<4D6963726F736F667420576F7264202D20CF20D8C7D1DE20DAC8CF20C7E1D3C7CFC9> Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 45   MOLECULAR CHARACTERIZATION OF Rhizoctonia solani ISOLATED FROM PEPPER PLANTS IN IRAQ BY USING PCR . Kareem T. A.* M. S. Hassan *Department of Plant protection- College of Agriculture- University of Baghdad- Republic of Iraq. tariqask@yahoo.com ABSTRACT Rhizoctonia solani is one of the most important vegetables plants pathogens which are distributed in soil worldwide. Three isolates are selected from eight R. solani isolates pathogenic for Pepper plants from different area in Baghdad – Iraq, according to the pathogenicity test on Radish seeds. The sequences of rDNA- ITS region of the Iraqi pepper isolates (IQ- 34, IQ-39 & IQ- 40) were showed variation in similarity. Phylogenetic tree based on rDNA- ITS regions indicated that the IQ-34 and IQ- 40 isolates from pepper belonged to AG5 and IQ- 39 isolate belonged to AG4-HGIII. The nucleotide sequence data were sent to International GenBank to check and registered. The GenBank send accession number for each sequence at Jul.2013 as: KF372660, KF372661 and KF372662 respectively. Key words: Rhizoctonia solani, Pepper, pathogenicity, molecular characteristics, DNA sequencing, phylogenetic. INTRODACTION Genus Rhizoctonia is a highly heterogeneous group of filamentous fungi that share similarities in their anamorphic, sterile state. They do not produce asexual spores and sexual state occurs only rarely. The group contains several economically important and global plant pathogens like Rhizoctonia solani Kühn [telemorph Thanatephorus cucumeris (Frank) Donk] (Gonzáles García et al., 2006). R. solani is the most widely known and most studied species of genus Rhizoctonia. It was originally described by Julius Kühn from potato in 1858. R. solani is soilborne Basidiomycete occurring world-wide, with complex biology. Its highly destructive lifestyle as a non-obligate parasite causes necrosis and damping-off on numerous host plant species. Because of the lack of conidia and the scarcity of the sexual spores, R. solani exists as vegetative hyphae and sclerotia in nature. The fungus is dispersed mainly via sclerotia, contaminated plant material or soil spread by wind, water or during agricultural practices such ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Sept. 10 , 2013 . Accepted for publication Nov. 12 , 2013 . Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 46   as tillage and seed transportation. The fungus can stay in the soil as a saprophyte for long periods (Ogoshi, 1087; Ogoshi et al., 1990). Disease symptoms include leaf blights, leaf spots, damping-off, rots on roots, shoots and fruits, canker lesions on sprouts and stolon, and sclerotial diseases. However, some R. solani strains form symbiotic mycorrhizal relationships with orchid plants (Carling et al., 1999; Chang and Chou, 2007). The host range of R. solani is wide and it causes various diseases on important crop plants of the world , including plant species in the Solanaceae, Fabaceae, Asteraceae, Poaceae and Braccicaceae as well as ornamental plants and forest trees (Ogoshi, 1996). Hyphal fusion has been proved to be a reliable method for grouping R. solani strains into anastomosis groups (AGs) (Ogoshi, 1987). The concept has given rise to currently 14 AGs (Carling et al., 2002). AGs 1, 2, 3 and 4 are the biggest pathogen groups, characterized individually in three different geographical locations, in Europe (Carling, 1996), in the North America (Parmeter et al., 1969), and in Asia (Watanabe and Matsuda, 1966). The most informative DNA-based molecular technique for investigating diversity