Strawberry

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Establishment and Application of Strawberry Molecular Fingerprinting

SONG Hui, SHEN Lan, ZHANG Xiang-qin

2015, 30(S1): 196-199. https://doi.org/10.7668/hbnxb.2015.S1.035

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To investigate the strawberry cultivars by molecular markers,Hongjia,Zhangji,Ningxin(Xiangmei),Aerbi and Mengxiang were used to establish the molecular fingerprinting and cluster analysis by RAPD markers.Four markers were screened from 112 RAPD,which generated five polymorphic loci.These RAPD markers showed the polymorphism ratio of tested strawberry varieties were 3.6%.Hongjia,Aerbi and Mengxiang could be verified,the fingerprinting of Ningyu and Xiangjiao could be established and the variation of Mengxiang population could be detected by fingerprinting markers.Finally,the applicability of strawberry molecular fingerprinting and the certain instructive effect on cultivars spreading of cluster analysis were also discussed.

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Stability Assay of Three DNA Extraction Methods for Detection of Strawberry Vein Banding Virus

RAN Ce, CHEN Liu, LU Jia-lan, LIU Zheng-ping, WEI Yan-min, ZHAO Xiao-yan, SHANG Qiao-xia

2015, 30(3): 200-204. https://doi.org/10.7668/hbnxb.2015.03.034

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Different DNA isolation methods were used in researches due to the diversity of secondary metabolites in different plant tissues.The existing of large amounts of polysaccharides,proteins,polyphenols and pigments in strawberry plants influenced the extraction of high quality DNA from strawberry leaves and afterwards PCR reaction.In order to identify the DNA extraction method which was applied to the PCR detection for Strawberry vein banding virus (SVBV),total DNAs extracted from strawberry leaves by three different extraction methods to detect SVBV by PCR were tested and compared in this study.Strawberry leaves infected by SVBV were used as the materials from which CTAB kit method,high salt and low pH method (HSLPM) and modified SDS method were used to extract DNA in this paper.The quality and quantity of DNA extracted by these three different methods were analyzed by gel electrophoresis and UV spectrophotometers.SVBV was detected by PCR from extracted DNA to determine the effect of different DNA extraction methods on the stability of PCR detection.The results showed that all the three methods could isolate complete DNA fragments.Sequencing and sequence analysis of the PCR products from strawberry leaves infected by SVBV proved the reliability of the methods.DNA extracted by three methods from strawberry leaves could be detected the infection of SVBV showed the quality of DNA extracted by all the three methods could meet the specific demands of PCR detection of SVBV in strawberry leaves.Compared with the other two extraction methods,results showed the quality of DNA extracted by CTAB kit was the best in these three different DNA extraction methods in this paper.

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Cloning and Sequence Analysis of a DNA Encoding Ethylene ReportorErs1 from Starwberry

LIU Si-si, MA Jun-lian, TANG Xia, ZHANG Zi-de, SONG Chun-li

2011, 26(5): 46-49. https://doi.org/10.7668/hbnxb.2011.05.010

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In this study a ripening related ethvlene recepor Ers1 gene in strawberry fruits was cloned for the following-up reseach on gene and improving varieties of strawberry through transgenic technology.A pair of primers were designed and synthesized based on the sequence reported of Chandler-Ers1.Using genome DNA extracted from All-star strawberry mature fruits as template the specific PCR product of Ers1 was obtained,then it was ligated to pEGM-T easy vector and sequenced.The sequencing data showed that the PCR product was 667 bp encoding 222 peedicted amino acid residues.Comparison with the cDNA sequence from Chandler-Ers1 indicated the homlogy was 98%,and amino acid identitise were 97%.

