A selective PCR-based method for the identification of Phytophthora hibernalis

Preharvest and postharvest brown rot of citrus fruit is responsible for important economic losses thoroughout the world. The disease is commonly caused by several species of Phytophthora. In Spain, citrus brown rot is mainly caused by P. citrophthora, but in the last years outbreaks of the disease in lemon and sweet orange caused by P. hibernalis Carne have been recorded in northwestern citrus-growing areas. In this work, a PCR method has been developed for the diagnostic of P. hibernalis either from isolated mycelia or directly from fruit lesions. One specific primer pair, PHIB1 and PHIB2, was designed from the nucleotide sequences of ITS1 and ITS2 regions. The two primers amplified a 407-bp fragment from the genomic DNA of P. hibernalis that was sequenced (Acc. No. AY827556). Results demonstrated that PCR amplification of ITS regions by primers PHIB1 and PHIB2 followed by DNA sequencing can provide a rapid, selective and reliable identification of P. hibernalis. Additional key words: Citrus, diagnostics, fruit brown rot, oomycetes, specific primers.

brown rot of fruits, although differences in incidence among important citrus-growing areas have been reported.In Florida (USA), citrus brown rot is caused primarily by P. palmivora, and secondarily by P. nicotianae (Timmer et al., 2000).In mediterranean citrus plantations, outbreaks of Phytophthora diseases in citrus fruits have been mostly attributed to P. citrophthora, P. citricola, P. hibernalis and P. syringae (Erwin and Ribeiro, 1996;Vicent et al., 2003;Vernière et al., 2004).
In Europe, citrus crops are cultivated only in mediterranean countries.The main citrus producers are Spain, Italy and Greece (FAO, 2004).Spain produces 6.3 millions tons of fruit, mainly of sweet oranges, mandarins and lemons, and the main citrus-growing areas are in the East coast of the country (MAPA, 2004).In Spanish orchards, brown rot of fruits is mainly caused by P. citrophthora (Páez et al., 2004), and usually appears in years with excessive rains (higher than 100 mm) continuing for two or three days during the autumn and winter (Tuset et al., 1984).
Small tree plantations and isolated trees can also be found in other Spanish regions with low or null risk of frosts.This is the case of Galicia (NW Spain), in which small plantations of lemons and near 120,000 isolated trees, mostly of lemon and sweet orange, are produced mainly in two provinces, A Coruña and Pontevedra.Despite the climate of Pontevedra is maritime mediterranean, with mild temperatures in winter and little interannual thermal variability, since 2003 sporadic outbreaks of citrus brown rot in lemon and sweet orange caused by P. hibernalis have been recorded at the province.
P. hibernalis is seldom found causing brown rot of fruits in mediterranean countries, and has only been reported in areas with cool wet winters (Timmer and Menge, 1988).
The identification of P. hibernalis is solely based on morphological characteristics of sporangia and sexual organs.P. hibernalis is included in Stamps and Waterhouse morphological group IV (Stamps et al., 1990).It is an homothallic fungus that produces semipapillate, caducous sporangia with long pedicel, and can form paragynous and amphyginous antheridia (Waterhouse and Waterston, 1964).No molecular method specific for this Oomycete has been developed.However, molecular methods based on the analysis of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (rDNA) have complemented and greatly improved the diagnostics of most Phytophthora species.Ristaino et al. (1998) have proved PCR-RFLP patterns, obtained with the restriction enzymes RsaI, MspI, and HaeIII, to be effective for identification of isolates of 14 Phytophthora species, including P. nicotianae, P. citrophthora, P. citricola, and P. palmivora.Ippolito et al. (2002) designed specific primers to amplify, by nested PCR, DNA from P. nicotianae and P. citrophthora isolated from citrus roots and soils.Specific primers were also used for detecting P. nicotianae and P. citrophthora in citrus roots and soils by real-time experiments (Ippolito et al., 2004).
The aim of the present work was to develop a molecular method for the detection and identification of P. hibernalis.

Isolation of P. hibernalis
Forty two fruits from 11 trees of Citrus sinensis (sweet orange) and from 10 trees of C. lemon (lemon) showing symptoms of citrus brown rot were collected in the province of Pontevedra (NW Spain) from 2003 to 2007.Fragments of lesions from the fruit surface were placed in 9 cm Petri dishes containing V8-agar medium supplemented with pimaricin 5 mg L -1 , rifampicin 25 mg L -1 , hymexazol 5 mg L -1 , and benomyl 10 mg L -1 .Petri dishes were kept at 16°C in the dark for one week.Identification of the isolates was based on morphological characteristics of sporangia and sexual organs (Waterhouse, 1963;Stamps et al., 1990;Erwin and Ribeiro, 1996).

