ارزیابی فعالیت آنزیم‌های مخرب دیواره سلولی گونه‌های Fusariumهمراه ریشه و بنه زعفران در استان خراسان جنوبی

نوع مقاله: مقاله علمی پژوهشی

نویسنده

استادیار، موسسه تحقیقات ثبت و گواهی بذر و نهال، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران

10.22048/jsat.2020.184121.1349

چکیده

هدف از این مطالعه شناسایی گونه‌های Fusarium همراه با ریشه و بنه زعفران و ارزیابی فعالیت آنزیم‌های مخرب دیواره سلولی تولید شده توسط آن­ها می باشد. به منظور بررسی گونه‌های Fusarium، طی فصل رشدی 1397 در مجموع 53 نمونه ریشه و بنه به طور تصادفی از مزارع زعفران استان خراسان جنوبی جمع­آوری شد. پس از شستشو نمونه­ها با آب مقطر استریل و ضدعفونی سطحی با هیپوکلریت سدیم یک درصد، قطعات کوچک ریشه و بنه روی محیط­های کشت‌ عمومی و اختصاصی کشت داده شدند. تهیه کشت خالص قارچ­ها به روش تک اسپور و نوک ریسه کردن روی محیط کشت‌ آب - آگار دو درصد انجام شد. شناسایی قارچ­ها بر اساس مشخصات میکروسکوپی و ریخت­شناختی با توجه به کلیدهای معتبر شناسایی انجام گرفت. با توجه به خصوصیات ریخت­شناختی، گونه های F. oxysporum، F. culmorum،F. proliferatum و F. graminearum شناسایی شدند. نتایج به دست آمده از تجزیه و تحلیل مولکولی با استفاده از آغازگرهای اختصاصی گونه، شناسایی ریخت­شناختی را تأیید کرد. نتایج نشان داد که 6/22 درصد از نمونه­های زعفران توسط گونه­های Fusarium آلوده بودند. بر اساس مشاهدات ریخت­شناختی و مولکولی، F. oxysporumبه­عنوان گونه‌ غالب با بیشترین درصد فراوانی در نمونه­ها به میزان 4/9 درصد پس از آن F. culmorum (7/5 درصد)،F. proliferatum (8/3 درصد) و F. graminearum (8/3 درصد) بود. نتایج نشان داد که تمام جدایه‌های Fusarium قادر به تولید آنزیم‌های مخرب دیواره سلولی و به طور عمده سلولاز و زایلاناز می‌باشند. سطوح فعالیت آنزیم­های سلولز، زایلاناز و پکتیناز در این مطالعه بالاتر از فعالیت لیپاز در همه جدایه­های مورد بررسی بود. تجزیه و تحلیل روند فعالیت آنزیم‌های مخرب دیواره سلولی نشان داد که برای بسیاری از جدایه‌های مورد بررسی حداکثر سطح فعالیت سلولاز، زایلاناز، پکتیناز و لیپاز به ترتیب در 72، 96، 144 و 192 ساعت پس از کشت در محیط مایع مشاهده شد. سپس فعالیت­های آنزیمی به تدریج با گذشت زمان کاهش یافت، تا اینکه در سطوح ثابتی باقی ماند. بر اساس منابع موجود F. graminearum، F. culmorum و F. proliferatum برای اولین بار در ایران از روی زعفران گزارش می شوند.

کلیدواژه‌ها


عنوان مقاله [English]

Evaluation of cell wall degrading enzymes of Fusarium species associated with root and corm of saffron in South Khorasan province

نویسنده [English]

  • Nima Khaledi
Assistant Professor, Seed and Plant Certification and Registration Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
چکیده [English]

