شناسايي مولكولي قارچ هاي توليد كننده ۸-كتو تريكوتسين همراه با بذر روناس و رديابي ژن هاي سنتزكننده دي اكسي نيوالنول در آنها با استفاده از PCR

Molecular Identification of 8-ketoTrichothecene Producing Fungi in Madder Seeds and Detection of Their Deoxynivalenol Synthesis Gene Using PCR

روناس (Rubia tinctorum) يكي از محصولات مهم زراعي با مصارف دارويي و صنعتي در استان يزد مي باشد. به منظور شناسايي قارچ هاي همراه با بذور روناس و توليد كننده توكسين، طي سال هاي زراعي ۹۱ لغايت ۹۳ از بذور روناس در مناطق مهم كاشت آن در استان يزد شامل اردكان و بافق نمونه برداري به عمل آمد. پس از كشت و خالص سازي جدايه هاي قارچي در محيط هاي كشت هاي سيب زميني دكستروز آگار (PDA) و برگ ميخك آگار (CLA)، شناسايي تكميلي با آغازگرهاي اختصاصي هر گونه با استفاده از واكنش زنجيره اي پليمراز صورت گرفت. رديابي قارچ هاي داراي پتانسيل توليد مايكوتوكسين ۸-كتوتريكوتسين از جمله دي اكسي نيوالنول (DON) از طريق آغازگرهاي اختصاصي ژن Tri۱۳ انجام شد. جهت تاييد پتانسيل توليد مايكوتوكسين DON در جدايه هاي فوزاريوم از كروماتوگرافي مايع با كارايي بالا (HPLC) استفاده شد. در مجموع ۲۴۹ جدايه قارچي از بذور روناس جداسازي شد كه متعلق به شش جنس قارچي شامل فوزاريوم، آسپرژيلوس، پنيسليوم، آلترناريا، رايزوكتونيا و رايزوپوس بودند كه بيشترين فراواني قارچ هاي جداسازي شده مربوط به فوزاريوم با ۷۱ درصد كل جدايه ها بود. در بين قارچ هاي فوزاريوم، گونه هاي F.solani (۵۵ جدايه) و F.oxysporum (۴۱ جدايه) بيشترين فراواني را داشتند. گونه هاي F. poae، F. semitectum و F. equiseti توانايي توليد دي اكسي نيوالنول را دارا بودند. ژن Tri۱۳موثر در توليد دي اكسي نيوالنول نيز در اين سه گونه رديابي گرديد و در آزمايش HPLC همه قارچ هاي فوزاريوم مورد بررسي پتانسيل توليد مايكوتوكسين مورد نظر را داشتند.

Introduction: Madder plant with the scientific name of Rubia tinctorum (a member of Family Rubiaceae) is one of the most important crops used for medical and industrial applications, is widely cultivated in Yazd province, Iran. Madder root is widely applied in the dyeing industry. Cultivation of these plants is rather common in Europe and Asia. In Iran madder was cultivated in ancient times in Tabriz, Urmia, Arak, Yazd, and Fars but nowadays it is cultivated mainly in different cities in Yazd province especially in Bafq and Ardakan. The madder plant's roots contain different polyphenolic compounds, such as 1,3-Dihydroxyanthraquinone (purpuroxanthin), 1,4-Dihydroxyanthraquinone (quinizarin), 1,2,4-Trihydroxyanthraquinone (purpurin) and 1,2- dihydroxyanthraquinone (alizarin). The madder can grow up to 1.5 m in height. Their leaves produced in whorls of 4-7 star like around the central stem with approximately 5-10 em long and 2-3 em wide. Its flowers are small with five pale yellow petals in dense racemes followed by red to blackberries. The roots can be over a meter long, up to 12 mm thick and the source of red dyes known as rose madder. Several factors such as fungal diseases during planting and harvesting period infect madder plants. These factors, in addition to causing damage to madder plants reduce the quality and quantity of color obtained from roots. Many of these fungi are associated with madder seeds and infect plant after cultivation. Some fungi infect leaves and stems of madder. Some of these fungi such as Fusarium, Aspergillus, and Penicillium are associated with madder seeds and in addition to a role as phytopathogenic fungi produce dangerous mycotoxin effective on color quality of madder. So far from the aerial parts of the madder plants such as leaves and stems, several fungi were reported. Septoriarubiae and Sporonema punctiforme were reported as leaf spot agents. Furthermore, P/eospora ch/amydospora and Sporonea punctiforme were isolated from Rubia spp. Fusarium species is one of the most abundant fungi that are able to produce hazardous mycotoxin especially trichothecenes in the different growth stage of Madder. Fusarium mycotoxins are among the main fungal mycotoxin contaminations in food and feed, and some human diseases, such as Kashi neck diseases and esophageal cancer, have been epidemiologically associated with consumption of trichothecenes. The most predominant trichothecenes found in crops are 8-keto trichothecenes (type B trichothecenes). Based on the production and chemical structures of different 8-keto trichothecenes, several trichothecene mycotoxins were identified that includes nivalenol (NIV), 4- acetylnivalenol ( 4-AcNIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON) and 15-acetyldeoxynivalenol (15-AcDON). There is a little knowledge about seed madder microtlora and given that the fungi with madder seeds can reduce the power of viability, seedling and ultimately damage the quality and quantity of products, the present study was aimed to the identification of fungi associated with seed madder and also fungi toxin-producing especially Fusarium species. Materials and Methods: During 2012, sampling was carried out form seeds of madder in important planting areas in Yazd province, including Bafq and Ardakan. After culturing, fungal isolate was purified in potato dextrose agar (PDA), carnation leaf agar (CLA), and CY20S (Czapek Yeast Extract Agar with 20 sucrose) media. Fusarium, and Aspergillus species were identified by valid keys identification. The Fusarium isolates were grown in liquid potato dextrose broth medium and total genomic DNA was extracted using the CTAB method. Molecular identification ofF equiesti, F. semitectum and F poae was performed using specific-species primers FEFI/2, SEM-1/2, and FpoF/R. Detection of fungi mycotoxins producing potential such as Deoxynivalenol (DON) using Tri 13 primers was done. High-performance liquid chromatography (HPLC) was used to confirm the produce NIV mycotoxins potential in Fusarium species. Results and Discussion: 249 fungal strains were isolated from madder seed belonging to 6 genera of fungi including Fusarium spp., Aspergillus spp., Penicillium spp., Alternaria spp., Rhizuctonia so/ani and Rhizopus spp., which Fusarium isolates with 71 percent was the most frequency among fungi isolated. Among Fusarium fungi isolated, F.solani (55 isolates) and F.oxysporum (41 isolates) were the most frequency. F. poae, F semitectum, and F. equiseti ability to produce mycotoxins such as Deoxynivalenol (DON) that are harmful to human health and animals as well as the effect on the yield of madder. Tri 13 gene involved in production DON was detected in three Fusarium species (F. poae, F. semi tectum and F. equiseti). The results of HPLC showed that all studied Fusarium fungi, have the potential to produce DON mycotoxins. The results of this study showed that fungi associated with seeds madder are able to produce 8-keto trichothecene mycotoxins that they can be dangerous for consumers. Conclusions: Given that, this is the first report of fungi mycotoxins producing on seeds madder in Yazd province, thus should be measured to control and reduce fungal agents in these products.