For. Snow Landsc. Res. 76, 3: 351–356 (2001) 351

Molecular identification and detection of 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, [email protected] 2 Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, , 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 importance were found, including P. palmivora on olive (Olea europaea) trees. Three Phytoph - 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 Existing, but previously unpublished, rapid, sensitive, DNA-based assays for P. cambivora and P. cinnamomi are described.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 -specific primers to amplify the rDNA from all Phytophthora spp. (and , 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 and 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, ), 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 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* 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. P. capsici (P.p. MF4) CH2 Italy Silverbush 52. P. capsici Peperone Italy Green pepper 53. P. citrophthora Spina Italy Citrus 54. P. cactorum UK* Strawberry crown 55. P. cactorum Noce Italy English walnut 56. P. palmivora PalmI Italy Olive roots 57. P. palmivora PalmII Italy Olive roots 58. P. palmivora P488 UK* Coconut 354 Santa Olga Cacciola et al.

Table 2. Primers used in nested PCR for the specific detection of Phytophthora species. Conditions for the various rounds of PCR are described in BONANTS et al. (1997).

Target organism(s) Forward Primer Reverse primer First round of nested PCR Peronosporales DC6 5’ GAGGGACTTTTGGGTAATCA 3’ ITS4 5’ TCCTCCGCTTATTGATATGC 3’ Second round of nested PCR P. fragariae DC1 5’ ACTTAGTTGGGGGCCTGTCT 3’ DC5 5’ CGCCGACTGGCCACACAG 3’ P. cambivora DC4 5’ TTAGTTGGGGGCTAGTCCC 3’ DC5 as above P. cinnamomi DC9 5’ AACTGAGCTAGTAGCCTCTC 3’ DC5 as above

3 Results and discussion 3.1 Identification The affinities of the Phytophthora isolates in the Italian collection, determined with refer- ence to the ITS-RFLP database, are shown in Figure 1. The dendrogram generated in Figure 1 is based on RFLP banding patterns, which, in comparison to sequence data, reveal only crude measures of phylogenetic relationships. Minor gel-to-gel variation in the ITS-RFLP patterns within a species is inevitable and should not necessarily be interpreted as intra- specific variation. This is certainly true for the four isolates of P. cambivora examined. If the identity of an isolate was not resolved by ITS-RFLP, then its PCR amplicon was sequenced and compared with the sequence database in order to confirm its identity. All isolates clustered with the reference isolates of known species of Phytophthora, although some host-pathogen combinations are uncommon. Noteworthy was the occurrence of P. palmivora on olive (Olea europaea), a first record for this host-pathogen combination. P. palmivora causes damaging and economically important diseases on a number of tropical hosts: black pod of cocoa, root and fruit rot of papaya, and bud rot of coconut (ERWIN and RIBEIRO 1996). Its occurrence on olive, an economically and ecologically important plant in the Mediterranean, must be a cause for concern as it causes olive root rot. Other new host-pathogen combinations were also found, including Phytophthora sp. “O- group” on peach (Prunus persica) and queen palm (Syagrus romanzoffianum) and another species very similar to P. gonapodyides on peach (pers. comm. C. Brasier, Forestry Commission, Alice Holt, U.K. and David Cooke). P. gonapodyides sensu stricto was recently isolated from stream-beds within chestnut (Castanea sativa) stands in Italy (VETTRAINO et al. 2001). Phytophthora sp. “O-group”, shortly to be described as a new species (Clive Brasier, pers. comm.; COOKE et al. 2000, SÁNCHEZ-HERNÁNDEZ et al. 2001), has not been reported previously from Italy. All three isolates recovered from European chestnut were identified as P. cambivora, a species already implicated in chestnut decline. Interestingly, all three P. cambivora isolates contained an ITS polymorphism, as revealed by the digestion with restriction enzyme MspI that occurs only in this species (COOKE et al. 2000) and in some isolates of the new pathogen of alders in Europe (BRASIER et al. 1999).

3.2 Detection The P. cambivora- and P. cinnamomi-specific primer sets were tested, together with the primer set for P. fragariae, against DNA extracted from nineteen Phytophthora species (Fig. 2). In each case the primers only amplified DNA from the species for which it had been designed. Both the P. cambivora and P. cinnamomi primers have also successfully detected the presence of these fungi in occasional tests on naturally infected woody material. Similar primer sets have been designed for other species: P. cryptogea/P. drechsleri, P. quercina, P. nicotianae, P. syringae and P. citricola, most of which infect woody hosts, including trees. As previously shown for P. fragariae (BONANTS et al. 2001), the primers for P. cambivora and For. Snow Landsc. Res. 76, 3 (2001) 355

P. cinnamomi could also be used for their sensitive and rapid detection in planting material, and in water and soil. The test for P. fragariae in strawberry is sufficiently sensitive to detect even less than 0.5% (w/w) infection in a sample of root material in less than two days; it is also effective on water and soil samples. There is every prospect that PCR tests for species affecting trees will also be as sensitive and rapid (NECHWATAL et al. 2001).

