ﺎﺳﺎﻨﺷ ﯽﯾ در وﯾﺑﺎﯽ ﺻﺎﺼﺘﺧا ﯽ Phytopthora Drechsleri
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ﺑﻴﻤﺎﺭﻱﻫﺎﻱ ﮔﻴﺎﻫﻲ / ﺟﻠﺪ ۵۱ / ﺷﻤﺎﺭﻩ ۴ / ﺳﺎﻝ ۱۳۹۴: ۵۵۳-۵۴۱ ﺷﻨﺎﺳﺎﯾﯽ و ردﯾﺎﺑﯽ اﺧﺘﺼﺎﺻﯽ P. cryptogea ،Phytopthora drechsleri و .P erythroseptica ﺑﺎ واﮐﻨﺶ زﻧﺠﯿﺮهاي ﭘﻠﯿﻤﺮاز 1 *1 رﺿﺎ ﻣﺴﺘﻮﻓﯽزاده ﻗﻠﻤﻔﺮﺳﺎ و ﺿﯿﺎءاﻟﺪﯾﻦ ﺑﻨﯽﻫﺎﺷﻤﯽ (ﺗﺎرﯾﺦ درﯾﺎﻓﺖ: 28/3/1394؛ ﺗﺎرﯾﺦ ﭘﺬﯾﺮش: 1394/10/8) ﭼﮑﯿﺪه ﮔﻮﻧﻪﻫﺎي P. cryptogea ،Phytophthora drechsleri و P. erythroseptica ﺑﯿﻤﺎرﮔﺮﻫﺎي ﮔﯿﺎﻫﯽ اُاُﻣﯿﺴﺘﯽ ﺧﻮﯾﺸﺎوﻧﺪﻧﺪ و از ﻧﻈﺮ رﯾﺨﺖﺷﻨﺎﺧﺘﯽ ﺑﻪ ﯾﮏدﯾﮕﺮ ﺷﺒﺎﻫﺖ دارﻧﺪ. ﺑﺮاي ﺗﻤﺎﯾﺰ اﯾﻦ آراﯾﻪﻫﺎ از ﯾﮏدﯾﮕﺮ و از ﺳﺎﯾﺮ ﮔﻮﻧﻪﻫﺎﯾﯽ ﮐﻪ ﺻﻔﺎت رﯾﺨﺖﺷﻨﺎﺧﺘﯽ ﻫﻤﮕﺮا دارﻧﺪ، ﺷﯿﻮهاي ﺑﺮ اﺳـﺎس واﮐﻨﺶ زﻧﺠﯿﺮهاي ﭘﻠﯿﻤﺮاز ﺳﺎده و ﺗﻮدرﺗﻮ اﺑﺪاع ﺷﺪ. ﺑﺪﯾﻦ ﻣﻨﻈﻮر ﻣﺠﻤﻮﻋﻪاي از ﺟﺪاﯾﻪﻫﺎ ﻣﺮﺑﻮط ﺑﻪ ﻣﯿﺰﺑﺎنﻫﺎي ﻣﺨﺘﻠﻒ، ﮐـﻪ ﻧﻤﺎ ﯾﻨـﺪه ي ﺗﻨـﻮع ﻣﻮﺟﻮد در ژنﻫﺎي ﻫﺴﺘﻪاي و ﻣﯿﺘﻮﮐﻨﺪرﯾﺎﯾﯽ اﯾﻦ ﮔﻮﻧﻪﻫﺎ ﺑﻮدﻧﺪ، ﺑﺮرﺳﯽ ﺷﺪ. ﺑﺮ اﺳﺎس ﺗﻮاﻟﯽ ﻓﻮاﺻﻞ ﺗﺮاﻧﻮﯾﺴﯽ ﺷﺪهي داﺧﻠـ ﯽ (آيﺗـ ﯽاس) و زﯾﺮواﺣﺪ 1 ﺳﯿﺘﻮﮐﺮوم اﮐﺴﯿﺪاز ﺳﯽ، ﺷﺶ ﻋﺪد آﻏﺎزﮔﺮِ واﮐﻨﺶ زﻧﺠﯿﺮهاي ﭘﻠﯿﻤـﺮاز اﺧﺘﺼﺎﺻـ ﯽ ﺑـﺮا ي P. drechsleri و ﻫﻤﭽﻨـ ﯿﻦ ﺑـﺮ اﺳـﺎس زﯾﺮواﺣﺪ 1 ﺳﯿﺘﻮﮐﺮوم اﮐﺴﯿﺪاز ﺳﯽ ﺳﻪ ﻋﺪد آﻏﺎزﮔﺮ اﺧﺘﺼﺎﺻﯽ ﺑﺮاي P. cryptogea و P. erythroseptica ﻃﺮاﺣﯽ و واﺳﻨﺠﯽ ﺷﺪ. واﮐﺎويﻫﺎ ﻧﺸﺎن داد ﮐﻪ ﺑﻬﺘﺮﯾﻦ ﻧﺎﻣﺰد ﺑﺮاي ﺷﻨﺎﺳﺎﯾﯽ ﺟﺪاﯾﻪﻫﺎي P. drechsleri ﻣﺠﻤﻮﻋﻪي ITS-DF2 و ITS-DR2 ﺑﻮد ﮐﻪ ﻣﺤﺼﻮﻟﯽ 567 ﺟﻔﺖ ﺑـﺎز ي را ﻓﺰونﺳﺎزي ﻣﯽﮐﺮد. اﺳﺘﻔﺎده از آﻏﺎزﮔﺮﻫﺎي COX-CF1 و COX-CR2 ﺑﻬﺘﺮﯾﻦ ﻣﺠﻤﻮﻋﻪ ﺑﺮاي ﺗﻤﺎﯾﺰ P. cryptogea/P. erythroseptica از ﺳـﺎ ﯾﺮ ﮔﻮﻧﻪﻫﺎ ﺑﻮد و ﻣﻮﺟﺐ ﻓﺰونﺳﺎزي ﻗﻄﻌﻪاي 415 ﺟﻔﺖﺑﺎزي ﺷﺪ. واﮐﺎوي ﻧﻘﺸﻪي آﻧﺰﯾﻢﻫﺎي ﺑﺮﺷﯽ اﯾﻦ دو ﮔﻮﻧﻪ ﻧﺸـﺎن داد ﮐـﻪ ﺟﺎ ﯾﮕـﺎه آﻧـﺰ ﯾﻢ ﺑﺮﺷﯽ ﻏﯿﺮﭘﺎﻟﯿﻨﺪروﻣﯽ Mnl I در ﻓﺰوﻧﻪي ﺟﺪاﯾﻪﻫﺎي P. erythroseptica ﻣﻨﺤﺼﺮ ﺑﻪ ﻓﺮد اﺳﺖ و از آن ﻣﯽﺗﻮان ﺑـﺮا ي ﺗﻤـﺎ ﯾﺰ اﯾـ ﻦ ﮔﻮﻧـﻪ از .P cryptogea اﺳﺘﻔﺎده ﮐﺮد. ﻧﺘﺎﯾﺞ اﯾﻦ ﻣﻄﺎﻟﻌﻪ ﻧﺸﺎن داد ﮐﻪ اﺳﺘﻔﺎده از واﮐﻨﺶ زﻧﺠﯿﺮهاي ﺗﻮدرﺗﻮ ﺑﺮاي ردﯾﺎﺑﯽ اﯾـ ﻦ ﮔﻮﻧـﻪ ﻫـﺎ ﺣـﺪاﻗﻞ 100 ﺑﺮاﺑـﺮ ﺣﺴﺎسﺗﺮ از روش ﺳﻨﺘﯽ اﺳﺖ. ﮐﻠﯿﺪواژه: Phytophthora erythroseptica ،Phytophthora cryptogea ،Phytophthora drechsleri، اُاُﻣﯿﮑﻮﺗﺎ، ﻓﺎﺻﻠﻪي ﺗﺮاﻧﻮﯾﺴﯽ ﺷﺪهي داﺧﻠﯽ، زﯾﺮواﺣﺪ 1 ﺳﯿﺘﻮﮐﺮوم اﮐﺴﯿﺪاز ﺳﯽ، ﺷﻨﺎﺳﺎﯾﯽ، ردﯾﺎﺑﯽ * ﻣﺴﺌﻮل ﻣﮑﺎﺗﺒﺎت، ﭘﺴﺖ اﻟﮑﺘﺮوﻧﯿﮑﯽ: [email protected] 1- ﺑﺨﺶ ﮔﯿ ﺎهﭘﺰﺷﮑﯽ داﻧﺸﮑﺪه ﮐﺸﺎورزي داﻧﺸﮕﺎه ﺷﯿﺮاز Mostowfizadeh-Ghalamfarsa and Banihashemi: Species-specific PCR identification and detection … Species-specific PCR identification and detection of Phytophthora drechsleri, P. cryptogea and P. erythroseptica R. Mostowfizadeh-Ghalamfarsa1* and Z. Banihashemi1 (Received: 18.6.2015; Accepted: 29.12.2015) Abstract Phytophthora drechsleri, P. cryptogea and P. erythroseptica are phylogenetically