Journal of Plant Pathology (2003), 85 (1), 15-25

Edizioni ETS Pisa, 2003

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RAPID AND SENSITIVE DETECTION OF ROSELLINIA NECATRIX IN ROOTS AND SOILS BY REAL TIME SCORPION-PCR*
L. Schena and A. Ippolito
Dipartimento di Protezione delle Piante e Microbiologia Applicata, Universit degli Studi, Via Amendola 165/A, 70126, Bari, Italy

SUMMARY Two primer pairs specific for Rosellinia necatrix, designed from the internal transcribed space (ITS) regions, were utilised to develop a molecular detection method suitable for large-scale analyses of soils and plant materials. One primer (R15) was modified to obtain a Scorpion primer for detecting a specific 71 bp amplicon by fluorescence emitted from a fluorophore through a self-probing PCR assay. Primer R15 Scorpion in combination with a reverse conventional primer (R18) produced a more intense fluorescent signal compared to a previously reported pair of Scorpion primers. Primer specificity was assessed both by means of BLAST (Basic Local Alignment Search Tool) analyses to exclude the presence of similar sequences in other microrganisms among available DNA databases (GenBank) and by using genomic DNA from a large number of R. necatrix isolates and other fungi from several hosts and different geographic areas. Simple, rapid, and effective procedures for direct DNA extraction from soil and plant materials were developed to yield DNA of purity and quality suitable for PCR assays. Combining these protocols and a single amplification with primer R15 Scorpion-R18 it was possible to detect the pathogen in naturally and artificially infected host tissues. To detect the pathogen from infested soils, and to improve the sensitivity on host tissue, a nested Scorpion-PCR with conventional (R3-R8) and Scorpion (R15 Scorpion-R18) primers was utilised. The reliability of the entire procedure was assessed using both artificially and naturally infested soils and plant materials from 26 different woody species. Compared to traditional detection methods based on baiting and fungal isolation on agar media, the molecular approach proved to be more rapid, sensitive, reliable, and enabled large scale analyses. Key words: white root rot, molecular detection, nested-PCR, ITS regions, real-time PCR, quantitative PCR.

INTRODUCTION Rosellinia necatrix Prill. (anamorph: Demathophora necatrix Hartig) induces root and crown rots (white root rot) on many fruit and forest tree species. The pathogen is widely distributed throughout temperate climates (Anonymous, 1987; Anselmi and Giorgetti, 1990). In Italy, R. necatrix is widespread and a recent extensive field survey has shown an increasing trend of attacks especially to sweet cherry and olive (Amenduni et al., 2001). The pathogen is disseminated by propagating materials and once in the soil it can survive for many years. Control strategies including cultural practices and soil disinfestations (Guillaumin, 1986), chemical treatments (Gupta, 1977), soil solarization (Freeman et al., 1990; LpezHerrera et al., 1999) and biological control (Freeman et al., 1986; Sztejnberg et al., 1987; Arakawa et al., 2002) are expensive and not always effective. Therefore, white root rot control largely depends on attempts to exclude the pathogen through the use of R. necatrix-free propagating material and planting in non-infested soils. Appropriate phytosanitary provisions have been issued by the European and Mediterranean Plant Protection Organization (EPPO) to prevent the spread of dangerous pathogens such as R. necatrix (Anonymous, 2002). In particular, EPPO activities encourage the development of detection methods effective and suitable for large-scale analyses. Current detection of R. necatrix involves isolation of the pathogen from infected tissues and baiting from infested soils (Sztejnberg et al., 1987). Both procedures are laborious, time consuming (1-3 weeks) and require skilled expertise to identify the pathogen after isolation (Petrini, 1992). These constraints have encouraged the search for alternative approaches. Recently, molecular techniques have contributed to the alleviation of some of the challenges associated with detection, control, and movement of plant pathogens. In particular, considerable success was obtained in the detection of soil borne pathogens by PCR-based techniques (Bridge and Spooner, 2001; Ippolito et al., 2002). Nevertheless, technical limitations related to post-amplification procedures still limit large-scale applications of PCR for plant pathogen diagnosis (Finetti and Gallitelli, 2001). Real time-PCR combines the sensitivity of conventional PCR with the generation of a specific fluo-

Corresponding author: L. Schena Fax. +39.080.5442911 E-mail: leonardo.schena@agr.uniba.it * Paper recipient of the Giovanni Scaramuzzi Award sponsored by the Italian Society for Plant Pathology.