in Rhizoctonia isolates has been sequence analysis of ribosomal ribonucleic acid (rRNA) genes (28S) and the internal transcribed spacer (ITS) region (Gonzalez et al., 2001). Analyses of these genes have not only shown the genetic relatedness of Rhizoctonia isolates, but have also confirmed some of the anastomosis groupings. In this study, detailed description of morphology characteristics, pathogenicity, and molecular characteristics of R. solani isolates from pepper plants root with a typical crown, which originate from different Baghdad regions- Iraq. MATERIALS AND METHODS Isolates Isolates used in this study were collected during vegetative period in 2011 from five regions (Abu Ghraib, Yusifiyah, Al Rashidiya, Tarmiya and Doura) in Baghdad, Iraq. Diseased pepper plants (Capsicum annuum L.) showing symptoms of infection by R.solani was surface sterilized in 0.5% hypochlorite for 1 min and then plated on water agar (WA) with 250µg/ml chloramphenicol. After incubation at 25 ± 1 °C for 2-3 days, culture resembling R.solani were transferred to fresh plates to ensure purity. Isolations were transferred to slant PDA and kept on 4°C until further investigation. Morphological characteristics Tested isolates were examined for macroscopic characteristics typical of R. solani, such as development and change of mycelial color, sclerotia formation, mycelial appearance, branching of hyphae and existence of Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 47   multinucleate cells were also determined (Parmeter and Whitney, 1970; Herr, 1979). Pathogenicity tests Pathogenicity of tested R. solani isolates was evaluated by colonized agar disks (7-10 mm) taken from the margins of 3 day-old cultures growing on PDA were transferred to the center of water agar (WA) plates and incubated for 3 days. Six pre-germinated seeds of radish (Raphanus sativus) were placed on the margins of the Rhizoctonia colonies in separate plates. The pathogenicity of the isolates was evaluated after a further 6 days for radish at 25± 1 °C. Disease severity was assessed visually and scored using a disease severity index (DSI) ranging from 0-5, where 0-1=<1 mm lesion; 2= 1-3 mm; 3=3-5 mm; 4=5-7 mm; 5=>7 mm or dead plant. Isolates causing no symptoms or very mild symptoms (0-0.3 DSI) were considered avirulent ; isolates causing mild symptoms (0.4-1.9 DSI) were considered low virulent; isolates causing moderate symptoms (2-2.9 DSI) were considered moderately virulent; isolates causing severe symptoms (3-3.9 DSI) were considered virulent and isolates causing very severe symptoms (4-5 DSI) were considered strongly virulent (Sneh et al.,2004). Molecular characteristics Each isolate from R.solani was grown in potato dextrose broth for 4- 7days at 25± 1 °C. Mycelial mats were harvested by filtration, dry in room chamber and ground to fine powder in liquid N and then stored in – 20 °C prior to DNA isolation. Genomic DNA was extracted from 100 mg ground fungal tissue using the fungal DNA Kit (EZ-10 spin column fungal genomic DNA, Bioneer corporation , Korea) and following the protocol recommended by the manufacturer. Internal transcribed spacer region of ribosomal DNA was amplified using ITS1 F (TCC GTA GGT GAA CCT GCG G) and ITS4 R (TCC TCC GCT TAT TGA TAT GC) set of primers that were described by Hsiang and Dean (2001). The 20 µL reaction mixture for PCR amplification consisting of 5 µL of PCR PreMix (Bioneer Corporation, Korea), 5 µL of DNA template, 3 µL of ITS1, 3 µL of ITS4 and 4µL of PCR distilled water. The amplification was performed in PCR thermal cycler (My Genie 32 Thermal Block, Bioneer, Korea). The cycle parameters were: An initial denaturation (95°C, 