest extension time were found out for the three pairs of specific primers screened in Example 1, which made the amplification process faster and the amplification result bands clearer.

The final PCR reaction conditions determined by screening are as follows:

The PCR amplification system of the trichothecenes Tri5H1-F/R uses a 25 μL Mix reaction system, as follows: 1 μL of each primer (10 μmol/L), 12.5 μL of Mix, 1 μL of DNA template (100 ng/μL), supplemented with ddH ₂ O to a final volume of 25 μL.

The PCR amplification procedure of trichothecenes Tri5H1-F/R is as follows: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 40s, 35 cycles; extension at 72°C for 10 min; 4 Store at ℃.

The PCR amplification system of zearalenone ZEA13H1-F/R uses a 25 μL Mix reaction system, as follows: 1 μL of each primer (10 μmol/L), 12.5 μL of Mix, 1 μL of DNA template (100 ng/μL), supplemented with ddH ₂ O to a final volume of 25 μL.

The PCR amplification procedure of zearalenone ZEA13H1-F/R was as follows: pre-denaturation at 95 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, 35 cycles; extension at 72 °C for 10 min; storage at 4 °C .

The PCR amplification system of fumonisin FUM8H1-F/R uses a 25 μL Mix reaction system, as follows: 1 μL of each primer (10 μmol/L), 12.5 μL of Mix, 1 μL of DNA template (100 ng/μL), supplemented with ddH ₂ O to Final volume 25 μL.

The PCR amplification procedure of fumonisin FUM8H1-F/R was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min; storage at 4°C.

Example 3 Establishment of PCR detection method for chemotypes of Fusarium alfalfa root rot pathogenic toxins

According to the specific primers determined by screening in Example 1 and the PCR reaction conditions determined by screening in Example 2, the present embodiment provides a PCR detection method for the chemotype of the pathogenic toxin of Fusarium alfalfa root rot, as follows:

(1) extracting the genomic DNA of the Fusarium to be tested;

(2) Using the extracted Fusarium genome DNA as a template, Tri5H1-F/R (SEQ ID NO.1-2), ZEA13-H1F/R (SEQ ID NO.3-4), FUM8H1-F/ R (SEQ ID NO.5-6) is a primer for PCR amplification:

The PCR amplification system of the trichothecenes Tri5H1-F/R uses a 25 μL Mix reaction system, as follows: 1 μL of each primer (10 μmol/L), 12.5 μL of Mix, 1 μL of DNA template (100 ng/μL), supplemented with ddH ₂ O to a final volume of 25 μL. The PCR amplification procedure was as follows: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 40s, 35 cycles; extension at 72°C for 10 min; storage at 4°C.

The PCR amplification system of zearalenone ZEA13H1-F/R uses a 25 μL Mix reaction system, as follows: 1 μL of each primer (10 μmol/L), 12.5 μL of Mix, 1 μL of DNA template (100 ng/μL), supplemented with ddH ₂ O to a final volume of 25 μL. The PCR amplification program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 30s, 35 cycles; extension at 72°C for 10 min; storage at 4°C.

The PCR amplification system of fumonisin FUM8H1-F/R uses a 25 μL Mix reaction system, as follows: 1 μL of each primer (10 μmol/L), 12.5 μL of Mix, 1 μL of DNA template (100 ng/μL), supplemented with ddH ₂ O to Final volume 25 μL. The PCR amplification procedure was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min; storage at 4°C.

(3) Spot 5 μL of PCR products for electrophoresis. After electrophoresis, put the gel into EB solution for 9 minutes, rinse with distilled water, and take pictures under the UV gel imaging system to check whether there is a DNA band that meets the size of the target band. , to determine whether it has related genes and the ability to produce related toxins.

Example 4 Application of PCR detection method of Fusarium alfalfa root rot pathogenic toxin chemotype

Genomic DNA was extracted from 224 strains of Fusarium (Table 1) that were isolated and purified from the collected alfalfa root rot plants in our laboratory from 2013 to 2018, and the detection method provided in Example 3 was used to carry out Fusarium alfalfa root rot. Detection of pathogenic toxin chemotypes.

