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Pdf 387.79 K رﺳﺘﻨﻴﻬﺎ Rostaniha 11(2): 151-162 (2010) ( 1389 ) 151 -162 :( 11)2 Molecular phylogeny of three desert truffles from Iran based on ribosomal genome ﻓﻴﻠﻮژﻧﻲ ﻣﻮﻟﻜﻮ ﻟﻲ ﺳﻪ ﮔﻮﻧﻪ دﻧﺒﻼن ﻛﻮﻫﻲ از اﻳﺮان ﺑﺮاﺳﺎس ژﻧﻮم رﻳﺒﻮزوﻣﻲ درﻳﺎﻓﺖ /2/8: 1389 / ﭘﺬﻳﺮش /Received: 26.04.2010 / Accepted: 23.10.2010 1389 :1/8 . رﺿﺎ ﻣﺴﺘﻮﻓﻲ زاده ﻗﻠﻤﻔﺮﺳﺎ : اﺳﺘﺎدﻳﺎر ﺑﺨﺶ ﮔﻴﺎه ﭘﺰﺷﻜﻲ، ,.R Mostowfizadeh-Ghalamfarsa : Assistant Prof Department of Plant Protection, School of داﻧﺸﻜﺪه ﻛﺸﺎورز ي، داﻧﺸﮕﺎه ﺷﻴﺮاز، ﺑﺎﺟﮕﺎه، ﺷﻴﺮاز، Agriculture, Shiraz University, Bajgah, Shiraz 7144133111, Iran ﻛﺪ ﭘﺴﺘﻲ E-mail: [email protected]) .7144133111) (E-mail: [email protected]) S. Jamali: Ph.D. student,, Department of Plant ﺻﻤﺪ ﺟﻤﺎﻟﻲ : داﻧﺸﺠﻮي دﻛﺘﺮي ﺑﺨﺶ ﮔﻴﺎه ﭘﺰﺷﻜﻲ، داﻧﺸﻜﺪه Protection, School of Agriculture, Shiraz ﻛﺸﺎورزي، داﻧﺸﮕﺎه ﺷﻴﺮاز، ﺑﺎﺟﮕﺎه، ﺷﻴﺮاز، ﻛﺪ ﭘﺴﺘﻲ University, Bajgah, Shiraz 7144133111, Iran Z. Banihashemi: Prof., Department of Plant .7144133111 Protection, School of Agriculture, Shiraz ﺿﻴﺎءاﻟﺪﻳﻦ ﺑﻨﻲ ﻫﺎﺷﻤﻲ: اﺳﺘﺎد ﺑﺨﺶ ﮔﻴﺎه ﭘﺰﺷﻜﻲ، داﻧﺸﻜﺪه University, Bajgah, Shiraz 7144133111, Iran ﻛﺸﺎورزي، د اﻧﺸﮕﺎه ﺷﻴﺮاز، ﺑﺎﺟﮕﺎه، ﺷﻴﺮاز، ﻛﺪ ﭘﺴﺘﻲ Abstract The ITS region including the 5.8S gene of rDNA .7144133111 ﭼﻜﻴﺪه of three desert truffle species were amplified using ITS4 ﻧﻤﻮﻧﻪ ﻫﺎﻳﻲ از ﺳﻪ ﮔﻮﻧﻪ دﻧﺒﻼن ﻛﻮﻫﻲ از ﻣﻨﺎﻃﻖ ﻣﺨﺘﻠﻒ and ITS1 primers. The ITS sequences were compared to ﺟﻐﺮاﻓﻴﺎﻳﻲ اﻳﺮان ﺟﻤﻊ آوري ﺷﺪ . ﻣﻨﻄﻘﻪ ITS ﺷﺎﻣﻞ ژن S 5/8 از those of other related authentic sequences obtained from GenBank. Among 12 specimens studied, seven isolates rDNA ﺑﺎ اﺳﺘﻔﺎده از آﻏﺎزﮔﺮﻫﺎي ﻋﻤﻮﻣﻲ ITS4 و ITS1 ﺗﻜﺜﻴﺮ و corresponded to Terfezia claveryi reported by other ﺗﻮاﻟﻲ ﻳﺎﺑﻲ ﺷﺪ . ﺗﻮاﻟﻲ ﻫﺎي ﺑﻪ دﺳﺖ آﻣﺪه ﺑﺎ ﺳ ﺎﻳﺮ آراﻳﻪ ﻫﺎي ﻣﻌﺘﺒﺮِ authors. Iranian T. claveryi specimens had an average ﻣﺴﺘﺨﺮج از ﺑﺎﻧﻚ ژن ﻣﻘﺎﻳﺴﻪ .ﺷﺪ در ﻣﻴﺎن 12 ﻧﻤﻮﻧﻪ ﻣﻄﺎﻟﻌﻪ ﺷﺪه، similarity of 99.4% (range 98.7–100%) among ﻫﻔﺖ ﺟﺪاﻳﻪ ﺑﺎ ﻧﻤﻮﻧﻪ ﻫﺎي Terfezia claveryi ﮔﺰارش ﺷﺪه ﺗﻮﺳﻂ ﺳﺎﻳﺮ themselves, while all T. claveryi sequences analyzed had ﻣﺤﻘﻘﺎن، ﻣﻄﺎﺑﻘﺖ داﺷﺖ . ﻧﻤﻮﻧﻪ ﻫﺎي T. claveryi اﻳﺮاﻧﻲ ﺑﻪ ﻃﻮر an average of 95.2% (range 87.2–100%) similarity. Four ﻣﻴﺎﻧﮕﻴﻦ /4 99 % ( ﺑﺎ داﻣﻨﻪ /7 -98 100 %) ﺑﺎ ﻳﻜﺪﻳﮕﺮ ﺗﺸﺎﺑﻪ داﺷﺘﻨﺪ، specimens corresponded to Trimania pinoyi , being a در ﺣﺎﻟﻲ ﻛﻪ ﻣﻴﺎﻧﮕﻴﻦ ﺗﺸﺎﺑﻪ ﻛﻠﻴﻪ T. claveryi ﻫﺎي ﻣﻮرد ﻣﻄﺎﻟﻌﻪ sister taxon to Tirmania nivea , of which only one /2 95 % ( ﺑﺎ داﻣﻨﻪ /2 -87 100 %) ﺑﻮد . ﭼﻬﺎر ﻧﻤﻮﻧﻪ ﻣﻄﺎﺑﻖ ﺑﺎ specimen could be