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rescent signal, which can be measured throughout the procedure, providing real time-analysis of the reaction kinetics and allowing quantification of specific DNA targets (Schmittgen, 2001). Among PCR-based real time techniques, Taq-Man (Lee et al., 1993), Molecular beacons (Tyagi and Kramer, 1996), and Scorpion-PCR (Whitecombe et al., 1999) are the most promising and there are increasing reports of their use in plant pathology (Weller et al., 2000; Bates et al., 2001; Cullen et al., 2001). Scorpion-PCR seems to be more rapid and sensitive (Thelwell et al., 2000) and it has been utilised both in human pathology to detect HIV virus (Saha et al., 2001) and in plant pathology to identify and detect antagonistic (Schena et al., 2000, 2002a) or pathogenic fungi (Ippolito et al., 2000; Bates and Taylor, 2001; Nigro et al., 2002), viruses (Finetti et al., 2000), and nematodes (Ciancio et al., 2000). In a previous study, several R. necatrix specific primers were identified in the internal transcribed spacer (ITS) regions and two conventional (R2-R8) and Scorpion (R10 Scorpion-R7) pairs were utilised to identify and detect R. necatrix in artificially infested soils (Schena et al., 2002). In the present work, primer R2 was replaced by primer R3 (Schena et al., 2002) and a new pair of Scorpion primers (R15 Scorpion-R18) was designed. These primers were utilised to develop a reliable, rapid, and sensitive detection method suitable for routine analyses of soils and plants.

Fig. 1. Schematic representation of R15 Scorpion probe. R15 Scorpion is composed of a primer element (R15), a non amplifiable monomer (HEG), a quencher (MR), a closed stem, a probe element and a fluorophore (FAM) (Whitecombe et al., 1999).

MATERIALS AND METHODS Primer selection and specificity. The internal transcribed spacer regions (ITS1 and ITS2) of two Italian isolates of R. necatrix were amplified using the universal primers ITS5-ITS4 (White et al., 1990) and PCR products sequenced by MWG-Biotech (Florence, Italy). Two reported primers (R3-R8) (Schena et al., 2002) and a new pair of primers (R15-R18), designed from the ITS regions, were utilised to specifically amplify DNA fragments from R. necatrix. The primer R15 (5-CCATAGGCGAGATGAGAAATC-3) was modified according to Whitcombe et al. (1999) to obtain a Scorpion primer (Fig. 1) and utilised in conjunction with primer R18 (5CAGCCCCTCGAAGTCAGT-3) for real time identification of R. necatrix. Primer pairs R3-R8 amplified a DNA fragment of 388 bp comprising the amplified regions of R15-R18 (71 bp). This primer localization enabled the development of a nested-PCR providing a first amplification with primers R3-R8 and a second amplification with primers R15 Scorpion-R18 (nested Scorpion-PCR). Specificity of primer pairs R3-R8 and R15 ScorpionR18 was preliminarily assessed by means of Basic Local Alignment Search Tool (BLAST) analyses to explore all of the available sequence DNA databases and exclude the presence of similar sequences in other microrganisms. Furthermore, specificity of the selected primers

was assessed as previously described (Schena et al., 2002) using genomic DNA from a large number of fungal (Table 1) and R. necatrix (Table 2) isolates from several hosts and different geographic areas. To compare primer pairs R15 Scorpion-R18 with the previously reported primers R10 Scorpion-R7 (Schena et al., 2002), genomic DNA was extracted from a pure culture of R. necatrix, diluted to 100 ng l-1 and amplified in triplicate with both Scorpion primers. Water was utilised as a control. Melting curves from 30 to 95C were constructed on the amplified reaction mixtures from both primer pairs using the following cycling conditions: initial denaturation for 5 min at 95C, cooling to 30C for 3 s and melting from 30 to 95C with a 0.2C transition rate every 7 s. Fluorescence was measured continuously during the melting phase. Conventional and real-time PCR reactions as well as data analyses were performed using a spectrofluorometric Bio-Rad thermal cycler as previously described (Schena et al., 2002), except that the annealing-extension temperature was reduced from 60 to 58C. The relative normalised fluorescence (Rn) (Lockey et al., 1998) was utilised to calculate the average background fluorescence emission in the initial PCR cycles before fluorescence increased. Threshold fluorescence intensity was established at 10 fold above the standard deviation in the initial PCR cycles and any sample that reached a fluorescence value exceeding the fluorescence threshold value was considered as positive. The PCR cycle at which fluorescence exceeded the threshold was defined as the cycle threshold (Ct). Therefore, data from molecular analyses of plant and soil samples were recorded as Ct. DNA extraction from host tissues and soils. Total DNA was extracted directly from small roots or bark pieces collected from large roots and crown. Samples were washed with running tap water, dried on blotting