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Isolation and Express Analysis of FaCO-2, a CO Homologue from Strawberry

LIU Yue-xue, ZOU Dong-mei, LI He, ZHANG Zhi-hong, MA Yue, DAI Hong-yan

2012, 27(4): 1-6. https://doi.org/10.3969/j.issn.1000-7091.2012.04.001

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The CO homologue, FaCO-2,was isolated from strawberry （Fragaria x ananassa cv. Huaji） with the method of PCR and RT-PCR. Its express pattern in different tissues and organs was checked with qRT-PCR. The full length of its DNA sequence is 1 882 bp and its CDS is 1 146 bp in length. FaCO-2 codes a predicted protein of 382 amino acids. The amino acid identity compared with other CO homologues is highly conserved. The protein molecular weight was 42 021.69 Da with an isoelectric point （PI） of 5.49. The real-time RT-PCR result showed that FaCO-2 mainly transcripts differ in different tissues and floral organs. It was detected its highest expression in the expanded leaf. In the four flower organs, it was only detected in sepal, while no transcription was detected in other flower organs.

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Cloning, Sequence Analysis and Prokaryotic Expression of Coat Protein Gene of Strawberry Mild Yellow Edge Virus

YU Cui-mei, ZHANG Zhi-hong, LIU Yue-xue, MA Yue, LI He, DAI Hong-yan

2012, 27(1): 30-34. https://doi.org/10.3969/j.issn.1000-7091.2012.01.006

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The objective of this study is to clone and analyze the sequence characterization of strawberry mild yellow edge virus CP gene, then construct its prokaryotic expression vector and express its protein in Escherichia coli Rosetta-gami B(DE3). Virus detection primers was used to detect the strawberry plantlets infected with SMYEV by RT-PCR, and a pair of primers were designed according to the uploaded sequence of SMYEV CP gene in NCBI to isolate the gene of SMYEV isolated in Shenyang region(SY05). The CP gene of SY05 was subcloned into expression vector pGEX-6p-1 and then transferred into Escherichia coli Rosetta-gami B(DE3) pLysS, finally this CP protein was induced by IPTG to express. Sequence analysis indicates that the SMYEV CP gene from SY05 is 729 bp in length and encodes 242 amino acid residues. The nucleotide sequence of this CP gene shares 80. 1%-97. 4% homology with other CP genes of SMYEV isolated from different plants. The deduced protein shares 92. 1%-99. 6% with the other homologous genes, including a 98. 3% homology with that isolated from SY03. The CP gene was subcloned into the expression vector pGEX-6P-1 and transferred into prokaryotic cells. Induced with IPTG, a 52. 0 kDa induction expression band of fusion protein was found in SDS-PAGE detection. All the results showed that we have isolated the CP gene of SMYEV in SY05 and prokaryotic expression of this gene was realized. This research provides a foundation for the serology research of SMYEV detection in strawberry.

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High Density Genetic Map of Cultivated Strawberry by SLAF-seq

DONG Hui, YANG Li, LI Li, ZHANG Jianjun, FAN Jingfang, SONG Shijia, YANG Lei, LI Haishan

2020, 35(6): 52-57. https://doi.org/10.7668/hbnxb.20191341

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For the development of a large number of SNP for the construction of high quality genetic map of cultivated strawberry(Fragaria×ananassa,2n=8X=56),Hongxing×Benihoppe crossing populations were sequenced by SLAF-seq that yielded 565 919 066 reads, with Q30 value of 95.36% and GC content of 39.68%. 717 881 SLAFs and 2 136 939 SNPs were identified.Total 56 237 SNPs showed high quality for genetic map construction, of which 14 412 were successfully mapped to 28 linkage groups spanning a total of 4 022.16 cM with an average interval genetic distance of 0.28 cM. It was showed that the greater part of each individual was derived from the parents by evaluating the monomer source of the genetic map. The results of heat map showed that the sequence of most markers on each linkage group was consistent with the genome, the sequence of markers was correct, which could show that the map was of high quality.A high-density genetic map of cultivated strawberry by SLAF-seq could be laying a foundation for genetic research and molecular marker-assisted breeding of cultivated strawberry.