The aim of this study is to identify  Fusarium species associated with root and corm of saffron and evaluate extracellular enzyme activity secreted by them. In order to study the species of Fusarium, a total of 53 root and corm samples were randomly collected from saffron fields of the South Khorasan province during 2017 growing season. After washing the samples with sterile water and disinfected the surface with 1% sodium hypochlorite, small pieces of root and corm were cultured on general and specific culture media. Purification of fungal colonies was performed using single spore and hyphal tip methods on 2% water agar medium. Identification of fungi based on the microscopic and morphological characteristics was performed with valid identification keys. The F. oxysporum, F. graminearum, F. culmorum and F. proliferatum species were identified based on morphological characters. The results obtained from molecular analysis using species-specific primers confirm morphological identification. The results reveal that 22.6% of saffron samples are contaminated by Fusarium species. F. oxysporum is the predominant species with the highest isolation frequency in samples of 9.4% followed by F. culmorum (5.7%), F. proliferatum (3.8%) and F. graminearum (3.8%) based on morphological and molecular observations,

کلیدواژه‌ها [English]

  • Cellulase
  • Identification
  • molecular
  • Morphological
  • Xylanase
Abdel-Razik, A.A. 1970. The parasitism of white Sclerotium cepivorum berk. the incitant of white rot of onion. PhD thesis, Agriculture Faculty, Assiut University, Assiut, Egypt.
Ahmad, M., and Sagar, V. 2007. Integrated management of corm/tuber rot of saffron and Kalazeera. Horticulture Mini Mission-1, Indian Council for Agricultural Research (ICAR), India, 22 p.
Ahrazem, O., Rubio-Moraga, A., Castillo-López, R., Trapero-Mozos, A., and Gómez-Gómez, L. 2010. Crocus sativus pathogens and defence responses. Functional Plant Science and Biotechnology. Global Science Book Isleworth, UK, pp. 81-90.
Asoufi, H., Hameed, K.M., and Mahasneh, A. 2007. The cellulase and pectinase activities associated with the virulence of indigenous Sclerotinia sclerotiorum isolates in Jordan Valley. The Plant Pathology Journal 23: 233-238.
Bailey, M.J., Biely, P., and Poutanen, K. 1992. Interlaboratory testing of methods for assay of xylanase activity. Journal of Biotechnology 23: 257-270.
Booth, C. 1977. Fusarium Laboratory Guide to Identification of Major Species. Common wealth Mycological Institute. Kew, Surrey, England, 55 p.
Colowich, S.P. 1995. Methods in Enzymology. London, Academic Prees INC.
Dhar, A.K. 1992. Bio-ecology and control of corm rot of saffron (Crocus sativus L.). MSc thesis, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, India, 109 p.
Di Primo, P., and Cappelli, C. 2000. Preliminary study on vegetative compatibility groups of Fusarium oxysporum f. sp. gladioli isolates obtained from saffron in Italy. Informatore Fitopatologico 50: 35-38. (In Italian).
Ebadzadeh, H.R., Ahmadi, K., Mohammadnia Afrouzi, S., Abbas Taghani, R., Abbasi Islami, M., and Yari, S. 2017. Agricultural Statistics of 2016: Economic and Planning Adjutancy, Information and Communication Technology Center. Iran's Ministry of Agriculture Press. 401 p. (In Persian).
Fisher, N.L., Burgess, L.W., Toussoun, T.A., and Nelson, P.E. 1982. Carnation leaves as a substrate and for preserving cultures of Fusarium species. Phytopathology 72: 151-153.
Gibson, D.M., King, B.C., Hayes, M.L., and Bergstrom, G.C. 2011. Plant pathogens as a source of diverse enzymes for lignocellulose digestion. Current Opinion in Microbiology 14 (3): 264-270.
Hajihasani, A., Hajihassani, M., and Khaghani, S. 2012. Incidence and distribution of seed-borne fungi associated with wheat in Markazi province, Iran. African Journal of Biotechnology 11 (23): 6290-6295.
