Molecular characterization of Phytophthora nicotianae associated with diseases of horticultural crops by RFLP of PCR internal transcribed spacer region of ribosomal DNA and AFLP fingerprints. P. Chowdappa1 Central Plantation Crops Research Institute Regional Station, Vittal- 574 243, India E-mail: pallem @iihr.ernet.in ABSTRACT Molecular characterization of Phytophthora isolates from tobacco, tomato, carnation, gerbera, crossandra and periwinkle was carried out using internal transcribed spacer(ITS) regions of rDNA gene repeat and amplified fragment length polymorphism (AFLP). All the isolates of Phytophthora were identical in morphology and ITS- RFLP patterns, indicating P. nicotianae and P. parasitica are synonymous. However, based on AFLP, four sub groups were evident within population of P. nicotianae (P. parasitica): Group I, includes isolates from tomato, tobacco and periwinkle. Isolates from carnation represents Group II. Isolates from gerbera falls under group III. The crossandra isolates formed a Group IV. Thus, present study showed the existence of four molecular sub groups within population of P. nicotianae (P. parasitica) in India. Key words: Phytophthora nicotianae, AFLP, ITS-RFLP 1Present address: Division of Plant Pathology, Indian Institute of Horticultural Research, Bangalore – 560 089, India INTRODUCTION Phytophthora parasitica ( sensu Dastur,1913) and P. nicotianae (sensu Waterhouse, 1963), are pathogenic to a wide range of hosts ranging from tree crops to herbaceous plants(Oudemans and Coffey, 1991). Phytophthora nicotianae has been reported as causal agent of diseases of tobacco, crossandra, carnation, gerbera and periwinkle and P. parasitica on tomato in India (Mehrotra and Aggarwal, 2001). These species were mainly identified based on morphological features (Stamps et al, 1990; Sarma et al, 2002)Morphological criteria have limitations in understanding genetic diversity and genetic distance between isolates of P. nicotianae and P. parasitica. Protein patterns (Erselius and de Vallavielle, 1984), serological analysis (Morton and Dukes, 1967), DNA - RFLP patterns (Forster and Coffey, 1991) and isozyme patterns (Oudemans and Coffey, 1991) were found to be useful in understanding genetic divergence between isolates belonging to P. nicotianae and P. parasitica. Endonuclease restriction digestion analysis of the ITS region of ribosomal DNA (ITS-RFLP) (Cooke and Duncan,1997; Crowford et al, 1996; Chowdappa et al, 2003a, b, c; Chowdappa and Sarma, 2003) and AFLP finger prints were successful to separate species and also differences. The objective of the present study is to understand molecular diversity of Phytophthora isolates from tobacco, tomato carnation, gerbera, crossandra and periwinkle in India based on ITS-RFLP and AFLP fingerprinting. MATERIAL AND METHODS Fungal isolates Phytophthora nicotianae isolates from tobacco, tomato, perwinkle, gerbera (05 isolates each), carnation and crossandra (04 isolates each) collected from Bangalore, Mysore, Tumkur and Hassan districts of Karnataka were used in this study. Reference cultures of P. nicotianae (IMI 3406160 and IMI 235604) were also included for comparison. Morphological studies Colony morphology of isolates, on carrot agar medium(CA) after three days of incubation in the dark at 24 ± 1ºC, was