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Inhibition of Strawberry FaEtr2 Gene Expression by RNA Interfering

SONG Chun-li, MA Jun-lian, TANG Xia, ZHANG Zi-de, ZHAO Cong-zhi, LI Jing

2014, 29(1): 28-30. https://doi.org/10.7668/hbnxb.2014.01.006

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In order to delay the ripening and softening process of strawberry,RNA interference technology was used to inhibit strawberry ethylene receptor gene expression. Two pairs of primers containing restriction enzyme sites were designed and used to amplify sequenced plasmid. PCR products and the plasmid pBI121 were digested by the corresponding restricted enzymes respectively,and linked directionally. Then the shRNA expression vector can be designed. After transcription,the hairpin mRNA obtained due to the inverted-repeat DNA fragment. The constructed expression vector was transformed into Agrobacterium LBA4404 for the following-up genetic transformation research. Four strains of transgenic plants were detected by PCR and GUS histochemical staining.

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Effect of Fruit Water Content on Fruit Property of Strawberry

LIU Mingchi, KOJIMA Takayuki, TANAKA Munehiro, CHEN Hang

2002, 17(3): 114-117. https://doi.org/10.3321/j.issn:1000-7091.2002.03.023

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With the decreasing of fruit water content,the fresh size and titratable acidity decrease and fruit hardness,soluble solid-acid ratio,soluble sugar content and color indexes increased.The effect of fruit water content on sucrose,glucose and fructose was different.Fruit water content has a significant correlation with soluble solid content (r=-0171)and soluble solid-acid ratio(r=-0161).

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Study on Tissue Culture and Regeneration Plantlet from Strawberry Leaf

SUN Rui-fen, LI Tian-ran, LI Kun, DENG Xiang-lan, SHI Hui-qin, ZHANG Ying-li, JIA Li-min

2002, 17(4): 49-53. https://doi.org/10.3321/j.issn:1000-7091.2002.04.011

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The medium of regeneration plantlet from shoot tip of strawberry creeping stem supplemented with different hormones was worked out.The protocol of tissue culture and rapid propagation has been developed. The influences of hormone condition, genotypes on shoot regeneration from leaf disc of strawberry were examined.By further studying, the effects of genotypes and explants on shoot induction were also examined when basal medium was MS+ B₅ supplemented with 2.253 mg/L 6-BA and 1.752 mg/L IAA. An efficient and higher frequence method of direct shoot regeneration has been established. Meanwhile, a suitable receptor material of genetic transformation.

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Cloning and Expression Analysis of Auxin Response Gene FvIAA17 in Strawberry

SU Liyan

2019, 34(4): 46-52. https://doi.org/10.7668/hbnxb.201751591

Abstract (376) PDF (92) RichHTML

Aux/IAA protein plays an important role in regulating plant development as a transcriptional inhibitor. In order to identify the function of FvIAA17 gene in strawberry development, it was cloned by RT-PCR from strawberry. The sequence feature of FvIAA17 gene was analyzed by bioinformatics methods, and its expression patterns were analyzed by Real-time quantitative PCR(qPCR)in different tissues and under different phytohormone treatments. The results showed that FvIAA17 contained a 594 bp open reading frame and encoded 197 amino acid, with a molecular mass of 21.85 ku and an isoelectric point of 7.56. The amino acid sequence alignment showed that FvIAA17 had the typical structure domain and conservative basic sequence of Aux/IAA gene families. Promoter analysis revealed that the sequence of FvIAA17 contained the related response elements to auxin, ABA, MeJA and other stresses. Phylogenetic tree analysis indicated that FvIAA17 was most closely related to MdIAA1 from Malus domestica. FvIAA17 was an auxin response gene and was located in periplasmic space according to subcellular localization. Real-time PCR analysis showed FvIAA17 had specific expression differences in different organs, and it could be induced by exogenous IAA and ABA significantly, indicating that FvIAA17 might play an important role in auxin and abscisic acid signal transduction during strawberry development. This research provided a theoretical basis for further studying the function and molecular regulation mechanism of FvIAA17.