closely related Oomyceteous plant pathogens which are morphologically similar. In order to discriminate these taxa from each other and from species with convergent morphological characteristics a simple as well as a nested-PCR based method was developed. A collection of isolates of each species from different hosts representing world-wide diversity of species were examined for unique regions of nuclear as well as mitochondrial genes. Six candidate PCR primers were designed and calibrated for species-specific amplification of P. drechsleri based on the DNA sequences of rDNA internal transcribed spacer regions and the cytochrome c oxidase subunit I, and also three candidate PCR primers specific for P. cryptogea and P. erythroseptica were designed and calibrated based on cytochrome c oxidase subunit I. Studies showed that the best primer set for identification of P. drechsleri was the combination of ITS-DF2 and ITS-DR2, which amplified a 567 bp band. The combination of COX-CF1 and COX-CR2 was the best set for discrimination of P. cryptogea/P. erythroseptica from other species which amplified a 415 bp product from both species. A restriction map analysis of P. cryptogea/P. erythroseptica indicated that the non-palindromic Mnl I enzyme restriction site was unique to amplicons of P. erythroseptica isolates and could be employed to distinguish this species from P. cryptogea. Based on this study, nested-PCR was at least 100 times more sensitive than conventional PCR for detection of these species. Keywords: Phytophthora drechsleri, Phytophthora cryptogea, Phytophthora erythroseptica, Oomycota, Internal transcribed spacer, cytochrome c oxidase subunit I, identification, detection * Corresponding Author, Email: [email protected] 1. Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz, Iran 542 Iran. J. Plant Path., Vol. 51, No. 4, 2015: 541-553 Introduction objective of this study was therefore to develop diagnostic molecular tools for identification of P. The identification and discrimination of drechsleri, P. cryptogea and P. erythroseptica Phytophthora cryptogea Pethybridge & Lafferty isolates using species-specific PCR primers. (1919) and Phytophthora drechsleri Tucker (1931) has been a matter of controversy for more than 75 years. These two soil-borne plant pathogenic Material and Methods oomycetes are morphologically similar and phylogenetically related species which were Origin