The results showed that 102 of the 224 Fusarium strains contained the target band of the toxigenic-related gene Tri5, and the fragment size was 448 bp (part of the detection results are shown in Figure 2). Among the strains containing the target band, 93 strains of F. equiseti, 4 strains of F. oxysporum, 2 strains of F. solani, and F. tricinctum 1 strain, 1 strain of F. proliferatum, 1 strain of F. incarnatum.

Thirty-three of the 224 Fusarium strains contained the target band of the toxigenic-related gene Pks13, and the fragment size was 273 bp (part of the detection results are shown in Figure 3). Among the strains containing the target band, 29 strains of F. equiseti, 2 strains of F. oxysporum, 1 strain of F. solani, and F. tricinctum 1 strain.

Eleven of the 224 Fusarium strains contained the target band of the toxigenic-related gene Fum8, with a fragment size of 742 bp (partial detection results are shown in Figure 4). Among the strains containing the target band, 1 strain of F. equiseti, 1 strain of F. oxysporum, 6 strains of F. proliferatum, and F. incarnatum) 1 strain, F. redolens 1 strain, F. commune 1 strain.

Among them, using the D27-1 strain as the template, the Tri5 gene fragment sequence obtained by using the Tri5H1-F/R primer to amplify is shown in SEQ ID NO. The sequence of the added Pks13 gene fragment is shown in SEQ ID NO. 17, and the sequence of the Fum8 gene fragment amplified by using the FUM8H1-F/R primer using the KLX1 strain as a template is shown in SEQ ID NO. 18.

Example 5 Accuracy analysis of PCR detection method for chemotypes of Fusarium alfalfa root rot pathogenic toxin

According to the detection results of the PCR method of Example 4, three strains of Fusarium spp. were selected as positive for trichothecenes, zearalenone and fumonisin genes, and the disease index was relatively large, using The detection accuracy of PCR method was verified by liquid phase-mass spectrometry detection method. The strain information is shown in Table 5:

Table 5 Fusarium species used to verify the accuracy of PCR detection methods

1. Detection of trichothecenes toxins produced by strain QZ3 (F.oxysporum)

The formula of trichothecenes seed medium is as follows: per 1000mL solution: sucrose 10g, ammonium nitrate 2g, magnesium sulfate 0.5g, ferric sulfate 0.2g, potassium dihydrogen phosphate 2g, peptone 4g, yeast extract 2g, Distilled water to 1000mL, autoclaved at 121°C for 30min.

The formula of the trichothecenes fermentation broth is as follows: per 1000 mL of solution: 1 g of ammonium nitrate, 3 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 4 g of sodium chloride, 20 g of sucrose, 10 mL of glycerol, 0.02 g of zinc sulfate, Add distilled water to dilute to 1000mL, and sterilize by autoclaving at 121°C for 30min.

The bacterial strain QZ3 was inoculated into the PDA medium for activation, and after the bacterial species had grown for 5 days, two bacterial cakes with a diameter of 6 mm were inoculated into a triangular flask containing 250 mL of trichothecenes seed culture solution, and the culture solution was placed on a shaking table. Shaking was performed at 140 rpm and a temperature of 28°C. After 3 days, inoculate the trichothecenes seed culture solution into the trichothecenes fermentation culture solution on the ultra-clean workbench, and inoculate 50 mL of the trichothecenes seed culture solution per 1000 mL triangular flask. The trichothecenes toxin fermentation broth was also placed in a shaker with a rotating speed of 140 rpm and a temperature of 28° C. for fermentation and culture, and the toxin was extracted after 15-20 days. Shake the fermented medium evenly, and use a Buchner funnel to perform suction filtration to obtain its liquid crude toxin. Add ethyl acetate and liquid crude toxin respectively to the separating funnel, shake up and down evenly, and release the lower aqueous phase after the extraction is complete. The ethyl acetate fraction was rotary evaporated to obtain a crude extract of trichothecenes.