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Prediction of Antigens of the Coat Protein of the Strawberry mild yellow edge virus

DENG Zibing, MA Jianzhong, WANG Yonggang, YANG Jumei, WEI Yan, PAN Bo, LI Yinwu

2016, 31(S1): 106-111. https://doi.org/10.7668/hbnxb.2016.S1.019

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An epitope of antigen is a key part recognized and combined by antibodies. Epitope prediction is helpful to recombinant antigen design and rapid screening,and to preparation of high activity antigen. In this paper,the coat protein of the Strawberry mild yellow edge virus was analyzed and predicted for its secondary structure and physicochemical and biological properties by programs including DNAStar,ExPASy,TMHMM Sever2.0,and IEDB. The results showed that there was no transmembrane regions in the SMYEV coat protein. The average hydrophility of the coat protein was -0.252,which belonged to a soluble protein. There were 5 potential antigen epitopes,of which values of the average antigen index range from 0.026 5 to 0.077 3. Antigen activities of the above predicted epitopes,however,are still to be verified by experiments.

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Cloning and Sequence Analysis of a DNA Encoding Ethylene Receptor Etr2 from Chiyoda Strawberry

SONG Chun-li, MA Jun-lian, TANG Xia, ZHANG Zi-de, LIU Yong-ju

2008, 23(6): 46-49. https://doi.org/10.7668/hbnxb.2008.06.010

Abstract (322) PDF (320) RichHTML

In this study a ripening related ethylene receptor etr2 gene in strawberry fruits was cloned for the following-up research on gene funct ion and improving variet ies of strawberry through transgenic technology. A pair of primers were designed and synthesized based on the sequence reported of Chandler -Etr2. Using genome DNA extracted from Chinyoda strawberry mature fruits as template the specific PCR product of etr2 was obtained, then it was ligated to pGEM-T easy vector and sequenced. The sequencing data showed that the PCR product was 1 049 bp encoding 349 predicted amino acid residues. Comparison with the cDNA sequence from Chandler - etr2 indicated the homlogy was 99%, and amino acid identities were 98%.

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Expression Vector Construction of Strawberry FaEtr2 Gene

SONG Chun-li, ZHAO Cong-zhi, MA Jun-lian

2010, 25(6): 71-73. https://doi.org/10.7668/hbnxb.2010.06.013

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Two fragments of All-star strawberry FaEtr2 gene were cloned using All-star strawberry as materials.Sense and antisense expression vector were constructed for improving storage and transport varieties of strawberry through transgenic technology.According to the restriction enzyme sites of expression vector and cloning sequence of strawberry FaEtr2 gene, two pairs of primers containing restriction enzyme sites were designed and used to amplify sequenced plasmid.PCR products and the plasmid pBL221 were digested by the corresponding restricted enzymes respectively, and linked directionally.Then the sense and antisense expression vector can be designed.The constructed expression vector was transformed into Agrobacterium LBA4404 for the following-up research.

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Analysis of Quality Composition in a New Strawberry Cultivar Shimei 10

DONG Hui, YANG LI, YANG Lei, LI Li, ZHANG Jianjun, FAN Jingfang

2017, 32(S1): 125-129. https://doi.org/10.7668/hbnxb.2017.S1.022

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Took strawberry cultivar Shimei 10 and Daselect as material to analyze and assess the nutritional quality of Shimei 10.The main nutritional components of Shimei 10 and Daselect were investigated.The results showed that the content of ash, soluble solid and tss-acid ratio in Shimei 10 was higher than that in Daselect(P<0.05) with value of 0.68%±0.21%,7.53%±0.06%and 9.41±0.23,respectively;the content of ascorbic acid in both of them were high with content were about 50.00 mg/100g;the anthocyanin in Shimei 10 was 35.78 mg/100g,which was higher than Daselect(P<0.05).The fruit surface of Shimei 10 had a deep red color and the texture properties was better than Daselect(P<0.05) in hardness,elasticity and chewiness.Five aroma constituents were found in Shimei 10 were better than Daselect(P<0.05),which make it good smelling. In conclusion,this investigation indicated that Shimei 10 had good nutritional quality, which can be used as the fine processing varieties in production.