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Table 1. Fungal species utilized to evaluate primer specificity.
Alternaria spp. (6)* Alternaria brassicicola Alternaria citri Aspergillus spp. (2)* Aspergillus niger Botryosphaeria sp. Botryosphaeria ribis (2)* Botrytis cinerea (3)* Camarosporium sp. Cephalosporium sp. Cladosporium sp. Colletotrichum sp. Cylindrocarpon sp. Cytospora sp. Endothia parasitica Eutypa lata Fomitiporia punctata Fusarium spp. (7)* F. roseum Fusicoccum amygdali Gliocladium spp. (2)* Gliocladium roseum Gloeosporium sp. Macrophomina sp. Mycocentrospora cladosporioides Myrothecium roridum Penicillium sp. Penicillium funiculosum Penicillium digitatum (2)* Penicillium italicum Phaemoniella chlamydospora Phialophora sp. Phialophora parasitica Phoma sp. Phomopsis diospyri Phomopsis viticola Phyllosticta sp. Phytophthora spp. (4)* Phytophthora boehmeriae Phytophthora cactorum Phytophthora capsici Phytophthora cinnamomi Phytophthora citricola Phytophthora citrophthora Phytophthora cryptogea Phytophthora erythroseptica

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Phytophthora heveae Phytophthora nicotianae Pleurotus ostreatus Pythium spp. (3)* Rhizoctonia solani Rosellinia aquila Rosellinia limoniispora Rosellinia mammiformis Rosellinia millegrana Rosellinia reticulispora Rosellinia sanguinolenta Sclerotinia spp. (2)* Septoria tritici Stemphylium sp. Trichoderma spp. (4)* Trichoderma harzianum Trichoderma koningii Trichoderma pseudokoningii Trichoderma viride Trichothecium sp. Ulocladium sp Verticillium albo-atrum Verticillium dahliae (2)*

* Number of isolates analysed.

Table 2. R. necatrix isolates utilized to evaluate primer specificity.

Host Sweet cherry Olive Peach Almond Walnut Pear Grapevine Pistachio Narcissus Apple Avocado N. isolates 27 20 9 6 6 1 2 1 1 4 2 Origin Southern Italy Southern Italy Southern Italy Southern Italy Southern Italy Southern Italy Southern Italy (1), Japan (1) Southern Italy Netherlands (CBS 267.30)* Southern Italy (2), Israel (1), Argentina (1) (CBS 349.36)* Israel (1) and Spain (1) (CECT 2817)** Central Italy (1) and New Zealand (9) Japan Spain (CECT 2818)**

Poplar 10 Japanese pear 3 Unknown 1

* CBS = Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands) ** CECT = Coleccion Espaola de Cultivos Tipo (CECT, University of Valencia, Valencia, Spain).

paper and cut to yield pieces of 3-4 cm. Two to three g of tissue were ground with liquid nitrogen and 0.1 g of the powder was transferred to 2-ml screw-cap tubes containing an equal volume of polyvinylpyrrolidone (PVP) together with two 5 mm stainless steel ball bearings, 0.5 g acid-washed glass beads (425-600 m diameter), and 1.5 ml of extraction buffer [200 mM Tri-HCl

(pH 7.7), 250 mM NaCl, 25 mM EDTA, 0.5% SDS]. The extraction mixture was blended in a FastPrep FP120 Instrument (Qbiogene, France) at 4 m s-1 for 40 s and centrifuged at 13000 x g for 5 min at 4C. The upper phase (approximately 800 l) was extracted twice with 1 ml of phenol/chloroform (1:1) and 700 l of chloroform, respectively. DNA was precipitated with an equal volume of isopropanol for 1 h at 5C, washed with 70% cold ethanol (-20C), dried and resuspended in 50 l of sterile distilled water. Before amplification, DNA was purified using a polyvinylpolypyrrolidone (PVPP) spin column. A small hole was pierced at the bottom of 0.5 ml Eppendorf tubes from which the cap was removed. These tubes were held in 2 ml tubes and filled with a drop of glass beads (autoclaved and stored in TE pH 8 buffer) and dry PVPP powder. Columns were conditioned by two sequential additions of 400 and 200 l of dH2O, each followed by a 3 min centrifugation at 3000 x g. DNA solutions (50 l) were added to the top of the PVPP powder and columns were centrifuged for 3 min at 3000 x g. Purified eluate was collected in a sterile 1.5 ml tube, quantified by spectrophotometry and stored at 20C. DNA from soil was extracted as previously reported (Schena et al., 2002), using hexadecyltrimethylammonium bromide (CTAB) buffer. DNA was purified with Sepharose CL-6B (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) using a spin column (Bramwell et al., 1995), quantified by spectrophotometry, and stored at 20C. DNA from soils and plant tissues was extracted in

