For. Snow Landsc. Res. 76, 3: 351–356 (2001) 351 Molecular identification and detection of Phytophthora species on some important Mediterranean plants including sweet chestnut Santa Olga Cacciola1, Naomi A. Williams2, David E. L. Cooke2 and James M. Duncan2 1 Dipartimento di Scienze Entomologiche Fitopatologiche, Microbiologiche Agrarie e Zootecniche – Sezione di Patologia Vegetale e Microbiologia Agraria 90128 Palermo, University of Palermo, Italy [email protected] 2 Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, U.K. [email protected] Abstract PCR amplification of the internal transcribed spacer regions and 5.8S gene of rDNA and sequencing or restriction digestion of the resultant product were used to confirm the identities of a collection of Phytophthora isolates from a variety of hosts in Italy. All isolates were of previously described species, but a number of new host-pathogen combinations of potential economic import ance were found, including P. palmivora on olive (Olea europaea) trees. Three Phyto ph - thora isolates from chestnut (Castanea sativa) trees were identified as P. cambivora, a known pathogen of chestnut. Existing, but previously unpublished, rapid, sensitive, DNA-based assays for P. cambivora and P. cinnamomi are described. In combination with similar diagnostics for other species such as P. cactorum, these assays could be used to detect nearly all important Phytophthora species known to occur in chestnut. Keywords: Phytophthora, ITS-RFLP, specific primers, diagnosis, Castanea sativa, Italy 1 Introduction The genus Phytophthora comprises approximately 60 species, nearly all of which are pathogens that damage plants and some of which are among the most important pathogens worldwide (ERWIN and RIBEIRO 1996). They cause economically devastating rots and blights of a wide range of crops and natural hosts, including many trees, but their identi- fication by classical criteria, chiefly morphology and physiology, is difficult and requires con- siderable expertise. Recently, advances in the molecular characterisation via the polymerase chain reaction (PCR) and the subsequent sequencing or restriction digestion of parts of the genomic ribosomal RNA gene repeat (rDNA) (COOKE et al. 2000, 2001) have allowed more rapid and objective identification of many Phytophthora taxa. The internal transcribed spacer (ITS) ITS1, the 5.8S gene and ITS2 can be amplified from pure cultures using the “Universal” primers, ITS6 (a version of ITS5 modified to give improved amplification from Ooymcota according to COOKE et al. [2000]) and ITS4 (WHITE et al. 1990) and the approx. 900 bp amplicon sequenced directly using the same primers. Alternatively, the same PCR product can be digested with restriction enzymes to yield RFLPs that are characteristics of a species or of small groups of closely related species. The sequences and RFLP patterns are compiled in databases against which unidentified isolates can be compared (COOKE et al. 2001). At present, the sequence database of the Scottish Crop Research Institute (SCRI) contains the sequences of >400 Phytophthora isolates of nearly all known species, and the RFLP database the combined digest patterns of many more. The methodology of this latter approach and the species identification profiles obtained with it have been made available on the worldwide web ‹www.phytID.org›. 352 Santa Olga Cacciola et al. Alignments of rDNA sequences have also been used to develop specific PCR primers for the diagnosis of disease and the detection of individual Phytophthora taxa in plant tissue (LACOURT et al. 1996, BONANTS et al. 1997). The sensitive species-specific detection proto- cols are based on a nested PCR, the first stage of which uses generic Peronosporales-specific primers to amplify the rDNA from all Phytophthora spp. (and Pythium, Peronospora, etc.) in a sample. As the amplicon from this first “generic” round contains complete ITS1 and ITS2 regions of any Phytophthora present in the sample, individual species of interest can be detected in the second round by using species-specific primers located in ITS1 (the forward primer) and/or in ITS2 (the reverse primer). The power of such an approach is that different combinations of second round primers can be used to test for more than one Phytophthora species in the same sample. This nested PCR has been used extensively and successfully to test for P. fragariae and P. cactorum within field samples of strawberry and raspberry plants entered for plant health certification (BONANTS et al. 2001). At SCRI, specific second-round primers have been designed against at least eight other Phytophthora species, including P. cambivora and P. cinnamomi, but have yet to be subjected to the same extensive laboratory and field testing. However, given the similarity of the primers for these two species to the primers for P. fragariae, it can be supposed that, in the second round of nested PCR, they will have comparative specificity and sensitivity. In P. fragariae the sensitivity is approx. 