40 50 60 70 80 90 100 Restriction digest enzyme Isolate Species AluI MspI TaqI 1 P. mega sperma 2 “ “ 3 “ “ 4 “ “ 5 R. gonapodyides-like 6 P. asparagi sp. nov. 7 Phytophthora sp.‘O’ 8 “ “ “ 9 “ “ “ 10 P. sojae 11 “ “ 12 “ “ 13 “ “ 14 “ “ 15 P. cambivora 16 “ “ 17 “ “ 18 “ “ 19 P.cinnamomi 20 “ “ 21 “ “ 22 “ “ 23 “ “ 24 P. nicotianae 25 “ “ 26 “ “ 27 “ “ 28 “ “ 29 “ “ 30 “ “ 31 “ “ 32 “ “ 33 “ “ 34 “ “ 35 P. cryptogea 36 “ “ 37 “ “ 38 P. drechsleri 39 P. cryptogea 40 “ “ 41 “ “ 42 “ “ 43 P. drechsleri 44 P. cryptogea 45 P. drechsleri 46 P. cryptogea 47 “ “ 48 P. citrophthora 49 P. citricola 50 P. capsici 51 “ “ (MF4) 52 “ “ 53 P. citrophthora 54 P. cactorum 55 “ “ 56 P. palmivora 57 “ “ 58 “ “

Fig. 1. ITS-RFLP patterns of the Italian Phytophthora isolates after clustering with those of SCRI refer- ence isolates. The PCR (primers ITS6 and ITS4) amplicons were digested with each of three restriction enzymes (AluI, MspI and TaqI) and the three profiles combined end-to-end in a single gel strip. Cluster analysis grouped isolates according to the degree of similarity in the banding patterns of their gel strips. 356 Santa Olga Cacciola et al.

A

1 2 3 4 5 6 7 8 9 L 10 11 12 13 14 15 16 17 18 19 B Fig. 2. Amplification of the DNA of: 1 Phytophthora fragariae var. fragariae; 2 Phytophthora fragariae var. rubi; 3 P. cambivora; 4 P. cinnamomi; 5 P. sojae; 6 P. cryptogea; 7 P. drechsleri; 8 P. gonapodyides; 1 2 3 4 5 6 7 8 9 L 10 11 12 13 14 15 16 17 18 19 9 P. megasperma v meg; 10 P. citricola; 11 P. capsici; C 12 P. citrophthora; 13 P. palmivora; 14 P. megakarya; 15 P. ilicis; 16 P. nicotianae; 17 P. infestans; 18 P. idaei; 19 P. cactorum with: A primers DC1 and DC5 designed to amplify rDNA of P. fragariae; B primers DC4 and DC5 for P. cambivora; C primers DC9 and DC5 for 1 2 3 4 5 6 7 8 9 L 10 11 12 13 14 15 16 17 18 19 P. cinnamomi.

4 References

BONANTS, P.J.M.; HAGENAAR-DE VEERDT, M.; VAN GENT-PELZER, M. P.; LACOURT, I.; COOKE, D.E.L.; DUNCAN, J.M., 1997: Detection and Identification of Phytophthora fragariae Hickman by the Polymerase Chain Reaction. Europ. J. Plant Pathol. 103: 345–355. BONANTS, P.J.M.; VAN GENT-PELZER, M.P.; HAGENAAR-DE VEERDT, M., 2001: Characterization and detection of Phytophthora fragariae in plant, water and soil by molecular methods. OEPP/EPPO Bull. 30: 525–531. BRASIER, C.M.; COOKE, D.E.L.; DUNCAN, J.M., 1999: Origin of a new Phytophthora pathogen through interspecific hybridization. Proceedings of the National Academy of Science of of America. 96: 5878–5883. COOKE, D.E.L.; DRENTH, A.; DUNCAN, J.M.; WAGELS, G.; BRASIER, C.M., 2000: A molecular phylogeny of Phytophthora and related . Fungal Genet. Biol. 30: 17–32. COOKE, D.E.L.; DUNCAN, J.M.; WILLIAMS, N.A.; HAGENAAR-DE WEERDT, M.; BONANTS, P.J.M., 2001: Identification of Phytophthora species on the basis of restriction enzyme fragment analysis of the Internal Transcribed Spacer regions of ribosomal RNA. OEPP/EPPO Bull. 30: 519–523. ERWIN, D.C.; RIBEIRO, O.K., 1996: Phytophthora Diseases Worldwide. APS Press, American Phytopathological Society, St Paul. 562 pp. LACOURT I.; BONANTS, P.J.M.; VAN GENT-PELZER, M.P.; COOKE, D.E.L.; HAGENAAR DE VEERDT, M.; SURPLUS, L.; DUNCAN, J.M., 1996: The use of nested primers in the polymerase chain reaction for the detection of Phytophthora fragariae and P. cactorum in strawberry. Acta Horticulturae (Proceedings of the Third International Strawberry Symposium, Veldhoven, The , 19–24 May 1996.) 493: 829–838. NECHWATAL, J.; SCHLENZIG, A.; JUNG, T.; COOKE, D.E.L.; DUNCAN, J.M.; OßWALD, W.F., 2001: A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak stands. Forest Pathol. 31: 85–97. SÁNCHEZ-HERNÁNDEZ, S.; MUÑOZ-GARCIA, M.; BRASIER, C.M.; TRAPERO-CASAS, A., 2001: Identity and pathogenicity of two taxa associated with a new root disease of olive trees. Plant Dis. 85: 411–416. VETTRAINO, A.M.; NATILI, G.; ANSELMI, N.; VANNINI, A., 2001: Recovery and pathogenicity of Phytophthora species associated with a resurgence of ink disease in Castanea sativa in Italy. Plant Pathol. 50: 90–96. WHITE, T.J.; BRUNS, T.; LEE, S.; TAYLOR, J., 1990: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: INNIS, M.A.; GELFAND, D.H.; SNINSKY, J.J.; WHITE, T.J. (eds) PCR Protocols: A guide to Methods and Applications. San Diego, Academic Press. 315–322. Accepted 19.2.02