شناسایی و ردیابی مولکولی Phytophthora melonis بر‌اساس ژنوم هسته‌ای و میتوکندریایی*

نوع مقاله : مقاله کامل پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی ارشد

2 استاد بیماری شناسی گیاهی و دانشیار اصلاح نباتات، دانشکده کشاورزی دانشگاه شیراز

3 دانش آموخته کارشناسی ارشد دانشکده کشاورزی دانشگاه شیراز

چکیده

گونه­ی بیمارگر گیاهی Phytophthora melonis از نظر ریخت­شناختی به برخی گونه­های بدون پستانک جنس Phytophthora به­خصوص P. drechsleri شباهت دارد وبنابراین تفکیک این آرایه­های هم­گرا دشوار است. این پژوهش به منظور طراحی آغازگرهای اختصاصی P. melonis بر اساس ژنوم هسته­ای و میتوکندریایی، بررسی اختصاصیت آن­ها در برابر سایر گونه­های همگرا، و بهینه­سازی استفاده از این آغازگرها برای ردیابی P. melonis انجام شد. برای طراحی آغازگر­های اختصاصی گونه­ی P. melonis نُه ژن­ هسته­ای و چهار ژن میتوکندریایی از نظر تفاوت نوکلئوتیدی مناسب بودند، بنابراین سیزده آغازگر اختصاصی طراحی و خصوصیات آن­ها بهینه‌سازی گردید. ردیابی P. melonis با استفاده از پنج جفت از آغازگر­های اختصاصی در بافت مایه­زنی شده­ی گیاهان میزبان آن شامل خیار، خربزه، هندوانه، چغندر­قند و پسته­ انجام شد. با استفاده از آغازگرهای طراحی شده در واکنش زنجیره‌ای پلیمراز تودرتو، تا سطح یک درصد مایه­ی بیماری­زا­ در خاک آلوده و زئوسپورهای بیمارگر تا غلظت­10 زئوسپور در میلی­لیتر در آب آلوده ردیابی شدند. با بررسی اختصاصیت و حساسیت آغازگر­های طراحی شده، کارامد­ترین آن­­­ها مجموعه­ی ITS-M2 (ترکیب آغازگرهای ITS-MF1 و ITS-MR2) در نظر گرفته شد. دمای هم­جوشی بهینه‌سازی شده برای این مجموعه 68 درجه­ی سلسیوس بود. به نظر می‌رسد استفاده از واکنش زنجیره­ای پلیمراز تودرتو با استفاده از مجموعه­ی ITS-M2 به همراه آغازگر­های عمومی ITS4 و ITS6 در نقش آغازگر­های خارجی در حدود 106 برابر حساس­تر از واکنش زنجیره­ای پلیمراز مستقیم است. ­آزمون زنجیره­ای پلیمراز چندگانه‌ی طراحی شده، قادر به ردیابی هم­زمان سه گونه­ی بیمارگر شامل P. melonis، P. drechsleri و P. nicotianae بود.آزمایش‌ها نشان داد که آغازگر­های طراحی شده ابزاری کارآمدی برای ردیابی P. melonis در بافت گیاه، خاک و آب آلوده هستند.

کلیدواژه‌ها

عنوان مقاله [English]

Molecular Identification and Detection of Phytophthora melonis Based on Nuclear and Cytoplasmic Genome

نویسندگان [English]

  • S. M. Zolanvari 1
  • R. Mostowfizadeh-Ghalamfarsa 2
  • A. Dadkhodaie 3
چکیده [English]

The plant pathogen Phytophthora melonis is morphologically similar to some other non-papillate Phytophthora spp. especially P. drechsleri and therefore it is difficult to discriminate these convergent taxa. This study was performed to design specific primers based on nuclear and cytoplasmic genome, examine their specificity against other convergent species, and optimize the specific primers’ conditions to detect P. melonis. Nine nuclear and four cytoplasmic genes were appropriate to design thirteen specific primers based on their nucleotide polymorphism. PCR conditions were optimized. Phytophthora melonis were detected by specific primers in inoculated plants such as cucumber, watermelon, melon, sugar beet and pistachio. All specific primers detected pathogen in 1:100 (pathogen: soil) inoculated soil.Up to 10 zoospores per milliliter were detected using nested polymerase chain reaction. ITS-MF1 and ITS-MR2 (ITS-M2 set, from internal transcribed spacers of rRNA gene) were selected as the most efficient primers based on their specificity and sensitivity. The optimized annealing temperature for this primer set was 68 °C. It seemed that nested PCR by ITS-M2 primer set together withthe universal primers ITS6 and ITS4 as external primers is at least 106 times more sensitive than simple PCR. Multiplex polymerase chain reaction simultaneously detected the three cucurbits’ pathogens including P. melonis, P. nicotianae and P. drechsleri. This study showed that the designed primers could be effective tools for detection of P. melonis isolates from infected tissues, and infested water and soil.

