三种重要水果褐腐病菌快速检测试纸的研制
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摘要
以水果上3种重要的褐腐病菌,即美澳型核果褐腐菌(Monilinia fructicola)、核果褐腐菌(M. laxa)和仁果褐腐菌(M.fructigena)为检测对象,研究开发适用于口岸水果检疫的快速检测试纸。以硝酸纤维素膜为固相载体,以病菌翻译延长因子基因(Translation elongation factor 1-alpha,EF-1α)为检测靶标,生物素标记的PCR扩增产物为检测标记物,采用DNA-DNA杂交方式对3种褐腐病菌进行试纸法快速检测。分别以褐腐病菌DNA和本研究构建的EF-1α重组质粒DNA为阳性标准品检验试纸的特异性和灵敏度。试验结果表明,本研究开发的检测试纸能够同时特异地检出以上3种褐腐病菌,整个检测流程(包括DNA提取和扩增过程)在2 h内即可完成,最低检测限达到10 fg/μL。其特异性强、灵敏度高、稳定性好,且检测结果可肉眼判定,无需依赖昂贵设备,便于向基层检疫实验室推广使用。
A rapid detection dipstick for fruit quarantine in port was studied and developed using three important brown rot pathogens(Monilinia fructicola,Monilinia laxa and Monilinia fructigena)on fruits. Using nitrocellulose membrane as the solid phase carrier,the translation elongation factor 1-alpha(EF-1α)of above 3 Monilinia species as detecting targets,and PCR amplification products of biotin marker as detecting markers,DNA-DNA hybridization was applied in the rapid detection of 3 Monilinia species with the developed dipstick.The specificity and sensitivity of the Monilinia dipstick(abbr. as MON dipstick below)were evaluated by using DNA of brown rot strains and the constructed recombinant plasmids of EF-1α as positive standard samples,respectively. The results showed that the MON dipstick specifically detected 3 Monilinia species;the whole detection process(including DNA extraction and amplification process)was completed in 2 h,and the lowest detection limit reached 10 fg/μL. In sum,the dipstick developed in this study is of high specificity,sensitivity and stability,and the detection result is visible by human eyes,thus it does not rely on expensive equipment and can be expanded and used in local quarantine laboratory.
A rapid detection dipstick for fruit quarantine in port was studied and developed using three important brown rot pathogens(Monilinia fructicola,Monilinia laxa and Monilinia fructigena)on fruits. Using nitrocellulose membrane as the solid phase carrier,the translation elongation factor 1-alpha(EF-1α)of above 3 Monilinia species as detecting targets,and PCR amplification products of biotin marker as detecting markers,DNA-DNA hybridization was applied in the rapid detection of 3 Monilinia species with the developed dipstick.The specificity and sensitivity of the Monilinia dipstick(abbr. as MON dipstick below)were evaluated by using DNA of brown rot strains and the constructed recombinant plasmids of EF-1α as positive standard samples,respectively. The results showed that the MON dipstick specifically detected 3 Monilinia species;the whole detection process(including DNA extraction and amplification process)was completed in 2 h,and the lowest detection limit reached 10 fg/μL. In sum,the dipstick developed in this study is of high specificity,sensitivity and stability,and the detection result is visible by human eyes,thus it does not rely on expensive equipment and can be expanded and used in local quarantine laboratory.
引文
[1] Byrde RJW, Willetts HJ. The brown rot fungi of fruit:Their biology and control[M]. Oxford:Pergamon Press, 1977:1-171.
[2] Berrie AM, Holb I. Brown rot diseases//Compendium of apple and pear diseases and pests[M]. St. Paul:APS Press, 1990:43-45.
[3] Batra LR. World species of Monilinia(fungi):Their ecology,biosystematics and control[J]. Mycologia Memoir, 1991, 16:1-246.
[4] Ogawa JM, Zehr EI, Bird GW, et al. Brown rot//Compendium of stone fruit diseases[M]. St. Paul:APS Press, 1995:7-10.
[5] Hrusti?J, et al. Genus Monilinia on pome and stone fruit species[J]. Pesticidi I Fitomedicina, 2012, 27(4):283-297.
[6]中华人民共和国农业部和国家质量监督检验检疫总局.中华人民共和国进境植物检疫性有害生物名录.农业部第862号公告[S].北京:2007.
[7] Sonoda RM, Ogawa JM, Manji BT. Use of interactions of cultures to distinguish Monilinia laxa from M. fructicola[J]. Plant Dis, 1982,66(1):325-326.
[8] van Leeuwen GCM, van Kesteren HA. Delineation of the three brown rot fungi of fruit crops(Monilinia spp.)on the basis of quantitative characteristics[J]. Can J Bot, 1998, 76(12):2042-2050.
[9] Lane CR. A synoptic key for differentiation of Monilinia fructicola,M. fructigena and M. laxa, based on examination of cultural characters[J]. EPPO Bulletin, 2002, 32(3):489-493.