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Cloning，Bioinformatics and Expression Analysis of FvARF5 Gene in Strawberry

SU Liyan

2019, 34(3): 16-22. https://doi.org/10.7668/hbnxb.201751324

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Auxin response factor ARF is a new class of transcription factors. They play an important role during plant development as an activation or inhibition in auxin-related gene expression. In order to identify the function of FvARF5 gene during strawberry development, FvARF5 gene was cloned by RT-PCR from Fragaria vesca and analyzed by bioinformatics. Furthermore, Real-time quantitative PCR(qPCR) assay was performed to investigate the tissue-specific expression of FvARF5 gene, and its expression patterns under various phytohormone treatments. The results showed that the open reading frame of FvARF5 gene was 2 745 bp, encoding a protein of 914 amino acids. The molecular weight of FvARF5 protein was 100.65 ku, and the theoretical pI was 5.25. Multiple sequence alignment and phylogenetic tree analysis showed that FvARF5 was closely related to ARF5 from Rosa chinensis with 95% identity. FvARF5 was predicated to be the auxin response gene that was located in the nucleus. Promoter analysis revealed that the sequence of FvARF5 contained multiple hormone response elements. Real-time PCR analysis showed that FvARF5 had specific expression difference in different organs, and it had a higher expression in stem and green fruit. Also, FvARF5 had significant response to exogenous IAA and GA treatment. The results showed that the FvARF5 might play an important role in auxin and GA signal transduction during strawberry development. This research provided a theoretical basis for further study of the function and molecular regulation mechanism of FvARF5.

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Inheritance Analysis Research of Several Quantitive Characters of Strawberry Fruit

YANG Lei, YANG Li, LI Li, HAO Bao-chun

https://doi.org/10.7668/hbnxb.2009.S2.021

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181 seedlings of 8 strawberry cross combinations were used to study the inheritance trend of main fruit quantitive characters. The result indicated that weight of the first grade fruit, weight of the second fruit, the biggest fruit weight, the soluble solid content, fruit firmness, vitamine C were quantitative characters with succession variance. As concerning F_1, the first grade fruit weight, the second grade fruit weight, the biggest fruit weight, the soluble solid content and Vc content trended mainly to be smaller, hut fruit firmness trended mainly to be bigger.

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A New Method for Determination of the Contents of Nutrition in intact Strawberry-Near Infrared Spectrometry

Jin Tongming, Cui Hongchang

1994, 9(2): 120-123. https://doi.org/10.3321/j.issn:1000-7091.1994.02.024

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In this paper a fast non-destructive analytical method to measure various nutrients in the intact strawberry--Near infrared Spectrometry(NIRs)was introduced.Using this method the contents of six nutrients including sucrose,glucose, fructose,citric acid,malic acid and vitamin C were determined simultaneously.The multiple correlation coefficients between these values and those obtained on high performance liquid chromatography (HPLC) were 0 9920, 0.9914, 0.9888, 0.9954, 0.9949 and 0.9980,respectively.The standard’errors of calibration were 0.021, 0.035, 0.065,0.044, 0.029,0.727, respectively.These results showed that NIRs had a similiar accuracy to HPLC.However since it is faster and non-destructive,and dose not require any sample preparation and expensive chemicals, NIRs can be favourably introduced and applied to the nutrient analysis and quality determination of the intact strawberry.