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triplicate and 1 l of purified DNA was amplified by Scorpion-PCR and nested Scorpion-PCR. In the latter, 1 l of amplified product from conventional PCR was utilised as a template in the real time amplification. Detection of R. necatrix from host tissues. Detection in artificially infected plants and plant parts. Healthy and artificially inoculated soils, obtained using Roselliniacolonised grains as described by Sztejnberg and Madar (1980), were utilised to grow plantlets of 11 different species (Fig. 4) in 2 litre plastic pots. Pots were maintained in a greenhouse at 252C (day), 152C (night), and watered once a week. Samples (roots, crown, and trunk base) were collected after the appearance of decline symptoms (1-3 months later, depending on the species) and processed to extract DNA. Moreover, a practical approach based on the use of artificially infected cutting was developed to validate the detection method on a large number of hosts. Cuttings with a diameter of approximately 10 mm were collected from 26 different woody species (Table 3), cut to segments of 15 cm, and inserted for half their length in an artificially infested soil in plastic boxes. Cuttings stuck in a non-inoculated soil served as negative controls. For each species and treatment 5 replicate cuttings were used. Soils were moistened, covered with a lid and maintained at 24C and 9598% RH. After 40 days in the dark, bark was collected from infected and non-infected cuttings (half inserted in the soil) and used for DNA extraction. Detection in naturally infected plants. Potentially infected plants with and without symptoms of decline were collected from fields in which R. necatrix had been previously isolated. Samples included roots and crown, and were collected from sweet cherry (3 samples), olive (3 samples), and walnut trees (6 samples). Six healthy plants (2 per species) collected from uncontaminated fields were utilised as negative controls. For each sample 3-4 g of bark and/or roots were used for DNA extraction. The remaining parts of each sample were carefully washed with tap water, surface sterilised with Na-hypochlorite (2 min in a 1% solution), rinsed with distilled water, dried under a laminar hood and utilised to isolate the pathogen. Isolations were carried out on potato dextrose agar (PDA) with addition of streptomycin sulphate and ampicillin (250 mg l-1 each). After 5 days of incubation at 24C in the dark, the percentage of bark and/or root segments yielding R. necatrix was determined. Identification was performed on 15 day-old mycelium by microscope observation of the pear-shaped swellings adjacent to septa typical of R. necatrix (Francis, 1985). Detection of R. necatrix in soil. Detection in naturally infested soils. Ten potentially infested soils were collected from the base of declining walnut trees from which the pathogen had been previously isolated. Five

soils collected from fields in which non-infected plants were observed over a 10-year period of inspections were utilised as negative controls. From each soil (approximately 1 kg) triplicate samples of 0.5 g were used for DNA extraction. The remaining soil was analysed by means of a baiting test as described by Sztejnberg et al. (1987). Briefly, soils were placed in plastic boxes with 100 avocado leaf discs (10 mm in diameter) serving as traps for R. necatrix. These containers were incubated under light conditions at 25C and after 15 days the percentage of leaf discs colonised by R. necatrix was recorded. Sensitivity of detection. To compare sensitivity of the molecular detection system with traditional baiting method an artificially infested soil was serially diluted with a healthy soil to have 10 mixtures with a progressively lower concentration of pathogen (Fig. 7). From each soil mixture (1 kg) after accurate mixing, three soil sample replicates of 0.5 g were collected for DNA extraction. The remaining mixture was analysed by baiting using avocado leaves as described above. The data from molecular analyses were subjected to 1st degree regression analysis (Snedecor and Cochran, 1980) to evaluate the correlation between the percentage of infested soil (Log scale) and the PCR cycles at which fluorescence arose above the fixed threshold level (Ct). Persistence of R. necatrix DNA in soil. The persistence of R. necatrix DNA in soils was assessed by analysing an artificially infested soil treated at different temperatures to kill the pathogen. Uniform samples of 3 kg held in 5 l Erlenmeyer flasks were closed with a waterproof cotton plug and treated at 60, 80, or 100C for 1 h. An untreated infested soil sample and a healthy sample were utilised as positive and negative controls, respectively. All soils were maintained at 24C in the dark for 30 days. From each sample 1 kg of soil was collected after 1, 5, and 15 days of incubation and analysed by means of molecular and baiting tests as already described.