Hassan, M., and Devi, L.S. 2003. Corm rot diseases of saffron in Kashmir valley. Indian Phytopathology 56: 122.
Hossainnia, A., and Mohammadi, A. 2018. Investigation of Alternaria alternata pathogenicity on corm and leaves of saffron in vitro and greenhouse conditions. Saffron Agronomy and Technology 6: 61-72. (In Persian with English Summary).
Husaini, A.M., Hassan, B., and Ghani, M.Y. 2010. Saffron (Crocus sativus L. Kashmirianus) cultivation in Kashmir: practices and problems. Functional Plant Sciences Biotechnology 4: 108-115.
Kalha, C.S., Gupta, V., and Gupta, D. 2007. First report of sclerotial rot of saffron caused by Sclerotium rolfsii in India. Plant Disease 91: 1203-1206
Khairy, E.M., Sammour, H.M., Ragheb, A., Ghandour, M.F., and Aziz, K. 1964. A Laboratory Manual of Practical Chemistry. Cairo, Egypt: Dar El-Nahda El-Arabia, pp.1-142.
Khaledi, N., Taheri, P., and Falahati-Rastegar, M. 2017. Identification, virulence factors characterization and analysis virulence together with aggressiveness of Fusarium spp., causing wheat head blight in Iran. European Journal of Plant Pathology 147: 897-918.
Khaledi, N., Taheri, P., and Tarighi, S. 2015. Antifungal activity of various essential oils against Rhizoctonia solani and Macrophomina phaseolina as major bean pathogens. Journal of Applied Microbiology 118: 704-717.
Kikot, G.E., Hours, R.A., and Alconada, T.M. 2009. Contribution of cell wall degrading enzymes to pathogenesis of Fusarium graminearum: a review. Journal of Basic Microbiology 49: 231-241.
Klotz, L.V., Nelson, P.E., and Toussoun, T.A. 1988. A medium for enhancement of chlamydospore formation in Fusarium species. Mycologia 80: 108-109.
Koocheki, A., and Seyedi, S.M. 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period. Journal of Saffron Research 3: 134-154. (In Persian with English Summary).
Koocheki, A., Siahmarguee, A., Aziz, G., Jahan, M., Alimiradi, L., 2009. The effect of plant density and depth on agronomic characteristic of saffron. 3rd International Symposium on Saffron. Forth coming Challenges in Cultivation, Research and Economics, 20-23 May 2009. Korokos, Kozani, Greece.
Leslie, J.F., and Summerell, A.B. 2006. The Fusarium laboratory manual. Ames: Blackwell Publishing Professional. 388 p.
Ma, L.J., Geiser, D.M., Proctor, R.H., Rooney, A.P., O’Donnell, K., Trail, F., Gardiner, D.M., Manners, J.M., and Kazan, K. 2013. Fusarium pathogenomics. Annual Review of Microbiology 67: 399-416.
MacMillan, J.D., and Voughin, R.H. 1964. Purification and properties of a polyglacturonic acid- transeliminase produced by Clastridium multiformentans. Biochemistry 3: 564-572.
Martinez-Soto, D., Robledo-Briones, A.M., Estrada-Luna, A.A., and Ruiz-Herrera, J. 2013. Transcriptomic analysis of Ustilago maydis infecting Arabidopsis reveals important aspects of the fungus pathogenic mechanisms. Plant Signaling and Behavior 8: 25-59.
Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31: 426-428.
Mishra, P.K., Fox, R.T.V., and Culham, A. 2003. Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria. Federation of European Microbiological Societies Microbiology Letters 218: 329-332.
Najari, G., Nourollahi, K., and Piri, M. 2018. The first report of (Fusarium oxysporum) causal agent of wild saffron corm rot disease in Iran. Saffron Agronomy and Technology 6: 119-123. (In Persian with English Summary).
Nash, S.M., and Snyder, W.C. 1962. Quantitative and estimations by plat counts of propagules of the bean rot Fusarium in field soils. Phytopathology 73: 458-462.
Nelson, P.E., Toussoun, T.A., and Marasas, W.F.O. 1983. Fusarium Species, An Illustrated Mannual for Identification. Pennsylvania State University Press, University Park. 193 p.