studied. Iranian T. pinoyi specimens Trimania pinoyi ، ﺑﻪ ﺻﻮرت آراﻳﻪ ﺧﻮاﻫﺮي Tirmania nivea ﺧﻮد را had an average of 99.9% similarity (range 99.8–100%) ﻧﺸﺎن دادﻧﺪ ﻛﻪ از ﮔﻮﻧﻪ اﺧﻴﺮ ﺗﻨﻬﺎ ﻳﻚ ﻧﻤﻮﻧﻪ ﻳﺎﻓﺖ ﺷﺪ . ﻧﻤﻮﻧﻪ ﻫﺎي (among themselves, and 97.2% (range 93.1–100% T. pinoyi اﻳﺮان داراي /9 99 % ﻣﻴﺎﻧﮕﻴﻦ ﺗﺸﺎﺑﻪ ( ﺑﺎ داﻣﻨﻪ between all T. pinoyi sequences compared. All T. nivea /8 -99 100 %) در ﺑﻴﻦ ﺧﻮد و /2 97 % ( ﺑﺎ داﻣﻨﻪ /sequences, including Iranian specimen, had an average of (% 100 93- 1 ﻣﻴﺎﻧﮕﻴﻦ ﺗﺸﺎﺑﻪ در ﺑﻴﻦ ﻛﻠﻴﻪ ﺗﻮاﻟﻲ ﻫﺎي ﻣﻮرد ﻣﻘﺎﻳﺴﻪ ﺑﻮدﻧﺪ . ﻛﻠﻴﻪ similarity (range 92.2–99.8%). T. claveryi and 96.6% T. nivea ﺗﻮاﻟﻲ ﻫﺎي ، از ﺟﻤﻠﻪ ﻧﻤﻮﻧﻪ اﻳﺮان داراي ﻣﻴﺎﻧﮕﻴﻦ ﺗﺸﺎﺑﻪ T. pinoyi are widespread in the country and are mostly /6 96 % ( ﺑﺎ داﻣﻨﻪ /2 /8-92 99 %) ﺑﻮدﻧﺪ . T. claveryi و T. pinoyi در .associated with Helianthemum salicifolium and Carex sp ﻗﺴﻤﺖ ﻫﺎي ﻣﺨﺘﻠﻒ ﻛﺸﻮر ﻳﺎﻓﺖ ﺷﺪه، اﻏﻠﺐ ﻫﻤﺮاه ﺑﺎ Iranian truffles flora resembles those of Mediterranean Helianthemum salicifolium و .Carex sp ﺑﻮدﻧﺪ . ﻓﻠﻮر دﻧﺒﻼن ﻫﺎي اﻳﺮاﻧﻲ ﺷﺒﻴﻪ ﺑﻪ .region and South coast of the Persian Gulf دﻧﺒﻼن ﻫﺎي ﻣﺪﻳﺘﺮاﻧﻪ و ﻛﺮاﻧﻪ ﻫﺎي ﺟﻨﻮﺑﻲ ﺧﻠﻴﺞ ﻓﺎرس اﺳﺖ . واژه ﻫﺎي ﻛﻠﻴﺪي: آﺳﻜﻮﻣﻴﻜﻮﺗﺎ، ﻓﻴﻠﻮژﻧﻲ، ﻗﺎرچ رﻳﺸﻪ ﻫﺎي ﺑﺮوﻧﻲ، Keywords: Ascomycota , Phylogenetics, ectomycorrhizal fungi, internal transcribed spacer of rDNA ﺗﻮاﻟﻲ ﻫﺎي ﺟﺪاﻛﻨﻨﺪه ﻧﺴﺨﻪ ﺑﺮداري ﺷﺪه داﺧﻠﻲ 152 / 152 Mostowfizadeh-Ghalamfarsa et al. / Molecular phylogeny of three desert truffles from Iran …/ Rostaniha 11(2), 2010 Introduction Desert truffles are hypogeous ascocarps of morphological characters of hypogeous Ascomycetes can Ascomycetes occurring in arid and semi-arid ecosystems be unreliable (Ferdman et al. 2005). Other molecular of Mediterranean region and Middle Eastern countries studies on truffles have led to the reclassification of some (Díez et al. 2002). They belong to Pezizales (Trappe genera or species (O’Donnell et al. 1997, Hansen et al. 1979) and mostly the genera Terfezia and Tirmania 2001). (Morte & Honrubia 1995, Moawad et al. 1997, Shavita Almost all molecular studies on truffle & Volk 2007). However, there are other truffle genera in identification were based on comparison of sequences or Pezizales such as Picoa and Mattirolomyces which restriction fragment length polymorphisms (RFLPs) of occasionally could be found in these regions (Alsheikh & the internal transcribed spacers (ITS) of rDNA (e.g. Trappe 1983, Moreno et al. 2000, 2001). Apart from Henrion et al. 1994, Paolocci et al. 