Identification of Phomopsis Species on Some Ornamental and Forest Plants in Iran on the Basis of the Morphological and Molecular Characteristics
محورهای موضوعی : مجله گیاهان زینتیSurena Bavand Savadkuhi 1 , Safarali Mahdian 2 , Valiollah Babaeizad 3 , Mohammadali Tajik Ghanbari 4
1 - MSc. in Plant Pathology, Sari Agricultural and Natural Resources University, Sari, Iran
2 - Associate Professor, Department of Plant Protection, Sari Agricultural Sciences and Natural Resources University, Sari, Iran
3 - Associate Professor, Department of Plant Protection, Sari Agricultural Sciences and Natural Resources University, Sari, Iran
4 - Associate Professor, Department of Plant Protection, Sari Agricultural Sciences and Natural Resources University, Sari, Iran
کلید واژه: Molecular identification, Morphological identification, <i>Phomopsis</i>,
چکیده مقاله :
Phomopsis is a genus of imperfect plant pathogenic fungus whose hosts comprise several species in different regions of the world, such as grapes, soybean, acacia, hollyhock, velvetleaf, and several other plants. In this study, samples were collected from hollyhock, velvetleaf, purple bauhinia, and acacia plants suspected to be infected with Phomopsis fungi. They were, then, cultured and subjected to the separation and purification steps, and morphological characteristics of the derived isolates were determined. Among the isolates, five representatives (P1 to P5) were selected from Mazandaran province and their DNAs were extracted. The TEF and ITS regions of DNAs were amplified and sequenced. The ITS (4 and 5) region from ribosomal DNA and a part of the TEF gene were amplified and sequenced. The nucleotide sequence of the isolates was compared with the corresponding sequences available in the GeneBank, and the phylogenetic tree of the isolates was constructed. Results showed that the sequence of the isolates P1, P2, and P4 had the highest similarity with Phomopsis malvacearum species. The sequence of the isolate P3 was very similar to Phomopsis loropetali, and the sequence of the isolate P5 was very similar to Phomopsis theicola. The average size of pycnidia produced on three culture media, i.e. CDA, CMA, and PDA, was measured 16 days after incubation at 20-25°C. Sizes were 256 × 85 μm in acacia, 230 × 68 μm in velvetleaf, 230 × 69 μm in hollyhock and 193 × 47 μm in purple bauhinia isolates. Morphological and molecular features of the identified species were matched. The species identified here are reported from Iran for the first time. Symptoms of Phomopsis infection varied with host and identified species.
شبه جنس Phomopsis از جمله قارچهای ناقص بیماریزای گیاهی است که گونههای آن در نقاط مختلف دنیا میزبانهای متعددی همچون انگور، سویا، اقاقیا، ختمی، گاوپنبه و چندین گیاه دیگر دارند. در این بررسی از گیاهان ختمی (Alcea rosea)، گاوپنبه (Abutilon theophrasti)، بهیمه (Phanera purpurea) و اقاقیا (Robinia pseudoacacia var. Umbraculifera) مشکوک به آلودگی Phomopsis نمونهبرداری شد. پس از کشت نمونهها، جداسازی و خالص سازی جدایههای قارچی انجام شد و خصوصیات ریختشناسی جدایههای بهدست آمده مورد بررسی قرار گرفت. از بین جدایهها پنج جدایه بهعنوان نماینده انتخاب و DNA آنها استخراج شد. ناحیه ITS از DNA ریبوزومی و بخشی از ژن TEF تکثیر و توالییابی شدند. توالی نمونهها با توالیهای موجود در ژن بانک (NCBI) مقایسه شد. نتیجه نشان داد توالی جدایههای P1 از ختمی، P2 از گاوپنبه و P4 از اقاقیا بالاترین شباهت را با گونه Phomopsis malvacearum داشتند. توالی جدایه P3 از روی بهیمه بیشترین شباهت را با Phomopsis loropetaliو توالی جدایه P5 از روی اقاقیا بیشترین شباهت را با گونه Phomopsis theicolaنشان دادند. میانگین ابعاد پیکنیدهای تولید شده جدایهها روی سه محیط کشت CDA، CMA و PDA بعد از 16 روز در جدایههای اقاقیا 256 × 85، گاوپنبه 230 × 68، ختمی 230 × 69 و بهیمه 193 × 47 میکرومتر اندازه‌گیری شد. مشخصات ریختشناسی و مولکولی گونههای شناسایی شده با هم تطابق داشتند. این گونهها از ایران برای اولین بار گزارش میشوند. علایم بیماری ناشی از Phomopsis بسته به میزبانهای مورد مطالعه و گونههای مختلف بهصورت متفاوت مشاهده شد.