and Maintenance of Isolates considered as a species complex with ambiguous species boundaries (Erwin et al. 1983, Erwin & Details of the isolates examined in this study are Ribeiro 1996, Mills et al. 1991). Recent findings listed in Table 1. The isolates were sourced from however showed that these species are distinct taxa the culture collections of the authors. Isolates were (Cooke et al. 2000, Kroon et al. 2004, Blair et al. stored on cornmeal agar (CMA; ground corn −1 −1 2008, Mostowfizadeh-Ghalamfarsa et al. 2010) extract 40 g l , agar 15 g l ) slopes at 15 °C. with three lineages distinguished among P. Routine stock cultures for research studies were cryptogea isolates (Mostowfizadeh-Ghalamfarsa et also grown on CMA at 20 °C. al. 2010). Studies based on multiple gene genealogy analysis also showed that the DNA Extraction homothallic species, Phytophthora erythroseptica Isolates were grown in 50 ml still culture of pea Pethybridge (1913), appears to have evolved from broth (boiled extract of 125 g frozen green peas in within one group of P. cryptogea (Mostowfizadeh- 1000 ml distilled water at pH 6.2) at 20 ºC. After Ghalamfarsa et al. 2010). vacuum filtration, the mycelia was washed with Given its morphological similarity, P. sterilized distilled water, freeze-dried and stored at drechsleri is traditionally discriminated from P. -20 °C. Freeze-dried mycelia were homogenized cryptogea by its ability to grow well at and above using sea sand (Fluka, Germany) and a plastic 35 °C (Tucker 1931). Other studies however show disposable pestle. DNA was extracted from that the high-temperature criterion does not always homogenized preparation using a Puregene DNA correlate with the other identifying features extraction kit, Flowgen (Lichfield, England) (Klisiewicz & Beard 1976, Banihashemi & Ghaisi according to the manufacturer’s instructions. The 1993) and as a result, some isolates were described amount of DNA obtained was estimated by a as intermediate between both species (Flowers et NanoDrop spectrophotometer (NanoDrop al. 1973, Shepherd & Pratt, 1973, Klisiewicz, Technologies, USA). 