CN111876514B - Rapid detection method for gibberellin miniascape generated in bakanae disease germ of rice - Google Patents

Abstract

A rapid detection method for gibberellin miniascapes generated in rice bakanae disease germs belongs to the technical field of plant disease control and plant pathogen detection. The method aims at Fusarium vine canum, fusarium layering, fusarium verticillium and Fusarium verticilliumF.andiyaziThe specific primer is designed according to the specific gene difference of the (2), and the specific primer is used for carrying out multiplex PCR amplification by taking the DNA of the sample to be detected as a template, and the gibberellin seed is generated in the bakanae disease germ of the rice according to the amplification result. The method is suitable for the rapid and reliable detection and identification of the bakanae disease germ of the rice, and is suitable for fusarium graminearum, fusarium layering and fusarium verticilliumF.andiyaziThe prevention and the control of the bakanae disease of the rice caused by the bakanae disease have important effectPractical application value.

Description

Rapid detection method for gibberellin miniascape generated in bakanae disease germ of rice

Technical Field

The invention belongs to the technical field of plant disease control and plant pathogen detection, and particularly relates to a rapid detection method for gibberellin miniascape generated in rice bakanae disease.

Background

Gibberellin (GA) is one of five plant hormones, is a high-efficiency plant growth regulator, has multiple physiological functions for plant growth and development, and can regulate plant seed germination, stem and leaf elongation, fruit development and the like. Currently, 136 species of gibberellins have been found from plants, fungi and bacteria, most of which are present in higher plants, one part being present in fungi or bacteria and the other part belonging to fungi and plants in common. Gibberellins are tetracyclic diterpenoids whose basic backbone consists of four isoprene units. Different gibberellins are formed due to the difference in the number and positions of double bonds, hydroxyl groups, and the presence or absence of lactone rings. Gibberellins are classified into C19 and C20 gibberellins according to the number of carbon atoms in the gibberellin molecule. The former includes more kinds than the latter, and the physiological activity is also higher than the latter. Among them, gibberellins having biological activity mainly include GA1, GA3, GA4, GA7, etc.

In fungi, gibberellin biosynthesis genes were found earlier in the model strain Fusarium canum. 7 genes involved in gibberellin biosynthesisDES、P450-4、P450-1、P450-2、GGS2、CPS/KSAndP450-3the gene clusters are closely linked and exist on the 4 th chromosome. The biosynthesis pathway of gibberellin is that acetyl-CoA is obtained by mevalonate monoacyl-CoA, mevalonic acid, isopentenyl pyrophosphate, isopentenyl diphosphate, geranyl pyrophosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate. The gibberellin biosynthesis process is mainly divided into 3 stages: the first stage is carried out in cytosol with geranylgeranyl pyrophosphate as substrate; the second stage is to convert the kaurene from the inner root into GA 12-aldehyde in the endoplasmic reticulum through the catalysis of various enzymes; the last stage is completed in cytoplasm, and GA 12-aldehyde generates different kinds of gibberellins under the combined action of GA 3-oxidase (GA 3 ox), GA 20-oxidase (GA 20 ox), GA 2-oxidase (GA 2 ox) and the like. Wherein GA20ox is late in gibberellin synthesis pathwayThe key enzyme belongs to the subfamily of soluble dioxygenase 2OG-Fe (II) and is responsible for degrading gibberellin of class C20. GA20ox is capable of oxidizing GA12 and GA53, which are not biologically active in the GA synthesis pathway, to GA20 and GA9, which are biologically active, maintaining a balance between gibberellin and intermediates, which are biologically active.

Bakanae disease of rice is a worldwide disease and is mainly distributed in Guangdong, hunan, jiangsu and Zhejiang places in China. The symptoms of the disease are mainly marked by overgrowth, fading, dwarfing and death of seedlings, and the yield loss of the rice can reach more than 40% when the disease is serious. In recent years, with the popularization of light cultivation techniques such as mechanical transplanting, seedling throwing, direct seeding and the like, bakanae disease caused by bakanae bacteria has drug resistance to chemical agents, and the occurrence degree of bakanae disease of rice is increased year by year. The bakanae disease of rice is mainly transmitted by seeds, and pathogenic bacteria causing the disease comprise: fusarium vineFusarium fujikuroiFusarium roseum (L.) kuntzeF. proliferatumFusarium pseudoverticillium (L.) F.F. verticillioides、F. andiyazi [Wulff E G, Sorensen J L, Lübeck M, et al. Fusarium spp. associated with rice bakanae: ecology, genetic diversity, pathogenicity and toxigenicity. Environ Microbiol, 2010, 12: 649-657.]. The four pathogenic bacteria have the characteristics of being belonging to the composite species of the gibberella canescensGibberella fujikuroi specificity complexes, GFC). The Saccharum sinensis Roxb complex contains at least 11 mating types which are completely different in inheritance, are named as MP-A to MP-K respectively, are different in cooperation, and can produce different secondary metabolites such as fusaric acid, fusarium, gibberellin, moniliformin, fumonisin and the like. Wherein, the fusarium in the rattan warehouse belongs to mating type MP-C, the fusarium in the layer belongs to mating type MP-D, and the fusarium verticillatum is mating type MP-A. The colony morphology (color, growth rate), spore type (megaspore, microspore) and spore-forming cell structure of the four fusarium species are very similar, and the traditional identification means not only requires a great deal of expertise, but also is complicated and takes a long time. Currently, identification of gibberella caner complex species, based on traditional morphological observations, has become a current trend in combination with molecular biology methods to distinguish these problematic species. Such as applicationsITS、28S rDNA、mt SSUrDNA、β-tubulinSecond group of RNA polymeraseRPB2) Protein translation elongation factorEF-1αAnd analysis of calmodulin and other polygenic sites to determine the inner members of the akabane fungus complex species. For example, muleo et al (2004) have been able to identify Fusarium verticillium, fusarium layering [ Muleg G, susca A, stea G, moretti A.A patterns-specific PCR assay based on the calmodulin partical gene for identification of ] by designing specific primers using partially conserved sequences of calmodulin genesFusarium verticillioides, F. proliferatum and F. subglutinas. Eur J Plant Pathol, 2004, 110: 495-502.]. In addition, yuantian and the like (2018) and Rong Zhenyang and the like (2018) can mark 4 bakanae disease germs based on a loop-mediated isothermal amplification technology [ Yuan days, rong Zhenyang, she Wenwu and the like ], and the bakanae disease germs carried by Jiangsu rice seeds are detected by applying the loop-mediated isothermal amplification technology; rong Zhenyang, yuan days, zeng Dandan, et al, rapid diagnosis by Loop-mediated isothermal amplificationF. andiyaziPlant pathology report, 2018, 48 (2): 256-262.]。

Fusarium graminearum is the main infection source of bakanae disease of rice, has the strongest pathogenicity, and only Fusarium graminearum can secrete gibberellin GA3 with a cyclic alcohol structure. Although Fusarium layering contains all gibberellin biosynthesis gene clusters, it loses gibberellin synthesis capacity. Based on the complex species of the Saccharum sinensis Roxb which causes bakanae disease of rice, the morphology is difficult to determine, the rapid and accurate detection technology is researched and explored, and the method has important application value for preventing the transmission and the spread of bakanae disease of rice, taking prevention and control countermeasures as soon as possible and ensuring the safe production of rice in China. The invention relates to fusarium graminearum, fusarium layering, fusarium verticillatum and fusarium graminearumF. andiyaziComparison of gibberellin biosynthesis gene clusters to find Fusarium rosenbergii and Fusarium rosenbergiiF. andiyaziLarge fragment deletions of the middle gibberellin biosynthesis gene cluster; further carrying out whole genome comparison on fusarium graminearum and fusarium layering, and finding gibberellin synthesis genesGA20oxDeletion in fusarium layering. Thus, it is possible toThe rice bakanae disease caused by fusarium graminearum is rapidly identified by using gibberellin biosynthesis and regulatory genes. At present, no report of detecting fusarium graminearum based on the difference of gibberellin biosynthesis and regulatory genes is yet seen at home and abroad. Meanwhile, the research result can provide an original experimental material for improving the gibberellin product content in the microorganism by utilizing a molecular genetic method to improve the gibberella canescens strain, and has important practical significance for further applying gibberellin to improve crops.

Disclosure of Invention

Aiming at the problems existing in the prior art, the application is based on fusarium graminearum, fusarium layering and fusarium verticillatum andF. andiyazithe specific genes of the medium gene difference are obtained through bioinformatics means, and the technical scheme of the rapid detection method for producing gibberellin miniascape in rice bakanae disease germs is designed and provided.

The invention aims at realizing the following steps:

the specific gene is aimed at fusarium graminearum, fusarium layering and fusarium verticillatum in bakanae disease of riceF. andiyaziIs applied to detection of the (C).

Comparison of these four gibberellin-synthesizing gene clusters revealed that the gibberellin-synthesizing gene clusters of Fusarium canum appeared in Fusarium layering, but in Fusarium verticillium and Fusarium pseudolarimF. andiyaziIn the gibberellin biosynthesis gene clusterP450-1（SEQ ID NO.22）、P450-2（SEQ ID NO.23）、GGS2（SEQ ID NO.24）、CPS/KS(SEQ ID NO. 25) andP450-3the (SEQ ID NO. 26) gene was completely deleted. According to gibberellin synthesis in fusarium graminearumCPS/ KSThe gene design specific primer is characterized by having nucleotide sequences of SEQ ID NO.59 and SEQ ID NO.60, carrying out PCR amplification of four small species genome DNA of the bakanae disease germ, and separating 1327bp specific fragments in fusarium graminearum and fusarium layering by agarose gel electrophoresis.

Further comparing the whole genome sequences of Fusarium vine caner and Fusarium layering, the method finds out that gibberellin biosynthesis is regulatedGA20oxThe gene (SEQ ID NO. 1) was deleted in Fusarium layering. According to vineIn Fusarium canumGA20oxThe gene design specific primer is characterized by having nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.3, carrying out PCR amplification of four small species genome DNA of the bakanae disease germ, and separating 848bp fragments in fusarium graminearum by agarose gel electrophoresis.