RESULTS Primer specificity. Alignments obtained among ITS regions of R. necatrix and database sequences (GenBank) of other species revealed differences of at least three bases in the target sequences of the selected primers (R3, R8, R15, and R18) except for Rosellinia arcuata (accession number AB017660) which had 100% homology. Previously reported primer R2 (Schena et al., 2002) was shown to be less specific compared to primer R3, since similar sequences (differences of only two bases) were found in database sequences of some Xylaria and Nemania species. ITS sequences of Italian isolates were identical to the same regions to two other Japanese isolates of R. necatrix (accession numbers AB017657 and AB017658). Primer specificity was confirmed by the amplification of a DNA fragment of the

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expected size (primer R3-R8) and the increase of a specific fluorescence signal (primer R15 Scorpion-R18), for all the isolates of R. necatrix (Table 2) but not for the other fungi (Table 1) (data not shown). Comparison between primer pairs R15 ScorpionR18 and the previously reported R10 Scorpion-R7 showed a relative normalised fluorescence (Rn) approximately 5 times higher for the first couple of primer (Fig. 2A, B). Melting curve analyses showed a greater difference between fluorescence of positive (R. necatrix) and negative (water) samples for primer R15 Scorpion-R18 (Fig. 3A, B). At the annealing-extension temperature of 58C the differences between the fluorescence of positive and negative samples were 480 and 950, respectively, for R10 Scorpion-R7 (Fig 3A) and R15 Scorpion-R18 (Fig 3B).

Fig. 3. Comparison between fluorescence melting curves of primer pair R10 Scorpion-R7 (A) and R15 Scorpion-R18 (B) from 30 to 95C. Melting curves were evaluated on the amplified product obtained with R. necatrix DNA or water (control) at an annealing-extension temperature of 58C.

The molecular approach was preliminarily utilised to detect R. necatrix in artificially infected roots and/or bark. All samples caused an increase of fluorescence after both Scorpion-PCR and nested Scorpion-PCR, whereas no fluorescence increase was detected in noninoculated healthy plant samples (data not shown). The cycle number at which the fluorescence signal arose above the fixed threshold value (Ct) ranged from 30 to 35 in Scorpion-PCR and from 9.8 to 15.1 in nested Scorpion-PCR (Fig. 4).

Fig. 2. Comparison between relative normalised fluorescence (Rn) of primer pair R10 Scorpion-R7 (A) and R15 Scorpion R18 (B) using DNA from R. necatrix. Water was used as a control.

Detection of R. necatrix from host tissues. The protocol utilized to extract DNA from host tissue enabled the extraction of total nucleic acid suitable for PCR amplification in 3-4 h. DNA yields ranged from 2 to 3 g per g of plant material. Before purification with PVPP spin column the extraction solutions had a dark colour ranging from brown to black and were not amplifiable. The PVPP spin column clarified all DNA solutions regardless of the host species and/or tissue type utilised for the extraction.

Fig. 4. Detection of R. necatrix in roots and/or bark of different plant species grown in artificially infested soils as assessed by cycle threshold (Ct) values after Scorpion-PCR ( ), and nested Scorpion-PCR ( ). Each column represents the average of 3 Ct values from different DNA extractions and amplifications. Bars represent the standard deviation.

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Table 3. Cycle threshold values obtained with Scorpion and nested Scorpion-PCR in the detection of R. necatrix in cuttings from 26 woody species artificially inoculated with the pathogen. Non-inoculated cuttings were utilised as controls.
Species Sweet cherry Sour cherry Mahaleb cherry Peach Almond Plum Apricot Apple Pear Quince Pomegranate Loquat Grapevine Olive Oleander Walnut Japanese persimmon Mulberry Fig Carob Avocado Poplar Bay Rose Palm Chestnut Scorpion-PCR Inoculated 32.0 31.8 32.2 34.9 30.7 32.4 36.2 33.2 29.0 29.2 30.1 32.2 33.4 34.1 29.7 30.5 34.3 33.3 31.8 32.3 32.3 35.5 33.4 Non-inoculated Nested Scorpion-PCR Inoculated Non-inoculated 10.4 12.3 11.2 10.4 14.5 11.4 11.8 12.0 10.6 10.9 15.1 7.7 10.2 11.5 12.9 13.1 12.4 10.9 9.5 10.3 10.8 12.2 10.4 10.6 10.4 10.6 -