100 attograms (10-16 g) of DNA, or less than is contained in one zoospore (BONANTS et al. 2001). In this paper, the rDNA-based identification of a large collection of Phytophthora isolates from chestnut and other hosts in Italy is described. Specific primers for P. cambivora and P. cinnamomi are a pre-requisite for the molecular detection and diagnosis of these species on chestnut trees by nested PCR. The specificity of second round primer sets for these two species has been determined against the DNAs of a wide range of Phytophthora species in order to test whether they are suitable in nested PCR to diagnose these pathogens in chest- nut trees. 2 Materials and methods The isolates of Phytophthora examined in this study are listed in Table 1. They were grown in still culture in 20 ml of pea-broth (COOKE et al. 2000). After vacuum filtration, the mycelium was freeze-dried for extended storage at –20 ºC. DNA was extracted from mycelium using Puregene DNA extraction kit, (Flowgen, Lichfield, England), amplified by PCR using the universal primers ITS6 and ITS4, and digested with the restriction enzymes AluI, MspI and TaqI (COOKE et al. 2001). The resultant banding patterns were combined into one pattern using GelCompar software (Applied Maths, Kortrijk, Belgium), and this was analysed with reference to a database containing the ITS-RFLP patterns of approx. 1000 Phytophthora isolates, representing all known species (‹www.phytID.org›). Dendrograms showing clustering of banding patterns among the isolates were also generated by the above software (COOKE et al. 2001). The extraction of the DNA from the plant sample was as described by BONANTS et al. (2001). Primers used for nesting PCR are listed in Table 2. A standard first round of PCR with the “Peronosporales” primer set DC6 and ITS4 was followed by a second round using species-specif- ic primer sets. These were DC1 and DC5 for P. fragariae, DC4 and DC5 for P. cambivora and DC9 and DC5 for P. cinnamomi. Note that in each case the reverse primer is DC5. These three related species are similar in the ITS sequences, especially in ITS2. Primer DC5, which is located in ITS2, discriminates this group from other groups of species. Discrimination within the group resides in the forward primers, in each case located in ITS1 (see Table 2). Again in each case, the procedures and conditions for the second round of nested PCR are as described for P. fragariae (BONANTS et al. 1997), except that in the second round the annealing temperatures were 65 °C for P. fragariae, 60 °C for P. cambivora and 61 °C for P. cinnamomi. For. Snow Landsc. Res. 76, 3 (2001) 353 Table 1. Phytophthora isolates examined in this study. *Reference isolates from the Collection of SCRI. No. Species Isolate Country Host 1. P. megasperma Olivo corteccia Italy Olive bark 2. P. megasperma Udine 3CB Italy Peach root 3. P. megasperma Udine 3CE Italy Peach root 4. P. megasperma MEG1 UK* Raspberry root 5. P. gonapodyides PescoRC Italy Peach bark 6. P. asparagi sp. nov. Asp. Grosseto Italy Asparagus 7. Phytophthora sp. 'O' Udine-4CT Italy Peach 8. Phytophthora sp. 'O' Vivai Paternó Italy Palm 9. Phytophthora sp. 'O' PescoM Italy Peach 10. P. sojae A902051A Italy Soya 11. P. sojae A901213A Italy Soya 12. P. sojae A911412 Italy Soya 13. P. sojae A90205A Italy Soya 14. P. sojae A891112A Italy Soya 15. P. cambivora CAM1 UK* Raspberry root 16. P. cambivora P21FA3 Italy European Chestnut 17. P. cambivora P26F6 Italy European Chestnut 18. P. cambivora 218 Italy European Chestnut 19. P. cinnamomi MirtoA Italy Myrtle 20. P. cinnamomi Fuerte Italy Avocado 21. P. cinnamomi IMI77377 France Walnut 22. P. cinnamomi 11G5B Italy Avocado 23. P. cinnamomi CIN8 UK* Avocado 24. P. nicotianae 24STA Italy Buckthorn 25. P. nicotianae HybiscusB Italy Hibiscus 26. P. nicotianae Pn peperone Italy Green pepper 27. P. nicotianae C88 Italy Jojoba 28. P. nicotianae TL8V Italy Lavender 29. P. nicotianae RC=nicl Italy Citrus roots 30. P. nicotianae C92 Italy Hibiscus 31. P. nicotianae SR1 Italy Spider flower 32. P. nicotianae PnKVB Italy Kentia palm 33. P. nicotianae P22-IMI208688 UK* Citrus roots 34. P. nicotianae HY-A Italy Hibiscus 35. P. cryptogea Maria 2 Italy African daisy 36. P. cryptogea Climax 1 Italy African daisy 37. P. cryptogea CRY3 UK* Tomato 38. P. drechsleri DRE1 UK* Raspberry roots 39. P. cryptogea TGCAT Italy African daisy 40. P. cryptogea Pomodoro Acate Italy Tomato 41. P. cryptogea CH4 Italy Silverbush 42. P. cryptogea IMI21278 USA Callistephus hortensis 43. P. drechsleri CBS292.35 Italy Sugar beet 44. P. cryptogea Pomodora Ac B Italy Tomato 45. P. drechsleri EB1 Italy Escallonia 46. P. cryptogea Dimorfoteca Italy Cape marigold 47. P. cryptogea T2 Italy Pistachio tree 48. P. citrophthora IMI332632 UK* (Chile) Kiwi fruit plant 49. P. citricola P282 UK* Yew tree, soil 50. P. capsici P255 UK* Black pepper 51.