A specific primer is designed according to calmodulin genes in fusarium pseudoverticillatum, and is characterized by having nucleotide sequences of SEQ ID NO.69 and SEQ ID NO.70, performing PCR amplification on four small species of genome DNA of bakanae disease of rice, and separating 578bp fragments from the fusarium pseudoverticillatum only by agarose gel electrophoresis.

Therefore, for the rice bakanae germ to be detected, whether the fusarium graminearum exists can be obtained by identifying whether sequences shown as SEQ ID NO.1 and SEQ ID NO.25 exist in the sequence of the genome of the strain; if SEQ ID NO.25 is present but the sequence shown in SEQ ID NO.1 is not present, judging that the strain is Fusarium layering; if the specific fragment exists by PCR amplification with the specific primers of the calmodulin of the fusarium pseudoverticillium, judging that the strain belongs to the fusarium pseudoverticillium; if the genome sequence does not contain SEQ ID NO.1, SEQ ID NO.25 and Fusarium pseudoverticillium calmodulin gene sequences, the strain can be judged to beF. andiyaziAs shown in fig. 1.

Accordingly, the inventors of the present invention have achieved the following means.

The rapid detection method for generating gibberellin seed in rice bakanae disease germ comprises fusarium graminearum, fusarium verticillatum and fusarium graminearumF. andiyaziIs characterized in that aiming at fusarium graminearum, fusarium layering and fusarium verticillatumF. andiyaziThe specific primer is designed according to the specific gene difference of the (2), and the specific primer is used for carrying out multiplex PCR amplification by taking the DNA of the sample to be detected as a template, and the gibberellin seed is generated in the bakanae disease germ of the rice according to the amplification result.

The rapid detection method for gibberellin seed generation in rice bakanae disease germ is characterized in that fusarium graminearum, fusarium verticillium and fusarium graminearum are adoptedF. andiyaziThe specific genes of (a) are: as shown in SEQ ID NO.1Gibberellin synthesis regulationGA20oxGene, gibberellin biosynthesis as shown in SEQ ID No.25CPS/KSGenes and calmodulin genes.

The rapid detection method for gibberellin miniascapes generated in rice bakanae disease germs is characterized in that the specific primers comprise: gibberellin synthesis regulation of nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3GA20oxA gene specific primer; gibberellin biosynthesis with the nucleotide sequences shown as SEQ ID No.59 and SEQ ID No.60CPS/KSA gene specific primer; calmodulin gene specific primer with the nucleotide sequence shown as SEQ ID No.69 and SEQ ID No. 70.

The rapid detection method for gibberellin miniascapes generated in rice bakanae disease germs is characterized in that the multiplex PCR amplification conditions are as follows:

the multiplex PCR reaction system is as follows: 100ng of sample genome DNA, three pairs of forward and reverse primers of 10 mu M each of 1 mu L and three pairs of forward and reverse primers comprisingGA20oxA gene specific primer,CPS/KSGene-specific primer and calmodulin gene-specific primer, 2.5mM dNTPs 4. Mu.L, 10 XPCR buffer 2. Mu.L, 5U/. Mu.L TaqPolymerase 0.4. Mu.L, add ddH ₂ O makes up 20. Mu.L;

the PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, elongation at 72℃for 80s,35 cycles; finally, the extension is carried out for 10min at 72 ℃.

The rapid detection method for gibberellin miniascapes generated in rice bakanae disease is characterized in that the judgment result is as follows: when the PCR amplification product presents two bands of 1327bp and 848bp, judging that the sample is fusarium graminearum; when the PCR product presents a 1327bp single band, judging that the sample is fusarium layering; when the PCR product presents 578bp single band, judging that the sample is fusarium verticillium; when the PCR product has no band, the sample is judged to beF. andiyazi。

The rapid detection method for gibberellin miniascapes generated in rice bakanae disease germs is characterized by comprising the following steps of:

1) Extracting whole genome DNA of a sample to be detected;

2) Multiplex PCR amplification is carried out by taking DNA of a sample to be detected as a template:

the multiplex PCR reaction system is as follows: 100ng of sample genome DNA, three pairs of forward and reverse primers of 10 mu M each of 1 mu L and three pairs of forward and reverse primers comprisingGA20oxA gene specific primer,CPS/KSGene-specific primer and calmodulin gene-specific primer, 2.5mM dNTPs 4. Mu.L, 10 XPCR buffer 2. Mu.L, 5U/. Mu.L TaqPolymerase 0.4. Mu.L, add ddH ₂ O makes up 20. Mu.L;

the PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, elongation at 72℃for 80s,35 cycles; finally, the mixture is extended for 10min at 72 ℃;

the saidGA20oxThe specific primers of the genes are shown as SEQ ID NO.2 and SEQ ID NO.3,CPS/KSThe specific primers of the gene are shown as SEQ ID NO.59 and SEQ ID NO.60, and the specific primers of the calmodulin gene are shown as SEQ ID NO.69 and SEQ ID NO. 70;

3) Judgment by amplification results

Separating the amplified product by 1.5% agarose gel electrophoresis, staining the separated product under an ultraviolet lamp by ethidium bromide according to the size judgment result of the amplified product, and judging that the sample is fusarium graminearum when the PCR amplified product presents two bands of 1327bp and 848 bp; when the PCR product presents a 1327bp single band, judging that the sample is fusarium layering; when the PCR product presents 578bp single band, judging that the sample is fusarium verticillium; when the PCR product has no band, the sample is judged to beF. andiyazi。

The said processGA20oxA gene specific primer,CPS/KSApplication of gene specific primer and calmodulin gene specific primer in rapid detection of gibberellin-producing species including Fusarium graminearum, fusarium layering and Fusarium verticilliumF. andiyazi。

The said processGA20oxA gene specific primer,CPS/KSThe application of the gene specific primer and the calmodulin gene specific primer in early diagnosis of bakanae disease of rice.

The method is suitable for the rapid and reliable detection and identification of the rice bakanae disease germ, and is suitable for fusarium graminearum and sickle layeringFusarium species, fusarium pseudoverticillium, and methods of producing the sameF. andiyaziThe prevention and treatment of the bakanae disease of the rice caused by the method has important practical application value. Compared with the prior art, the invention has the following technical advantages and positive effects:

1. the specificity is strong: the detection method of the invention obtains the specific genes of the species based on the sequence information of the genes of the four fusarium which cause bakanae disease of rice, and detects pathogenic bacteria through the specific nucleic acid sequences of the species, and the detection primer has good specificity and can amplify the strip with specific size from the target sample.

2. The practicability is good: the specific gene, the primer and the detection method can be used for rapidly, simply, conveniently and accurately identifying fusarium graminearum, fusarium verticillium and fusarium graminearum which cause bakanae disease of riceF. andiyaziThe method can be applied to detection of DNA of the pathogenic tissues of the bakanae disease of rice and detection of DNA of fungi separated from the pathogenic tissues.

3. The operation is simple, convenient and quick: the serial specific primer combination used in the invention can judge the result after completing a reaction system of multiplex PCR, PCR amplification and conventional agarose gel electrophoresis for four small species detection in the gibberella caner complex species, and generally the whole PCR detection process is completed within a few hours.

Drawings

FIG. 1 is an agarose gel electrophoresis diagram of a triple PCR detection technology after amplification of a PCR detection sample of rice bakanae disease germ; the swimming band M is a DNA standard substance (100 bp DNA Ladder Marker), and 1-14 are identified strains; wherein 1-5 are fusarium graminearum; 6-7 is fusarium; 8-12 are Fusarium verticillium; 13-14 isF. andiyazi。

FIG. 2 is a view of Fusarium protein translational elongation factorEFCarrying out agarose gel electrophoresis after PCR detection of sample amplification by using universal primers of genes; the swimming band M is a DNA standard substance (100 bp DNA Ladder Marker), and 1-14 are identified strains; wherein 1-5 are fusarium graminearum; 6-7 is fusarium; 8-12 are Fusarium verticillium; 13-14 isF. andiyazi。

FIG. 3 shows fusarium graminearum gibberellin biosynthesis genesCPS/KSPCR detection of sample amplification by specific primers of (2)Agarose gel electrophoresis pattern after increasing; the swimming band M is a DNA standard substance (100 bp DNA Ladder Marker), and 1-14 are identified strains; wherein 1-5 are fusarium graminearum; 6-7 is fusarium; 8-12 are Fusarium verticillium; 13-14 isF. andiyazi。

FIG. 4 shows the fusarium graminearum gibberellin control synthesis genesGA20oxPerforming agarose gel electrophoresis after PCR detection of sample amplification; band M is DNA standard (100 bp DNA Ladder Marker), wherein 1-14 are identified strains; wherein 1-5 are fusarium graminearum; 6-7 is fusarium; 8-12 are Fusarium verticillium; 13-14 isF. andiyazi。

FIG. 5 is an agarose gel electrophoresis diagram of a sample amplified by PCR detection of a specific primer of the Fusarium verticillium calmodulin gene; the swimming band M is a DNA standard substance (100 bp DNA Ladder Marker), and 1-14 are identified strains; wherein 1-5 are fusarium graminearum; 6-7 is fusarium; 8-12 are Fusarium verticillium; 13-14 isF. andiyazi。

FIG. 6 is Fusarium vine canum, fusarium layering, fusarium verticillium and Fusarium verticilliumF. andiyaziComparison of gibberellin biosynthesis gene clusters.

FIG. 7 is an agarose gel electrophoresis chart of a field collected rice bakanae disease germ after PCR detection sample amplification; m is DNA standard (100 bp DNA Ladder Marker), and 1-14 are strains to be tested.

Detailed Description

The invention will be further illustrated with reference to specific examples, which are to be understood as illustrative only and are not to be construed as limiting the scope of the invention.