Standard preparation: Dissolve the standard (1 mg) in 1 mL of chromatographic grade methanol, take 500 μL of an organic filter membrane with a pore size of 0.22 μm, and place it in an ultrasonic cleaner for 10 minutes to remove air bubbles and promote dissolution. The sample preparation was the same as that of the standard product. The crude toxin of QZ3 was dissolved in chromatographic grade methanol to form a 0.22 μm organic filter membrane, and it was placed in an ultrasonic cleaner for 10 minutes to remove air bubbles and promote dissolution. Mobile phase preparation: Chromatographic grade methanol and water were passed through a filter membrane with a diameter of 50 mm and a pore size of 0.2 μm, and then ultrasonicated for 30 min to remove air bubbles. Chromatographic conditions: Agilent ZORBAX SB-C18 4.6×150mm 5-Micron column, detection wavelength 218nm, mobile phase methanol-water (20:80, v/v), column temperature: 40°C, flow rate: 1.0mL/ min, the injection volume was 20 μL.

After optimization, when the mobile phase is methanol-water (20:80, v/v), the toxin peak time and peak shape are better. Under this condition, the peak of trichothecenes standard toxins The retention time was 5.020min, and the sample had an obvious peak at 5.396min, indicating that the crude toxin of strain QZ3 contained trichothecenes.

2. Detection of zearalenone produced by strain B1-12-3 (F.equiseti)

The formula of zearalenone fermentation broth is as follows: glucose 60g, peptone 20g, yeast extract 1.0g, sodium nitrate 6.0g, dipotassium hydrogen phosphate 1.0g, potassium nitrate 1.5g, potassium chloride 0.5g, magnesium sulfate 0.5g , ferric sulfate 0.025g, add distilled water to make up to 1000mL, and sterilize by autoclaving at 121°C for 30min.

The bacterial strain B1-12-3 was inoculated into the PDA medium for activation, and after the growth of the bacterial species for 5 days, inoculation of 4 bacterial cakes with a diameter of 6 mm to a 1000 mL conical flask was connected and shaken, the rotating speed was 92 rpm, and the temperature was 22.9 ° C. Shakers were used for fermentation and culture, and toxins were extracted after 15-20 days. Shake the fermented medium evenly, and use a Buchner funnel to perform suction filtration to obtain its liquid crude toxin. Add ethyl acetate and liquid crude toxin respectively to the separating funnel, shake up and down evenly, and release the lower aqueous phase after the extraction is complete. The ethyl acetate fraction was rotary evaporated to obtain a crude extract of zearalenone.

Standard preparation: Dissolve all zearalenone standard (1 mg) in 1 mL of chromatographic grade methanol, take 500 μL of an organic filter membrane with a pore size of 0.22 μm, place it in an ultrasonic cleaner for 10 minutes, and remove air bubbles. Promote dissolution. The sample preparation was the same as that of the standard product. The crude toxin B1-12-3 was dissolved in chromatographic grade methanol to form a 0.22 μm organic filter membrane, and it was placed in an ultrasonic cleaner for 10 minutes to remove air bubbles and promote dissolution. Mobile phase preparation: Chromatographic grade methanol and water were passed through a filter membrane with a diameter of 50 mm and a pore size of 0.2 μm, and then ultrasonicated for 30 min to remove air bubbles. Chromatographic conditions: the column is Agilent ZORBAX SB-C18 4.6×150mm 5-Micron, the detection wavelength is 236nm, the mobile phase is methanol-water (60:40, v/v); the column temperature is 40°C, and the flow rate is 1.0mL /min, and the injection volume was 20 μL. After optimization, when the mobile phase is methanol-water (60:40, v/v), the peak time and peak shape of the toxin are better. Under this condition, the peak retention time of the zearalenone standard is 5.020 min, while the sample B1-12-3 had an obvious peak at 5.396 min, indicating that the crude toxin of strain B1-12-3 contained zearalenone toxin.

3. Detection of fumonisin B ₁ produced by strain KLX2 (F. proliferatum)

The formula of the fumonisin fermentation medium is as follows: take 160 g of corn, add it to a 1000 mL conical flask, add an equal volume of distilled water, and sterilize it by autoclaving at 121° C. for 30 minutes before use.