DNA extraction
Genomic DNA was extracted either directly from 10 to 40 mg of fragments of fruit lesions or from 10 to 40 mg of isolated mycelia.In case of mycelial samples, pieces of mycelium were removed with a sterile scalpel from the border of colonies actively growing in plates.For both types of material (fruit lesions and isolated mycelia), fungal genomic DNA was extracted with the EZNA Fungal DNA Miniprep kit (Omega Biotek), following the manufacturer's instructions with the exception of RNase or mercaptoethanol, which were not used.The DNAs were electrophoresed on 0.8% agarose gels buffered with 0.5 X TBE (0.89 M Tris base, 0.89 M boric acid, and 0.02 M EDTA pH 8.4) (BioRad) (Sambrook et al., 1989) and stained with ethidium bromide.
PCR reactions were carried out in microcentrifuge tubes each containing one PuReTaq Ready-To-Go™ PCR Bead (Amersham Biosciences).For both PCRs, each reaction tube further contained 1 µL genomic DNA, 0.3 µL of each primer (10 µM), and sterile water up to a f inal volume of 25 µL.DNA amplif ication reactions were carried out in a PCR-Express thermocycler (Thermo Hybaid) under the following conditions: one cycle at 94°C for 3 min; 30 cycles at 94°C for 30 s, 55ºC for 30 s, 72°C for 1 min; and a f inal cycle at 72°C for 10 min.Negative and positive controls were included in all experiments.PCR products were electrophoresed on 2% agarose gel as above described.The size of PCR products was calculated by comparison with a 100 bp standard ladder (Marker XIV, Roche Diagnostics), using the program ID-manager (TDI, Madrid).

RFLPs (restriction fragment length polymorphism)
Seven microliters from PCR reactions amplified by primers ITS4 and ITS6 were digested with the restriction endonucleases AluI, MspI and TaqI (Roche Diagnostic).Restriction digests were run on 3% agarose gel.A standard ladder of 100 bp was also run to accurate sizing of the restriction fragments.Restriction patterns were compared, through a telematic application, against a library elaborated by Cooke and Smith (2000) that includes patterns of over 40 Phytophthora spp.other than P. hibernalis.

DNA sequencing and design of specific primers for P. hibernalis
Amplicons obtained with primers ITS4 and ITS6 were purified using High Pure PCR Product Purification columns (Roche Diagnostics), according to the manufacturer's protocol.DNA sequences were obtained by automated DNA sequencing with fluorescent terminators using an ABI Prism Sequencer (Applied Biosystems).DNA sequences were analysed with the program Sequencing Analysis 5.1 (AP Biotech) and the BLAST algorithm (http:www.ebi.ac.uk).
Sequences were aligned by using the program Clustal X (Thompson et al., 1997).Seven candidate primers, internal to the universal primers ITS4 and ITS6, were identified and evaluated for melting temperature (Tm), G+C content, primer secondary structure and primer dimer formation, with the Beacon Designer software (Premier Biosoft International).Two primers were finally selected and designated PHIB1 (forward) and PHIB2 (reverse).
Further experiments were conducted to assess the sensitivity of both primers.Serial dilutions of genomic DNA from all P. hibernalis isolates, from 1 µg to 1 fg µL -1 , were prepared to determine the smallest amount of DNA template needed to obtain a PCR product with the primers.Negative controls (no DNA template) were also included.

Results
In culture, isolates from symptomatic fruits were homothallic and formed slowly growing hyaline fine mycelia, which were irregularly branched, without hyphal swellings or chlamydospores.Isolates produced spherical oogonia 22 to 56 µm in diameter, with antheridia mainly amphyginous (some paragynous were also observed), and yellow-orange plerotic oospores.Sporangia were elongated, ellipsoidal to ovoid, caducous and semipapillate, with long pedicels.These morphological characters indicated that all fruits were infected by P. hibernalis.
Since no molecular method had been previously reported for identification of P. hibernalis, techniques previously described for molecular identification of other pathogenic Phytophthora species were initially used.Primers ITS4 and DC6 yielded a single 1,300 bp band from all DNA templates, confirming that isolates were Oomycetes.
The universal primers ITS4 and ITS6, that have been successfully used to identify over 40 Phytophthora species (not including P. hibernalis) by RFLP-PCR analysis, amplified a single fragment of approximately 900 bp from all isolates, that was subsequently used for restriction enzymatic digestion.After digestion of DNA templates from our isolates of P. hibernalis, three different restriction patterns were obtained with endonucleases AluI, MspI and TaqI (Fig. 1).Five DNA fragments were found with the enzyme AluI (67, 78, 120, 180, 396 bp), 4 with MspI (81, 122, 324, 378 bp) and 5 with TaqI (89,104,159,195,292 bp).When matching these experimental RFLP patterns against the library available at a telematic application, considering a band width of 10% and lower cut-off length of 10%, the best match was P. lateralis with a 59% similarity.
In contrast with the inefficacy of the RFLP-PCR method for the identification of P. hibernalis, when the sequence of DNA amplified by primers ITS4 and ITS6 from citrus isolates in Pontevedra was matched against the GenBank library, a 100% homology with the corresponding sequences of P. hibernalis was found.This sequence was deposited in the GenBank database as Acc.No. AY827556.
To develop specific primers for P. hibernalis, multiple sequences of the ITS region of P. hibernalis and another 15 Phytophthora species were aligned.Two primers were designed: PHIB1 (5'-TCGGGTCTGAGCTAGTA GTCTT-3') and PHIB2 (5'-CTTCCACAACCAATTC CATTATGC-3').The primers amplify a 407 bp product that contains parts of ITS1, ITS2 and the whole 5.8S  subunit.The primer sequences were matched against those of all Phytophthora species included in GenBank, no homology being found except for P. hibernalis.Primers PHIB1 and PHIB2 were then tested to amplify DNA of P. hibernalis mycelial samples and fruit lesions from Pontevedra, culture type specimens of P. hibernalis, and collection isolates of seven other Phytophthora species and Pythium spp., yielding an expected 400 bp band for P. hibernalis and no band for the rest, with the only exception of a light band for P. citricola DNA.These PCR results were obtained with an annealing temperature of 64°C.In experiments where the temperature was increased up to 66-69°C, that band disappeared.It must be outlined that primers PHIB1 and PHIB2 gave no cross reaction with P. ramorum.All sequences of DNA amplified by these two primers matched those of P. hibernalis available at GenBank library.
Experiments on primer sensitivity showed that the smallest concentration of DNA template needed to obtain a PCR product was 1 pg µL -1 , that corresponds to a concentration of 40 fg µL -1 in the reaction tube (Fig. 2).