Example 1 molecular identification of four Fusarium species of Pyricularia oryzae

Extracting genome DNA of fusarium isolated from bakanae disease sample of rice, translating elongation factor by fusarium proteinEFAnd (3) carrying out molecular identification on universal primers designed by the gene conserved sequence.

Step 1: strain culture

The strain was inoculated on a potato dextrose agar solid medium (potato 200g, glucose 18g, agar 20g, distilled water 1L, PDA), a mass of 5d was taken and transferred into a100 mL potato dextrose liquid medium (potato 200g, glucose 18g, distilled water 1L, PDB), and after shaking culture for 5d at 28℃in a constant temperature shaker, mycelia were collected.

Step 2: quick extraction of strain DNA

(1) Pretreatment of a sample: 20 mg mycelia were placed in a2 mL centrifuge tube and 500 μ L Extraction Buffer buffer (1M KCL,100 mM TrisHCl,10 mM EDTA,pH =8) was added;

(2) Grinding: placing the centrifuge tube in a rapid nucleic acid extraction instrument, setting 60 HZ, and grinding for 1min;

(3) And (3) centrifuging: centrifuging at 12000rpm for 10min at 4deg.C;

(4) Precipitation: transferring 400 mu L of supernatant to a new centrifuge tube, adding isopropyl alcohol precooled in equal volume, gently mixing, standing at room temperature for 10min, and centrifuging at 12000rpm for 10min;

(5) Washing: removing supernatant, adding 700 mu L of 70% ethanol, shaking and mixing uniformly, centrifuging at 12000rpm for 5min at 4 ℃, and washing for 2 times;

(6) Dissolving: vacuum drying for 10min, dissolving DNA in ddH ₂ O, stored at 4 ℃.

Step 3:EFmolecular validation of genes

Protein translation elongation factor according to different species of fusariumEF) The conserved sequence of the gene, O 'Donnell (1998), designed the general primer EF1/EF2 for Fusarium [ O' Donnell, K., cigelnik, E., nirenberg, H.I. Molecular systematics and phylogeography of the)Gibberella fujikuroispecies complex. Mycologia, 1998, 90, 465–493.]EF1 and EF2 have the nucleotide sequences of SEQ ID NO.4 and SEQ ID NO.5, respectively, and PCR amplification is carried out on different Fusarium species. The PCR reaction system is as follows: 50ng of strain genome DNA, 1. Mu.L of forward and reverse primers (10. Mu.M), 4. Mu.L of dNTPs (2.5 mM), 2. Mu.L of 10 XPCR buffer,Taqpolymerase (5U/. Mu.L) 0.3. Mu.L, ddH was added ₂ O makes up 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 55℃for 30s, extension at 72℃for 40s,30Cycling; finally, the extension is carried out for 10min at 72 ℃. As shown in FIG. 2, the identified strains each amplified a fragment of about 700 bp. The PCR amplified product is cut from agarose gel, and the specific fragment is recovered and purified by using a DNA recovery kit and then connected to a pMD18-T carrier to be transformed into escherichia coliE. coliThe recombinant plasmid showing positive clones was sent to Shanghai bioengineering company for sequence sequencing.

Sequencing results in Fusarium TEF sequence database (FUSARIUM-ID v 1.0) [ Geiser D M, jimenez-Gasco M, kang S, et al FUSARIUM-ID v 1.0: A DNA Sequence Database for Identifying ]Fusarium. Eur J Plant Pathol, 2004, 110, 473-479 ]The submitted sequences are aligned to determine their small type. Five strain protein translation elongation factorsEFThe genes respectively have nucleotide sequences of SEQ ID NO. 6-SEQ ID NO.10, and are judged to be fusarium graminearum; 2 strainsEFThe genes respectively have nucleotide sequences of SEQ ID NO.11 and SEQ ID NO.12, and the genes are judged to be Fusarium layering; five strainsEFThe genes respectively have nucleotide sequences of SEQ ID NO. 13-SEQ ID NO.17, and the genes are judged to be Fusarium pseudoverticillium; two strainsEFThe genes have nucleotide sequences of SEQ ID NO.18 and SEQ ID NO.19 respectively, and are judged asF. andiyazi。

By combining the traditional morphological method, the shape and the implantation mode of megaspore and microspore of the strain, the characteristics of conidiophore, the growth color of bacterial colony, the existence of pigment and the like are observed, and finally, the strain No.1 to No.5 to be detected is identified as fusarium gracilis, the strain No.6 to No.7 is identified as fusarium gracilis, the strain No. 8 to No.12 is identified as fusarium verticillium, and the strain No.13 to No. 14 are identified as fusarium gracilisF. andiyazi。

Example 2 comparison of four Fusarium Saccharum species gibberellin biosynthesis Gene clusters

Wiemann et al (2013) have published sequences of gibberellin synthesis gene clusters in Fusarium canum [ Wiemann P, sieber C M, von Bargen K W, et al Deciphering the cryptic genome:genome-wide analyses of the rice pathogen ]Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog, 2013, 9(6): e1003475]. According to gibberellin biosynthesis gene clustersDES（SEQ ID NO.20）、P450-4（SEQ ID NO.21）、P450-1（SEQ ID NO.22）、P450-2（SEQ ID NO.23）、GGS2（SEQ ID NO.24）、CPS/KS(SEQ ID NO. 25)P450-3(SEQ ID NO. 26) Gene sequence three pairs of specific primers were designed at the front, middle and rear of each gene respectively (Table 1), for Fusarium layering, fusarium verticillium and Fusarium pseudolarix in example 1F. andiyaziIs subjected to PCR amplification. The PCR reaction system is as follows: 50ng of strain genome DNA, 1. Mu.L of forward and reverse primers (10. Mu.M), 4. Mu.L of dNTPs (2.5 mM), 2. Mu.L of 10 XPCR buffer,Taqpolymerase (5U/. Mu.L) 0.3. Mu.L, ddH was added ₂ O makes up 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, extension at 72℃for 1min/1kb,30 cycles; finally, the extension is carried out for 10min at 72 ℃. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the presence or absence of a DNA band was observed. Identified, fusarium layering contains complete gibberellin biosynthesis gene clusters, while Fusarium pseudoverticillatum and Fusarium pseudolarisF. andiyaziContaining onlyDESAndP450-4the gene is used for the gene expression,P450-1、P450-2、GGS2、CPS/KSandP450-3the genes were deleted (FIG. 6). FIG. 3 is a schematic view ofCPS/KSAnd (3) according to an agarose gel electrophoresis diagram after the specific primer of the gene is amplified, the primer sequences respectively have nucleotide sequences of SEQ ID NO.59 and SEQ ID NO.60, and the fusarium graminearum and fusarium graminearum can amplify a 1327bp specific fragment.

TABLE 1 Gene sequence of Fusarium canum gibberellin synthesis Gene Cluster and primer sequence thereof

。

Example 3 comparison of Fusarium vine and Fusarium layering complete genome

For comparison of functional genomes of Fusarium vine caner and Fusarium layering, fusarium layering Fp9 strain is downloaded from NCBI database in consideration of availability, representativeness and importance of dataSRA access: PRJNA 517537) and fusarium graminearum 58289 strain (ANFV 00000000), including genes, protein sequences, and gene annotation information. The two Fusarium whole genome sequences are aligned pairwise by utilizing mGENERATORS on-line analysis software (http:// bioinfo-mml.sjtu.edu.cn/mGS /)EThe value is set to 1e ^-5 ) Screening to obtain the specific gene for encoding GA 20-oxidase in late stage of gibberellin synthesis pathGA20oxThe gene is present in the genome of Fusarium canum (GenBank No. FFUJ_12912) but not in Fusarium layering, and has the nucleotide sequence of SEQ ID No. 1.

Example 4 differentiation of Fusarium vine and Fusarium layering

By Fusarium graminearum of example 3GA20oxThe gene was designed with specific primers having the nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.3, respectively, and four Fusarium species of example 1 were PCR amplified. The PCR reaction system is as follows: 50ng of strain genome DNA, 1. Mu.L of forward and reverse primers (10. Mu.M), 4. Mu.L of dNTPs (2.5 mM), 2. Mu.L of 10 XPCR buffer,Taqpolymerase (5U/. Mu.L) 0.3. Mu.L, ddH was added ₂ O makes up 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, extension at 72℃for 50s,30 cycles; finally, the extension is carried out for 10min at 72 ℃. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the presence or absence of a DNA band was observed. As shown in FIG. 4GA20oxThe agarose gel electrophoresis diagram of the amplified gene specific primer can amplify a specific fragment of 848bp in fusarium binthini, but does not amplify specific fragments in other three fusarium species.

EXAMPLE 5 Fusarium roseum and Fusarium pseudolarisF. andiyaziIs of (3)

To distinguish Fusarium roseum from Fusarium roseumF. andiyaziAccording to Muleg et al (2004) [ Muleg, susca A, stea G, moretti A. A patterns-specific PCR assay based on the calmodulin partical gene for identification of ]Fusarium verticillioides, F. proliferatumand F. subglutinas. Eur J Plant Pathol, 2004, 110: 495-502.]Fusarium verticillium calmodulin gene specific primers VER1 and VER2 PCR amplification of the four Fusarium species of example 1, VER1 and VER2 have the nucleotide sequences SEQ ID NO.69 and SEQ ID NO.70, respectively. The PCR reaction system is as follows: 50ng of strain genome DNA, 1. Mu.L of forward and reverse primers (10. Mu.M), 4. Mu.L of dNTPs (2.5 mM), 2. Mu.L of 10 XPCR buffer,Taqpolymerase (5U/. Mu.L) 0.3. Mu.L, ddH was added ₂ O makes up 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, extension at 72℃for 40s,30 cycles; finally, the extension is carried out for 10min at 72 ℃. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the presence or absence of a DNA band was observed. As shown in FIG. 5, which shows agarose gel electrophoresis of calmodulin-encoding gene-specific primer (VER 1/VER 2) after amplification, a 578bp PCR product was produced only in Fusarium pseudolaris, whereas no specific fragment was amplified in other strains.

