شناسایی اختصاصی نماتد ریشه ‎گرهی Meloidogyne incognita رایج در باغات کیوی‌فروت استان‌های گیلان و مازندران با استفاده از توالی ژن sec-1

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه گیلان، رشت، ایران.

2 موسسه تحقیقات علوم باغبانی، پژوهشکده مرکبات و میوه‌های نیمه‌گرمسیری، سازمان تحقیقات، آموزش و ترویج کشاورزی، رامسر، ایران.

3 گروه علوم باغبانی، دانشکده کشاورزی، دانشگاه گیلان، رشت، ایران.

چکیده

در سال­های اخیر، شیوع بیش از پیش نماتدهای ریشه‎ گرهی باعث ایجاد خسارت جدی در مناطق کشت کیوی‌ فروت در شمال ایران شده است. با توجه به رواج چند گونه از این جنس در کشور، شناسایی دقیق گونه­های این نماتد در صنعت تولید محصول حائز اهمیت است. در این تحقیق، تلاش شد شناسایی مولکولی نماتد ریشه‎ گرهی به کمک واکنش زنجیره‌ای پلی­مراز، با استفاده از آغازگرهای اختصاصی ژن کدکننده یکی از پروتئین­های غدد مری (pharyngeal gland secretory protein, sec-1) در جهت تأیید شناسایی­های مبتنی بر ویژگی‌های ریخت­­ شناختی صورت گیرد. بدین منظور، پس از انجام نمونه برداری و جداسازی نماتدها از باغ‌های کیوی‌ فروت آلوده در استان‌های گیلان و مازندران، اقدام به شناسایی اولیه با استفاده از خصوصیات ریخت ­شناختی گردید. جفت آغازگرهای Inc-K14 برای تکثیر ناحیه SCAR و جفت آغازگرهای SEC-1 برای تکثیر ژن sec-1 به منظور تشخیص اختصاصی گونه Meloidogyne incognita از سایر گونه‌ها مورد استفاده قرار گرفتند. در همه نمونه‌های مورد بررسی، قطعه‌ای 399 جفت بازی توسط جفت آغازگرهای Inc-K14 و قطعه‌ای 502 جفت بازی توسط جفت آغازگرهای SEC-1 تکثیر گردید که قطعات مورد انتظار برای شناسایی گونه M. incognita می‌باشند. انجام آزمون PCR با استفاده از آغازگرهای Rjav/Fjav، Rar/Far و Rh/Fh که به ترتیب آغازگرهای اختصاصی گونه­های M. javanica، M. arenaria و M. hapla هستند، در این مطالعه منجر به تکثیر باند نشد. با توجه به نتایج به دست آمده، آغازگر SEC-1 جهت شناسایی سریع و دقیق M. incognita در باغ‌های آلوده کیوی‌ فروت (در این مطالعه) مناسب بوده و مشکلات تکثیر غیراختصاصی با آغازگرهای Inc-K14 که ناحیه SCAR را تکثیر می­ کنند، نخواهد داشت.

کلیدواژه‌ها

عنوان مقاله [English]

Specific identification of root-knot nematode Meloidogyne incognita common in kiwifruit orchards of Guilan and Mazandaran provinces using sec-1 gene sequence

نویسندگان [English]

  • Seyedeh Najmeh Banihashemian 1
  • Salar Jamali 1
  • Morteza Golmohammadi 2
  • Mahmood Ghasemnejad 3
1 Plant Protection Department, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran.
2 Horticultural Science Research Institute, Citrus and Subtropical Fruits Research Center, Agricultural Research Education and Extension Organization (AREEO), Ramsar, Iran,
3 Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran.
چکیده [English]

In recent years, the increasing prevalence of root knot nematodes has caused serious damage to kiwifruit cultivation in northern Iran. Due to the prevalence of several species of this genus in the country, accurate identification of species of this nematode in the kiwifruit industry is important. In this study, we attempted to identify the root knot nematode species by polymerase chain reaction, using specific primers of the gene encoding one of the pharyngeal glands secretory proteins (sec-1) to confirm the morphological identifications. After sampling and isolation of the nematodes from kiwifruit orchards in Guilan and Mazandaran provinces, initial identification was performed using morphological characteristics. The Inc-k14 primer pair was used to amplify the SCAR region and the SEC-1 primer pair was used to amplify the sec-1 gene for specific identification of Meloidogyne incognita from the other species. In all samples, a 399 bp was amplified by Inc-K14 primers and a 502 bp fragment was amplified by SEC-1 primers, which are expected as specific bands for M. incognita. The PCR test using Rjav/Fjav, Rar/Far, and Rh/Fh primers, which are specific primers of M. javanica, M. arenaria and M. hapla, respectively, did not lead to band amplification in this study. According to the results, SEC-1 primer is suitable for rapid and accurate identification of M. incognita in infested kiwifruit orchards (in present study) and will not have non-specific band amplification problems using Inc-K14 primers that amplify the SCAR region.

