vestnik-bio-msuВестник Московского университета. Серия 16. БиологияVestnik Moskovskogo universiteta. Seriya 16. Biologiya0137-0952Lomonosov Moscow State University, School of Biologyvestnik-bio-msu-709Research ArticleОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕRESEARCH ARTICLEПЦР-ИДЕНТИФИКАЦИЯ ПАТОГЕННЫХ АГРОБАКТЕРИЙ, ВЫЯВЛЕННЫХ НА ВИНОГРАДНИКАХ КРАСНОДАРСКОГО КРАЯ, ПО ТИПУ СОДЕРЖАЩИХСЯ В НИХ Ti-ПЛАЗМИДPCR IDENTIFICATION OF PATHOGENIC AGROBACTERIES DETECTED ON VINEYARDS OF KRASNODAR TERRITORY BY THE TYPE OF CONTAINED Ti PLASMIDSМакаркинаМ. В.MakarkinaM. V.

Макаркина Марина Викторовна – младший научный сотрудник лаборатории сортоизучения и селекции винограда

350901, г. Краснодар, ул. имени 40-летия Победы, д. 39

40 years of Victory street, 39, Krasnodar, 350901

konec_citatu@mail.ruИльницкаяЕ. T.IlnitskayaE. T.

Ильницкая Елена Тарасовна – кандидат биологических наук, заведующая лабораторией сортоизучения и селекции винограда

350901, г. Краснодар, ул. имени 40-летия Победы, д. 39

40 years of Victory street, 39, Krasnodar, 350901

ilnitskaya79@mail.ruТокмаковC. В.TokmakovS. V.

Токмаков Сергей Вячеславович – кандидат биологических наук, научный сотрудник лаборатории генетики и микробиологии

350901, г. Краснодар, ул. имени 40-летия Победы, д. 39

40 years of Victory street, 39, Krasnodar, 350901

ad-a-m@mail.ruСеверо-Кавказский федеральный научный центр садоводства, виноградарства, виноделияРоссияNorth-Caucasian federal scientific center of horticulture, viticulture, wine-makingRussian Federation2019190320197415058Copyright © Макаркина М.В., Ильницкая Е.T., Токмаков C.В., 20192019Макаркина М.В., Ильницкая Е.T., Токмаков C.В.Makarkina M.V., Ilnitskaya E.T., Tokmakov S.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestnik-bio-msu.elpub.ru/jour/article/view/709

Agrobacterium vitis и некоторые штаммы A. tumefaciens и A. rhizogenes вызывают опасное хроническое заболевание на виноградных растениях – бактериальный рак. С использованием молекулярно-генетического метода проведена работа по идентификации и изучению разнообразия патогенных агробактерий, вызывающих бактериальный рак на виноградниках Краснодарского края. В качестве материала использовали 76 образцов молодых и одревесневших опухолевидных наростов бактериального рака винограда, собранных с растений в четырнадцати виноградарских хозяйствах Краснодарского края и имеющих различное происхождение посадочного материала (Италия, Франция, Австрия, Сербия, Болгария, Россия). Во всех исследуемых образцах, по данным ДНК-анализа с помощью тест-системы PGF/PGR, идентифицировано присутствие A. vitis. При изучении разнообразия агробактерий по типу содержащихся в них Ti-плазмид определены 4 группы, содержащие Tiплазмиды: октопинового (59,5%), октопин/витопинового (13,5%), витопинового (13,5%) и нопалинового (13,5%) типов. На основе полученных данных сделан вывод, что на виноградниках Краснодарского края бактериальный рак винограда вызывается агробактериями A. vitis, преимущественно октопинового типа.

