Molecular Diagnostic Tools for the Detection and Characterization of Phoma tracheiphila M. Russo1, F.M. Grasso1, P. Bella1, G. Licciardello2, A. Catara1, 2 and V. Catara1 Department of Phytosanitary Sciences and Technologies, University of Catania, Via S. Sofia 100, 95123 Catania, Italy 2 Laboratory of Phytosanitary Diagnosis and Biotechnologies, Science and Technology Park of Sicily, Catania, Italy 1 Keywords: Citrus, lemon, “mal secco” disease, quantitative PCR, fAFLP Abstract In the recent years PCR-based techniques for the identification and detection of Phoma tracheiphila, the causal agent of citrus mal secco disease, have been evaluated aiming to provide tools for biological and epidemiological studies. A wide collection of P. tracheiphila strains was used to evaluate and validate diagnostic protocols and a fAFLP method for fungal characterization. Conventional and realtime PCR protocols were successfully tested for the specific identification of P. tracheiphila and its detection in planta. A further improvement of the real-time PCR protocol and the DNA extraction methods allowed the quantification of the fungus both from naturally infected and artificially inoculated citrus species as well from soil. The protocol was proved diagnostic flexible, rapid and more sensitive than any other (0.1 pg fungal DNA). Real-time PCR from leaf and stem samples taken at various time after inoculation allowed quantitative monitoring of P. tracheiphila spread in planta. Variable DNA concentrations were detected and quantified by real-time in naturally contaminated soil sampled beneath infected lemon trees in four different citrus groves from spring to autumn. In other experiments, representative P. tracheiphila isolates were used to develop a fluorescent-AFLP protocol to detect intraspecific variability. fAFLP were able to differentiate fungal isolates but no correlation with the geographic origin, isolate morphology or virulence was observed. To our knowledge, the protocol makes available a tool to discriminate isolates of P. tracheiphila, useful for epidemiological and population studies. INTRODUCTION Phoma tracheiphila (Petri) Kanchaveli & Ghikashvili is the causal agent of citrus “mal secco” disease (reviewed in Migheli et al., 2009). No effective method is currently available to control the disease. Preventive measures, phytosanitary programs, and early diagnosis are the most effective ways to limit the introduction and further spread of the fungus. P. tracheiphila is, by the fact, of quarantine concern to most regional Plant Protection services worldwide (APPPC, CPPC, COSAVE, EPPO, IPSC, NAPPO) (EPPO CABI, 1997). EPPO diagnostic protocol is mainly based on the isolation of the fungus on agar media, identification of cultural and morphological characters, and in the absence of sporulation by either PCR or PAGE analysis of mycelia proteins. The entire process take up to 15 days in case of PAGE analysis (EPPO/OEPP, 2007). The PCR protocol is suggested also for analysis of DNAs extracted from symptomatic samples with a process that could take about 6 h. Molecular detection methods described to date rely either on a specific DNA probe described 20 years ago (Rollo et al., 1987) or on the alignment of fungal ITS sequences (Balmas et al., 2005; Ezra, 2007). The method currently used in our laboratory was developed from the precursor cloned probe described by Rollo et al. (1987) and rely on a PCR assay targetted to on that probe (Rollo et al., 1987; Albanese et al., 1998; Gentile et al., 2000; Coco et al., 2004; Licciardello et al., 2006). More recently, we proposed a new assay based on real-time PCR (Licciardello et al., 2006; Russo, 2008). Molecular tools used to date, such as microsatellites, RAPD analysis and ITS Proc. IInd IS on Citrus Biotechnology Eds.: A. Gentile and S. La Malfa Acta Hort. 892, ISHS 2011 207 sequencing didn’t differentiate P. tracheiphila isolates (Balmas et al., 2005; Ezra, 2007), Authors attributed this failure to the scarce variability of P. tracheiphila and the lack of sexual reproduction in this fungal species. In recent years our research has aimed at developing flexible diagnostic molecular tools that can contribute new insights into the biology and epidemiology of the fungus. The main purposes are: i) molecular typing methods to