کلیدواژه‌ها [English]

  • Detection
  • identification
  • Oomycota
  • root rot
  • specific primers

مراجع

Banihashemi, Z., and Fatehi, J. 1989. Reaction of cucurbit cultivars to Phytophthora drechsleri and P. capsici in greenhouse. In Proceedings of the 9th Iranian Plant Protection Congress, (pp. 9-14). Mashhad, Iran.
Blair, J. E., Coffey, M. D., Park, S. Y., Geiser, D. M., and Kang, S. 2008. A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences. Fungal Genetics and Biology 45(3): 266-277.
Bonants, P., Hagenaar-de Weerdt, M., van Gent-Pelzer, M., Lacourt, I., Cooke, D., and Duncan, J. 1997. Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction. European Journal of Plant Pathology 103(4): 345-355.
Chang, H. S. 1983. Crop diseases incited by Phytophthora fungi in Taiwan. Plant Protection Bulletin, Taiwan 25(4): 231-237.
Chen, Q., Li, B., Liu, P., Lan, C., Zhan, Z., and Weng, Q. 2013. Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis. European Journal of Plant Pathology 137(3): 597-607.
Cooke, D. E. L., Drenth, A., Duncan, J. M., Wagels, G., and Brasier, C. M. 2000. A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology 30(1): 17-32.
Cooke, D. E. L., Kennedy, D. M., Guy, D. C., Russell, J., Unkles, S. E., and Duncan, J. M. 1996. Relatedness of group I species of Phytophthora as assessed by randomly amplified polymorphic DNA (RAPDs) and sequences of ribosomal DNA. Mycological Research 100(3): 297-303.
Cooke, D. E., and Duncan, J. M. 1997. Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycological Research 101(06): 667-677.
Drenth, A.,Wagels, G., Irwin, J. A. G., Liew, E. C. Y., and Maclean, D. J. 1999. DNA based methods for the detection of Phytophthora species. Australia Patent Number 16330/97.
Drenth, A., Wagels, G., Smith, B., Sendall, B., O’Dwyer, C., Irvine, G., and Irwin, J. A. G. 2006. Development of a DNA-based method for detection and identification of Phytophthora species. Australasian Plant Pathology 35(2): 147-159.
El-Helaly, A. F., Assawah, M. W., Elarosi, H. M., and Wasfy, E. E. H. 1968. Fruit rots of vegetable marrow in Egypt (United Arab Republic). Phytopathologia Mediterranea 7: 107-115.
Ershad, J., and Mostofipour, P. 1969. Root rot of cucurbits in Iran. Iranian Journal of Plant Pathology 5: 38-45.
Esmaili, S. E., and Banihashemi, Z. 2008. The role of phytophthora melonis and P. drechsleri in cucurbit root rot in Iran. Iranian Journal of Plant Pathology 44: 54-72.
FAOSTAT. 2013. FAO Statistics Division 2012. Retrieved from http://faostat.fao.org/site/567/ default.aspx#ancor, 01 May 2014.
Gallegly, M.E., ChuanXue, H. and Hong, C. 2008. Phytophthora: identifying species by morphology and DNA fingerprints. APS Press, 158 pp. USA.
Grote, D., Olmos, A., Kofoet, A., Tuset, J. J., Bertolini, E., and Cambra, M. 2002. Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR. European Journal of Plant Pathology 108(3): 197-207.
Ho, H. H. 1986. Phytophthora melonis and P. sinensis synonymous with P. drechsleri Mycologia 78: 907-912.
Ho, H. H., Jiayun, L., and Longyin, G. 1984. Phytophthora drechsleri causing blight of Cucumis species in China. Mycologia 76: 115-121.
Ippolito, A., Schena, L., and Nigro, F. 2002. Detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils by nested PCR. European Journal of Plant Pathology 108(9): 855-868.
Jeffers, S.N. and Martin, S.B. 1968. Comparison of two media selective for Phytophthora and Pythium species. Plant Disease 70: 1035-1043.
Katsura, K. 1968. On Phytophthora melonis n. sp. of cucumber. Annals of Phytopathogical Society of Japan 34: 167.
Kong, P., Hong, C. X., and Richardson, P. A. 2003. Rapid detection of Phytophthora cinnamomi using PCR with primers derived from the Lpv putative storage protein genes. Plant Pathology 52(6): 681-693.
Kroon, L. P. N. M., Bakker, F. T., Van Den Bosch, G. B. M., Bonants, P. J. M., and Flier, W. G. 2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences. Fungal Genetics and Biology 41(8): 766-782.
Langrell, S. R., Morel, O., and Robin, C. 2011. Touchdown nested multiplex PCR detection of Phytophthora cinnamomi and P. cambivora from French and English chestnut grove soils. Fungal Biology 115(7): 672-682.
Lee, S. B., White, T. J., and Taylor, J. W. 1993. Detection of Phytophthora species by oligonucleotide hybridization to amplified ribosomal DNA spacers. Phytopathology 83(2): 177-181.
 Li, M., Asano, T., Suga, H., and Kageyama, K. 2011. A multiplex PCR for the detection of Phytophthora nicotianae and P. cactorum, and a survey of their occurrence in strawberry production areas of Japan. Plant Disease  95(10): 1270-1278.