studied. Sporangia were produced on CA using mycelium-agar-disk-in-water method (Al- Hedaithy and Tsao,1979). Observations were made on length and breadth of sporangia, presence of papilla, caducity and shapes. Observations on chlamydospore production were made on CA following incubation at 18ºC in the dark. The mating types of isolates were determined by taking 5 mm dia. discs from advancing margins of 3- day- old cultures of opposite mating types and placed them 20 mm apart on CA. The inoculated plates were incubated J. Hort. Sci. Vol. 2 (1): 58-62, 2007 at 20OC in the dark for 15-20 days. The influence of temperature on vegetative growth on CA was determined in the dark at temperature ranging from 6 to 39ºC with an increment of 3OC. Molecular studies The fungal mycelium was grown in 100 ml of V8 juice in a 250 ml flask for 3 days at 25°C on orbital shaker at 100 rpm. DNA was extracted from freeze dried vegetative mycelium following a method of Raeder and Broda (1985). PCR amplification of the ITS region was performed using with universal primer pair ITS1 and ITS 4 (White et al ,1990). Each 50 µl reaction mixture contained 100 ng of DNA, 50 pmol of each primer, 200 mm each dNTP, 5 µl of 10 x PCR buffer and 10 units of Taq DNA polymearse enzyme. PCR was carried out with following cycling parameters. A 4 minute denaturation at 94°C, followed by 34 cycles of 60s at 94°C, 60s at 55°C, and 1.5 min at 72°C and then a 5 min extension at 72°C. PCR products (5µl ) were electrophoresed in a 2 % agarose gel with Tris-Borate- EDTA (TBE) buffer, stained with ethidium bromide and visualised under UV illumination. PCR product was digested with restriction enzymes namely Hinf I, Msp I, Alu I, Hae III, Rsa I, Taq I and EcoR V. Restriction digestions were performed in 10 µl reaction containing 5 µl PCR product, 1 µl 10 x restriction buffer, 3.6 µ l PCR grade water, 0.1 µl Bovine serum albumn and restriction enzyme (3 U/reaction) at 37°C for overnight (16h). Digestion products were electrophoresed in 2.5% LE agarose. The size of restriction fragments was determined by comparison of fragments migration with that of known marker fragments (100bp molecular size ladder). Amplified fragment length polymorphism (AFLP) analysis was carried out as per the method of Muller et al (1996). Fungal genomic DNA ( 500 ng) was digested by Pst (20 units), ligated to Pst adaptor at 37°C for 4h and pre- amplification was carried out at 94ºC for 4 min for initial denaturation followed by 34 cycles of denaturation for 1 min at 94oC, annealing for 1 min at 60ºC, extension for 1.5 min at 72ºC and a final extension step of 5min at 72ºC. Pre - amplified PCR products were then diluted 1:100 in TE buffer and amplified with five AFLP primers, A(5’- GACTGCGTACATGCAGGT3’,B(5’GACTGCGTA CATGCAGGA3’), C,5’GACTGCGTACATGCAGGC3’), D (5’GACTGCGTACATGCAGAC3’), E(5’GACTGCGT ACATGCAGCG-3’) each with two selective bases at its 3’ end underlined, adopting above thermal cycling parameters. The PCR products were electrophoresed in 2% agarose gels in Tris-Borate-EDTA (TBE) buffer and visualized under UV after staining with ethidium bromide RESULTS AND DISCUSSION Phytophthora nicotianae isolates isolated from tobacco, tomato, periwinkle, carnation, crossandra and gerbera displayed no distinctive colony morphology (dense whitish aerial