[10] Fulton CE, Brown AE. Use of SSU rDNA group-I intron to distinguish Monilinia fructicola from M. laxa and M.fructigena[J]. FEMS Microbiol Lett, 1997, 157(2):307-312.
[11] Snyder CL, Jones AL. Genetic variation between strains of Monilinia fructicola and Monilinia laxa isolated from cherries in Michigan[J]. Can J Plant Pathol, 1999. 21(1):70-77.
[12] F?rster H, Adaskaveg JE. Early brown rot infections in sweet cherry fruit are detected by Monilinia-specific DNA primers[J].Phytopathology, 2000, 90(2):171-178.
[13] Hughes KJD, Fulton CE, McReynolds D, et al. Development of new PCR primers for identification of Monilinia species[J]. EPPO Bulletin, 2000, 30(3-4):507-511.
[14] Ioos R, Frey P. Genomic variation within Monilinia laxa,M. fructigena and M. fructicola, and application to species identification by PCR[J]. Eur J Plant Pathol, 2000, 106(4):373-378.
[15] Boehm EWA, Ma Z, Michailides TJ. Species-specific detection of Monilinia fructicola from California stone fruits and flowers[J].Phytopathology, 2001, 91(5):428-439.
[16] Gell I, Cubero J, Melgarejo P. Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp.in stone fruit trees[J]. J Appl Microbiol, 2007, 6:2629-2637.
[17] Miessner S, Stammler G. Monilinia laxa, M. fructigena and M.frutticola:Risk estimation of resistance to Qol fungicides and identification of species with cytochrome b gene sequence[J]. J Plant Dis Protect, 2010, 117(4):162-167.
[18] Hily JM, Singer SD, Villani SM, et al. Characterization of the cytochrome b(cyt b)gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance[J]. Pest Manage Sci, 2011, 67(4):385-396.
[19] Ma Z, Luo Y, Michailides TJ. Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California[J]. J Phytopathol, 2003,151(6):312-322.
[20] C?téMJ, Tardif MC, Meldrum AJ. Identification of Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma on inoculated and naturally infected fruit using multiplex PCR[J].Plant Dis, 2004, 88(11):1219-1225.
[21] Hu MJ, et al. Monilinia species causing brown rot of peach in China[J]. PLoS One, 2011, 6(9):e24990.
[22] Luo Y, Ma Z, Reyes HC, et al. Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using realtime PCR[J]. Eur J Plant Pathol, 2007, 118(2):145-154.
[23]程颖慧,王颖,章桂明,等.应用TaqMan MGB探针检测美澳型核果褐腐病菌[J].广东农业科学, 2009(7):196-198.
[24] van Brouwershaven IR, et al. A real-time(TaqMan)PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops[J]. Plant Pathol, 2010, 59(3):548-555.
[25] Guinet C, et al. One-step detection of Monilinia fructicola, M.fructigena, and M. laxa on Prunus and Malus by a multiplex realtime PCR assay[J]. Plant Dis, 2016, 12:2465-2474.
[26] Garcia-Benitez C, Melgarejo P, de Cal A. Detection of latent Monilinia infections in nectarine flowers and fruit by qPCR[J].Plant Dis, 2017, 101(6):1002-1008.
[27] Papavasileiou A, Madesis PB, Karaoglanidis GS. Identification and differentiation of Monilinia species causing brown rot of pome and stone fruit using High-Resolution Melting(HRM)analysis[J].Phytopathology, 2016, 106(9):1055-1064.
[28] Vilanova L, Usall J, TeixidóN, et al. Assessment of viable conidia of Monilinia fructicola in flower and stone fruit combining propidium monoazide(PMA)and qPCR[J]. Plant Pathol,2017, 66(8):1276-1287.
[29] EPPO. EPPO Standard PM 7/18(2)Diagnostic protocols for regulated pests Monilinia fructicola[J]. EPPO Bulletin, 2009,39, 337-343.
[30] Riccioni L, Valente MT. Comparison of different PCR tests to identify Monilinia fructicola[J]. EPPO Bulletin, 2015, 1:33-40.
[31] OndejkováN, HudecováM, BacigálováK. First report on Monilinia fructicola in the Slovak Republic[J]. Plant Protection Science,2010, 46(4):181-184.
[2] Berrie AM, Holb I. Brown rot diseases//Compendium of apple and pear diseases and pests[M]. St. Paul:APS Press, 1990:43-45.
[3] Batra LR. World species of Monilinia(fungi):Their ecology,biosystematics and control[J]. Mycologia Memoir, 1991, 16:1-246.
[4] Ogawa JM, Zehr EI, Bird GW, et al. Brown rot//Compendium of stone fruit diseases[M]. St. Paul:APS Press, 1995:7-10.
[5] Hrusti?J, et al. Genus Monilinia on pome and stone fruit species[J]. Pesticidi I Fitomedicina, 2012, 27(4):283-297.
[6]中华人民共和国农业部和国家质量监督检验检疫总局.中华人民共和国进境植物检疫性有害生物名录.农业部第862号公告[S].北京:2007.