Example 6 multiple PCR detection method for bakanae disease germ of rice

(1) Multiplex PCR amplification: multiplex PCR amplification was performed using the DNA of the strain to be tested in example 1 as a template.

(2) The multiplex PCR reaction system is as follows: sample genomic DNA100ng, three pairs of forward and reverse primers (10. Mu.M) each 1. Mu.L (comprisingGA20oxGenes (gene),CPS/KSSpecific primers for the gene and calmodulin gene), dNTPs (2.5 mM) 4. Mu.L, 10 XPCR buffer 2. Mu.L,Taqpolymerase (5U/. Mu.L) 0.4. Mu.L, add ddH ₂ O makes up 20. Mu.L. The PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, elongation at 72℃for 80s,35 cycles; finally, the extension is carried out for 10min at 72 ℃.

(3) Separating the amplified product by 1.5% agarose gel electrophoresis, and then staining the separated product by ethidium bromide under an ultraviolet lamp to judge the result according to the size of the amplified product. As shown in FIG. 1, samples No.1 to No.5 to be detected all contain target bands of 1327bp and 848bp, and are judged to be Fusarium graminearum; the sample No. 6-No. 7 to be detected contains a 1327bp target band, and the sample is judged to be Fusarium layering; the samples to be detected No. 8-12 contain 578bp target bands, and are judged to be Fusarium verticillium; judging whether the sample to be tested is strip or not in the samples No. 13-No. 14F. andiyazi. The multiplex PCR detection results and those based on example 1EFGene pair four sicklesThe molecular identification results of the knife fungus micro-species are consistent.

Example 7 multiple PCR detection of bakanae disease in Rice tissue

Step 1: isolation of rice bakanae disease strains: the rice bakanae disease-causing stalks are collected from a test base of rice in Fuyang areas of Hangzhou, zhejiang province. Cutting diseased tissue into 3mm ³ The left and right small blocks are inoculated on a water agar (20 g of agar, 1L of distilled water) culture medium for 48 hours, and then the top hypha is picked up and transferred into a potato dextrose agar PDA culture medium to be used as a rice bakanae germ strain to be detected.

Step 2: culturing the strain to be tested: step 1 as in example 1.

Step 3: extracting DNA of a strain to be detected: step 2 as in example 1.

Step 4: identification of the production of gibberellin Fusarium species:

(1) Multiplex PCR amplification: and (3) performing multiplex PCR amplification by taking DNA of the strain to be detected as a template. The multiplex PCR reaction system is as follows: 100ng of strain genome DNA, 1. Mu.L of each of three pairs of forward and reverse primers (10. Mu.M) (comprisingGA20oxGenes (gene),CPS/KSSpecific primers for the gene and calmodulin gene), dNTPs (2.5 mM) 4. Mu.L, 10 XPCR buffer 2. Mu.L,Taq0.4. Mu.L of polymerase (5U/. Mu.L) plus ddH ₂ O makes up 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, elongation at 72℃for 80s,35 cycles; finally, the extension is carried out for 10min at 72 ℃.

(2) Separating the amplified product by 1.5% agarose gel electrophoresis, and then staining the separated product by ethidium bromide under an ultraviolet lamp to judge the result according to the size of the amplified product. As shown in FIG. 7, the sample to be tested contains two target bands of 1327bp and 848bp, which indicates that all identified strains are Fusarium canum, and the Fusarium canum is possibly the dominant species of bakanae disease of rice in the collection area. If desired, further judgment can be made by recovery sequencing of the band of interest from electrophoresis, or based on elongation factorsEFBlast comparison after gene sequencing and cluster analysis after polygenic sequencing.

The multiplex PCR reaction system of the invention can simultaneously and rapidly and specifically detectFour species of fusarium graminearum, fusarium graminearum and fusarium graminearum causing bakanae disease of rice are measuredF. andiyazi。

The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications could be made by those skilled in the art without departing from the principles of the invention, which modifications would also be considered to be within the scope of the invention.

Sequence listing

<110> China institute of Rice

<120> method for rapidly detecting gibberellin miniascape produced in rice bakanae disease

<160> 70

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1067

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 1

atgccttctg ccattgacag catcccaatt gcctcctttg agacaatcaa ctactccgct 60

cttgagagac gagatgcgaa agagattgaa aagctcatgg gtgcaagccg tacagccgga 120

ttcttctacc tagactttga ccgtagtggt gccgctggtc tacccaagaa gaagaaagaa 180

gttctcaagg caatgaagga atatttcagt cagcctgatg acatcaagca acttgacagc 240

aaaggcgttc ctacgcgtgg gtaagtagac cgcaattatg aatggcacag tgctgattag 300

tttcaagata tgtcaagaaa ggcaccttca ccgccgttga ccccagccgc cctgacgaat 360

cttttgaaca tcttgctgta agccatatga gcggttcggc agattcatga ctgacttcaa 420

agcagattgg aacgcacgat ctgggaacta atatagcttc cacgctccct gcagtgttca 480

agaaggctgg aacgctcatc cccgactacg tagctctttg tgagcgtgta gtcgacgttc 540

tgctggactg ctactcacag gctctaggcg tccctggtca gttcctggag taccacgacc 600

acgaaaaacc atccgatacc atccttgcca tgctgagcta tcctggaaag ctcacccatc 660

aaaagcatac cgatctggga tccttgactg ttctcttcag tgatgagtgg ggacttcagg 720

ttgttgaacc cagcaacggc aattgggagt gggtcgagcc gcgggagaac gacgccgtca 780

tcaatgttgg cgacactctt cgcttcctgt cgggaaaaac cttgtattcc tgtgttcaca 840

gggtcatcag agatggtcgt gctagcgatg aggggcatag atactccatc gcatatctgc 900

tgcgtcctgg tgatgacgtg agctttgtcg acgccgatgg gtccaggatc accgcccagt 960

cctttgccgg tatcaagtac aaagcttatt ctgcggacca tgctgagcag gacaagaata 1020

cggtcctgac tggtggaatg gaacaggtac ttggtgtccg cgcctag 1067

<210> 2

<211> 16

<212> DNA

<213> primer (primer)

<400> 2

caaggcaatg aaggaa 16

<210> 3

<211> 16

<212> DNA

<213> primer (primer)

<400> 3

ccagtcagga ccgtat 16

<210> 4

<211> 20

<212> DNA

<213> primer (primer)

<400> 4

atgggtaagg aggacaagac 20

<210> 5

<211> 21

<212> DNA

<213> primer (primer)

<400> 5

ggaagtacca gtgatcatgt t 21

<210> 6

<211> 646

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 6

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga caacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcgctcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata agctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 7

<211> 646

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 7

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga caacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcgctcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata acctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 8

<211> 646

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 8

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga caacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcgctcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata agctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 9

<211> 646

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 9

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga caacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcgctcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata agctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 10

<211> 646

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 10

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga caacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcgctcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata agctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 11

<211> 646

<212> DNA

Fusarium (F. Proliferatum) is layered <213

<400> 11

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga cgacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcactcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata agctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 12

<211> 646

<212> DNA

Fusarium (F. Proliferatum) is layered <213

<400> 12

tgatatgtta gtatgaataa gtagaatgaa gcatgagcga caacatacca atgacggtga 60

catagtagcg aggagtctcg aacttccaga gagcaatatc gatggtgata ccacgctcac 120

gctcggcctt gagcttgtca agaacccagg cgtacttgaa ggaaccctta ccgagctcag 180

cggcttccta ttgtcgaatg gttagtttga cacgtgacaa tgcgctcatt gaggttgtgg 240

acaggaaagg gcaaaacgcg cccatcactc gagtggcggg gtaaatgccc caccaaaaaa 300

aattacggtc atatcgcaaa atttttggtc tcgagcgggg tagcaggcac gtttcgaatc 360

gtaagggaaa tcggtgggca aaggacgcgc gatcgaaggg aaagtgacta accttctcga 420

acttctcgat ggttcgcttg tcgataccac cgcactggta gatcaagtga ccggtctgtg 480

aagcgatgtc agcatattgt cttctgagat ataccccgcc agatcttggt caggatcacg 540

atgacagata agctcatcgt cgagggtagt actcacagtg gtcgacttgc cagagtcaac 600

gtggccgatg acgacgacgt taaggtgagt cttgttccct tttccc 646

<210> 13

<211> 652

<212> DNA

<213> Fusarium verticillium (F. Verillioides)

<400> 13

tgatgtgtta gtaataggag atatagaacg gagtaagagc gacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcgata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgga tggttagtga ctgcttgaca cgtgacgatg agctcagtga 240

ggttgtggaa tgggagaggg cagaaacgcg ccgctcgagt ggcggggtaa atgccccacc 300

agaaaaaatt acggtcgtat cgcaaaattt ttgggctcga gcggggtagc gggtacgttt 360

cgagtcgtag gggggaatcg atgggcaaag aacgcgcgat agaaggaaaa gtgactaacc 420

ttctcgaact tctcgatggt tcgcttgtcg ataccaccgc actggtagat caagtgaccg 480

gtctgtgaag cgatatcagt atgttttctt tgagaaatcc cccgccaggt cttggccggg 540

tttacgatgg ccgataagct catcgtcaag ggtagtactc acagtggtcg acttgccaga 600

gtcaacgtgg ccgatgacga cgacgttaag gtgagtcttg ttcccttttc cc 652

<210> 14

<211> 652

<212> DNA

<213> Fusarium verticillium (F. Verillioides)

