بررسی تأثیر عصاره‌های سیر و آویشن شیرازی بر روی بیماری کپک سبز (Penicillium digitatum) و بعضی از مکانیسم‌های دفاعی در پرتقال

نوع مقاله: علمی پژوهشی-فارسی

نویسندگان

1 استادیار دانشگاه اردکان

2 دانشجوی کارشناسی‌ارشد دانشگاه آزاد اسلامی واحد ورامین

3 استادیار دانشگاه آزاد اسلامی واحد ورامین

چکیده

استفاده از سموم شیمیایی در طی دهه ­ها باعث ایجاد آلودگی محیط ­زیست و همچنین ایجاد باقیمانده بر روی محصولات غذایی شده است. در این پژوهش از عصاره­ های آبی و متانولی آویشن ­شیرازی (Zataria multiflora Boiss.) و سیر (Allium sativum L.)جهت کنترل بیماری کپک سبز پرتقال با عامل .Penicillium digitatum Sacc، در آزمایشگاه و در انبار 15 درجه سلسیوس استفاده شد. نتایح آزمون دیسک­ گذاری در محیط کشت نشان داد که، عصارۀ آبی و الکلی سیر (غلظت 60 میلی ­گرم در میلی ­لیتر)با 26 و 32 میلی‌متر قطر ممانعت از قارچ بیمارگر، دارای بیشترین اثر کنترل‌ کنندگی و عصارۀ آبی و الکلی آویشن شیرازی (غلظت 15 میلی ­گرم در میلی ­لیتر) با 10 و 14 میلی‌ متر قطر ممانعت از قارچ بیمارگر، دارای کمترین اثر کنترل ­کنندگی بود. در آزمون اختلاط با محیط کشت، عصاره­ های آبی و الکلی سیر در غلظت­ 600 میلی ­گرم در لیتر به ترتیب با 25/65 و 26/75 درصد ممانعت از قارچ بیمارگر بهترین کنترل‌ کنندگی بیمارگر را نشان دادند. در آزمون انبار 15 درجۀ سلسیوس، سطح لکۀ ایجاد شده در تیمار عصاره­ های آبی و الکلی سیر و آویشن شیرازی با غلظت­ شش در هزار، cm2 30/5 (آبی) و cm226/3 (الکلی) برای سیر، cm2 44/7 (آبی) و  cm215/4 (الکلی) برای آویشن شیرازی، کمترین میزان خود را در مقایسه با سایر تیمارها وهمچنین تیمار شاهد (با میزان cm247/30) داشتند. نتایج بررسی فعالیت آنزیم‎ های پراکسیداز و پلی ­فنل ­اکسیداز نشان داد که بیشترین فعالیت هر دو آنزیم نه روز بعد از نمونه برداری و در تیمار شش در هزار عصارۀ سیر مشاهده شد. میزان نسخه­ برداری سه ژن کدکنندۀ آنزیم ­های پراکسیداز، پلی ­فنل ­اکسیداز و فنیل ­آلانین ­­آمونیالیاز روندی مانند میزان فعالیت سه آنزیم در طول 12 روز نمونه برداری داشت و در روز نهم، بیشترین میزان نسخه ­برداری این سه آنزیم به ترتیب با مقدار عددی 23/15، 80/11 و 36/12 برابر شاهد سالم بودند. بر اساس نتایج آزمون ­های آزمایشگاهی و انباری، عصارۀ هر دو گیاه سیر و آویشن شیرازی به عنوان ترکیبات طبیعی با پتانسیل کنترل‌ کنندگی معرفی می ­شوند.