کلیدواژه‌ها [English]

  • Molecular identification
  • SEC-1 primer
  • SCAR region

مراجع

Abad P, Favery B, Rosso MN, Castagnone-Sereno P, 2003. Root-knot nematode parasitism and host response: Molecular basis of a sophisticated interaction. Molecular Plant Pathology 4: 217–224.
Akyazi F, Felek A, 2013. Molecular identification of root-knot nematode Meloidogyne incognita from kiwi fruit orchards in Ordu province, Turkey. Türkiye Entomoloji Dergisi 37 (4): 449–456.
Adam MAM, Phillips MS, Blok VC, 2007. Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.). Plant Pathology 56: 190–197.
Alvarez-Ortega S, Brito JA, Subbotin S, 2019. Multigene phylogeny of root-knot nematodes and molecular characterization of Meloidogyne nataliei Golden, Rose and Bird, 1981 (Nematoda: Tylenchida). Scientific Reports 9 (1): 1–11.
Arzanlou M, Ghasemi Esfahlan S, Khodaei S, Tavakoli M, Babaei Ahari A, 2016. Molecular diagnostics of Biscogniauxia mediterranea, the causal agent of charcoal rot disease on oak using species-specific primers. Journal of Applied Research in Plant Protection 5 (2): 175–186.
Barros AF, Campos VP, Souza LN, Costa SS, Terra WC, et al., 2018. Morphological, enzymatic and molecular characterization of root-knot nematodes parasitizing vegetable crops. Horticultura Brasileira 36 (4): 473–479.
Blok VC, Powers TO, 2009. Biochemical and molecular identification. In: Perry RN, Moens M, Starr JL (eds). Root-knot Nematodes. CABI Publishing, Wallingford, UK. Pp. 98–118.
Calderon-Urrea A, Vanholme B, Vangestel S, Kane SM, Bahaji A, et al, 2016. Early development of the root-knot nematode Meloidogyne incognita. BMC Development Biology 16: 1–14.
Chen P, Peng DL, Zheng JW, 1990. Description of a new root-knot nematode Meloidogyne fanzhiensis sp. Journal of Shanxi Agricultural University 10: 55–60.
Chitwood BG, 1949. Root-knot nematodes, part I. A revision of the genus Meloidogyne göldi, 1887. Proceedings of the Helminthological Society of Washington 16: 90–104.
Da Silva AT, Penna JCV, Goulart LR, Santos MA, Arantes NE, 2000. Genetic variability among and within races of Heterodera glycines ichinohe assessed by RAPD markers. Genetic Molecular Biology 23: 323–329.
Devran Z, Sogut MA, 2009. Distribution and identification of root-knot nematodes from Turkey. Journal of Nematology 41: 128–133.
Dong K, Dean RA, Fortnum BA, Lewis SA, 2001. Development of PCR primers to identify species of root knot nematodes: Meloidogyne arenaria, M. hapla, M. incognita and M. javanica. Nematropica 31: 271–280.
Eisenback JD, Triantaphyllou HH, 1991. Root-knot nematodes: Meloidogyne species and races. In: Nickle WR (ed). Manual of Agricultural Nematology. Marcel Dekker, Inc., USA. Pp. 191–274.
Eskandarzadeh N, Moslehi Sh, Vaez N, 2020. Evaluation of the effects of Thorne-apple and henbane on egg hatching and mortality of juveniles of Meloidogyne javanica. Journal of Applied Research in Plant Protection 9 (2): 45–60.
FAO, FAOSTAT, Production, 2020. http://www.Fao.org
Fourie H, Zijlstra C, Mcdonald A, 2001. Identification of root-knot nematode species occurring in South Africa using the SCAR-PCR technique. Nematology 3 (7): 675–680.
Gasser RB, 2001. Identification of parasitic nematodes and study of genetic variabilitusing PCR approaches. In: Kennedy MW, Harnett W (eds). Parasitic Nematodes: Molecular Biology, Biochemistry and Immunology. CABI, UK. Pp. 53–82.
Ghasemi Esfahlan S, Arzanlou M, Babayahri A, Torbati M, Narmani A, 2019. Morphological and molecular characterization of endophytic fungi from oak trees in arasbaran forests. Journal of Applied Research in Plant Protection 8 (1): 1–17.
Golmohammadi M, Ghasemi M, Banihashemian SN, 2019. The study of tolerance or sensitive of kiwifruit varieties to the Bacterial Canker. 1st Iranian Plant pathology Congress.Tehran-Karaj. Proceedings of the 1th Iranian plant pathology Congress, Karaj, Iran, P. 342. (In Persian).
Hajihassani A, Lawrence KS, Jagdale GB, 2018. Plant parasitic nematodes in Georgia and Alabama, In: Subbotin SA, Chitambar JJ (eds). Plant Parasitic Nematodes in Sustainable Agriculture of North America, Vol. 2 Springer International Publishing, USA. Pp. 357–391.
Hartman K, Sasser JN, 1985. Identification of Meloidogyne species on the basis of differential hosts test and perineal pattern morphology. In: Barker KR, Carter CC, Sasser J.N (eds). An advanced Treatise on Meloidogyne. Volume II: Methology. North Carolina State University, USA. Pp. 69–77.