<400> 14

tgatgtgtta gtaataggag atatagaacg gagtaagagc gacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcgata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgga tggttagtga ctgcttgaca cgtgacgatg agctcagtga 240

ggttgtggaa tgggagaggg cagaaacgcg ccgctcgagt ggcggggtaa atgccccacc 300

agaaaaaatt acggtcgtat cgcaaaattt ttgggctcga gcggggtagc gggtacgttt 360

cgagtcgtag gggggaatcg atgggcaaag aacgcgcgat agaaggaaaa gtgactaacc 420

ttctcgaact tctcgatggt tcgcttgtcg ataccaccgc actggtagat caagtgaccg 480

gtctgtgaag cgatatcagt atgttttctt tgagaaatcc cccgccaggt cttggccggg 540

tttacgatgg ccgataagct catcgtcaag ggtagtactc acagtggtcg acttgccaga 600

gtcaacgtgg ccgatgacga cgacgttaag gtgagtcttg ttcccttttc cc 652

<210> 15

<211> 652

<212> DNA

<213> Fusarium verticillium (F. Verillioides)

<400> 15

tgatgtgtta gtaataggag atatagaacg gagtaagagc gacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcgata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgga tggttagtga ctgcttgaca cgtgacgatg agctcagtga 240

ggttgtggaa tgggagaggg cagaaacgcg ccgctcgagt ggcggggtaa atgccccacc 300

agaaaaaatt acggtcgtat cgcaaaattt ttgggctcga gcggggtagc gggtacgttt 360

cgagtcgtag gggggaatcg atgggcaaag aacgcgcgat agaaggaaaa gtgactaacc 420

ttctcgaact tctcgatggt tcgcttgtcg ataccaccgc actggtagat caagtgaccg 480

gtctgtgaag cgatgtcagc atgttttctt tgagaaatcc cccgccaggt cttggccggg 540

tttacgatgg ccgataagct catcgtcaag ggtagtactc acagtggtcg acttgccaga 600

gtcaacgtgg ccgatgacga cgacgttaag gtgagtcttg ttcccttttc cc 652

<210> 16

<211> 652

<212> DNA

<213> Fusarium verticillium (F. Verillioides)

<400> 16

tgatgtgtta gtaatagaag atatagaacg gaataagagc gacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcgata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgga tggttagtga ctgcttgaca cgtgacgatg agctcagtga 240

ggttgtggaa tgggagaggg cagaaacgcg ccgctcgagt ggcggggtaa atgccccacc 300

agaaaaaatt acggtcgtat cgcaaaattt ttgggctcga gcggggtagc gggtacgttt 360

cgagtcgtag gggggaatcg atgggcaaag aacgcgcgat agaaggaaaa gtgactaacc 420

ttctcgaact tctcgatggt tcgcttgtcg ataccaccgc actggtagat caagtgaccg 480

gtctgtgaag cgatgtcagc atgttttctt tgagaaatcc cccgccaggt cttggccggg 540

tttacgatgg ccgataagct catcgtcaag ggtagtactc acagtggtcg acttgccaga 600

gtcaacgtgg ccgatgacga cgacgttaag gtgagtcttg ttcccttttc cc 652

<210> 17

<211> 652

<212> DNA

<213> Fusarium verticillium (F. Verillioides)

<400> 17

tgatgtgtta gtaataggag atatagaacg gagtaagagc gacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcgata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgga tggttagtga ctgcttgaca cgtgacgatg agctcagtga 240

ggttgtggaa tgggagaggg cagaaacgcg ccgctcgagt ggcggggtaa atgccccacc 300

agaaaaaatt acggtcgtat cgcaaaattt ttgggctcga gcggggtagc gggtacgttt 360

cgagtcgtag gggggaatcg atgggcaaag aacgcgcgat agaaggaaaa gtgactaacc 420

ttctcgaact tctcgatggt tcgcttgtcg ataccaccgc actggtagat caagtgaccg 480

gtctgtgaag cgatgtcagc atgttttctt tgagaaatcc cccgccaggt cttggccggg 540

tttacgatgg ccgataagct catcgtcaag ggtagtactc acagtggtcg acttgccaga 600

gtcaacgtgg ccgatgacga cgacgttaag gtgagtcttg ttcccttttc cc 652

<210> 18

<211> 651

<212> DNA

<213> F. andiyazi(F. andiyazi)

<400> 18

tgacatgtta gtaagaagag atgtagaatg aagcatgagc gacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcaata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgaa tgtttagtga ctgcttgaca cgtgacgatg cgctcagtga 240

ggttgtggaa tgggagaggg caaaaacgcg ccgctcgagt ggcggggtaa ataccccacc 300

aaaaaaatta cggtcatatc gcaaaatttt tggactcgag cggggtagcg ggcacgtttc 360

gagtcgtagg gagaaatcgg tggacaaagg acgcgcgatc gaagggagtg tgactaacct 420

tctcgaactt ctcgatggtt cgcttgtcga taccaccgca ctggtagatc aagtgaccgg 480

tctgtgaagc gatgtcagca tgttttcttt tgaaaatacc ccgccaggtc ttggtcggga 540

atacgatggc cgataagctc atcgtcaagg gtagtactca cagtggtcga cttgccagag 600

tcaacgtggc cgatgacgac gacgttaagg tgagtcttgt tcccttttcc c 651

<210> 19

<211> 652

<212> DNA

<213> F. andiyazi(F. andiyazi)