2 min), 35 cycles of denaturation (94°C, 30s), annealing (55°C, 1 min) and extension (72°C, 1 min). Final extension was at 72°C for 10 min (Hsiang and Dean, 2001). DNA sequencing and data analysis After the amplification of the ITS region of the rDNA, each product was purified using the AccuPrep® PCR Purification Kit and protocol (Bioneer Corporation, Korea). Purified rDNA was sending to sequenced in DNA Sequencing Facility at Bioneer Corporation, Korea. Analysis of ITS sequences Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 48   was performed using on-line software CLUSTALW. Sequence data base of National Centre for Biotechnology Information-NCBI) – GenBank, which was entered via web page www.ncbi.nlm.nih.gov, was used for information on R. solani isolates. Phylogenetic analysis, all sequences obtained in this study and some available at GenBank were aligned with multiple aliment program ClustalW. A tree showing the phylogenetic relatedness between isolates constructed from Maximum Composite Likelihood by the neighbor – joining method, using the computer software package MEGA5.2. the tree was rooted with an isolate of AG 6HGI ( accession number : DQ301740) as out-group. RESULTS AND DISCUSSION Morphological characteristics A total of 8 Rhizoctonia spp. isolates were obtained from 28 samples, from Pepper plants roots with typical symptoms of Rhizoctonia root rot was isolated (Table 1). All isolates (IQ-18 , IQ-24 , IQ- 25 , IQ- 26 , IQ- 28 , IQ-34 , IQ- 39 & IQ-40) showed typical features of R. solani complex including brown pigmentation of hyphae, branching near distal septum, constriction of hyphae and formation of septum short distance from the place of branching, the presence of dolipore septa and multinuclear cells in young vegetative hyphae (Parmeter and Whitney, 1970). Table 1. Pathogenicity testing examined on Radish seeds to determine disease severity index of R. solani isolates used in this study. Rhizoctonia spp. Isolate code Regions Baghdad-Iraq Disease severity index IQ- 18 Yusifiyah 2.6 IQ- 24 Yusifiyah 2.5 IQ- 25 Yusifiyah 2.3 IQ- 26 Yusifiyah 2.9 IQ- 28 Yusifiyah 2 IQ- 34 Yusifiyah 5 IQ- 39 Yusifiyah 5 IQ- 40 Yusifiyah 5 Pathogenicity tests At the end of all pathogenicity experiments, re-isolations were done from all examined plant species with typical symptoms. R. solani was successfully re- isolated to confirm Koch’s postulates. The results of the research showed three examined isolates proved to be strong virulent on radish seeds (IQ-34, IQ- 39 & IQ- 40). This isolates were selected for depth research in this study (table 1). Also, isolates with the lowest pathogenicity (IQ- 18, IQ- 24, IQ- 25, IQ- 26 & IQ- 28) on radish seeds test were taken for further research. Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 49   Molecular characteristics of isolates The selected isolates sequences were compared between themselves and with 30 isolates (ClustalW), randomly chosen representative of R. solani AGs-1 to 12 and AG BI, whose sequences were downloaded from GenBank (Table 2). Similarity of these test sequences showed that homology ranged from 57 to 90%. ITS sequences of isolates IQ- 34, IQ- 39 & IQ-40 had the highest sequence similarity with Indiana isolate JF701784 that belong to AG 5 (81%), Australian isolate AF153795 that belong to AG8ZGI-1 (66%) and Indiana isolate JF701784 that belong to AG 5 (90%) respectively. All sequencing results for R.solani isolates (IQ- 34, IQ- 39 & IQ- 40) were sent to International GenBank to check and registered. The GenBank send accession number for each sequence at Jul.2013 as: KF372660, KF372661 & KF372662 respectively (available now on www.ncbi.nlm.nih.gov). This