mycelium). Most isolates produced spherical, ovoid, ellipsoid and persistent sporangia on irregular / loose sympodial sporangiophore (Fig1). Sporangial sizes ranged from 36.70 to 46.50 x 24.92 to 32.25 µ m (Table1). L:B ratios of sporangia ranged from 1.31 to 1.53. Chlamydospores were produced readily by all isolates on CA incubated in the dark at 18°C. Chlamydospores were single, spherical, terminal or intercalary and pale yellow in colour (Fig 1). The mean chlamydospores diameter ranged from 20 to 30 µ m (Table 2). None of the isolates produced sexual structures. When paired with isolates of P. capsici of known mating types, all isolates produced sexual Table 1. Sporangial measurement in P. nicotianae isolates Isolate No Length*(µm) Breadth (µm) L/B ratio(µm) Mating type Tobacco OH 37 41.50 ± 1.21 29.50 ± 0.77 1.40 ± 0.77 A2 OH 38 43.07 ± 0.93 31.92 ± 0.61 1.33 ± 0.02 A2 OH 39 41.00 ± 1.08 30.20 ± 0.71 1.37 ± 0.02 A2 OH 40 36.70 ± 1.21 24.92 ± 0.54 1.47 ± 0.36 A2 OH 41 40.93 ± 1.91 27.80 ± 0.69 1.41 ± 0.04 A2 Tomato OH 42 38.50 ± 1.67 28.50 ± 1.14 1.30 ± 0.06 A2 OH 43 42.50 ± 0.86 32.25 ± 0.74 1.30 ± 0.02 A2 OH 44 40.00 ± 1.30 29.57 ± 0.83 1.37 ± 0.05 A2 OH 45 40.50 ± 0.77 29.00 ± 0.63 1.39 ± 0.03 A2 OH 46 39.00 ± 1.71 25.50 ± 1.00 1.38 ± 0.08 A2 Periwinkle OH 47 41.66 ± 1.45 28.54 ± 1.23 1.43 ± 0.03 A2 OH 48 42.75 ± 1.14 30.25 ± 0.96 1.41 ± 0.06 A2 OH 49 41.25 ± 1.53 28.75 ± 0.78 1.43 ± 0.02 A2 OH 50 41.66 ± 1.63 30.41 ± 1.09 1.36 ± 0.03 A2 OH 51 39.58 ± 1.81 27.91 ± 1.37 1.41 ± 0.06 A2 Carnation OH 52 38.61 ± 1.52 27.22 ± 1.32 1.41 ± 0.02 A2 OH 53 38.50 ± 1.67 28.50 ± 1.14 1.30 ± 0.06 A2 OH 54 44.44 ± 1.39 30.55 ± 1.51 1.46 ± 0.06 A2 OH 55 38.75 ± 1.39 27.25 ± 1.20 1.45 ± 0.48 A2 OH 56 46.50 ± 2.74 31.50 ± 1.53 1.46 ± 0.03 A2 Crossandra OH 57 43.25 ± 1.17 28.75 ± 1.28 1.53 ± 0.06 A2 OH 58 45.00 ± 1.32 31.11 ± 1.42 1.46 ± 0.05 A2 OH 59 40.00 ± 1.23 27.00 ± 0.93 1.48 ± 0.04 A2 OH 60 46.38 ± 2.28 30.31 ± 1.34 1.53 ± 0.05 A2 OH 61 40.55 ± 1.70 27.77 ± 1.32 1.46 ± 0.05 A2 Gerbera OH 62 45.50 ± 1.88 30.25 ± 1.24 1.49 ± 0.04 A2 OH 63 41.66 ± 1.63 30.41 ± 1.09 1.36 ± 0.03 A2 OH64 41.25 ± 1.53 28.75 ± 0.78 1.43 ± 0.02 A2 OH 65 44.07 ± 1.00 30.47 ± 0.90 1.44 ± 0.04 A2 OH 66 42.91 ± 1.49 28.33 ± 1.27 1.51 ± 0.05 A2 *Mean of 100 sporangia; ± standard deviation J. Hort. Sci. Vol. 2 (1): 58-62, 2007 59 Fig 1. Morphology of P. nicotianae isolates, Top left, sporangia, Top right, chlamydospores, Bottom left, oogonia, Bottom right, oospores Table 2. Dimensions of chlamydospores , oogonia and oospores of P. nicotianae isolates Isolate Chlamydospores Oogonia Oospores (µm) (µm) (µm) Tobacco OH 37 24.38 ± 1.75* 28.34 ± 2.10 25.90 ± 2.00 OH 38 22.78 ± 2.30 27.26 ± 1.90 26.24 ± 1.75 OH 39 28.20 ± 2.60 28.75 ± 2.00 25.34 ± 2.30 OH 40 23.34 ± 3.24 25.20 ± 1.95 24.37 ± 2.30 OH 41 23.28 ± 1.95 27.32 ± 2.60 25.20 ± 2.60 Tomato OH 42 27.34 ± 2.02 29.75 ± 2.72 26.37 ± 2.27 OH 43 28 .28 ± 2.78 27.14 ± 2.02 25.40 ± 2.50 OH 44 23.38 ± 2.50 27.30 ± 2.50 26.32 ± 2.71 OH 45 26.72 ± 2.31 28.14 ± 2.60 26.37 ± 2.80 OH 46 24.32 ± 1.31 27.20 ± 1.90 25.27 ± 1.70 Periwinkle OH 47 20.24 ± 1.32 28.14 ± 2.60 25.32 ± 2.75 OH 48 26.32 ± 2.34 28.20 ± 2.80 26.97 ± 2.60 OH 49 26.78 ± 2.14 27.20 ± 1.97 24.32 ± 2.32 OH 50 28.24 ± 3.21 27.30 ± 2.30 25.84 ± 2.00 OH 51 24.32 ± 2.20 27.10 ± 2.02 26.80 ± 2.72 Carnation OH 52 28.45 ± 2.10 28.32 ± 2.00 27.40 ± 1.72 OH 53 30.25 ± 1.78 27.20 ± 1.80 26.68 ± 2.34 OH 54 28.75 ± 2.24 27.45 ± 2.20 27.35 ± 2.40 OH 55 25.62 ± 3.14 28.47 ± 2.15 26.40 ± 1.80 OH 56 25.34 ± 2.45 27.90 ± 2.70 25.21 ± 2.30 Crossandra OH 57 26.25 ± 2.41 29.30 ± 1.60 24.00 ± 2.26 OH 58 20.35 ± 2.35 26.40 ± 2.30 25.34 ± 1.60 OH 59 20.75 ± 2.48 25.30 ± 1.60 26.73 ± 1.70 OH 60 24.34 ± 1.82 28.17 ± 1.20 27.40 ± 1.20 OH 61 25.75 ± 2.35 26.30 ± 2.00 24.37 ± 1.78 Gerbera OH 62 27.32 ± 1.32 27.34 ± 2.06 26.75 ± 1.80 OH 63 26.31 ± 2.71 26.48 ± 2.68 25.30 ± 2.07 OH 64 28.25 ± 2.24 27.84 ± 2.50 25.24 ± 2.01 OH 65 26.32 ± 2.18 26.34 ± 1.85 25.70 ± 2.07 OH 66 22.35 ± 2.14 27.84 ± 2.00 26.87 ± 2.80 *Mean of 100 reproductive structures ± standard deviation structures (Fig 1). Size of oogonia ranged from 26 to 29 µm and oospores varied from 24 to 27 µm (Table 2). Minimum temperature for growth of all isolates was 9°C; optimum was 24 - 30°C; and maximum was 33°C. Thus, the present study showed that sporangial, chlamydospore, oogonial and oospore characteristics of P. nicotianae isolates from periwinkle, carnation, gerbera, tobacco and crossandra are similar to those of P. parasitica isolates from tomato. Waterhouse (1963) created two morphological varieties; P. nicotianae var. nicotianae and P. nicotianae var. parasitica, which were based on Ashby (1928) microspore and macrospora, respectively. Further morphological studies of Ho and Jong (1989) in isolates from tobacco and other hosts clearly showed that var. nicotianae and var. parasitica could not be differentiated. Separation of the varieties nicotianae and parasitica based on morphological criteria was also questioned (Gallegly,1983). Oudemans and Coffey (1991) and Hall (1993) reported that oogonia , oospore and sporangial dimensions are similar among two groups. When DNA was amplified with universal primer pair (ITS1 and ITS4), there was single band of 920 bp (Fig 2) for all isolates of P. nicotianae and reference cultures of P. nicotianae (IMI 3406160 and IMI 235604). All isolates had identical ITS - RFLP patterns when PCR product was digested with enzymes Hinf 1, Msp I, Alu I, Hae III and Rsa I (Fig 3). Restriction fragment sizes of the ITS region of rDNA digested with different enzymes are also identical. Fig 2. PCR amplification of ITS region of r DNA Lane1, Marker, 100bp ladder, Lane2, Periwinkle, Lane 3, gerbera, Lane,4,carnation, Lane,5, crossandra 600bp J. Hort. Sci. Vol. 2 (1): 58-62, 2007 60 Chowdappa Fig 3. ITS-RFLP patterns generated with Hinf I (Lane 2-5)and TaqI(Lane 6-9). Lane 1and 10, Marker (100bp) Lane2 and 6,periwinkle, Lane 3and 7, gerbera, Lane,4 and 8,carnation, Lane,5 and 9, crossandra Protein profiles (Erselius and de Vallavielle,1984), serological reactions (Morton and Dukes, 1967), RFLP- DNA patterns ( Forster and Coffey,1991) of P. nicotianae isolates from tobacco were almost identical to those of P. nicotianae isolates from other crops. ITS- RFLP data obtained in the present study also showed that P. nicotianae isolates from tobacco, tomato, periwinkle, carnation, gerbera and crossandra were identical. As varieties nicotianae and parasitica are identical based on morphological and molecular criteria, many researchers proposed (Ho and Jang,1989; Oudemans and Coffey,1991; Hall,1993) that the name P. nicotianae be