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Cloning and Sequence Analysis of a DNA Encoding Ethylene Reportor Etr1 from Starwberry

WANG Li, MA Junlian, TANG Xia, ZHANG Zide, SONG Chunli

2010, 25(5): 68-71. https://doi.org/10.7668/hbnxb.2010.05.014

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In this study a ripening related ethylene recepor Etr1 gene in strawberry fruits was cloned for the following-up reseach on gene and improving varieties of strawberry through transgenic technology. A pair of primers were designed and synthesized based on the sequence reported of Chandler-Etr1. Using genome DNA extracted from All-star strawberry mature fruits as template the specific PCR product of Etr1 was obtained,then it was ligated to pEGM-T eas y vector and sequenced. The sequencing data showed that the PCR product was 617 bp encoding 205 predicted amino acid residues. Comparison with the cDNA sequence from Chandler-Etr1 indicated the homlogy was 98% ,and amino acid identitise were 97% .

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Variation and Correlation Analysis of Main Quantitative Characters Among Strawberry Germplasms

YANG Li, LI Li, SUN Li-min, YANG Lei, YANG Qiu-ye, LIU Tie-zheng, HAO Bao-chun

2005, 20(3): 22-24. https://doi.org/10.3321/j.issn:1000-7091.2005.03.006

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Variation condition, distribution rule and correlation of main quantitative character of 82 open ground strawberry varieties(or lines)were studied and analyzed. The results showed that: Fruit firmness, soluble solid content, mean single fruit weight, max single fruit weight and yield of per plant were normal distribution respectively. The variation of different character is differrent. Correlation analysis showed there were notable positive relationship between fruit firmness and mean single fruit weight, and definitely positive relationship between fruit firmness and yield of per plant, and definitely positive relationship between mean single fruit weight and max single fruit weight and yield of per plant, and definitely positive relationship between max single fruit weight and yield of per plant. The research provided basis for preselection of parent and hybrid offspring of strawberry breeding.

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Dynamic Regular Studies about Important Characters of Strawberry Fruit

YANG Lei, YANG Li, LI Li, LI Hai-shan, HAO Bao-chun

2010, 25(S1): 96-99. https://doi.org/10.7668/hbnxb.2010.S1.022

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In order to ascertain growth developmental rule of strawberry fruit and dynamic rule of the nutrition ingredient change,the experiment material are Daselect,shimei 4,shimei 5 and shimei 6. It was studied that fruit growth developmental rule and rule of the nutrition ingredient change from flower latter 3 days to fruit mature. The result showed that the fruit length and width continuously is steadily growing during the whole growth developmental process; The fruit weight and the fruit volume of different culvars fast grows after flowers latter 15 days; The sugar content assumes the rising tendency in the overall during the strawberry fruit growth process; The acid content starts to increase from flower latter 3 days,and achieves biggest to half red period,then begin to drop in mature period; vitamins C content continuously increases from the young fruit period to the mature period,but the vitamin C content achieves the maximum value on turing red period,maturing period drops a little.

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The Influence of the Salicylic Acid for Membrane Lipid Peroxidation under Water Stress on Strawberry Young Plants

LIU Yan, CHEN Guilin, LI Xiaoyan, LIU Jingxiu

2010, 25(5): 127-131. https://doi.org/10.7668/hbnxb.2010.05.026

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Effects of salicylic acid in plant drought resistance were investigated. We take strawberry young plants as the materials and use the pretreatment of different concentrations of salicylic acid,each processing strawberry leaves were determined plasmalemma peroxide index,protective enzymes activity and photosynthetic characteristics. under water stress,the reactive oxidative species(ROS) of strawberry leaves appears two different peaks. The antioxidant enzymes obvious increase after the first peak,and the membrane lipid peroxidation maintain lower level; with the stress time prolong,ROS accumulates again,and membrane lipid peroxidation increase immediately with it. Suitable concentration salicylic acid treatments promote the ROS earlier period accumulation and effectively stimulate antioxidant enzymes activity,and alleviate membrane lipid peroxidation damage,also leaves maintain high photosynthesis activity; but high concentration salicylic acid treatments promote membrane lipid peroxidation damage. salicylic acid promote oxidative burst and activate protective enzyme activity and alleviate membrane lipid peroxidation damage to improve plant water stress resistance.