Annesi, T., Luongo, L., Vitale, S., Galli, M. and Belisario, A. 2016. Characterization and pathogenicity of Phomopsis theicola anamorph of Diaporthe foeniculina causing stem and shoot cankers on sweet chestnut in Italy. Journal of Phytopathology, 164 (6): 412-416.
Babaeizad, V. and Sayari, M. 2012. Morphological and molecular identification of stem canker hibiscus in Iran. In: Proceedings of the 20th Congress of Iranian Plant Protection, College of Agriculture, Shiraz University, p. 356.
Banihashemi, Z. and Javadi, A.R. 2009. Further investigation on the biology of Phomopsis cinerascens the cause of fig canker in Iran. Phytopathologia Mediterranea, 48: 454-460.
Barnett, H.L. and Hunter, B.B. 1998. Illustrated genera of imperfect fungi. 4th ed. APS Press, USA.
Boerema, G.H., Gruyter, J., de Noordeloos, M.E. and Hamers, M.E.C. 2004. Phoma identification manual. Differentiation of specific and infra-specific taxa in culture. CABI Publishing.
Doyle, J.J. and Doyle, J.L. 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.
Farr, D.F., Castlebury, L.A. and Rossman, A.Y. 2002. Morphological and molecular characterization of Phomopsis vaccinii and additional isolates of Phomopsis from blueberry and cranberry in the eastern United States. Mycologia, 94: 494-504.
Gomes, R.R., Glienke, C., Videira, S.I.R., Lombard, L., Groenewald, J.Z. and Crous, P.W. 2013. Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia, 31: 1-41.
Harrington, T.C., Steimel, J., Workneh, F. and Yang, X. B. 2000. Molecular identification of fungi associated with vascular discoloration of soybean in the north central United States. Plant Disease, 84: 83-89.
Hibbett, D.S. and Taylor, J.W. 2013. Fungal systematics: Is a new age of enlightenment at hand? Nature Reviews, Microbiology, 11: 129-133.
Jurkovic, D.K., Vrandecic, K., Cosic, J., Riccioni, L. and Duvnjak, T. 2007. Morphological identification of Diaporthe/Phomopsis sp. Isolated from Xanthium italicum. Poljoprivreda, 13 (2): 10-14.
Mirabolfathi, M., Hoseinian, L. and Moghadam, A.M. 2013. First report of Phomopsis amygdali (DEL.) Tuset Portilla causing galls on common hazel (Corylus avellana) twigs in Iran. Iranian Journal of Plant Pathology, 49: 43-45.
Mousavi, H., Babaeizad, V. and Sayari, M. 2012. Incidence of stem canker caused by Phomopsis longicola in Iran. In: Proceedings of the 20th Congress of Iranian Plant Protection, College of Agriculture, Shiraz University, Iran. p. 355.
Rehner, S.A. and Buckley, E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: Evidence for cryptic diversification and links to Cordyceps teleomorph. Mycologia, 97: 84-98.
Santos, J.M., Correia, V.G. and Philips, J.L. 2010. Primers for mating-type diagnosis in Diaporthe and Phomopsis: Their use in teleomorph induction in vitro and biological species definition. Fungal Biology, 114: 255-270.
Santos, J.M. and Phillips, A.J.L. 2009. Resolving the complex of Diaporthe (Phomopsis) species occurring on Foeniculum vulgare in Portugal. Fungal Diversity, 34: 111-125.
Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W. and Consortium, F.B. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proceedings of the National Academy of Sciences of the United States of America, 109: 6241–6246.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30: 2725-2729.
Udayanga, D., Castlebury, L.A., Rossman, A.Y., Chukeatirote, E. and Hyde, K.D. 2014. Insights into the genus Diaporthe: Phylogenetic species delimitation in the D. eres species complex. Fungal Diversity, 67: 203-229.
Udayanga, D., Liu, X., Crous, P.W., McKenzie, E.H.C., Chukeatirote, E. and Hyde, D. 2012. A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis). Fungal Diversity, 56: 157-171.
Uecker, F.A. 1988. A world list of Phomopsis names with notes on nomenclature, morphology and biology. Mycologia Memoir, 13: 231 pp.
Uecker, F.A. and Johnson, D.A. 1991. Morphology and taxonomy of Phomopsis on Asparagus. Mycologia, 83: 192-199.
Vedashree, S., Sateesh, M.K., Chowdappa, P. and Nirmalkumar, B.J. 2015. Species-specific PCR-based assay for identification and detection of Phomopsis (Diaporthe) azadirachtae causing die-back disease in Azadirachta indica. Journal of Phytopathology, 163: 818-828.
Vrandecic, K., Cosic, J., Riccioni, L., Duvnjak, T. and Jurkovic, D. 2004. Phomopsis longicola- new pathogen on Abutilon theophrasti in Croatia. Plant Pathology, 53: 251. DOI: 10.1111/j.0032-0862.2004.00992.x.