Nicholson, P., Simpson, D.R., Weston, G., Rezanoor, H.N., Lees, A.K., Parry, D.W., and Joyce, D. 1998. Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiology and Molecular Biology of Plants 53 (1): 17-37.
Nirenberg, H. 1976. Unterstructure uber die morphologische und biologische differnzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologischen Bundesansatlt fur Landund Forstwirtschaft, Berlin-Dahlem 169: 11-17.
Omidi, H., Naghdibadi, H.A., Golzad, A., Torabi, H., and Fotoukian, M.H. 2009. The effect of chemical and biofertilizer source of nitrogen on qualitative and quantitative yield of saffron (Crocus sativus L.). Journal of Medicinal Plants 8: 98-109. (In Persian with English Summary).
Ortega, L.M., Kikot, G.E., Astoreca, A.L., and Alconada, T.M. 2013. Screening of Fusarium graminearum isolates for enzymes extracellular and deoxynivalenol production. Journal of Mycology 358140: 1-7.
Phalip, V., Delande, F., Carapito, Ch., Goubet, F., Hatsch, D., Leize-Wagner, E., Dupree, P., Van Dorsselaer, A., and Jetsch, J.M., 2005. Diversity of the exoproteome of Fusarium graminearum grown on plant cell wall. Current Genetics 48: 366-379.
Rezvani Moghaddam, P., Khorramdel, S., Amin Ghafori, A., and Shabahang, J. 2013.  Evaluation of growth and yield of saffron (Crocus sativus L.) affected by spent mushroom compost and corm density. Saffron Agronomy and Technology 1: 13-26. (In Persian with English Summary).
Saeedizadeh, A. 2014. Identification of some saffron corm rot fungi and their control. Saffron Agronomy and Technology 3: 205-213. (In Persian with English Summary).
Schilling, A.G., Moller, E.M., and Geiger, H.H. 1996. Polymerase chain reaction-based assays for speciesspecific detection of F. culmorum, F. graminearum and F. avenaceum. Phytopathology 86: 515-522.
Steinkellner, S., Mammerler, R., and Vierheilig, H. 2008. Germination of Fusarium oxysporum in root exudates from tomato plants challenged with different Fusarium oxysporum strains. European Journal Plant Pathology 122: 395-401.
Thakur, R.N., Singh, C., and Kaul, B.L. 1992. First report of corm rot in Crocus sativus L. Indian Phytopathology 45: 278-282.
Trane, U. 1990. Grouping Fusarium section Discolor isolates by statistical analysis of quantitative high performance liquid chromatographic data on secondary metabolite production. Journal of Microbiological Methods 12: 23-39.
Tsedaley, B., and Adugna, G. 2016. Detection of fungi infecting maize (Zea mays L.) seeds in different storages around jimma, southwestern Ethiopia. Journal of Plant Pathology and Microbiology 7: 338.
Vakalounakis, D.J. 1996. Root and stem rot of cucumber caused by Fusarium oxysporum f. sp. radicis-cucumerinum. Journal of Plant Disease 81: 313-316.
Voigt, C.A., Schäfer, W., and Salomon, S. 2005. A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals. The Plant Journal 42: 364-375.
Wani, A. 2004. Studies on corm rot of saffron (Crocus sativus L.). PhD thesis, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, India, 108 p.
Wani, M.A.S., and Dar, G. 2004. Studies on Corm Rot of Saffron (Crocus sativus L.). University of Kashmir.
Withanage, G.S., Murata, H., Koyama, T., and Ishiwata, I. 2001. Agonistic and antagonistic effects of zearalenone, an etrogenic mycotoxin, on SKN, HHUA, and and HepG2 human cancer cell lines. Veterinary and Human Toxicology 43: 6-10.
Wood, T.M., and Bhat, M. 1998. Methods for measuring cellulase activities. Methods in Enzymology 160: 87-112.
Yilmaz, A., Nyberg, N.T., Mølgaard, P., Asili, J., and Jaroszewski, J.W. 2010. 1H NMR metabolic fingerprinting of saffron extracts. Metabolomics 6: 511-517.
Zhao, Z., Liu, H., Wang, C., and Xu, J.R. 2013. Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi. BMC Genomics 14: 274.