1995, 1999). The these genera, some other desert truffles inhabit in semi- major phylogenetic studies on Pezizales (Spatafora 1995, arid ecosystems of other continents. For instance, Landvik et al. 1997, Harrington et al. 1999), specially Mycoclelandia with two species is a part of Australian truffles (O’Donnell et al. 1997, Roux et al. 1999, Díez mycoflora (Trappe & Beaton 1984) and the genera et al. 2002, Ferdman et al. 2005) were also rooted in Kalaharituber and Eremiomyces were reported from these regions. Different parts of the rRNA genes present South Africa (Ferdman et al. 2005). different levels of conservation which can be exploited to These fungi are economically and ecologically analyze any desired phylogenetic level (Hillis & Dixon valuable (Halasz et al. 2005). They are considered to be 1991). For example, the monophyletic origins of the one of the oldest human foods in their origin countries, genera Terfezia and Tirmania are illustrated through appreciated for their nutritional value and as flavouring analysis of ITS region (Díez et al. 2002). (Hussain & Al-Ruqaie 1999), and also antimicrobial Many researchers have studied various aspects of effects, especially in treating skin and eye desert truffles in the Mediterranean region and the diseases (Janakat et al. 2004). Additionally, these Middle East (Morte & Honrubia 1995, Moawad et al. ectomycorrhizas promote plants growth and productivity 1997, Hussain & Al-Ruqaie 1999) but no such studies through different mechanisms such as enhancement of have been carried out in Iran. Few older studies, report nutrient uptake, production of growth hormones and the presence of truffles in various parts of Iran (Chatin protection of plants rhizosphere against soil-borne 1897, Petrak & Esfandiari 1941). A recent morphological diseases (Hayman 1983, Smith & Read 1997, Buscot study reported the collection of Tirmania pinoyi and et al. 2000). Desert truffles establish ectomycorrhizal Terfezia leonis in Fars, Hormozgan and E Azerbaijan symbioses with members of the Cistaceae , mainly provinces adjacent to Artemisia and Helianthemum Helianthemum species (Awameh 1981, Morte & species (Daneshpazhuh 1991). Unfortunately the Honrubia 1995, Morte et al. 2000, Kovacs et al. 2003, locations of the collected samples were not specified. Dikie et al. 2004). Terfezia boudieri also has been reported from Tarom in Morphological characters have been used