<400> 19

tgacatgtta gtaagaagag atgtagaacg gagcataagc cacaacatac caatgacggt 60

gacatagtag cgaggagtct cgaacttcca gagagcaata tcgatggtga taccacgctc 120

acgctcggcc ttgagcttgt caagaaccca ggcgtacttg aaggaaccct taccgagctc 180

agcggcttcc tattgtcgaa tggttagtga ctgcttgaca cgtgacaatg cgctcagtga 240

ggttgtggaa tgggagaggg cagaaacgcg ccgctggagt ggcggggtaa atgccccacc 300

aaaaaaatta cggacatatc gcaaaatttt tggactcgag cggggtagcg ggcacgtttc 360

gagtcgtagg gagaaatcgg tggccaaagg acgcgcgatc gaagggattg tgactaacct 420

tctcgaactt ctcgatggtt cgcttgtcga taccaccaca ctggtagatc aagtgaccgg 480

tctgtgaagc gatgtcagca tgttttcttt tgagaaacac ctcgccaggt cttgggcggg 540

tttacgatgg ccgataagct catcgtcaag ggtagtactc acagtggtcg acttgccaga 600

gtcaacgtgg ccgatgacga cgacgttaag gtgagtcttg ttcccttttc cc 652

<210> 20

<211> 1029

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 20

atgcctcata aagataatct tcttgaatcg ccagtgggca agagtgtcac tgctactata 60

gcctaccata gcggaccggc tcttccaacc tccccgatcg ctggtgtcac tacgctccaa 120

gactgcactc agcaggccgt agcagtgact gatatccgcc cttcagtctc gtcctttacc 180

ctagatggta acggcttcca ggttgtcaaa catacatcgg cggtaggctc tccgccgtat 240

gatcactcgt cgtggacaga tccagtcgtt cgcaaggaag tgtatgaccc cgaaatcatt 300

gaactggcaa agtctctcac tggagccaag aaggtcatga ttctacttgc ttcgtctcgg 360

aatgttccct tcaaggagcc agagctcgcc cctccttatc ccatgcctgg caaatcaagc 420

agcggcagca aggaaaggga agccatccca gctaatgagc tccctactac aagggcaaaa 480

ggtttccaaa aaggcgaaga ggaaggccca gtacgaaagc ctcataagga ctggggtcca 540

tccggtgcgt ggaacactct ccggaactgg agccaagagc tcattgatga ggctggcgat 600

atcatcaagg ctggcgatga ggctgcaaag ctgccagggg gcagagcaaa gaactaccaa 660

ggcagacgat gggccctgta tactacctgg cgtccactga aaactgtcaa gcgggatccc 720

atggcctatg tagactactg gacagctgat gaggaagatg gcgtgagctt ctggcgtaac 780

ccgccagggg tgcatgggac atttgagtcg gatgtactac ttaccaaggc taatccaaag 840

cataagtggt actggatcag tgaccagact ccggatgagg ttctcctcat gaagatcatg 900

gacaccgaga gtgagaagga cgggagtgaa atagcgggag gggttcacca ctgttcattt 960

catctgccgg gaactgagaa ggaggaagtg agagagagca ttgagaccaa gttcattgca 1020

ttctggtag 1029

<210> 21

<211> 1655

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 21

atgccgctaa tggacgttca ctggctgatc tacgtggcct ttggcgcttg gttatgctct 60

tatgtcatcc atgtcctatc gtcctcttct acagtcaaag tgcccgtcgt aggctaccgc 120

agcgtctttg agcctacatg gcttctccgt ttgcgctttg tttgggaagg gggatctatc 180

atcggccaag gctacaacaa agtaagcgat tccttctaca gccatgtaac catatctcat 240

ctatctatag tttaaagact ctatcttcca ggtgcgaaag cttggtaccg atatcgtcat 300

catcccgcca aactacatcg atgaggtcag aaagctgtcc caagacaaga ctcgctcggt 360

cgagcccttc atcaatgact ttgcgggaca gtatacacgg ggcatggtct ttctgcaaag 420

tgatttgcag aaccgtgtga ttcagcagcg gttgacgcca aaactcgtat cgttgacaaa 480

ggtaatgaag gaggagcttg actatgcctt gaccaaagag atgcctgaca tgaagagtga 540

gaatgtctcg acttttgaac tagatcaata aatgctgatg gttcttagat gatgaatggg 600

ttgaagtcga catttcttcc atcatggtca ggctcatatc acgcatctca gccagagtgt 660

ttctcggtcc agagcactgc cgcaaccaag aatggttgac gaccactgca gagtacagcg 720

agagcctgtt cataactggc tttattctcc gcgttgtccc ccatattcta agaccattca 780

tagccccgct gctaccctcc tacagaacac tacttcgcaa cgtctcgtca ggtcgaagag 840

ttattggaga catcattcgc tcccagcaag gtgatggcaa cgaggacatc ctgtcatgga 900

tgagggatgc tgcgacaggg gaagaaaagc aaattgacaa cattgcccag cggatgctta 960

tcctgagtct cgcgtctatt cacactacgg caatgacgat gacgcatgct atgtatgact 1020

tatgtgcttg ccctgagtac atagagcctc ttagagatga ggtcaaaagt gtcgttggcg 1080

ctagtggttg ggacaagacg gcgttgaatc gattccacaa actcgacagc tttctcaaag 1140

agtcacaacg cttcaacccc gtgttcctct gtaagtcctt ctccatcttt agctcttcta 1200

ggatctggct gaaacctata gtaacgttca atcgcattta tcaccaatcc atgacactct 1260

cagatggcac caacatccca tcaggcactc gcatcgcggt tccctctcac gcgatgcttc 1320

aggactcagc gcatgtccca ggcccgacgc caccaaccga gtttgatgga tttagatact 1380

caaagattcg ctcagactca aactatgcac agaaatatct cttctccatg actgattcta 1440

gtaacatggc gtttgggtat gggaaatacg cctgcccagg gcggttctat gcatctaatg 1500

agatgaagct gactttggcg atactccttt tacaatttga gttcaagttg ccagatggga 1560

aaggaagacc acgaaatatc actattgata gtgacatgat acctgatccg agagctaggc 1620

tgtgcgttag gaagcgatca ctgagagatg aatga 1655

<210> 22

<211> 1479

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 22

tcaaatcgca atctcctcct ttctgcgacg aacagataac tttgcagttg ggttggcatt 60

catgttgagg ccatacttcc tcggctccat gctcgacccc tcaacaggct tgaagtcgta 120

cttgagcaga atgtgagaca gtgcaatctt gatctcctca gacgcaaaga accgtccagg 180

acaggcatga agcccgtacc cgaagcccat gtgatccgga gtggcactca cgagctgtgc 240

tttgctctct ttgcctggct cgcgccgcat gttgaagaaa cggtatccat cgaacttcag 300

gggatccttg tagtactctg gatcccagtg ttgatgggct gatacaagcg tgagtttgtt 360

cttgggtagg atgacgccat cggataactt gacgttgtgc gttgtgaatc ggcgcatgct 420

tgctagtact gttttttagt gcgtgctatc accccaagct aggaactgga tacgtaccaa 480

tggctatggg cttcagtcgt tgactctctt tcaggacgct gtccatgagc ttcaggttgt 540

acagcgagtt ctttgaccat ccctgcttcc ccaaaacagc tatgatctcc tcgcgcaaag 600

gctcgataag ctccggattc tgcgcaatgt caaacatgac ctgggtaaag aaatcagtag 660

tactatgtaa agcagccact gagagcgaga gctgagcaca agcggggtca tatccaacac 720

ccttttcacg cgcaaggtca tccaaccact caacagcatc gttgtaggtg accttctctc 780

cagttctctc agcctcagct ttctcttcgc gtcgccgctc aagaagcggg ttgataaggt 840

ctcttgcttc ttggactagg gctcgggact gtgtgcagtg cggcatgaac cactggacta 900

caggccggag ccacgatggc cagagacgca gttcttctac tgcacggaag gcgatgacgg 960

cgtaggtaga tgtaattcta agccactggg ggtttcgaca catctcctta ccgaggaata 1020

ctcgagaggt gatgcgggcc atgagtttca tgtttgcgtc tttggcggtg atgtcgtgcc 1080

attctgcgag actgatttag cgactgactc ttgcaagcag cgcatggacc atacctggac 1140

tatctgtgta cacatctttc aacaccaagg cgcactcttc agagactgct cccgtcacca 1200

gagtcaactg atgagtcaat tgatgtcgcg caaccagctt catgatgtga ctctcgttgg 1260

taccctctct gaagccctca aacccaggaa gatgagcata aaaccactat tctcagacgt 1320

cagccacatc ttgatcccgt actttgagct actcaccttg aaagctgcca tggtgaagct 1380

gagcttttca ttattgcgaa cctcgtaggc gtactttggc ggcaggatgt gcagctcccc 1440

gacatcgccc atgattcgaa atggcttgtc ggggctcat 1479

<210> 23

<211> 1903

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 23

tcaaattgct tccaaatcca actcaactga ctcccgacgc ttgatcaaga tgtccgtgac 60

agggctggac ttggcaatca tgcccctggt atcaggcttg gtctccgtgt cagggcacag 120

cttccaatca tacttgacca agatgtggca tagcgctact ttgatctcat tcgcagcgaa 180

gaaacgccct gggcatgagt gctgaccgtg gccgaagccc atatggtttg agccagttga 240

tactagctgt gccccatggt ctttcccggc ttcggagcgc atgtcgtaga agcgataagg 300

attgtaaacc tcggggttat cgtagatttt gggatcgtcg aggcgtctgt tgtcaacgtt 360

gaggcgggtc ccttttttga gggctaggca aaggaagggt tagtcagaaa tgcaggtgga 420

atgtgggatc tactaactca ggccgctaga gagggtgatg tcttcggtta cgtagcgacg 480

catggtaact gtattgcgag tgtcagtagt actcaagaaa ccgttggaaa gtccatacct 540

atgcttccag gcttcattcg ctgagactct ttgatagcac tgtcaaggag cttcatcttg 600

aaaagcgttg ttttcttcca accttcttca cgaagaagtt gaacaacttc ctgtctaaga 660

ggctcgatat actctgggtg gcgaccaagg tcaatcatcg tttgctggag gagatcatac 720

gtcgtatgaa ttgccagaag agagagcgta agctggaaga tcacggggtc aaacgaggcc 780

cctgtgcctg cagcttctgc ctcctgttcc gaccagtcaa tagcgtcgtg gaacacaggc 840

agaggctgac cagctgcgat cgcagctctt cgaagctcac ggcggcgctc aatcagtggc 900

gtaataatac cgattgcatc cttgcgctcc tgtcgaagct ttctgcattc ggggaggaac 960

cagtgggcga gaggacggat cgatcgagga aacattcgga ggttggtaga tgcagtgtag 1020

aagttggtgg tgtatgtctt tgtgatcttc agccaagctt cgttgcggca tagttggtcg 1080

ccgagataga ttctggacga gatgcgggcg atgatgtcta gaattgcggg cttgaggcgt 1140

atcgctcgcc attctgtatt tttggtgggt tagtgatagc tcttggctga tgagcatcgg 1200

aaagagactt gcctgttgtc tctccaaagt tgagcgacac tgcgagggtg gactctctag 1260

atagtggctc tatgacagcg gcttcttctt gttagtgtat ggtcctcact cgctagggat 1320

gaaaggtctc acaaagatgc ttggtcaact gctttcgggc caccttctga ataagttggt 1380

cttcacgacc caccagggca acagtctcaa agccaggtat tccagcatga ttatcctatg 1440

gcacaaggtt ttgtattcag cgctccagaa atccaaggtg ttgttagggg tacttacctg 1500

catcgccgcc ttggagaagc tcagtctcgg atcatttcta atctcatctg caaagtctgg 1560

ggggagaatg agaacctcac cccagtccgt aatcaagcga aatggctcat tagggaacaa 1620

gctcctcgct ttagcgagaa tttctctact aagcttgaca aaactcctct atagacaaag 1680

tcagtttccg cttggttgtc tcgtcaacag atggtcagac cctggtttta ccggtgccga 1740

acagtgagtc gggcgggttc gcaagtggca ccactgagcc cttgcgcaat tctagccaag 1800

agaagcggat tgcccatggg actaaagtaa agacgaatat ggcgatgtag aagggcaaaa 1860