eliminated and that all isolates from tobacco and other hosts should be merged under the name P. parasitica. Among the isolates of P. nicotianae, four AFLP finger print groups were evident based on the combined results of five primers (A,B,C,D and E). The AFLP finger prints generated with primer E is shown in Fig 4. Group I includes isolates from tomato, tobacco and periwinkle. Isolates from carnation represents Group II. Isolates from gerbera falls under group III. The crossandra isolates formed a Group IV. Although there was no intra specific ITS variation among isolates of P. nicotianae, host specialized groups from tobacco, tomato, periwinkle, gerbera, carnation, and crossandra were found based on AFLP fingerprints. There is considerable amount of evidence to show that some isolates of P. nicotianae are host-specific. For instance, isolates from okra and sesame were host specific, while isolates from Peperomia obtusifolia and Saintpaulia ionatha had different host ranges (Ho and Jong,1989). Isolates from carnation and tomato were more virulent on Fig 4. AFLP finger prints of Phytophthora nicotianae isolates Lane 1, bp ladder(size marker), Lane2,periwinkle, Lane 3, gerbera, Lane,4,carnation, Lane,5, crossandra carnation and tomato respectively than other hosts. Thus, the present study clearly indicated that P. nicotianae isolates from tobacco, periwinkle, carnation, gerbera and crossandra and P. parasitica isolates from tomato were identical. The AFLP finger prints were useful in identifying host specialized groups within P. parasitica (P. nicotianae). While screening the accessions for disease resistance, host specialized AFLP molecular groups should be used rather than morphologically defined isolates. ACKNOWLEDGEMENT The author is grateful to the Indian Council of Agricultural Research, New Delhi for financial assistance in the form of National Network Project on Phytophthora diseases of horticultural crops. He is also thankful to Dr. N. Ramachandran, Head, Division of Plant Pathology, Indian Institute of Horticultural Research, Bangalore, for Phytophthora cultures from gerbera, carnation and crossandra, and, to Dr. M. M. Shenoi, Head, CTCRI Regional Station, Hunsur, for Phytophthora infected samples of tobacco. REFERENCES Al-Hedaithy, S. S. A. and Tsao, P. H.1979. Sporangium pedicel length in Phytophthora species and the consideration of its uniformity in determining sporangium caducity. Trans. Brit. Mycol. Soc.,72: 1-13. Ashby, S. F.1928. The oospores of Phytophthora nicotianae Br.de.Haan with notes on the taxonomy of P.parasitica Dastur. Trans. Brit. Mycol. Soc., 13: 86-95 600bp 600bp J. Hort. Sci. Vol. 2 (1): 58-62, 2007 61 Molecular characterization of Phytophthora Chowdappa, P., Brayford, D., Smith, J. and Flood, J. J.2003a. Molecular discrimination of Phytophthora isolates on cocoa and their relationship with coconut, black pepper and bell pepper isolates based on r DNA repeat and AFLP finger prints. Curr. Sci., 84: 1235 – 1238. Chowdappa, P., Brayford, D., Smith, J. and Flood J. J. 2003b. Identification of Phytophthora species affecting plantation crops by RFLP of PCR amplified ITS region of ribosomal RNA. Curr. Sci., 85: 34-36. Chowdappa, P., Brayford, D., Smith, J. and Flood, J. J. 2003c. Identity of Phytophthora associated with arecanut and its relationship rubber and cardamom isolates based on RFLP of PCR- amplified ITS region of r DNA and AFLP fingerprints. Curr.Sci., 85: 585- 587. Chowdappa, P. and Sarma, Y. R. 2003. Application of molecular markers for resolving taxonomic complexities in Phytophthora . J. Plantn. Crops.,31: 1-12 Cooke, D. E. L . and Duncan, J. M. 1997. Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycol.Res.,101: 667-677 Crowford, A. R., Bassam, B. J., Drenth, A., Maclean, D. J. and Irwin, J. A. G . 1996. Evolutionary relationships among Phytophthora species deduced from r DNA sequence analysis. Mycol. Res., 100: 437-443. Dastur,J. F.1913. Phytophthora parasitica n.sp, a new disease of castor oil plant. Mem. Dep. Agric. Bot. Ser., 5: 177-231. Erselius, L. J. and de Vallavieille, C. 1984. Variation in protein profiles in Phytophthora : comparision of six species. Trans. Brit. Mycol. Soc., 83: 463-472 Forster, H. and Coffey, M. D. 1991. Approaches to the taxonomy of Phytophthora using polymorphisms in mitochondrial and nuclear DNA. p164-183. In: Phytophthora. Leucas, J.A. Shattock, R.C. Shaw.D.S. and Cooke, L.R.(ed.) Cambridge Uni. Press, Cambridge, UK Gallegly, M. E. 1983. New criteria for classifying Phytophthora and critique of existing approaches.p167-172. In: Phytophthora: its Biology, Taxonomy, Ecology and Pathology. Erwin, D.C. Bartnicki-Garcia, S. and Tsao, P.H.(ed.) American Phytopathological Society, St. Paul. Hall, G. 1993. An integrated approach to the analysis of variation in Phytophthora nicotianae and a redescription of the species. Mycol. Res.,97 :559-574. Ho, H. H and Jong, S. C.1989. Phytophthora nicotianae (P. parasitica). Mycotaxon, 36 : 243-276. Mehrotra, R. S. and Aggarwal, A. 2001.Phytophthora diseases in India. SMPS, Dehradun, p 322 Morton, D. J. and Dukes, P. D.1967. Serological differentiation of Pythium aphanidermatum from Phytophthora parasitica var.nicotianae and Phytophthora parasitica. Nature, 213: 923. Mueller, U. G., Lipari, S. E. and Milgroom, M. G. 1996. Amplified fragment length polymorphism finger printing of symbiotic fungi cultured by fungus- growing ant Cyphomyrex minutus. Mol. Ecology, 5: 119-122. Oudemans, P. and Coffey, M. D. 1991. A revised systematics of twelve papillate Phytophthora species based on isozyme analysis. Mycol. Res., 95: 1025- 1046 Raeder, U. and Broda. P. 1985. Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbiol., 1: 17-20. Sarma, Y R., Chowdappa, P. and Anandaraj. M. 2002. Phytophthora disease of plantation crops in India.p149-187. In: IPM system in Agriculture: Key pathogens and diseases .Vol. 8. Upadhyay, R.K. Arora, D. K. and Dubey, O. P. (ed.). Aditya Books Pvt. Ltd., New Delhi Stamps, D. J., Waterhouse, G. M., Newhook, F. J and Hall, G. S .1990. Revised tabular key to the species of the Phytophthora, Mycological papers 162, CAB International Mycological Institute, Kew, London, p 28 Waterhouse, G. M. 1963. Key to the species of Phytophthora de Bary; Mycological papers (CMI), 92 : 1-22 White, T. J., Bruns, T., Lee, S. and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. P 315-322. In:PCR protocols- a guide to methods and applications .Innis, M A. Gelfand, D. H. Sninsky, J. J. and White, T. J(ed. ). Academic Press, San Diego. (MS Received 3 November, 2006, Revised 2 June 2007) J. Hort. Sci. Vol. 2 (1): 58-62, 2007 62 Chowdappa