identify isolates at intraspecific level; ii) sensitive, reliable and quick detection methods for diagnostic purposes; and, iii) quantitative detection methods to monitor P. tracheiphila in plant or in inoculum reservoirs. MATERIAL AND METHODS Fungal Isolates and Citrus Samples P. tracheiphila isolates obtained from mal secco infected plants in Italy and Greece, identified by morphology and PCR (Grasso and Catara, 2006; Grasso, 2008) were used throughout the study. Infected citrus plants were obtained either from the field or were artificially inoculated. A suspension of phialoconidia (106 conidia ml-1) was used as inoculum source. Leaves were inoculated by deposing a drop of conidial suspension on the leaf blades and puncturing a secondary vein with three needles. Disease was monitored using an arbitrary scale from 0 to 4 (Interaction phenotype, IP; Luisi et al., 1977). Leaf disks ( 6 mm) were removed from the inoculation points 21 days post inoculation and the DNA was extracted. Six, 12 and 24 month-old sour orange and Troyer citrange seedlings were inoculated by injection of a phialoconidia suspension (106 conidia ml-1) in a hole in the bark just above the trunk collar. Twigs showing dieback of the distal portion were sampled from mal secco affected groves located in Sicily. Serial transverse sections of twigs or stems were taken and subjected to DNA extraction. DNA Extraction Total DNA was isolated from 60 mg of fresh P. tracheiphila mycelium and from 20 mg of leaves or wood of plants artificially inoculated with P. tracheiphila using the Puregene Genomic DNA isolation Kit (Quiagen) as described by Licciardello et al. (2006). A quick DNA extraction method slightly modified from that of Wang et al. (1993) (NaOH method) was also used for mycelium of 3-days-old fungal culture and for symptomatic woody samples according to Licciardello et al. (2006). Total soil genomic DNA was extracted using the MoBio Ultraclean isolation kit (MoBio Laboratories, Solana Beach, CA, USA) according to the manufacturer’s instructions from 250 mg field soil samples. DNA samples were stored at -20°C for the duration of the experiment. fAFLP Analysis Restriction, ligation, amplification and capillary electrophoresis were performed as previously described (Oliveri et al., 2008). The AFLP® Core Reagent Kit (Invitrogen™) was used according to the manufacturer's instructions with minor modifications. Pre-amp Primer Mix I (Invitrogen™), containing adapter complementary AFLP primers, each with one selective nucleotide (M-C; E-A), was used in the preamplification reaction. Selective amplification was performed using a EcoRI primer with two selective nucleotides (AT) labeled at 5’ ends with Cy5 fluorophore (MWG Biotech) in combination with a number of primers containing 2 or 3 selective nucleotides (Grasso, 2008). Data was exported in binary format with “1” for the presence of a peak and “0” for its absence. Dendrograms were constructed by the unweighted pair group method using arithmetic averages (UPGMA) from the PHYLIP® software package (Nei and Li, 1979; Felsenstein, 2004). PCR and Real-Time PCR Conditions PCR amplifications were performed either with primers GR70/GR71 (Rollo et al., 208 1990) or GR70/GL1 using previously described PCR conditions (Licciardello et al., 2006). Real-time PCR was performed with GR70/GL1 primers and probe described by Licciardello et al. (2006) with modification that the Black Hole Quencer1 (BHQ1) was used in probe labeling, instead of TAMRA, as quencher (PP2 probe; Russo, 2008). Realtime PCR assays were performed using IQTM supermix (BIO-RAD) with 400 nM of each primer, 200 nM fluorogenic probe and 1-2 μl of target DNA. Negative control reactions contained the same mixture, with sterile water replacing the DNA template. All PCR reactions were performed in 25 μl Smart Cycler reaction tubes (Cepheid) in a Smart Cycler TDII System (Transportable Device TD configuration; Cepheid). A standard curve for fungal DNA quantification in plant tissues and soil by real-time PCR was generated using P. tracheiphila DNA (100 μg ml-1) serially diluted in sterile distilled water (SDW). The standard curve was linear over seven log units of initial quantities of DNA template spanning from 1×102 to 1×10-4, with a correlation coefficient (R2) of 0.99. RESULTS AND DISCUSSION