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Effects of Ethyl Methyl Sulfonate on Physiology of Callus from Leaves of Strawberry

ZHOU Hou-cheng, LUO Jing, ZHAO Xia, WANG Yong-qing

2008, 23(3): 138-142. https://doi.org/10.7668/hbnxb.2008.03.033

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The calli of strawberry were used as materials to study the effects of Ethyl Methyl Sulfonate(EMS)on the activities of protecting enzyme and oxidizing enzyme,the contents of total phenol and MDA and the death rate of callus.The result showed that with the increase of EMS concentration and treatment time,the death rate of callus increased.With the increase of EMS concentration,the activities of SOD,CAT,PPO and POD increased at first,and decreased sharply afterwards.With the extension of treatment time,SOD activity decreased continuously,activities of CAT and PPO increased first and then decreased,POD activity decreased firstly and then increased,but decreased later. With the increase of EMS concentration and treating time,total phenol contents decreased firstly and then increased,while MDA contents increased continuously. During the culture of callus after EMS treatment,the activities of PPO and POD decreased sharply and then maintained a steady level,contents of total phenol and MDA decreased,and the contents of total phenol was positively correlated with the activities of PPO and POD.It was suggested that the better EMS treatment was 0.1%-0.2% and 1.0-1.5 h,and some measures should be taken for preventing callus browning in the first 12 h after EMS treatment.

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Effects of Different Nutrient Solutions Applied at Seedling Stage on Yield of Strawberry

Tan Xuewen

1996, 11(4): 123-125. https://doi.org/10.3321/j.issn:1000-7091.1996.04.025

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Effects of different nutrient solutions applied at seedling stage on yield of strawberry were studied in DFT Results showed that the early stage yield and total yield of strawberry were increased by the treatment of stopping nutrient solution to the strawberry seedlings after feeding three weeks.The solutions with the concenration of 10mS/cm was better for increasing the yield of strawberry than those with concentration of 0.5mS/cm.Results also indicated that the adaptability of different strawberry varieties to different solution treatments during the seedling stage was similar. Hydroponic culture was suitable for variety Chun Xiang.

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Establishment and Application of Visual LAMP Method for Rapid Detection of Sphaerotheca aphanis Causing Strawberry Powdery Mildew

BEN Haiyan, HAO Yongjuan, HUO Jianfei, YAO Yurong, GAO Wei, WANG Wanli

2022, 37(6): 182-190. https://doi.org/10.7668/hbnxb.20193522

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Strawberry powdery mildew caused by Sphaerotheca aphanis is one of the strawberry diseases with large incidence area and high incidence frequency in the protected cultivation areas, which usually occurs from seedling stage to fruiting stage. Therefore, the establishment of a rapid and efficient detection method of S. aphanis is quite important for the diagnosis and control of strawberry powdery mildew.A set of LAMP amplification primers including F3/B3、FIP/BIP and LF/LB were designed for the conserved sequence of ITS gene of Sphaerotheca aphanis as the target gene. Through the double judgment method of Real-time fluorescence curve and fluorescence color change, the main factors in the LAMP reaction system including the final concentrations of dNTPs, MgSO₄, BstDNA polymerase and betaine and amplification temperature were selected and optimized to increased etections ensitivity and specificity. Finally, the effects of field samples detection by optimized LAMP were evaluated.The results showed that the LAMP detection system of strawberry powdery mildew was established successfully. The optimized reaction conditions included dNTPs 1.6 mmol/L, MgSO₄ 8 mmol/L, BstDNA polymerase 320 U/moL and betaine 1.2 mmol/L and the amplification temperature was 65 ℃. Under such conditions, the minimum detection sensitivity was 3.2×10^-4 ng/μL within 60 min, and the efficiency of which was 100 times higher than the result of PCR amplification.The visual LAMP method could effectively detect the samples with no obvious symptoms infected by S. aphanis in the early stage. This method has the advantages of short detection time, direct observation with eyes, low requirements for operators and low detection cost. It has important guiding significance for the early diagnosis, monitoring, early prevention and determination of the best control time of strawberry powdery mildew.

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