کلیدواژه‌ها


عنوان مقاله [English]

The investigation of the effect of garlic and thyme extracts on orange green mold (Penicillium digitatum), defense enzymes and genes expression

نویسندگان [English]

  • J. Gholamnezhad 1
  • Sh Arsalani 2
  • M. Maleki 3
1 Assisstant professor, Ardakan University
2 M.Sc. Student, Islamic Azad University of Varamin-Pishva
3 Assisstant professor, Islamic Azad University of Varamin-Pishva
چکیده [English]

Background and Objectives
The use of chemical pesticides has caused environmental hazards as well as the creation of residues on food products. In this research, the extracts of Zataria multifloraBoiss. and Garlic (Allium sativum L.) were used for controlling the green mold of orange caused by Penicillium digitatum, invitro and invivo at 15 ̊C.
Materials and Methods
In this study, the effect of plant extracts against the pathogen was evaluated using two methods, included the paper disc and mixing with culture media. Then, the effect of these extracts on the fruit was studied against the pathogen. The level of activity of the enzymes including peroxidase, catalase and polyphenol oxidase, as well as total phenol content were measured. Finally, the expression of catalase, peroxidase and polyphenol oxidase genes was evaluated using the Real time PCR method.
Results
The results of disc test showed that aqueous and alcoholic extracts of garlic had the highest effect on fungal mycelium growth with 60 mg at ml concentration and 32.20 mm and 26.12 mm diameter of inhibition zone respectively. The mixing culture test showed that aqueous and alcoholic extracts of garlic with 65.25 and 75.26 percent of the inhibitory effect on the pathogen, respectively, showed the best control compared to the control in 600 mg at L concentration. The results of invivo assays indicated that 6×1000 concentration of aqueous and alcoholic garlic and thyme extracts had the lowest of decay area with 5.3 and 3.26 cm2 respectively (for garlic) and 7.44 and 4.15 cm2 for thyme. Results of enzyme activity of peroxidase and polyphenol oxidase showed that the highest activity of both enzymes was 9 days after sampling and in the treatment of 6×1000 garlic extract.
Discussion
Based on the results of laboratory and storage tests, the extract of both garlic and thyme are introduced as natural constituents with controlled potentials. The genes expression levels of enzymes as peroxidase, polyphenol oxidase, and phenylalanine ammonialis had the same trend to the activity of two enzymes over a period of 12 days. Based on the results of invivo and invitro tests, the extract of garlic and thyme are introduced as natural compounds with high controlling potentials. 

کلیدواژه‌ها [English]