Efficacy of the detection method was further assessed using artificially inoculated cuttings of 26 plant species. DNA extracted from non-infected cuttings did not induce any increase of fluorescence after both ScorpionPCR and nested Scorpion-PCR (data not shown). An increase in fluorescence was obtained from most infected cuttings after Scorpion-PCR and from all cuttings after nested Scorpion-PCR. Ct values ranged from 29.0 to 36.2 in Scorpion-PCR and from 7.7 to 15.1 in nested Scorpion-PCR (Table 3). R. necatrix was detected in naturally infected roots and/or bark samples of olive, sweet cherry, and walnut trees. Conventional isolations of the pathogen on PDA made it possible to assess the presence of R. necatrix on 7 out of 12 potentially infected samples, with a percentage of infected root and/or bark segments ranging from 2 to 50% (Fig. 5). The same samples were also found to be infected when assayed by Scorpion-PCR (Fig. 5A), whereas in nested Scorpion-PCR 2 additional samples from which the pathogen was not isolated on agar medium (OL3 and WN1) were also found positive (Fig. 5B). Ct values ranged from 28.0 to 34.3 in Scorpion-PCR and from 9.1 to 20.7 in nested Scorpion-PCR. From healthy control samples R. necatrix was not isolated on agar medium and fluorescence did not arise above the threshold level.

Detection of R. necatrix from soils. The protocol utilized to extract DNA from soils enabled the extraction of 0.5-1.5 g of total nucleic acids per g of soil in a 2-3 h period. Sepharose spin columns clarified all DNA solutions. Potentially infested soils collected at the base of infected plants were analysed by means of baiting and molecular detection. The baiting method enabled the detection of R. necatrix from two soils (3 and 4) out of 15 tested, with a percentage of infected leaf discs of 5 and 3%, respectively (Fig. 6). Scorpion-PCR produced no increase of fluorescence, whereas in nested ScorpionPCR an increase of fluorescence, in at least 1 of the 3 replicates, was observed in 7 out of the 10 potentially infested soils tested. For these samples, fluorescence arose above fixed background level after a number of PCR cycles ranging from 17.3 to 35.4 (Fig. 6). R. necatrix was not isolated from 5 healthy control soils and fluorescence did not arise above the threshold level (Fig. 6). Dilution series of an artificially infested soil with a healthy soil were utilised to compare the sensitivity of molecular and baiting analyses. The detection limit of baiting was when the mixture contained 6.3% of infested soil or above (Fig. 7A), whereas nested ScorpionPCR enabled the detection of R. necatrix in mixtures containing 1.6% of infested soil or above, with an average Ct value of 28.3 (Fig. 7B). A high and significant

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Fig. 5. Detection of R. necatrix on 12 potentially infected and 6 healthy samples (controls) of olive (OL), sweet cherry (SC), and walnut (WN). For each sample, columns represent Ct values from different DNA extractions and amplifications after Scorpion-PCR (A) and nested Scorpion-PCR (B). The percentage of cuttings infected by the fungus as assessed by isolation on PDA from each sample is reported on the top of the corresponding histogram. Samples in which fluorescence did not arise above the threshold level have no histogram.

Fig. 7. Comparison between sensitivity of conventional baiting test (% of infected avocado leaf discs) (A) and molecular analyses (nested Scorpion-PCR) (B) using soil mixtures with different concentrations of R. necatrix. In B each histogram represents the average of 3 Ct values from different DNA extractions and amplifications. Bars represent standard deviation. Samples in which fluorescence did not arise above the threshold level have no histogram.

Fig. 6. Detection of R. necatrix in 10 potentially infested and 5 healthy soils (controls) as assessed by cycle threshold (Ct) values after nested Scorpion-PCR. For each sample, histograms represent Ct values from the different DNA extractions and amplifications. The percentage of avocado leaf disc colonised by R. necatrix as assessed by baiting test is reported on the top of the corresponding histogram. Samples in which fluorescence did not arise above the threshold level have no histogram.

Fig. 8. Relationship between the percentage of infested soil (see Fig. 7) and the corresponding cycle threshold (Ct) value as assessed by nested Scorpion-PCR. Linear regression equation of Ct (x) on Log (% infested soil + 1) (y) was: y = 4.3880.108x; r = -0.962; correlation coefficient was significant at P0.001.