is the first detailed report representing the characteristic of R. solani on Pepper plants in Baghdad - Iraq with regard to its morphological, pathogenicity and phylogenetic analysis. Diseases caused by R. solani is worldwide of host plant species include Pepper plants (Sneh et al., 1991). Integrated research of morphological, pathogenic and molecular characteristics serves for the determination of groups and subgroups in R. solani. The importance of correct determination of anastomosis groups within R. solani complex is very important because of different virulence levels present at different anastomosis groups (Carling et al., 2002). Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 50   Table 2. R.solani sequences recovered from the GenBank (National Center for Biotechnology Information – NCBI) and sequence comparison with Iraqi Pepper isolates IQ-34, IQ-39 & IQ-40 using Clustal W. AG & Subgroup Host and geographic origin GenBank Accession number Sequence similarity (%) (Pepper) IQ-34 IQ-39 IQ-40 AG 1-IA Oryza sativa, Japan AB000017 70 62 71 AG 1-IB  Beta vulgaris, Japan AB000038 62 63 72 AG 1-IC  Beta vulgaris, Japan AB122142 72 62 80 AG 2-1  Solanum tuberosum, USA AB000026 65 60 77 AG 2-2IIIB  Beta vulgaris, USA AB054857 62 61 70 AG 2-2 IV  Beta vulgaris, USA AB054859 64 61 70 AG 2-3  Glycine max, Japan AB054870 62 64 75 AG 3 Beta vulgaris, USA AB019006 64 61 73 AG 3PT Solanum tuberosum, USA AB019013 63 61 73 AG 3TB Nicotiana tabacum, USA AB000001 67 64 81 AG 4HGI Beta vulgaris, Japan AB000028 64 61 71 AG 4HGII Beta vulgaris, Japan AB000033 63 60 70 AG4HGIII Beta vulgaris, USA AF354075 65 60 77 AG 5 Pea spp., India JF701784 81 63 90 AG 6 Pterostylis acuminata, Australia AF153784 71 65 82 AG 6GV Soil, Japan AF354101 74 64 80 AG 7 Soil, Japan AB000003 70 61 78 AG 8 Triticum aestivum, Australia AB000011 71 63 78 AG8ZGI-1 Soil, Australia AF153795 70 66 82 AG8ZGI-2 Soil, Australia AF153797 72 64 80 AG8ZGI-3 Hordeum vulgare, Australia AF354068 67 63 79 AG8ZGI-4 Hordeum vulgare, Scotland AF354066 69 60 76 AG 9 Solanum tuberosum, USA AF354109 64 60 74 AG 9TX Solanum tuberosum, USA AB000037 64 60 78 AG 9TP Solanum tuberosum, USA AB000046 64 61 78 AG 10 Hordeum vulgare, Australia AF354071 62 57 73 AG 11 Glycine max, USA AF354114 69 63 76 AG 12 Pterostylis acuminata, Australia AF153803 72 60 79 AG BI Soil, Japan AB000044 61 60 69 The R. solani diversity between Iraqi Pepper isolates and global isolates maybe back to this might occur due to climatic changes and global increase of temperature. The Neighbor-joining phylogeny test based on sequences differences in the ITS – rDNA region (Fig.1) illustrates estimates of phylogenetic relationships among all AGs of R.solani , including Iraqi isolates (IQ- 34, IQ- 39 & IQ- 40). Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 51   AB000037-AG9-TX AB000046-AG9-TP AF354109-AG9 AB000026-AG2-1 AF354071-AG10 AB000044-AG-BI AB000001-AG3-TB AB019006-AG3 AB019013-AG3-PT AF153784-AG6 AF354101-AG6-GV AF153795-AG8-ZGI-1 AF153797-AG8-ZGI-2 AB000011-AG8 AF354068-AG8-ZGI-3 AF354066-AG8-ZGI-4 AB000028-AG4-HGI AB000033-AG4-HGII AF354075-AG4-HGIII IQ-39-R-Pepper AB000038-AG1-IB AB122142-AG1-IC AB000017-AG1-IA AB000003-AG7 AF153803-AG12 JF701784-AG5 IQ-34-R-Pepper IQ-40-R-Pepper AB054857-AG2-2-IIIB AB054859-AG2-2-IV AB054870-AG2-3 AF354114-AG11 DQ301740-AG6-HGI 98 97 89 76 75 99 56 56 50 44 39 36 36 34 21 20 15 15 15 23 95 18 13 13 8 8 9 2 17 1 Fig 1. Neighbor - joining tree illustrating relationships estimates of phylogenetic relationships of test isolates (IQ – 34, IQ -39 & IQ – 40) and all other AGs of R. solani. The number below each branch indicates the percentage of congruent clusters in 1000 bootstrap trials when values were greater than 50%. Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 52   During the phylogenetic analysis in this study, the IQ- 39 isolate was found in a clustered with AG4- HGIII and IQ- 34, IQ- 40 isolates were clustered with AG5. Sequencing and phylogenetic analysis of the ITS region has been confirmed to reliably divide isolates of R. solani into distinct groups and subgroups which correspond to the different anastomosis groups (carling et al. 2002). REFERENCES Carling D.E. .1996. Grouping Rhizoctonia solani by hyphal anastomosis reaction. In. Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control. Eds. 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Ribosomal DNA systematics of Ceratobasidium and Thanatephorus with Rhizoctonia anamorphs. Mycologia 93: 1138–1150. Herr L. J. 1979. Practical nuclear staining procedures for Rhizoctonia like fungi. Phytopathology 69:958 – 961. Hsiang T. and J.D.Dean.2001.DNA sequencing for anastomosis grouping of Rhizoctonia solani isolates from Poa annua. International Turfgrass Society Research Journal V9:674-678. Ogoshi, A. 1987. Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kühn. Annual Review of Phytopathology 25: 125–143. Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 53   Ogoshi, A., R. J. Cook and Bassett, E. N. 1990. Rhizoctonia species and anastomosis groups causing root rot of wheat and barley in the Pacific Northwest. Phytopathology 80: 784–788. Ogoshi A .1996. Introduction – the genus Rhizoctonia. In. Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control. Eds. Sneh B, Jabaji-Hare S, Neate S and Dijst G. Kluwer Academic Publishers, The Netherlands. pp. 1-9. Parmeter, J.R.Jr., R.T. Sherwood and W.D Platt.1969. Anastomosis Grouping among isolates of Thanatephorus cucumeris. Phytopathology 59:1270-1278. Parmeter J.R. Jr., and H.S. Whitney. (1970). Taxonomy and nomenclature of the imperfect state. In: Biology and pathology of Rhizoctonia solani(Eds.: Parmeter Jr. JR). University of California Press, Berkeley, pp.7-19. Sneh, B., L. Burpee and A. Ogoshi . 1991. Identification of Rhizoctonia Species. American Phytopathological Society Press: St.Paul. MN, USA. D.R. Sumner and S.C. Phatak. 2003. First report of Rhizoctonia solani AG-2-4 on carrot In Georgia. Plant Disease 87:1264. Sneh B., E. Yamoah and A. Stewart.2004. Hypovirulent Rhizoctonia spp. Isolates from New Zealand soils protect Radish seedlings against Damping-off caused by R. solani. New Zealand Plant Protection 57:54-58. Watanabe B. and A. Matsuda. 1966. 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Kareem and Hassan Diyala Agricultural Sciences Journal, 5( 2 ) 45 – 54 ,2013 54   .PCR الخصائص الجزيئية لل R. solani استخدامبالمعزول من نباتات الفلفل في العراق محمد صادق حسن آريم طارق عبد السادة .جمهورية العراق – جامعة بغداد –آلية الزراعة –قسم وقاية النبات * مستخلصال والذي ينتشر بشكل واسع من الفطريات الممرضة المهمة على نباتات الخضر R. solani الفطر من ثالث عزالت ممرضة من الفطر من اصل ثمانية عزالت جمعت تم اختيار. في التربة حول العالم لعزالت . بذور الفجلاختبار امراضيتها على بعدو . العراق –من مناطق مختلفة في بغداد الفلفل نباتات النايتروجينية لمنطقة القواعد اظهر تسلسل rDNA- ITS الفلفل العراقية (IQ- 34, IQ-39 ، IQ- 40) . تشابة بانها غيرم الى ان العزالت rDNA- ITS اعتمادا على منطقة واشار اختبار شجرة االصل التطوري IQ- 39 الى تنتمي والعزلة AG5 IQ- 40 و IQ-34 تنتمي الى المعزولة من الفلفل للتدقيق الدولي   GenBank AG4-HGIII الى النيوآلوتيد تسلسل بيانات ارسلت. ،KF372662 : 2013في تموز العزالت تسلسالتوالذي ارسل ارقام تسجيل . والتسجيل .على التوالي KF372660 و KF372661 :الكلمات المفتاحية R. solani .األصل التطوري مراضية ، الخصائص الجزيئية، تسلسل الدنا ،، الفلفل ، اال