The strain KLX2 was inoculated into the PDA medium for activation, and after the strains covered the entire petri dish, a spore suspension of 10 ⁷ /mL was prepared. The spore suspension was inoculated into fumonisin fermentation medium, 8 mL per Erlenmeyer flask. After 2 weeks of incubation at 25°C, toxins were extracted after 2 weeks of incubation at 15°C. After stirring the culture medium evenly with a glass rod, add ethyl acetate to make up to 1000 mL, put it in an ultrasonic cleaner for 30 min, filter with four layers of gauze, evaporate the filtrate to dryness with a rotary evaporator, dissolve it with ethyl acetate and use it for later use. For crude toxin A. After drying the filter residue in a basin, it was extracted three times with methanol-water (3:1, v/v) according to the above method to obtain crude toxin B.

Standard preparation: Dissolve fumonisin standard (1 mg) in 1 mL of chromatographic grade methanol, take 500 μL of an organic filter membrane with a pore size of 0.22 μm, and place it in an ultrasonic cleaner for 10 minutes to remove air bubbles and promote dissolution. The sample preparation was the same as that of the standard product. KLX2 crude toxin B was dissolved in chromatographic grade methanol to form a 0.22 μm organic filter membrane, which was placed in an ultrasonic cleaner for 10 minutes to remove air bubbles and promote dissolution. Mobile phase preparation: Chromatographic grade methanol and water were passed through a filter membrane with a diameter of 50 mm and a pore size of 0.2 μm, and then ultrasonicated for 30 min to remove air bubbles. Chromatographic conditions: the chromatographic column was Zorbax Eclipse XDB-C18 column 150mm×2.1mm, 3.5mm, the mobile phase was methanol-water-formic acid (55:45:2, v/v/v); the column temperature was 30°C, and the flow rate was 0.2mL/min, the injection volume is 10μL. Mass spectrometry conditions: quadrupole time of flight mass spectrometer (Q-TOF) equipped with electrospray ionization (ESI) ion source, positive ion mode detection, fragmentation voltage: 150V, collision energy: 40eV. After optimization, when the mobile phase is methanol-water-formic acid (55:45:2, v/v/v), the peak time and peak shape of the toxin are better. Under these conditions, the fumonisin B ₁ standard has better The peak retention time is 3.589min, while the sample KLX2 has an obvious peak at 3.835min. The mass spectrum shows that the m/z of its [MH] ^-ion peak is 722.3922, and the [MH] ^-ion peak of the standard is 722.3957. The two are basically consistent with the reports in the literature (Li, C., Wu, YL, Yang, T., Huang-Fu, WG Rapid determination of fumonisins B ₁ and B ₂ in corn by liquid chromatography-tandem mass spectrometry with ultrasonic extraction . Journal of Chromatographic Science, 2012, 50(1): 57-63.), indicating that the crude toxin of strain KLX2 contains fumonisin B ₁ .

Example 6 Sensitivity determination of the chemotype detection method for the pathogenic toxin of Fusarium alfa root rot

The DNA of strains QZ3, B1-12-3 and KLX2 were extracted by the improved CTAB method in Example 1. After measuring the DNA concentration, the template DNA was diluted to 100ng/μL and 10ng/μL with ultrapure water by 10-fold concentration dilution method. , 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, 0.1pg/μL, using the above concentrations of DNA as templates, respectively, Tri5H1-F/R (SEQ ID NO.1-2), ZEA13-H1F Three pairs of primers /R (SEQ ID NO. 3-4) and FUM8H1-F/R (SEQ ID NO. 5-6) were tested for sensitivity, and a negative control was set at the same time. The PCR reaction system and reaction procedure were the same as those in Example 3. The above experiment was performed in triplicate. 6μL PCR product was sampled for electrophoresis detection, voltage 120V, current 150mA, electrophoresis time 30min, after electrophoresis, put the gel into EB solution for 9min staining, rinse with distilled water, take pictures under the UV gel imaging system, observe each Whether each lane has bands.