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correlation value (r = -0.962; P< 0.001) was found between the percentage of infested soil (Log scale) and Ct values (Fig. 8). The persistence of R. necatrix DNA in soils was assessed by analysing an artificially infested soil treated with different temperatures to kill the pathogen. One and 5 days after the treatment, DNA was detected by nested Scorpion-PCR in all soils whereas after 15 days target DNA was amplified only from the untreated infested soil (Fig. 9). Baiting method enabled always pathogen detection from untreated infested soil, although with a decreasing percentage of infected leaf discs over the duration of the test (Fig. 9). As to healthy control sample fluorescence did not arise above the fixed threshold level and R. necatrix was not isolated on avocado leaves.

Fig. 9. Persistence of R. necatrix DNA in soils treated for 1 h at different temperatures as assessed by nested Scorpion-PCR after 1, 5, and 15 days. An untreated infested soil and a healthy soil were used as positive and negative controls, respectively. Each histogram represents the average of 3 Ct values from different DNA extractions and amplifications. Bars represent standard deviation. Samples in which fluorescence did not arise above the threshold level have no histogram. The percentage of avocado leaf discs colonised by R. necatrix (baiting test) is reported on each corresponding histogram.

DISCUSSION The present paper describes a new pair of Scorpion primers showing a better performance than that previously reported (Schena et al., 2002). In fact these new primers produced a more intense fluorescent signal as verified by comparing the melting curves. This may indicate that the increased fluorescence output is due to differences in the properties of the two Scorpion primers rather than to differential PCR amplification conditions (Solinas et al., 2001). Specificity of primer R3-R8 and R15 Scorpion-R18 was assessed using a large number of fungal species, generally occurring in soil or as contaminants in Rosellinia-infected tissues, and numerous isolates of R.

necatrix from several hosts and different geographic areas. Moreover, BLAST analyses of DNA GenBank databases showed the presence of a sequence identical to that of the selected primers only in isolates of R. necatrix and in R. arcuata. Since R. arcuata has ITS1 and ITS2 sequences identical to R. necatrix, other genetic regions need to be analysed to differentiate these two species. However, from a practical point of view, the lack of specificity is not a major limitation considering that R. necatrix and R. arcuata have a quite different geographic distribution. In particular, R. arcuata seems to be limited to some equatorial zones planted with coffee, tea, and rubber (Anonymous, 1984) and does not occur in temperate climates where R. necatrix is widespread (Anonymous, 1987). The sequenced ITS1 and ITS2 regions of two Italian isolates of R. necatrix were identical to the same regions of two Japanese isolates. These data and the positive amplification obtained with R. necatrix isolates from a wide geographic origin, suggest that the primers we designed may be universally exploitable. Independent of the amplification system, the success and reliability of any PCR-based detection system largely depends upon obtaining high yields of target DNA from samples (Cullen et al., 2001). Environmental samples pose problems for PCR, since a variety of naturally occurring compounds (such as humic acids, tannins, and lignin associated compounds) can interfere with the reaction and inhibit amplification (Bridge and Spooner, 2001; Cullen and Hirsch, 1998). In the present work extraction methods based on the physical disruption of cells within the samples using a beadbeater were used to produce a high yield of DNA. The key steps for the removal of co-extracted humic/phenolic compounds from soil were the use of CTAB buffer and Sepharose gel filtration resins (Zhou et al., 1996; Miller, 2001). Similarly, the excess of polyphenols in the extraction from plant tissues was removed by the addition of PVP to the mixtures and the use of PVPP spin column chromatography (Martin-Laurent et al., 2001). These protocols have a small number of lysis and purification steps but maximize the yield and quality of recovered DNA, thus allowing rapid processing of many samples. R. necatrix was detected in artificially and naturally infected plant samples. As expected, in the first experiment series, the fluorescence signal increased after Scorpion and/or nested Scorpion-PCR, only for infected samples. These data confirm the efficacy of the extraction protocols (no PCR-inhibitors) and the specificity of the detection method (no amplification from healthy samples). In the experiments conducted with naturally infected plants, fluorescence increase was obtained from all samples that were positive for the isolation of R. necatrix and from two additional samples. This result seems to indicate a higher sensitivity of the molecular approach over the traditional isolation method. The presence of false positive for these two additional samples can be excluded because: (i) all positive samples