  • Plant extracts
  • Green mold
  • Defense enzymes activity
  • Defense gene expression
Abdolmaleki, M., Bahraminejad, S., Abassi, S., and Mahmodi, SB. 2010. Inhibitory effect of some plant extracts on mycelia growth of Rhizoctonia solani and Phytophthora drechsleri, sugar beet root rot agent. Journal of Sugar Beet, 5:193- 205.
Abdul Aziz, H., Omran, A., and Zakaria, W.R. 2010. H2O2 oxidation of pre-coagulated semi aerobic leachate. International Journal of Environmental Research, 4 (2), 209-216.
Agrios, G.N. 2005. Plant disease caused by fungi: disease caused by Ascomycetes and imperfect fungi. Science, 25th, 922. 
Alam, P., Mohammad, A., Ahmad, M.M., Khan, M.A., Nadeem, M., Khan, R., Akmal, M., Ahlawat, S., Abdin, M.Z., 2014. Efficient method for Agrobacterium mediated transformation of Artemisia annua L. Recent Patents Biotechnology, 8: 102-107.
Amini, M., Safaie, N., Salmani, M.J. and Shams-Bakhsh, M., 2012. Antifungal activity of three medicinal plant essential oils against some phytopathogenic fungi. Trakia Journal of Sciences, 10: 1-8.
Arras, G. 1996. Inhibitory action of microorganisms isolated from citrus fruit against Penicillium digitatum. Proceeding of the International Society of Citriculture, 456-460.
Arras, G. and Vsai, M. 2001. Fungitoxic activity of 12 essential oils against four postharvest citrus pathogens: chemical analysis of Thymus capitates oil and its effect in subatomospheric pressure condition. Journal of Food Protection, 64:1025-1029.
Azimi, A.A., Delnavaz H.B. and Mansour G.A. 2006. Antifungal effect of aqueous alcoholic and phenolic extracts of seed and leaves of Sorghum bicolor against Fusarium solani, Fusarium poae. Journal of Medicinal Plants, 6 (1): 26 – 32. (in Persian with English abstract)
Bahraminejad, S., Asenstorfer, R.E., Riley, I.T. and Schultz, C.J. 2008. Analysis of the antimicrobial activity of flavonoids and saponins isolated from the shoots oats (Avena sativa L.). Journal of Phytopathology, 156: 1-7.
Bakkali, F., Averbeck, S. and Averbeck, D. 2008.  Idaomar M. Biological effects of essential oils. A review. Food and Chemical Toxicology, 46: 446–475.
Barnett, H.L. and Hunter, B.B., 1998. Illustrated genera of imperfect fungi. Published by Amer Phytopathological Society.
Batta, Y.A. 2004. Effect of treatment with Trichoderma harzianum Rifai formulated in invert emulsion on postharvest decay of apple blue mold. International Journal of Food Microbiology, 96 (3): 281−288.
Belewu, M.A., Olatunde, O.A. and Giwa, T.A. 2009. Underutilized medicinal plants and spices: chemical composition and phytochemical properties. Journal of Medicinal Plant Research, 3(12): 1099 -1103. 
Boudet, A. 2007. Evolution and current status of research in phenolic compounds. Phytochemistry, 68, 2722–2735.
Bradford, M.M. 1976. A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
Burt, S. 2004. Essential oils: their antibacterial properties and potential applications in foods - a review. International Journal of Food Microbiology, 94: 223– 253.
Chaieb, K., Hajlaoui, H., Zmantar, T., Kahla-Nakbi, A.B., Rouabhia, M., Mahdouani, K. and Bakhrouf, A. 2017. The chemical composition and biological activity of clove essential oil Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytotherapy Research, 21: 501-506.
Cowan, M.M. 1999. Plant products as antimicrobial agents. Clinical Microbioligy Reviews, 12: 564 - 82.   
Droby, S., Vinokur, V., Weiss, B., Cohen, L., Daus, A., Gold-Schmidt, E.E. and Porat, R. 2002. Introduction of resistance to Penicillium digitatum in grapefruit by the yeast biocontrol agent Candida oleophila. Phytopathology, 92:393-399.
El-Ghaouth, A., Wilson, C.L. and Wisniewski, M.  1998. Ultrastructural  and      cytochemical  aspects  of  the biological  control  of  Botrytis cinerea by Candida  saitoana in apple fruit.  Phytopathology, 88: 282-291.
Ellis, M.B., Walker, J.M. 1974. Sclerotinia fuckelina (conidial state: Botrytis cinerea). CMI Descriptions of pathogenic fungi and bacteria. No. 431, CMI, Kew, Surrey, England.