Hasan ST, Abood ID, Abd Al Rasoul AM, 2020. Diagnostic of new species of root knot nematode (Meloidogyne cruciani) associted with egg plant in babylon governorate/Iraq based on morphological characters and molecular methods. Ecology, Environment and Conservation 26 (1): 69–74.
Hussey RS, Janssen GJW, 2002. Root-knot nematodes: Meloidogyne species. In: Starr JL, Cook R, Bridge J (eds). Plant Resistance to Parasitic Nematodes. CABI Publishing, Wallingford, UK. Pp. 43–70.
Hwang SM, Jang KS, Choi YH Choi GJ, 2016. Development of an efficient screening method for resistance of chili pepper plants to Meloidogyne incognita. Horticultural Science and Technology 34 (2): 282–293.
Janssen T, Karssen G, Verhaeven M, Coyne D, Bert W, 2016. Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution. Scientific Reports 6: 22591.
Jones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, 2013. Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology 14: 946–961.
Hewezi T, Baum TJ, 2013. Manipulation of plant cells by cyst and root-knot nematode effectors. Molecular Plant Microbe Interaction 26: 9–16.
Karssen G, 2002. The plant-parasitic nematode genus Meloidogyne goldi, 1892 (Tylenchida) in Europe. Brill, Leiden, Netherlands. 157 pp.
Kofoid CA, White WA, 1919. A new nematode infection of man. Journal of the American Medical Association 72: 567–569.
Neal JC, 1889. The root-knot disease of the peach, orange and other plants in Florida, due to the work of Anguillula. Bulletin 20, Division of Entomology, US Department of Agriculture.
Niu JH, Guo QX, Jian H, Chen CL, Yang D, Liu Q, Guo YD, 2011. Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots. Crop Protection 30 (8): 1063–9.
Quentin M, Abad P, Favery B, 2013. Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells. Frontiers in Plant Science 4: 53.
Randig O, Bongiovanni M, Carneiro RM Castagnone-Sereno P, 2002. Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species. Genome 45 (5): 862–870.
Ray C, Abbott, AG, Hussey, RS, 1994. Trans-splicing of a Meloidogyne incognita mRNA encoding a putative esophageal gland protein. Molecular and Biochemical Parasitology 68 (1): 93–101.
Salgado SML, Guimarães NMRB, Botelho CE, Tassone GAT, Marcelo AL, et al., 2015. Meloidogyne paranaensis e Meloidogyne exigua em lavouras cafeeiras na s. região sul de Minas Gerais. Coffee Science, Lavras 10 (4): 475–481.
Sellers GS, Jeffares DC, Lawson B, Prior T, Lunt DH, 2021. Identification of individual root-knot nematodes using low coverage long-read sequencing. Plos One 16 (12): e0253248.
Sikora R, Coyne D, Hallmann J, Timper P, 2018. Plant parasitic nematodes in subtropical and tropical agriculture. 3rd edition, CABI Publishing, Wallingford, UK. 888 pp.
Subbotin SA, Rius JEP, Castillo P, 2021. Systematics of root-knot nematodes (Nematoda: Meloidogynidae). Brill.
Tao Y, Xu C, Yuan C, Wang H, Lin B, et al., 2017. Meloidogyne aberrans sp. nov.(Nematoda: Meloidogynidae), a new root-knot nematode parasitizing kiwifruit in China. PloS One 12 (8): e0182627.
Tesarova B, Zouhar M, Rysanek P, 2003. Development of PCR for specific determination of root-knot nematode Meloidogyne incognita. Plant Protection Science 39: 23–28.
Thoden TC, Korthals GW, Termorshuizen AJ, 2011. Organic amendments and their influences on plant-parasitic and free-living nematodes: A promising method for nematode management. Nematology 13: 133–153.
Taylor DP, Netscher C, 1974. An improved technique for preparing perineal patterns of Meloidogyne spp. Nematologica 20 (2): 268–269.
Treub M, 1885. Onderzoekingen over sereh-ziek suikkeriet gedaan in s’Lands plantentium te Bbuitenzorg. Mededeelingen uit’s Lands Plantentium, Batavia 2: 1–39.
Yang Y, Hu X, Liu P, Chen L, Peng H, et al., 2021. A new root-knot nematode, Meloidogyne vitis sp. nov.(Nematoda: Meloidogynidae), parasitizing grape in Yunnan. Plos One 16 (2): e0245201.
Zijlstra C, Donkers-Venne DTHM, Fargette M, 2000. Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence characterised amplified region (SCAR) based PCR assays. Nematology 2: 847–853.
Zeng J, Zhang Z, Li M, Wu X, Zeng Y, et al., 2018. Distribution and molecular identification of Meloidogyne spp. parasitising flue-cured tobacco in Yunnan, China. Plant Protection Science 54: 183–189.
پژوهش های کاربردی در گیاهپزشکی
دوره 11، شماره 4
دی 1401
صفحه 63-72

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  • تاریخ دریافت: 25 دی 1400
  • تاریخ بازنگری: 16 بهمن 1400
  • تاریخ پذیرش: 13 فروردین 1401

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