1977, Stanghellini & Kronland 1982). Despite its convergent morphology, P. erythroseptica is a Primer Design homothallic species. However, P. drechsleri has occasional homothallic behavior (Waterhouse Sequenced regions of β-tubulin gene, translation 1963) and it seems that there are some intermediate elongation factor 1 α gene, elicitin gene, internal isolates of P. cryptogea which are also homothallic transcribed spacers 1, 2 and 5.8S gene of rDNA (David E. L. Cooke, unpublished data). These (ITS), and cytochrome c oxidase gene subunit I could be a source of error in the identification of (COX) from ca 72 Phytophthora species along these species. On the other hand, there are some with a collection of P. cryptogea, P. drechsleri, superficially similar taxa of Phytophthora that and P. erythrosepticae isolates from different hosts grow at or above 35ºC such as P. cajani (Amin et and matrices from previous studies al. 1978), P. melonis (Ho et al. 2007) and P. (Mostowfizadeh-Ghalamfarsa et al. 2010) were parsiana (Mostowfizadeh-Ghalamfarsa et al. recovered from GenBank using the Nucleotide 2008) that have been mistaken for either P. Sequence Search Program provided by the drechsleri or high-temperature tolerant P. National Center for Biotechnology Information cryptogea isolates. In the absence of clear (NCBI, http://www3.ncbi.nlm.nih.Gov/Entrez) morphological or physiological criteria for (Bethesda, MD, USA). Multiple sequence accurate identification of these species the alignment of each gene was made using ClustalX 543 Mostowfizadeh-Ghalamfarsa and Banihashemi: Species-specific PCR identification and detection … Table 1. Species-specific amplification of DNA sequences from different Phytophthora species by designed primer sets for Phytophthora drechsleri, P. cryptogea and P. erythroseptica. Isolate Amplification using Year of Host ITS- ITS- Cox- Cox- Cox- Species Local International Location a b c d e isolation (Matrix) D1 D2 D1 C1 C2 P. cactorum SCRP27 IMI296524 1985 Rubus idaeus Wales + - - - - P. cajani SCRP66 IMI320064 1987 Cajanus sp. India - - - - - P. cambivora SCRP67 IMI296831 1985 Rubus idaeus Scotland - - - + - P. capsici SCRP103 IMI352321 1989 Piper nigrum India + - - - - Chamaecyparis Netherlands P. cinnamomi SCRP115 CBS270.55 1993 lawsoniana - - - - - P. citricola SCRP130 1986 Rubus idaeus Scotland - - - - - P. IMI332632 Actinidia sp. Chile + - - - - citrophthora SCRP179 P. cryptogea Gerbera France * SCRP214 1973 - - - + + G I jamesonii SCRP205 IMI34684 ? Solanum Northern - - - + + tuberosum Ireland SCRP207 IMI045168 1951 Solanum New - - - + + lycopersicum Zealand SCRP206 ? ? England - - - + + SCRP212 1987 Solanum France lycopersicum - - - + + SCRP219 1983