gttggctacc cgcatagctg gtgatcatat tgaagatgct cat 1903

<210> 24

<211> 1445

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 24

atggctgaac aacagatctc caaccttctt tcaatgtttg atgcttctca cgcaagccag 60

aagttggaga ttacggttca gatgatggat acctaccatt acagagaaac tcctccagac 120

tcttcctctt cagaaggcgg ttccttatct cgctatgatg agcgacgggt ctcccttccg 180

ctctctcaca atgcagcctc cccagacata gtctcccagt tatgcttctc aacagctatg 240

agctcggagc tcaatcacag gtggaagtca cagcgcctca aggtgagtcg agatcgccct 300

catcccttta caatcacttg ctgagtatcc aggttgctga ctctccctac aactacatcc 360

tgactcttcc atctaaaggt attcgtgggg ctttcattga ctcactgaat gtctggctcg 420

aggtccccga agacgagacc tcggtgatca aagaggtgat tggcatgctc cacaactcgt 480

ctctcatgtt tgtatcccaa tctgttattg ctagagaccc tgctgacaat caagaatcga 540

tgacttccaa gacaactccc cacttcggcg gggcaagcca tctacacata ctgtcttcgg 600

tccagcacaa gcaatcaaca cagcaacata tgtcatcgtc aaggccatcg agaaaataca 660

ggatatcgtc ggtcacgatg cattggcaga tgtaactggc actataacca caatcttcca 720

gggtcaggca atggatctgt ggtggactgc taatgccatt gttccgtcta tccaagaata 780

tctcctgatg gtcaatgaca gtaagatgcc cctgtttatt gtggtgagac atagactaac 840

ttgcgttcct gcagagactg gtgccctgtt caggttatcg cttgaactac tggcgctgaa 900

ctctgaagca tccatcagtg acagcgcgct tgaatctctc agcagcgctg tctcactgct 960

cgggcagtat ttccagataa gagatgatta catgaatctc attgacaaca aggttcgatc 1020

cccacagccc acttcttgtc ttcggttacc agtaaaggag gtccacctct cggcactttg 1080

acctatctcc actatgttcc agaccctctg ctaaccactc tcagtatact gatcagaaag 1140

gattttgcga ggatctggac gaggggaaat actcgttgac tctaatccat gctctgcaga 1200

ccgactccag cgaccttctc accaacatct tatcgatgag aagagtccaa ggaaaactta 1260

cggcgcagca aaagatgctg gttttggaag tgatgaaaac aaacggcagc ttagactgga 1320

cgagcaaact ccttggcatg ctgcacacaa gagttgtagc cgagattgag agcctggaag 1380

ttagcacgaa gagggataac catgcattga gggctttagt ggaacgcctg aagctggaaa 1440

cctag 1445

<210> 25

<211> 3056

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 25

tcacttcatg ctgcttgaaa ggtccttgat cacgtaaagc tgatcataca agtcggtgac 60

atcacagaac agttttacaa tcttgagctt cctcatatcc ttagacccag cacgatcacc 120

tgcatcatca cgagactgtc gttccagagc ttcgagagca cgatcaagat aaccttgctc 180

atacgtagct atctttaaaa gtctctcttt cctttcatcg agattctgtg acgttccgtt 240

acataacgtg aactcgggga agtggatcga gttcacattc cgctcagcgt tatcgcgagc 300

tatggaccca aagtcgttgt acatacgaca catatttgta gcgtgacgca taacggatga 360

aatgagatat ttctgagtgc cggagggaaa tgcgtccttg ccttggagca gattcgctga 420

cattaggcag ttggagaaag cgaatgagta cgcgcaggcg acgtgactgc ctcctgtact 480

gttgacccat tggaagtacg attgttctgg ggaagagaaa gcgtcgctgg aggcctgctt 540

ggagaagcga ctgttgtcct ctatctgtgt gatatgggca tgcatgaacg ttctgaattc 600

tcggcgaaga gtgtcttggt cagaagagct tgaattcaga acatctttgt ggttgagcac 660

gctgttggtg aagcgagtga gagtatcctc aacttgtcca atgttgggtg actcatgctg 720

atgaccattg ccactgtgca cggtaccatt ggccctggct aagtttccca ttgtgttgtc 780

tatgaccttg tcgatggtct ggtgaagcag agagacatca ccgaataccg gcccagcgac 840

agcttccatg tactcatcgg tctggtagcc caggagggac aggtacatca tatcgtagag 900

ccatctgtta gatgcaaatg tgcgggatct gttgttgcat ccgacccaag taaaaggaat 960

gatgctcagg tatttgtcct cgtcgacctt gatgttgtcc ctcggataaa tctcaacgcg 1020

ctgagcttgt agaaggggca cgaagaacga ggactcaatg attgacgcca tgagtcccca 1080

ctcgtccagt ggcgagaaca aagcagtctt gcgcacaagc ctcatgtact tctccaagtc 1140

cgatgatgga accgccgagg tgacgctgtg gccaatagta gcggcgggca cctctaggct 1200

tgctgactga agggctgcta gcttgtacgc ttcagcaaca aacccaacct catatgctgt 1260

ctttgacgtc caagtgaggt cctgagaatg aaagctgcag gacttgagcc acgaaaagcc 1320

acggtcgacg caactctgta gcctgtccac catgtgagtg aagaaacata catggcgtgc 1380

ctgtacaaga gctagaatgg cgtagcatgt ttgctcgcga tatcctctcc aagatccgtc 1440

gttatcttgg gtgaggatga tacgtagtac agcctggaag attgacaggc cgatcttgca 1500

cttgaagctc tcgtcaaaga gactagaaag ctcgccacca tcaatgagat gaagtacctc 1560

ggtgaaagct tccaccagaa gcatggttgg gtatagatga ctcaaattct agaatagtta 1620

gcgagtgctt ctactagttg ggaagcagaa ctcacccatt tatctttgac gcaatggtcg 1680

ctgccccacc accatcggca agtgaacaac gtggtcttca ggatttgtgg gtggtattgc 1740

gatagattgg actgcttcag gagactcagc agcacatgga ggttggaagt caagctggga 1800

tcacgttcag atccgaacgt ggtgaagtga tcttttcctt caaagacctt gatcatgata 1860

tctgggctga caggttgatt gacaaggctc aatgccagaa gagcttttgc agtgtcatca 1920

acatcagcag tacggggtgc tgtactgtag ttagtctcca tggtacgata atgaaattgg 1980

ggcattctaa ccaaagccaa ttactccatt ctcgtccctc aacgcttcga gaagtatagt 2040

tgacaagccg cgcaagccat caccatcgat ttgtttcaaa gtgaagccac ccttcaggag 2100

tgtcgctatg atctaaaacc aaacttagca gccagaaggc gaaaataact agaacactta 2160

cccagctaca ctcaaaatgt gttgtaggaa aggtgcctga gatgccacca ttcccgtgac 2220

cagcaccatt tctcataacg tgcctcaggt aatcttcagc ctcgtcgtcc cacttggttg 2280

caccaatgag gtaggctgca gtggaagaag gtgaagccat catagagcca tgataaagat 2340

ggtgagaaag gcgatcaaag tcaagctttc caagaaaggc ttcaagggag tgaagtagtg 2400

aagacggctt gccatatact tgctccaaat cgaaatggcc gagcttctct ccgtgcatcc 2460

tctccaagat acttctgcaa ggaaactcaa aagatggcac gtcgagctct ttctcaagca 2520

tggaaagtaa agctggaata ataaactcga caccaatgtg attggtatct tcaacatcgt 2580

tccaaacagc tagctgtcgc ttcaaagaag ttaccccgtg ctcgatcctg agacccatct 2640

cgtctggcga gacgtccagt atctgcagag gctcttgtgc gtgacataag agtgcaagca 2700

cagctgaagc agtgtctaga ataccagcag tctgggtcgt ggggagacta ccccaactac 2760

catcagcggc ctgagtcttc agaagatagt ggaagcactc aggaaacagc cattgcttga 2820

cattgtctct cgtcttgggt atcatggcca cccaagcagt gtcatatacc tgacagctgg 2880

tggaacatag accatagtag ctatgatggc tcttgaaggc gcggtccagc agcgactttg 2940

ctgctgatac aaagtcattt cctgtcttga ggtctttggg ggtgccgttc tcgattttgc 3000

cacttggata ggttagaatg tgcatcagct aaaggtgtgt ttcatacgta cggcat 3056

<210> 26

<211> 1794

<212> DNA

<213> Fusarium vine bin (Fusarium fujikuroi)

<400> 26

tcatctcctt cgcatctcaa gaccagcttt ggtatccagc cgagtctccg tcccaacacg 60

catcaactca ggccctggct ttccatcctc aagtctaaaa tcatactttg ccaatagttc 120

tatgagtatc atcttgatct cagatatggc aaagaacctc ccagggcatg catgctttcc 180

atggttgaag atcagatagt caggacctgt agtggctgcc tggtgttgtg acccctgtcc 240

tttgatagat cggagattca agtaccggaa cccgtcaaag attcgaggag acgggttctc 300

gaagtcagag ctaaagtctg gcgagaatgt tggcgtttcc tccgacatgc tgttagatag 360

ttagtacagt aactattgac aaggggaagg gatggaaata ctggattgcg tgagcgggga 420

atgctataga agttcctctc tgtagcttga taccgtttga tagtgtcatt gacttcatga 480

cgcgtctgct tggagtcact gaaccattct gttagtgcct cttgcattac cagcatatgt 540

gctttagact taccaaatgt tgaagggcac cagcgctgga cctctctaat gaagctatcc 600

aacttgtgga gcctagaaag agcctctttg ttgatgcata tgtctggtga tacggcatca 660

ggtccaaaga catcttgcat ctcggttctc aacggctcga tgtactccgg ccgcttaaca 720

agctcccata ctacctttgt gagggccatg gctacccaag gtaacaagat cagaaacata 780

aacctagtat tgcggtttaa ataacttact cgtagtgtgg attgcggcga acgagaccag 840

catttgatct agcccgacct gctctggggt tctgagctcc tcaggtaagt gccgaagaag 900

ccatgagata aatgtacctc gcccttccgt ctgatccgct agaagtgtat ctgctcgatg 960

ttgcttttca tcttgtaaag cttgggagat cagaggagcc attatctctg ctgcgaagcg 1020

tagatgtctt cgcaccgaac ggacgctggg taaaaacggc ccaatgattg gtctcagcac 1080

cggactccag gtaaagagct gatcgcgaat tgagacacat tgaacagtgt agcctatgga 1140

agcttgtagc cagccttcat cgcgacacaa gggcagacca acaaacgctc tgccactcaa 1200

aagcgccacg gtccttagca tcatgtcata taaagataca gatgaccatt tgcttgttct 1260

aggccactga tcggaagcat atgttaactc atcctgtaat tccagtatga tgttgggcat 1320

atttcgagtc aagtcctggc gaatagtggc gggaatctca ggtctgatgg tggtaatgtg 1380

ggtgtacttg ccctgaaact tgtcttcatg ctccacaatg atggaaatat attcatttgg 1440

catgttgtgt atttcatgaa agacggacat tggtaggatg agcagtggtg ggcgtgatcc 1500

agccaagaag aagggcgagt cacgatactg cgctgtttag agacaagtga gtctggaaat 1560

catatgactt acctgctgca tccccttgat gaggttcttc tccaacgatt gtgcgtgggt 1620

atcgcctact acagggtatg gcaaggtcgg tttactcgaa gactgcttgt agatccacca 1680

agagaatgct aagagtcctg tagcgccgca aagggcggcg aacgccaccg cgatacgcaa 1740

aacccgtgag ccagtgagct ccagaggctc aatcaacgtc acaaaattac tcat 1794

<210> 27

<211> 16

<212> DNA

<213> primer (primer)

<400> 27

ggctcttcca acctcc 16

<210> 28

<211> 16

<212> DNA

<213> primer (primer)

<400> 28

gatggcttcc ctttcc 16

<210> 29

<211> 16

<212> DNA

<213> primer (primer)

<400> 29

ggctcttcca acctcc 16

<210> 30

<211> 16

<212> DNA

<213> primer (primer)

<400> 30

ccgctatttc actccc 16

<210> 31

<211> 16

<212> DNA

<213> primer (primer)

<400> 31

aggcccagta cgaaag 16

<210> 32

<211> 17

<212> DNA

<213> primer (primer)