Discussion
Phytophthora taxonomy has been based until recently on morphological characters of isolates in culture.Sporangia and sexual organs of Citrus isolates from Pontevedra resembled those described for P. hibernalis.However, this type of identification needs considerable experience and time of the researcher.Tools to unequivocally differentiate Phytophthora species are needed.Molecular methods are now considered useful, reliable and fast techniques for diagnostics of many Phythophthora species, although none had been described specifically for P. hibernalis.
In this work, the RFLP-PCR method proved to be not sufficient for a reliable molecular identification of P. hibernalis.The experimental RFLP patterns yielded by AluI, MspI and TaqI for P. hibernalis best matched, although poorly, with those from P. lateralis included in the library of Cooke and Smith (2000), and were also similar to those achieved with these enzymes for P. ramorum isolates from Camellia japonica and Viburnum tinus (Pintos et al., 2004).Our results are in agreement with previous works that have demonstrated the close relationship between P. ramorum, P. lateralis and P. hibernalis (Martin and Tooley, 2003;Ivors et al., 2004).In fact, differentiation of isolates of these Phytophthora species by traditional methods is difficult, since, as discussed by Martin and Tooley (2003), they share some morphological characteristics.Phytophthora hibernalis and P. lateralis are homothallic, and P. ramorum is heterothallic.P. ramorum and P. hibernalis have semipapillate caducous sporangia, whereas P. lateralis can be distinguished from them because it is in morphological group V and has non-papillate noncaducous sporangia.These three species also share some environmental requirements, as their growth is favoured by cool temperatures (Erwin and Ribeiro, 1996;Werres and Zielke, 2003).
In this work, two primers have been designed, namely PHIB1 and PHIB2, that amplify a product which sequence matches with a 100% homology those of P. hibernalis available at GenBank library and of two culture type specimens of the pathogen, with no cross reaction for P. ramorum.Primer sensitivity was similar to those reported for primers designed for P. nicotianae and P. citrophthora (Ippolito et al., 2002), and for P. capsici (Silvar et al., 2005).
Results demonstrate that PCR amplification of ITS regions by primers PHIB1 and PHIB2 followed by DNA sequencing can provide a rapid, selective and reliable identification of P. hibernalis either from fruit lesions or from isolated mycelia.The diagnosis of P. hibernalis in fruits can be a useful and fast tool to undertake prevention and appropriate management methods to control brown rot of Citrus fruits in the field.
There is only one previous record of P. hibernalis on sweet orange and lemon in NW Spain, identified by conventional culture methods (Urquijo et al., 1971), although the authors did not provide details on season of detection and climatic conditions, methods of isolation, or whether it was a preharvest or postharvest infection.In our study the fruits affected by P. hibernalis were collected from 2003 to 2007.In November 2003, precipitations were double those normally found in the Atlantic coast during that month, reaching 400 mm; maximum temperatures showed levels lower than average for the same month in other years (Xunta de Galicia, 2004).Although records for 2005 and afterwards have not been still published, precipitations in November 2005 and 2006 were also high and maximum temperatures rather low.These two anomalous climatic situations in November might explain the presence of P. hibernalis in the province of Pontevedra.It is widely known that intense precipitation favours the incidence of rot diseases caused by Phytophthora spp. in Citrus (in Spain particularly in October and November) (Tuset et al., 1984), and that low temperatures benefit growth of P. hiber-nalis (Erwin and Ribeiro, 1996).Severe weather events, such as precipitation anomalies and greater temperature variations, are predicted to increase due to global climate change (Rosenzweig et al., 2001).As outlined by Anderson et al. (2004), gradual change of climate can lead to disease emergence of pre-existing pathogens as major disease agents.This points out the need for a special attention to the incidence of P. hibernalis in citrus-growing areas, but also for developing research on its biology, host range, epidemiology and pathogenicity, aspects all poorly studied up to now.The specific primers PHIB1 and PHIB2 developed for the identification of P. hibernalis might be a useful tool in all these studies.
ramorum has a broader host range that is continuously increasing.