Agrobacterium vitis and some strains of A. tumefaciens and A. rhizogenes cause a dangerous chronic disease on grape plants – crown gall. Using molecular genetic methods, work has been carried out to identify and study the diversity of pathogenic agrobacteria that cause crown gall in the vineyards of the Krasnodar Territory. 76 samples of young and lignified tumor-like outgrowths of crown gall of grapes collected from plants in fourteen vineyards of the Krasnodar Territory and having a different origin of planting material (Italy, France, Austria, Serbia, Bulgaria, Russia) were used as material. In all studied samples, the presence of A. vitis was identified using the PGF/PGR test-system for DNA analysis. When studying the diversity of agrobacteria by the type of Ti plasmids contained therein, four groups were identified: containing octopine-type Ti plasmids (59.5%), octopine/vitopine (13.5%), vitopine (13.5%) and nopaline type (13.5%). The obtained data indicate that in the vineyards of the Krasnodar Territory crown gall of grapes is caused by agrobacteria A. vitis, mainly of the octopine type.

Agrobacterium vitisбактериальный рак виноградаполимеразная цепная реакцияTi-плазмидатипы опиновтест-системыAgrobacterium vitiscrown gall of grapepolymerase chain reactionTi plasmidopine-typestest-systemsРоссийский фонд фундаментальных исследований (проект № 16-34-00827 мол_а)ReferencesGelvin S.B. Agrobacterium-mediated plant transformation: The biology behind the “genejockeying” tool // Microbiol. Mol. Biol. Rev. 2003. Vol. 67. N 1. P. 16–37.Gelvin S.B. Agrobacterium-mediated plant transformation: The biology behind the “genejockeying” tool // Microbiol. Mol. Biol. Rev. 2003. Vol. 67. N 1. P. 16–37.Tarbah F.A., Goodman R.N. Rapid detection of Agrobacterium tumefaciens in grapevine propagating material and the basis for an efficient indexing system // Plant Dis. 1986. Vol. 70. P. 566–568Tarbah F.A., Goodman R.N. Rapid detection of Agrobacterium tumefaciens in grapevine propagating material and the basis for an efficient indexing system // Plant Dis. 1986. Vol. 70. P. 566–568Бурдинская В.Ф., Арестова Н.О. Латентная зараженность винограда бактериальным раком // Защ. карант. раст. 2010. № 10. С. 38– 39Бурдинская В.Ф., Арестова Н.О. Латентная зараженность винограда бактериальным раком // Защ. карант. раст. 2010. № 10. С. 38– 39Kerr A., Panagopoulos C.G. Biotypes of Agrobacterium radiobacter var. tumefaciens and their biological control // J. Phytopathol. 1977. Vol. 90. N 2. P. 172–179.Kerr A., Panagopoulos C.G. Biotypes of Agrobacterium radiobacter var. tumefaciens and their biological control // J. Phytopathol. 1977. Vol. 90. N 2. P. 172–179.Ophel K., Kerr A. Agrobacterium vitis sp. nov. for strains of Agrobacterium biovar 3 from grapevines // Int. J. Syst. Bacteriol. 1990. Vol. 40. N 3. P. 236–241.Ophel K., Kerr A. Agrobacterium vitis sp. nov. for strains of Agrobacterium biovar 3 from grapevines // Int. J. Syst. Bacteriol. 1990. Vol. 40. N 3. P. 236–241.Mousavi S.A, Osterman J, Wahlberg N., Nesme X., Lavire C., Vial L., Paulin L., de Lajudie P., Lindstrom K. Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov // Syst. Appl. Microbiol. 2014. Vol. 37. N 3. P. 208–215.Mousavi S.A, Osterman J, Wahlberg N., Nesme X., Lavire C., Vial L., Paulin L., de Lajudie P., Lindstrom K. Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov // Syst. Appl. Microbiol. 2014. Vol. 37. N 3. P. 208–215.Ramírez-Bahena M.H., Vial L., Lassalle F., Diel B., Chapulliot D., Daubin V., Nesme X., Muller D. Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid // Mol. Phylogenet. Evol. 2014. Vol. 73. P. 202– 207.Ramírez-Bahena M.H., Vial L., Lassalle F., Diel B., Chapulliot D., Daubin V., Nesme X., Muller D. Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid // Mol. Phylogenet. Evol. 2014. Vol. 73. P. 202– 207.Slater S.C., Goldman B.S., Goodner B. et al. Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria // J. Bacteriol. 2009. Vol. 191. N 8. P. 2501–2511.Slater S.C., Goldman B.S., Goodner B. et al. Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria // J. Bacteriol. 2009. Vol. 191. N 8. P. 2501–2511.Szegedi E. Opines in naturally infected crown gall tumors // Vitis. 2003. Vol. 42. N 1. P. 39–41.Szegedi E. Opines in naturally infected crown gall tumors // Vitis. 2003. Vol. 42. N 1. P. 39–41.Haas J.H, Moore L.W., Ream W., Manulis S. Universal PCR primers for detection of phytopathogenic Agrobacterium strains // Appl. Environ. Microbiol. 1995. Vol. 61. N 8. P. 2879– 2884.Haas J.H, Moore L.W., Ream W., Manulis S. Universal PCR primers for detection of phytopathogenic Agrobacterium strains // Appl. Environ. Microbiol. 1995. Vol. 61. N 8. P. 2879– 2884.Матвеева Т.В., Богомаз Д.И., Лутова Л.А. Способ диагностики биоматериалов на наличие в них агробактерий. Патент РФ на изобретение № 2458142. Дата публ.: 10.08.12. Бюл. №22/12.Матвеева Т.В., Богомаз Д.И., Лутова Л.А. Способ диагностики биоматериалов на наличие в них агробактерий. Патент РФ на изобретение № 2458142. Дата публ.: 10.08.12. Бюл. №22/12.Puławska J., Willems A., Sobiczewski P. Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR // Syst. Appl. Microbiol. 2006. Vol. 29. N. 6. P. 470–479.Puławska J., Willems A., Sobiczewski P. Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR // Syst. Appl. Microbiol. 2006. Vol. 29. N. 6. P. 470–479.Sawada H., Ieki H., Matsuda I. PCR detection of Ti and Ri plasmid from phytopathogenic Agrobacterium strains // Appl. Environ. Microbiol. 1995. Vol. 61. N 2. P. 828– 831.Sawada H., Ieki H., Matsuda I. PCR detection of Ti and Ri plasmid from phytopathogenic Agrobacterium strains // Appl. Environ. Microbiol. 1995. Vol. 61. N 2. P. 828– 831.Suzaki K., Yoshida K., Sawada H. Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers // J. Gen. Plant. Pathol. 2004. Vol. 70. N 6. P. 342–347.Suzaki K., Yoshida K., Sawada H. Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers // J. Gen. Plant. Pathol. 2004. Vol. 70. N 6. P. 342–347.Bini F, Kuczmog A., Putnoky P. Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens // Vitis. 2008. Vol. 47. N 3. P. 181–190.Bini F, Kuczmog A., Putnoky P. Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens // Vitis. 2008. Vol. 47. N 3. P. 181–190.Eastwell K.C., Kenneth C., Leslie Willis G., Cavileer D.T. A rapid and sensitive method to detect Agrobacterium vitis in grapevine cuttings using the polymerase chain reaction // Plant Dis. 1995. Vol. 79. N 8. P. 822–827.Eastwell K.C., Kenneth C., Leslie Willis G., Cavileer D.T. A rapid and sensitive method to detect Agrobacterium vitis in grapevine cuttings using the polymerase chain reaction // Plant Dis. 1995. Vol. 79. N 8. P. 822–827.Herlache T.C., Hotchkiss A.T., Burr T.J., Collmer A. Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum // Appl. Environ. Microbiol. 1997. Vol. 63. N 1. P. 338–346.Herlache T.C., Hotchkiss A.T., Burr T.J., Collmer A. Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum // Appl. Environ. Microbiol. 1997. Vol. 63. N 1. P. 338–346.Kawaguchi A., Sawada,H., Inoue, K., Nasu H. Multiplex PCR for the identification of Agrobacterium biovar 3 strains // J. Gen. Plant Pathol. 