<400> 32

tgaacagtgg tgaaccc 17

<210> 33

<211> 16

<212> DNA

<213> primer (primer)

<400> 33

atgccgctaa tggacg 16

<210> 34

<211> 16

<212> DNA

<213> primer (primer)

<400> 34

ttcgcacctg gaagat 16

<210> 35

<211> 18

<212> DNA

<213> primer (primer)

<400> 35

gtcaaagtgc ccgtcgta 18

<210> 36

<211> 16

<212> DNA

<213> primer (primer)

<400> 36

gtcgcagcat ccctca 16

<210> 37

<211> 16

<212> DNA

<213> primer (primer)

<400> 37

tcccatcagg cactcg 16

<210> 38

<211> 16

<212> DNA

<213> primer (primer)

<400> 38

cgcttcctaa cgcaca 16

<210> 39

<211> 17

<212> DNA

<213> primer (primer)

<400> 39

ctgcgacgaa cagataa 17

<210> 40

<211> 18

<212> DNA

<213> primer (primer)

<400> 40

cctacccaag aacaaact 18

<210> 41

<211> 20

<212> DNA

<213> primer (primer)

<400> 41

gtgagtttgt tcttgggtag 20

<210> 42

<211> 16

<212> DNA

<213> primer (primer)

<400> 42

tggtgacggg agcagt 16

<210> 43

<211> 16

<212> DNA

<213> primer (primer)

<400> 43

tgacggcgta ggtaga 16

<210> 44

<211> 15

<212> DNA

<213> primer (primer)

<400> 44

acaggttcgc aataa 15

<210> 45

<211> 16

<212> DNA

<213> primer (primer)

<400> 45

agggctaggc aaagga 16

<210> 46

<211> 16

<212> DNA

<213> primer (primer)

<400> 46

gtcgccaccc agagta 16

<210> 47

<211> 16

<212> DNA

<213> primer (primer)

<400> 47

gtcaatagcg tcgtgg 16

<210> 48

<211> 17

<212> DNA

<213> primer (primer)

<400> 48

tgagaccttt catccct 17

<210> 49

<211> 20

<212> DNA

<213> primer (primer)

<400> 49

tctccaaagt tgagcgacac 20

<210> 50

<211> 16

<212> DNA

<213> primer (primer)

<400> 50

tgccacttgc gaaccc 16

<210> 51

<211> 16

<212> DNA

<213> primer (primer)

<400> 51

cggttcctta tctcgc 16

<210> 52

<211> 16

<212> DNA

<213> primer (primer)

<400> 52

atggcattag cagtcc 16

<210> 53

<211> 16

<212> DNA

<213> primer (primer)

<400> 53

cttcggtcca gcacaa 16

<210> 54

<211> 16

<212> DNA

<213> primer (primer)

<400> 54

tccagatcct cgcaaa 16

<210> 55

<211> 18

<212> DNA

<213> primer (primer)

<400> 55

actatgttcc agaccctc 18

<210> 56

<211> 16

<212> DNA

<213> primer (primer)

<400> 56

atccctcttc gtgcta 16

<210> 57

<211> 17

<212> DNA

<213> primer (primer)

<400> 57

gattcgctga cattagg 17

<210> 58

<211> 16

<212> DNA

<213> primer (primer)

<400> 58

gtcggatgca acaaca 16

<210> 59

<211> 18

<212> DNA

<213> primer (primer)

<400> 59

cgctgacatt aggcagtt 18

<210> 60

<211> 16

<212> DNA

<213> primer (primer)

<400> 60

cgcaatacca cccaca 16

<210> 61

<211> 18

<212> DNA

<213> primer (primer)

<400> 61

agggagtgaa gtagtgaa 18

<210> 62

<211> 16

<212> DNA

<213> primer (primer)

<400> 62

ctatccaagt ggcaaa 16

<210> 63

<211> 16

<212> DNA

<213> primer (primer)

<400> 63

cctggtgttg tgaccc 16

<210> 64

<211> 16

<212> DNA

<213> primer (primer)

<400> 64

ccgttgagaa ccgaga 16

<210> 65

<211> 16

<212> DNA

<213> primer (primer)

<400> 65

cggcatcagg tccaaa 16

<210> 66

<211> 16

<212> DNA

<213> primer (primer)

<400> 66

gtggcagagc gtttgt 16

<210> 67

<211> 16

<212> DNA

<213> primer (primer)

<400> 67

aatagtggcg ggaatc 16

<210> 68

<211> 16

<212> DNA

<213> primer (primer)

<400> 68

ctggctcacg ggtttt 16

<210> 69

<211> 19

<212> DNA

<213> primer (primer)

<400> 69

cttcctgcga tgtttctcc 19

<210> 70

<211> 26

<212> DNA

<213> primer (primer)

<400> 70

aattggccat tggtattata tatcta 26

Claims (4)

1. A rapid detection method for generating gibberellin seed in rice bakanae disease germ, wherein the rice bakanae disease germ generating gibberellin seed is fusarium graminearumFusarium fujikuroi) Fusarium herb layerF. proliferatum) Fusarium speciesF. verticillioides) AndF. andiyaziis characterized in that aiming at fusarium graminearum, fusarium layering and fusarium verticillatumF. andiyaziIs of (1)Designing a specific primer by using the different gene difference, carrying out multiplex PCR (polymerase chain reaction) amplification by taking DNA of a sample to be detected as a template through the specific primer, judging that gibberellin miniascopetals are generated in bakanae disease of rice through an amplification result, and judging that the sample is fusarium graminearum when the PCR amplification product shows two bands of 1327bp and 848 bp; when the PCR product presents a 1327bp single band, judging that the sample is fusarium layering; when the PCR product presents 578bp single band, judging that the sample is fusarium verticillium; when the PCR product has no band, the sample is judged to beF. andiyazi；

Fusarium vine bin, fusarium layering, fusarium verticillatum and Fusarium verticilliumF. andiyaziThe specific genes of (a) are: gibberellin synthesis regulation as shown in SEQ ID NO.1GA20oxGene, gibberellin biosynthesis as shown in SEQ ID No.25CPS/ KSGenes and calmodulin genes;

the specific primer comprises: gibberellin synthesis regulation of nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3GA20oxA gene specific primer; gibberellin biosynthesis with the nucleotide sequences shown as SEQ ID No.59 and SEQ ID No.60CPS/KSA gene specific primer; calmodulin gene specific primer with the nucleotide sequence shown as SEQ ID No.69 and SEQ ID No. 70.

2. The rapid detection method for gibberellin miniascapes produced in bakanae disease of rice as claimed in claim 1, wherein the multiplex PCR amplification conditions are as follows:

the multiplex PCR reaction system is as follows: 100ng of sample genome DNA, 10 mu M of three pairs of forward and reverse primers of 1 mu L each, three pairs of forward and reverse primersGA20oxA gene specific primer,CPS/KSGene-specific primer and calmodulin gene-specific primer, 2.5mM dNTPs 4. Mu.L, 10 XPCR buffer 2. Mu.L, 5U/. Mu.L TaqPolymerase 0.4. Mu.L, add ddH ₂ O makes up 20. Mu.L;

the PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, elongation at 72℃for 80s,35 cycles; finally, the extension is carried out for 10min at 72 ℃.

3. Gibberellin miniascapes produced in bakanae disease of riceA rapid detection method is provided, wherein the rice bakanae germ for producing gibberellin fingerling is fusarium graminearumFusarium fujikuroi) Fusarium herb layerF. proliferatum) Fusarium speciesF. verticillioides) AndF. andiyazithe method is characterized by comprising the following steps of:

1) Extracting whole genome DNA of a sample to be detected;

2) Multiplex PCR amplification is carried out by taking DNA of a sample to be detected as a template:

the multiplex PCR reaction system is as follows: 100ng of sample genome DNA, 10 mu M of three pairs of forward and reverse primers of 1 mu L each, three pairs of forward and reverse primersGA20oxA gene specific primer,CPS/KSGene-specific primer and calmodulin gene-specific primer, 2.5mM dNTPs 4. Mu.L, 10 XPCR buffer 2. Mu.L, 5U/. Mu.L TaqPolymerase 0.4. Mu.L, add ddH ₂ O makes up 20. Mu.L;

the PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 54℃for 40s, elongation at 72℃for 80s,35 cycles; finally, the mixture is extended for 10min at 72 ℃;

the saidGA20oxThe specific primers of the genes are shown as SEQ ID NO.2 and SEQ ID NO.3,CPS/KSThe specific primers of the gene are shown as SEQ ID NO.59 and SEQ ID NO.60, and the specific primers of the calmodulin gene are shown as SEQ ID NO.69 and SEQ ID NO. 70;

3) Judgment by amplification results

Separating the amplified product by 1.5% agarose gel electrophoresis, staining the separated product under an ultraviolet lamp by ethidium bromide according to the size judgment result of the amplified product, and judging that the sample is fusarium graminearum when the PCR amplified product presents two bands of 1327bp and 848 bp; when the PCR product presents a 1327bp single band, judging that the sample is fusarium layering; when the PCR product presents 578bp single band, judging that the sample is fusarium verticillium; when the PCR product has no band, the sample is judged to beF. andiyazi。

4.GA20oxA gene specific primer,CPS/KSGene specific primer and calmodulin gene specific primer and application thereof in rapid detection of gibberellin-producing minispecies in rice bakanae disease germ, wherein gibberellin production is generatedThe bakanae disease germ of the rice of the mycin race is fusarium graminearumFusarium fujikuroi) Fusarium herb layerF. proliferatum) Fusarium speciesF. verticillioides) AndF. andiyazithe saidGA20oxThe specific primers of the genes are shown as SEQ ID NO.2 and SEQ ID NO.3,CPS/KSThe gene specific primers are shown as SEQ ID NO.59 and SEQ ID NO.60, and the calmodulin gene specific primers are shown as SEQ ID NO.69 and SEQ ID NO. 70.

Images