Etebarian, HR, Sholberg, PL, Eastwell, KC, Sayler, RJ. 2005a. Biological control of apple blue mold with Pseudomonas fluorescens. Microbiology, 51: 591-598.
Etebarian, H.R.,  Sholberg,  P.L.,  Eastwell,  K.C. and Sayler,  R.J. 2005b. Biological control of apple blue mold with Pseudomonas fluorescens.  Canadian Journal of Microbiology, 51: 591-598.
Farhang, V., Amini, J. and Javadi, T. 2015. The effect of garlic essential oil plants, Mancozeb and Metalaxyl-Mancozeb on damping-off diseases caused by Phytophthoraspp. Biological Control of Pests and Plant Diseases, 4 (1): 47-56.
Foroughi, M., Mohammadi, S. and Ghasemi, A. 2013.  Antifungal activity of five medical herbs on the plant pathogenic fungus Rhizoctonia solani. Journal of Microbial World, 5(4): 115-121.
Gholamnezhad, J., Etebarian, H. R. and Sahebani, N. 2010. Biological control of apple blue mold with Candida membranifaciens and Rhodotorula mucilaginosa. African Journal of Food Science, 4: 001-007.
Gholamnezhad, J., Etebarian, H.R.,  Roustaee, A. and Sahebani  N.  2009a. Biological control of apple blue mold by isolates of Saccharomyces cerevisiae.  Journal of Plant Protection Research, 49: 270-275.
Gholamnezhad, J., Etebarian, H.R., Sahebani, N.A. and Roustaee, A. 2009b. Characterization of biocontrol activity of two yeast strains from Iran against blue mould of apple in order to reduce the environmental pollution. Journal of International Environmental Application and Science, 4(1): 28-36.
Gholamnezhad, J. 2017. Effect of plant extracts against apple gray mold caused by Botrytis cinerea. Applied Microbiology In Food Industries, 3(1): 53-66.
Gholamnezhad, J., Sanjarian, F., Mohammadi goltapeh, E., Safaei, N. and Razavi, Kh. 2016a. Study of defense genes expression profile pattern of wheat in response to infection by Mycosphaerella graminicola. Iranian Journal of Plant Biology, 8(30): 43-55.
Gholamnezhad, J., Sanjarian, F., Mohammadi goltapeh, E., Safaei, N. and Razavi, Kh. 2016b. Effect of salicylic acid on enzyme activity in wheat in immediate early time after infection with Mycosphaerella graminicola. Plant Science, 47(1): 1-8.
Gholamnezhad, J., Sanjarian, F., Mohammadi goltapeh, E., Safaei, N. and Razavi, Kh. 2016c. Evaluation of housekeeping gene expression of wheat interaction against Mycosphaerella graminicola with Reverse northern dot blot method. Crop Biotechnology, 12:1-10. (In Farsi with English abstract).
Gong, M., Li, Y., Dai, X., Tian, M. and Li, Z. 1997. Involvement of calcium and calmodulin in the acquisition of HS inuced thermotolerance in maize seeding. Journal of Plant Physiology, 150: 615-621.
Hadian, S., Rahnama, K., Jamali, S. and Eskandari, A. 2011. Comparing Neem extract with chemical control on Fusarium oxysporum and Meloidogyne incognita complex of tomato. Advances in Environmental Biology, 5(8):2052-2057.
Ibtesam Badawy, F.M., Nashwa Sallam, M.A. Ibrahim A.R. and Asran, M.R. 2009. Efficacy of Some Essential Oils on Controlling Green Mold of Orange and their Effects on Postharvest Quality Parameters. Plant Pathology Journal, 10: 168-174.
Johnson, O.O., Ayoola, G.A. and Adenipekun, T. (2013). Antimicrobial activity and the chemical composition of the volatile oil blend from Allium sativum (Garlic Clove) and Citrus reticulate (Tangeeine Fruit). International Journal of Pharmaceutical Science and Drug Research, 5(4): 187-193.
Kamangar, H., Hemmati, R., Yazdinejad, A.R. and Movahedi Fazel, M., 2014. Study on antifungal effects of five plant species extracts against Fusarium solani and Rhizoctonia solani on bean. Iranian Plant Protection Science, 49: 49-58.
Khalil, A., and Dababneh, B.F.  2007. Inhibition of Phytopathogenic Fungi by Extracts from Medicinal Plants in Jordan. Journal of Biological Sciences, 7 (3): 579-581.
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.
Livak, K.L., and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 -∆∆CT Method. Methodes, 25: 402–408.
Martinez, M.C., Crozo, N. and Vilamiel M. 2007. Biological properties of onion and garlic. Trend in Food -Science and Technology, 18: 609-625.