2005. Vol. 71. N 1. P. 54–59.Kawaguchi A., Sawada,H., Inoue, K., Nasu H. Multiplex PCR for the identification of Agrobacterium biovar 3 strains // J. Gen. Plant Pathol. 2005. Vol. 71. N 1. P. 54–59.Lim S.H., Kim J.G., Kang H.W. Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains // Microbiol. Res. 2009. Vol. 164. N. 4. P. 451–460.Lim S.H., Kim J.G., Kang H.W. Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains // Microbiol. Res. 2009. Vol. 164. N. 4. P. 451–460.Canaday J., Gerard J.C., Crouzet P., Otten L. Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4 // Mol. Gen. Genet. 1992. Vol. 235. N 2–3. P. 292–303.Canaday J., Gerard J.C., Crouzet P., Otten L. Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4 // Mol. Gen. Genet. 1992. Vol. 235. N 2–3. P. 292–303.Szegedi E., Bottka S. Detection of Agrobacterium vitis by polymerase chain reaction in grapevine bleeding sap after isolation on a semiselective medium // Vitis. 2002. Vol. 41. N 1. P. 37–42.Szegedi E., Bottka S. Detection of Agrobacterium vitis by polymerase chain reaction in grapevine bleeding sap after isolation on a semiselective medium // Vitis. 2002. Vol. 41. N 1. P. 37–42.Szegedi E., Bottka S., Mikulás J., Otten L., Sule S. Characterization of Agrobacterium tumefaciens strains isolated from grapevine // Vitis. 2005. Vol. 44. N 1. P. 49–54.Szegedi E., Bottka S., Mikulás J., Otten L., Sule S. Characterization of Agrobacterium tumefaciens strains isolated from grapevine // Vitis. 2005. Vol. 44. N 1. P. 49–54.Bini F., Geider K., Bazzi C. Detection of Agrobacterium vitis by PCR using novel virD2 gene-specific primers that discriminate two subgroups // Eur. J. Plant Pathol. 2008. Vol. 122. N 3. P. 403–411.Bini F., Geider K., Bazzi C. Detection of Agrobacterium vitis by PCR using novel virD2 gene-specific primers that discriminate two subgroups // Eur. J. Plant Pathol. 2008. Vol. 122. N 3. P. 403–411.Argun N., Momol M.T., Maden S., Momol E.A., Reid C.L., Celek H., Burr T.J. Characterization of Agrobacterium vitis strains isolated from Turkish grape cultivars in the Central Anatolia region // Plant Dis. 2002. Vol. 86. N 2. P. 162–166.Argun N., Momol M.T., Maden S., Momol E.A., Reid C.L., Celek H., Burr T.J. Characterization of Agrobacterium vitis strains isolated from Turkish grape cultivars in the Central Anatolia region // Plant Dis. 2002. Vol. 86. N 2. P. 162–166.Lamovšek J., Zidarič I., Pleško I.M., Urek G., Trdan S. Comparative study of diagnostic methods used for monitoring of common grape vine (Vitis vinifera L.) crown gall (Agrobacterium vitis Ophel & Kerr) in Slovenia // Acta Agric. Slov. 2015. Vol. 103. N 2. P. 313–321.Lamovšek J., Zidarič I., Pleško I.M., Urek G., Trdan S. Comparative study of diagnostic methods used for monitoring of common grape vine (Vitis vinifera L.) crown gall (Agrobacterium vitis Ophel & Kerr) in Slovenia // Acta Agric. Slov. 2015. Vol. 103. N 2. P. 313–321.Genov I., Atanassov I., Tsvetkov I., Atanassov A. Isolation and characterization of Agrobacterium strains from grapevines in Bulgarian vineyards and wild grapes, V. vinifera ssp. Silvestris // Vitis. 2015. Vol. 45. N 2. P. 97– 101.Genov I., Atanassov I., Tsvetkov I., Atanassov A. Isolation and characterization of Agrobacterium strains from grapevines in Bulgarian vineyards and wild grapes, V. vinifera ssp. Silvestris // Vitis. 2015. Vol. 45. N 2. P. 97– 101.Winans S.C., Allenza P., Stachel S.E., McBride K.E., Nester E.W. Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6 // Nucleic Acids Res. 1987. Vol. 15. N 2. P. 825–837.Winans S.C., Allenza P., Stachel S.E., McBride K.E., Nester E.W. Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6 // Nucleic Acids Res. 1987. Vol. 15. N 2. P. 825–837.Tolba I.H., Zaki M.F. Characterization of Agrobacterium vitis isolates obtained from galled grapevine plants in Egypt // Ann. Agric. Sci. 2011. Vol. 56. N 2. P. 113–119.Tolba I.H., Zaki M.F. Characterization of Agrobacterium vitis isolates obtained from galled grapevine plants in Egypt // Ann. Agric. Sci. 2011. Vol. 56. N 2. P. 113–119.Kuzmanović N., Gašić K., Ivanović M., Prokić A., Obradović A. Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia. Arch. Biol. Sci. 2012. Vol. 64. N 4. P. 1487–1494.Kuzmanović N., Gašić K., Ivanović M., Prokić A., Obradović A. Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia. Arch. Biol. Sci. 2012. Vol. 64. N 4. P. 1487–1494.Rouhrazi K., Rahimian H. Characterization of Iranian grapevine isolates of Rhizobium (Agrobacterium) spp. // J. Plant Pathol. 2012. Vol. 94. N 3. P. 555–560.Rouhrazi K., Rahimian H. Characterization of Iranian grapevine isolates of Rhizobium (Agrobacterium) spp. // J. Plant Pathol. 2012. Vol. 94. N 3. P. 555–560.Filo A., Sabbatini P., Sundin G.W., Zabadal T.J., Safe G.R., Cousins P.S. Grapevine crown gall suppression using biological control and genetic engineering: a review of recent research // Am. J. Enol. Vitic. 2013. Vol. 64. N. 1. DOI 10.5344/ajev.2012.12038.Filo A., Sabbatini P., Sundin G.W., Zabadal T.J., Safe G.R., Cousins P.S. Grapevine crown gall suppression using biological control and genetic engineering: a review of recent research // Am. J. Enol. Vitic. 2013. Vol. 64. N. 1. DOI 10.5344/ajev.2012.12038.Doyle J.J., Doyle J.L. A rapid DNA isolation procedure for small quantities of fresh tissue // Phytochem. Bull. 1987. Vol. 19. P. 11– 15.Doyle J.J., Doyle J.L. A rapid DNA isolation procedure for small quantities of fresh tissue // Phytochem. Bull. 1987. Vol. 19. P. 11– 15.Makarkina M.V., Ilnitskaya E.T., Stepanov I.V. Identification of Agrobacteria on grape plants with symptoms of crown gall lesions in ampeloсenosises of Krasnodar Territory using the PCR method // Russ. Agric. Sci. 2017. Vol. 43. N 5. P. 402–405.Makarkina M.V., Ilnitskaya E.T., Stepanov I.V. Identification of Agrobacteria on grape plants with symptoms of crown gall lesions in ampeloсenosises of Krasnodar Territory using the PCR method // Russ. Agric. Sci. 2017. Vol. 43. N 5. P. 402–405.Макаркина М.В., Ильницкая Е.Т. Определение латентной стадии бактериального рака винограда методом полимеразной цепной реакции // Научные труды Северо-Кавказского федерального научного центра садоводства, виноградарства, виноделия. Т. 13. Краснодар, 2017. С. 100–104.Макаркина М.В., Ильницкая Е.Т. Определение латентной стадии бактериального рака винограда методом полимеразной цепной реакции // Научные труды Северо-Кавказского федерального научного центра садоводства, виноградарства, виноделия. Т. 13. Краснодар, 2017. С. 100–104.Ignatov A.N., Khodykina M.V., Vinogradova S.V., Polityko V.A., Kornev K.P., Mazurin E.S. First report of Agrobacterium vitis causing crown galls of wine grape in Russia // Plant Dis. 2016. Vol. 100. N. 4. P. 853.Ignatov A.N., Khodykina M.V., Vinogradova S.V., Polityko V.A., Kornev K.P., Mazurin E.S. First report of Agrobacterium vitis causing crown galls of wine grape in Russia // Plant Dis. 2016. Vol. 100. N. 4. P. 853.

The authors declare that there are no conflicts of interest present.