Loos, R., Husson, C., Andrieux, A., and Frey, P. 2005. SCAR–based PCR primers to detect the hybrid pathogen Phytophthora alni and its subspecies causing alder disease in Europe. European Journal of Plant Pathology 112(4): 323-335.
Lu, J. Y., and Gong, L. Y. 1982. Studies on idendification and biological characters of the causal organism of cucumber blight in Nanjing.Journal of Nanjing Agriculture College 3: 23-38.
Maden, S., and Karahan, O. 1980. New root and foot-rot disease of melons (Phytophthora dreschsleri Tucker) in central Anatolia and its pathogenicity on common melon cultivars in this region. Journal of Turkish Phytopathology 9: 49-55.
Martin, F. N. 2008. Mitochondrial haplotype determination in the oomycete plant pathogen Phytophthora ramorum. Current Genetics 54(1): 23-34.
Martin, F. N., and Coffey, M. D. 2012. Mitochondrial haplotype analysis for differentiation of isolates of Phytophthora cinnamomi. Phytopathology 102(2): 229-239.
Martin, F. N., Blair, J. E., and Coffey, M. D. 2014. A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora. Fungal Genetics and Biology 66: 19-32.
 Martin, F. N., Tooley, P. W., and Blomquist, C. 2004. Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology 94(6): 621-631.
Mihail, J. D., Rush, C. M., Singleton, L. L., Edited by Larry, L. S., Jeanne, D. M., and Charles, M. R. 1992. Methods for research on soilborne phytopathogenic fungi. St. Paul, MN, USA: APS Press.
Mirabolfathy, M., Cooke, D. E., Duncan, J. M., Williams, N. A., Ershad, D., and Alizadeh, A. 2001. Phytophthora pistaciae sp. nov. and P. melonis: the principal causes of pistachio gummosis in Iran. Mycological Research 105(10): 1166-1175.
Mirtalebi, M., and Banihashemi, Z. 2006. Reaction of safflower cultivars to Phytophthora drechsleri and P. melonis. In Proceedings of the 17th Iranian Plant Protection Congress, (p. 262). Karaj, Iran.
Mostowfizadeh-Ghalamfarsa, R. 2011. Molecular phylogenetics of Phytophthora cryptogea and P. drechsleri: a multiple gene genealogy approach. Germany: Lambert Academic.
Mostowfizadeh-Ghalamfarsa, R., and Banihashemi, Z. 2012. A revision of Iranian Phytophthora drechsleri isolates from cucurbits based on multiple gene genealogy analysis.InProceedings of the 20th Iranian Plant Protection Congress, (p. 399). Shiraz, Iran.
Mostowfizadeh-Ghalamfarsa, R., and Banihashemi, Z. 2015. Species-specific PCR identification and detection of Phytophthora drechsleri, P. cryptogea and P. erythroseptica. Iranian Journal of Plant Pathology 51(4): 541-553.
Mostowfizadeh-Ghalamfarsa, R., and Mirsoleimani, Z. 2013. Species-specific identification and detection of Phytophthora pistaciae, the causal agent of pistachio gummosis. Phytopathologia Mediterranea 52(1): 30-45.
Mostowfizadeh-Ghalamfarsa, R., Banihashemi, Z., and Cooke, D. E. L. 2005. Potato pink rot: a criterion for discrimination of Phytophthora melonis from P. drechsleri. Iranian Journal of Plant Pathology 41: 191-201.
Robideau G.P., de Cock A.W.A.M., Coffey M.D., Volgmayr H., Brouwer H., Bala K., Chitty D.W., Desaulniers N., Eggertson Q.A., Gachon C.M., Hu C.H., Kupper F.C., Rintoul T.L., Sarhan E., Verstappen E. C., Zhang Y., Bonants P. J., Ristaino J. B. and  Lévesque A. C. 2011. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer. Molecular Ecology Resources 11:1002-1011.
Rozen, S., and Skaletsky, H. 1999. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols 132: 365-386.
Schena, L, and Cooke D. E. L. 2006. Assessing the potential of regions of the nuclear and mitochondrial genome to develop a “molecular tool box” for the detection and characterization of Phytophthora species. Journal Microbiology Methods 67: 70-85.
Silvar, C., Duncan, J. M., Cooke, D. E. L., Williams, N. A., Díaz, J., and Merino, F. 2005. Development of specific PCR primers for identification and detection of Phytophthora capsici Leon. European Journal of Plant Pathology 112(1): 43-52.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. 1997. The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25(24): 4876-4882.
White, T. J., Bruns, T., Lee, S. J. W. T., and Taylor, J. W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis, M.A., Gelfand, D.H., Sninsky, J.J., & White T.J. (Eds.),PCR protocols: a guide to methods and applications, (pp. 315-322). Academic Press, Inc., New York. USA.
Winton, L. M., and Hansen, E. M. 2001. Molecular diagnosis of Phytophthora lateralis in trees, water, and foliage baits using multiplex polymerase chain reaction. Forest Pathology 31(5): 275-283.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, T. L. 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13(1): 1.
 
 
 
 
 
 
 
 

فایل ها

هم رسانی

ارجاع به این مقاله

آمار