The results showed that the sensitivity of the primer pair Tri5H1-F/R was 10pg/μL (as shown in Figure 5), and it could detect F. equiseti, F. oxysporum, F. solanacearum (F.solani), F. tricinctum (F. tricinctum), F. proliferatum (F. proliferatum), F. incarnatum (F. incarnatum) Tri5 gene fragment; the sensitivity of the primer to ZEA13-H1F/R is 100pg/μL (as shown in Figure 6), can detect F. equiseti, F. oxysporum, F. solani, and F. tricinctum ) fragment of the Pks13 gene; the sensitivity of the primer pair FUM8H1-F/R is 1 ng/μL (as shown in Figure 7), which can detect F. equiseti, F. oxysporum, Fragments of the Fum8 gene in F. proliferatum, F. incarnatum, F. redolens, and F. commune were layered.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

<110> China Agricultural University

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Claims (12)

1. Specific primers for detecting the chemical type of pathogenic toxins of fusarium graminearum root rot, which are characterized by comprising the following components:

SEQ ID NO.1：Tri5H1-F：AGTTGGGCCAAGGTTTCCAA；

SEQ ID NO.2：Tri5H1-R：AAGGAACTGCTTGCGCTCAT；

SEQ ID NO.3：ZEA13H1-F：AGTTGGAATACCCCGCTTGG；

SEQ ID NO.4：ZEA13H1-R：CCACGATCCAAGACCGTTGA；

SEQ ID NO.5：FUM8H1-F：TTGCATAGGGGTTGGTGCAA；

SEQ ID NO.6：FUM8H1-R：GGAACAATCCAGCAAGCGTC。

2. a kit comprising the specific primers of claim 1 for detecting the chemical type of a pathogenic toxin of fusarium graminearum.

3. Use of the specific primers of claim 1 or the kit of claim 2 for detecting the chemical type of a toxin causative of fusarium graminearum mycorrhizal rot.

4. Use of the specific primers of claim 1 or the kit of claim 2 for detecting the toxin-producing potential of fusarium alfalfa.

5. The use of the specific primers of claim 1 or the kit of claim 2 in alfalfa pathogen quarantine inspection or prevention and control of alfalfa fusarium root rot.

6. A method for detecting the chemical type of pathogenic toxin of fusarium graminearum rhizoctonia, which is characterized in that the specific primer of claim 1 or the kit of claim 2 is used for detection.

7. The detection method according to claim 6, characterized by comprising the steps of:

(1) extracting the genome DNA of fusarium graminearum root rot pathogen to be detected;

(2) performing PCR amplification by using the genomic DNA as a template and the specific primer of claim 1 or the kit of claim 2;

(3) judging the type of toxin genes contained in the fusarium graminearum root rot pathogen according to the PCR amplification product, and determining the chemical type of the toxin of the fusarium graminearum root rot pathogen.

8. The detection method according to claim 7, wherein the reaction procedure of PCR amplification comprises: pre-denaturation at 95 ℃ for 2-5 min; denaturation at 94 ℃ for 20-30 s, annealing at 52-54 ℃ for 30s, extension at 72 ℃ for 30-60 s, and 30-35 cycles; extension at 72 ℃ for 10 min.

9. The detection method according to claim 8, wherein when the Tri5H1-F/Tri5H1-R primer pair is used, the reaction procedure of the PCR amplification comprises: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 40s for 35 cycles; extending for 10min at 72 ℃;

when the primer pair ZEA13H1-F/ZEA13H1-R is adopted, the reaction program of the PCR amplification comprises the following steps: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 10min at 72 ℃;

when the primer pair FUM8H1-F/FUM8H1-R is adopted, the reaction program of the PCR amplification comprises the following steps: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.

10. the detection method according to any one of claims 7 to 9, wherein the 25. mu.L reaction system for PCR amplification comprises 1. mu.L of each of 10. mu. mol/L upstream and downstream primers, 12.5. mu.L of 2 × PCR reaction premix, 1. mu.L of 100 ng/. mu.L genomic DNA, and ddH complement₂O to 25. mu.L.

11. The detection method according to any one of claims 7 to 9, wherein the determination of the type of the toxin gene contained in the fusarium graminearum root rot pathogen according to the PCR amplification product is a determination of whether the fusarium graminearum root rot pathogen contains a trichothecene toxin according to the presence or absence and size of the electrophoretic bandTri5Gene zearalenonePks13Gene fumonisinFum8A gene.

12. The method according to claim 10, wherein the determination of the type of the toxin gene contained in the Fusarium graminearum root rot pathogen from the PCR amplification product is performed by determining whether the Fusarium graminearum root rot pathogen contains a trichothecene toxin according to the presence or absence and size of the electrophoretic bandTri5Gene zearalenonePks13Gene fumonisinFum8A gene.

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