[7] Sonoda RM, Ogawa JM, Manji BT. Use of interactions of cultures to distinguish Monilinia laxa from M. fructicola[J]. Plant Dis, 1982,66(1):325-326.
[8] van Leeuwen GCM, van Kesteren HA. Delineation of the three brown rot fungi of fruit crops(Monilinia spp.)on the basis of quantitative characteristics[J]. Can J Bot, 1998, 76(12):2042-2050.
[9] Lane CR. A synoptic key for differentiation of Monilinia fructicola,M. fructigena and M. laxa, based on examination of cultural characters[J]. EPPO Bulletin, 2002, 32(3):489-493.
[10] Fulton CE, Brown AE. Use of SSU rDNA group-I intron to distinguish Monilinia fructicola from M. laxa and M.fructigena[J]. FEMS Microbiol Lett, 1997, 157(2):307-312.
[11] Snyder CL, Jones AL. Genetic variation between strains of Monilinia fructicola and Monilinia laxa isolated from cherries in Michigan[J]. Can J Plant Pathol, 1999. 21(1):70-77.
[12] F?rster H, Adaskaveg JE. Early brown rot infections in sweet cherry fruit are detected by Monilinia-specific DNA primers[J].Phytopathology, 2000, 90(2):171-178.
[13] Hughes KJD, Fulton CE, McReynolds D, et al. Development of new PCR primers for identification of Monilinia species[J]. EPPO Bulletin, 2000, 30(3-4):507-511.
[14] Ioos R, Frey P. Genomic variation within Monilinia laxa,M. fructigena and M. fructicola, and application to species identification by PCR[J]. Eur J Plant Pathol, 2000, 106(4):373-378.
[15] Boehm EWA, Ma Z, Michailides TJ. Species-specific detection of Monilinia fructicola from California stone fruits and flowers[J].Phytopathology, 2001, 91(5):428-439.
[16] Gell I, Cubero J, Melgarejo P. Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp.in stone fruit trees[J]. J Appl Microbiol, 2007, 6:2629-2637.
[17] Miessner S, Stammler G. Monilinia laxa, M. fructigena and M.frutticola:Risk estimation of resistance to Qol fungicides and identification of species with cytochrome b gene sequence[J]. J Plant Dis Protect, 2010, 117(4):162-167.
[18] Hily JM, Singer SD, Villani SM, et al. Characterization of the cytochrome b(cyt b)gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance[J]. Pest Manage Sci, 2011, 67(4):385-396.
[19] Ma Z, Luo Y, Michailides TJ. Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California[J]. J Phytopathol, 2003,151(6):312-322.
[20] C?téMJ, Tardif MC, Meldrum AJ. Identification of Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma on inoculated and naturally infected fruit using multiplex PCR[J].Plant Dis, 2004, 88(11):1219-1225.
[21] Hu MJ, et al. Monilinia species causing brown rot of peach in China[J]. PLoS One, 2011, 6(9):e24990.
[22] Luo Y, Ma Z, Reyes HC, et al. Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using realtime PCR[J]. Eur J Plant Pathol, 2007, 118(2):145-154.
[23]程颖慧,王颖,章桂明,等.应用TaqMan MGB探针检测美澳型核果褐腐病菌[J].广东农业科学, 2009(7):196-198.
[24] van Brouwershaven IR, et al. A real-time(TaqMan)PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops[J]. Plant Pathol, 2010, 59(3):548-555.
[25] Guinet C, et al. One-step detection of Monilinia fructicola, M.fructigena, and M. laxa on Prunus and Malus by a multiplex realtime PCR assay[J]. Plant Dis, 2016, 12:2465-2474.
[26] Garcia-Benitez C, Melgarejo P, de Cal A. Detection of latent Monilinia infections in nectarine flowers and fruit by qPCR[J].Plant Dis, 2017, 101(6):1002-1008.
[27] Papavasileiou A, Madesis PB, Karaoglanidis GS. Identification and differentiation of Monilinia species causing brown rot of pome and stone fruit using High-Resolution Melting(HRM)analysis[J].Phytopathology, 2016, 106(9):1055-1064.
[28] Vilanova L, Usall J, TeixidóN, et al. Assessment of viable conidia of Monilinia fructicola in flower and stone fruit combining propidium monoazide(PMA)and qPCR[J]. Plant Pathol,2017, 66(8):1276-1287.
[29] EPPO. EPPO Standard PM 7/18(2)Diagnostic protocols for regulated pests Monilinia fructicola[J]. EPPO Bulletin, 2009,39, 337-343.
[30] Riccioni L, Valente MT. Comparison of different PCR tests to identify Monilinia fructicola[J]. EPPO Bulletin, 2015, 1:33-40.
[31] OndejkováN, HudecováM, BacigálováK. First report on Monilinia fructicola in the Slovak Republic[J]. Plant Protection Science,2010, 46(4):181-184.