to Zanjan province mostly associated with Carex species describe different species of truffles including spore size (Ammarellou et al. 2006) but also Kobresia bellardii and ornamentation, peridium morphology, gleba colour, (Ammarellou & Saremi 2008). However, the accuracy of and organoleptic characters (Trappe 1979, Moawad et al. the species identification merely on the basis of 1997). Due to limited morphological characters, these morphological features is questionable. fungi are difficult to identify at species level. Molecular In the present study we attempted to identify phylogenetic studies have recently demonstrated that desert truffles from various geographical regions of Iran, Mostowfizadeh-Ghalamfarsa et al. / Molecular phylogeny of three desert truffles from Iran …/ Rostaniha 11(2), 2010 153 / 153 extending from Sistan & Baloochestan (adjacent to Materials and Methods Pakistan) to E Azerbaijan provinces (adjacent to Turkey), - Organisms and specimen's conditions using morphological observation and molecular assays. The origins of fruiting bodies studied are listed in This study also reports the phylogenetic relationships of Table 1. Several visits were made to the truffle-bearing different species of the Iranian desert truffles based on areas of Iran during the months from February to May ribosomal RNA genome. This phylogenetic study could 2007–2009. Voucher specimens deposited in fungal provide a basis for designing molecular detection and collection of the Department of Plant Protection, Shiraz identification tools, which in turn could be used in University, Shiraz, Iran. Fresh samples were used for laboratory, soil, and symbiont rhizosphere of these cultivation and the rest were kept at –20 ° C for further truffles for biological studies. studies. Table 1. Origins of Iranian specimens studied and their GenBank sequence accession numbers for ITS sequences Specimen Year Province Locality Species GenBank Sa1 2007 Fars Sarvestan Terfezia claveryi EU519461 Si2 2007 Kerman Sirjan T. claveryi FJ197819 Sh1 2006 Fars Shiraz T. claveryi GQ888691 Fa1 2006 Fars Fasa, Mianjangal T. claveryi GQ888693 Da1 2006 Fars Darab, Ghale-Biaban T. claveryi GQ888694 Kh1 2009 Sistan & Baloochestan Khash T. claveryi GQ888692 Tb1 2009 E Azerbaijan Tabriz T. claveryi GQ888690 La1 2007 Fars Lar Tirmania pinoyi GQ231540 Kh2 2009 Sistan & Baloochestan
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