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were collected from fields in which the pathogen had been previously isolated; (ii) all positive samples showed root rot symptoms; and (iii) all negative controls collected from uninfested fields did not cause any fluorescence increase. Moreover, it is conceivable that in agar medium the presence of the pathogen was concealed by fungi, such as Trichoderma spp., Penicillium spp., Fusarium spp., etc., generally occurring as contaminants. Tests on naturally infested soils confirmed the suitability of the molecular detection method for practical applications and pointed out a higher sensitivity of nested Scorpion-PCR compared to the traditional baiting system. In fact, R. necatrix was found in two and seven soil samples by baiting and PCR, respectively. As to host tissues, the hypothesis of a false positive could be excluded since the positive samples were collected at the base of heavily infected plants and healthy controls did not show any increase of fluorescence. A higher sensitivity of the molecular detection method was also found using an artificially infested soil, serially diluted with a healthy soil. In this experiment a high and significant correlation (r = - 0.962; P 0.001) was found between the dilution factor (% of infested soil) and the cycle threshold, suggesting that Ct could be utilised to estimate the amount of the pathogen in the soil. In a previous report, Bates et al. (2001) converted the DNA quantification results in the percentage of Pyrenophora teres infected seeds by correlating the PCR tests with agar test results. A similar approach is more difficult for R. necatrix since specific selective media are not available and the pathogen occurs in nature mainly as mycelium, even if three different kinds of spores (ascospores, conidia, and chlamydospores) can be produced in culture (Guillaumin, 1988). To assess the persistence of R. necatrix DNA, the soil was exposed to different temperatures and target nucleic acids were subsequently detected by nested ScorpionPCR. One and five days after treatments R. necatrix DNA was detected in all soils tested; however, after 15 days, pathogens nucleic acids were detected only in the untreated soil in which the pathogen was still alive as assessed by baiting test. The absence of R. necatrix DNA in heat-treated soils after 15 days indicated that nucleic acids were degraded quite rapidly. These data are of interest since one of the major limitations of studies on soil fungi by purely molecular methods is the lack of discrimination between living and dead material (Bridge and Spooner, 2001). Moreover, these preliminary data regarding the persistence of R. necatrix DNA in the soil seems to exclude the risks of false positives due to the presence of dead cells. However, this result needs to be confirmed with other soils having a different composition and in different environmental conditions. Indeed, although several reports indicate that nucleic acids are quickly digested by DNases when introduced in the soil (England et al., 1998), other studies have shown that DNA persists in the soil for varying lengths of time by forming complexes with soil components that are protected from en-

zymatic degradation (England et al., 1997). In the present work the combination of two sequential amplifications with conventional and Scorpion primers allowed the detection of R. necatrix in soil and plant materials. The use of a labelled primer in the second amplification eliminated the requirement for postamplification steps such as gel electrophoresis and ethidium bromide staining and significantly reduced the time and labour of the analysis. The fluorescent detection of PCR products in conjunction with a built-in, 96well format thermal cycler, can greatly increase the throughput of PCR testing and is an important step towards a PCR-based automated diagnostic system. Our results could be utilised to develop a new diagnostic system for R. necatrix suitable for routine applications, not requiring specialised staff with specific knowledge of the pathogen. To date, the main problem is the requirement for dedicated, sophisticated spectrofluorometric thermal cyclers, which are expensive to purchase and to maintain. However, to this and, the recent results reported in the USA for the diagnosis of bacterial diseases by using a portable real-time PCR apparatus seems to be very encouraging (Schaad et al., 2002). In conclusion, the molecular technique developed in the present work: (i) is more sensitive and reliable than the traditional detection methods; (ii) is not affected by external factors, such as other fungal species that on agar media could conceal the presence of the pathogen; (iii) requires much less time (1 working day compared to 10-20 days); and (iv) does not require expertise for fungal identification. The molecular method developed for the diagnosis of R. necatrix should have a major role in the alleviation of some of the issues associated with the detection, control, and application of phytosanitary certification schedules. Furthermore, although additional efforts are needed, the development of a quantitative detection method will facilitate studies to determine the inoculum threshold levels that are necessary for the development of white root rot disease and to ascertain some still unclear epidemiological aspects of the pathogen (Anselmi e Giorgetti, 1990).

ACKNOWLEDGEMENTS Thanks are expressed to Mr. V. Maurantonio for his technical assistance. This work was supported by CEGBA (Centro di Eccellenza di Genomica in campo Biomedico e Agrario) research line n.7. REFERENCES
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Received 2 December 2002 Accepted 13 January 2003