Meliss, T.G.S., Sponia, M.S., Terezinha, G.F.M.B., Cardarelli, P. and Therezinha, C.B.T. 2005.  Studies on antimicrobial activity in vitro of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination. Mem inst Oswaldo Cruz Rio de Janerio, 100 (7): 779 - 82.
Mnayer, D., Sylvie, A., Tixier, F. and Petitcolas, E. 2014. Chemical composition, antibacterial and antioxidant activities of six essential oils from the Allium family. Molecules, 19: 20034-20053.
Nakhaei, M., Moin-Vaziri, V., Fata, A., Haghighi, A., Iranshahi, M., Abadi, A.R., Sartavi, K. and Jaffari, M.R. 2010. Effect of topical exudate and gel of Aloe vera in the course of Leishmanial major infection in susceptible Balb/C mice. thnational and the second regional congress of Parasitology and parasitic diseases in Iran. physical properties, Journal of Physics: Condensed Matter, 18: 35-66.
Nikpour, F., Mohammadi goltapeh, E. and Safaei, N. 2012. The effect of some essential oils of medicinal plant for control of Trichoderma disease of mushrooms. M.Sc. Thesis, Tarbiat Modares University.
Oxenham, S.K., Svoboda, K.P. and Walters, D.R. 2005. Antifungal Activity of leaf extracts and essential Oils of some medicinal plants against Didymella bryoniae.  Journal of Phytopathology, 148: 483-7.
Pitt, J. I. and Hocking, A. D. 1977. ‘Influence of solute and hydrogen ion concentration on the water relations of some xerophilic fungi’, Journal of General Microbiology, 101: 35- 40.
Prusky, D., Mcevoy, J. L., Saftner, R., Conway ,W. S. and Jones, R. 2004. Relationship between host acidification and virulence of Penicillium spp. on apple and citrus fruit. Phytopathology, 94: 44–51. 
Reuveni, R. 1995. Biochemical marker of disease resistance. p. 99–114. In: “Molecular Methods in Plant Pathology” (R.P. Singh, U.S. Singh, eds.). Boca Raton, CRC Press, Florida, USA. P. 507.
Razaghparast, A., Shams Ghahfarokh, M., Yadegari, M.H. and Razaghi Abyaneh, M. 2009. Antifungal effect of aqueous extract of garlic (Sativum allium) in isolation and in combination with fluconazole, israconazole and ketoconazole on pathogenic yeasts. Journal of Gorgan University of Medical Sciences, 11(1): 49-56. 
Shi, C., Dai, Y., Bingle, X., Xu, X., Xie, Y. and Liu, Q. 2001. The purification and spectral properties of polyphenol oxidase I from Nicotiana tabacum. Plant Molecular Biology Reporter, 19: 381–382.
Sirus, A. and Jamali Zavare, A.H. 2014. Effectiveness of celery leaf extract on the induction of resistance against cucumber Powdery Mildew. Plant Pathology, 50(2):151-161.
Taiz, L. and Zeiger, E. 2006. Plant Physiology. Sinauer Associates Inc. Sunderland, Massachusetts.USA. P. 690. 3rd edn.
Tripathi, P. and Dubay, N.K. 2004. Exploitation of natural products as an alternative sterategy to control postharvest fungal rotting of fruit and vegetables review. Postharvest Biology and Technology, 32: 235–245.
Viret, O., Keller, M., Jaudzems, V.G. and Cole, M. 2004. Botrytiscinerea infection of grape flowers: light and electron microscopical studies of infection sites. Phytopathology, 94(8): 850-857.
Vogt, T. 2010. Phenylpropanoid biosyntesis. Journal of Molecular Plant, 3: 2-20.
Wijewardane, R.M.N.A. and Guleria, S.P.S. 2009. Combined effect of Pre-cooling, application of natural extracts and packaging on the storage quality of Apple (Malus domestica) cv. Royal Delicious. Tropical Agricultural Research, 21: 10-20.
Wilkins, K.A., Bancroft, J., Bosch, M., Ings, J., Smirnoff, N. and Franklin-Tong, V.E. 2011. Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of Papaver. Plant Physiology, 156: 404–416. www. faostat.fao.org, 2015.
Yaghoubi, S. M. J., Ghorbani, G. R., Rahmani, H. R. and Nikkhah, A. 2007. In vitro manipulation of rumen fermentation by propolis flavonoids and monensin. Journal of Dairy Science, 90(Suppl. 1):105-106. (Abstract)
Zandi, Sh., Hemmati, R., Rezaee, S. and Movahedi Fazel, M. 2017. Study on the effect of some plant essential oils on two fungal causal agents of bean root rot, Fusarium solani and Rhizoctonia solani in Zanjan Province. Iranian Journal of Medicinal and Aromatic Plants, 33(3): 411-422.