P. tracheiphila is an important citrus pathogen. Thus, suitable tools for its detection and characterization are very important. Routinely laboratory assays and more recent studies permitted to develop protocols involving f-AFLP for fungal isolates identification and conventional or real-time PCR for detection and quantitative monitoring of the fungus. fAFLP Analysis A collection of 78 isolates from 75 groves in Italy, Greece and the Aegean islands identified according to their morphological and microscopic characteristics and by conventional PCR was analysed (Grasso, 2008). EcoRI+AT/Mse+AA and EcoRI+AT/Mse+CT were selected for the analyses and generated approximately 102 peaks, 75 of which were polymorphic (Fig. 1). Cluster analysis revealed small groups of isolates (2-8) that segregated according the Country of origin but in general isolates and small clusters were scattered through the dendrogram regardless of the Country of isolation (data not shown). Thus fAFLP has proved to be reliable for intraspecific characterization of P. tracheiphila. The species is indeed very uniform as shown by analysis of both microsatellites and RAPD that did not detected polymorphisms (Balmas et al., 2005; Ezra et al., 2007). Balmas et al. (2005), observed that RAPD and sequencing of ITS1-5.8SITS2 region together with microsatellite analysis did not allow to differentiate isolates isolated form different groves in different years. AFLP analysis is very discriminating and, depending on the choice of primers, it can increase or decrease the number of polymorphic fragments (Vos et al., 1995). This technique in plant pathology has been applied to study populations of both phytopathogenic bacteria and fungi, and to search and highlight possible markers of resistance to pathogens in several plant species. PCR and Real-Time PCR Detection PCR primers developed by Rollo et al. (1990) and the new pair developed by Licciardello et al. (2006) were tested against 20 isolates using three different methods of target preparation. All the isolates were positive either if a fungal hypha or DNA extracted either with NaOH quick extraction protocol or a commercial DNA extraction kit. PCR sensitivity was 10 pg of fungal DNA/reaction. All P. tracheiphila isolates were positive by real-time PCR using the primers and the probe described by Licciardello et al. (2006) further modified with the change of fluorophore and the quenching molecules. Although primers and probe concentration in the PCR reactions were increased, no fluorescence was detected for any of the other fungal species tested such as Aspergillus niger, Penicillium italicum, P. digitatum, Colletotrichum gloeosporioides, Alternaria citri, Fusarium solani, Phoma exigua, Phytophthora citrophthora, Verticillium albo-atrum, indicating that amplification had not 209 occurred. The real-time PCR assay for P. tracheiphila developed in our laboratory is more rapid and more sensitive assay than those previously described (Rollo et al., 1990; Balmas et al., 2005; Ezra et al., 2007; Demontis et al., 2008). The real-time PCR assay developed for the Smart Cycler II System can complete a run of 40 cycles in 30 min. The new probe PP2 allowed increasing the sensitivity to 0.1 pg of DNA. Quantitative Monitoring The real-time PCR has also proved a useful method for studying the biology and epidemiology of P. tracheiphila, as already observed for other plant pathogens (Schaad and Frederick, 2002; Okubara et al., 2005; Schena et al., 2004; Lopez et al., 2008). We quantified P. tracheiphila DNA in both asymptomatic and symptomatic citrus leaves and wood tissues. In leaves inoculated with 3 isolates showing different virulence assessed by Interaction Phenotype (IP) values, P. tracheiphila DNA was significantly reduced in leaves inoculated with the less virulent isolates. By the fact, the three strains showed an average IP of 3.0, 1.0, and 2.5 and DNA contents of 300, 20 and 50 pg, respectively (Grasso et al., 2008). Analogously inoculated leaf disks from plants of different varieties positively correlated (P<0.01) between IP and DNA amount. Concentration of P. tracheiphila DNA in the samples, estimated on the basis of the threshold cycles obtained in real-time PCR measurements, ranged from 100 to 660 ng (Fig. 2). The DNA of the fungus in each case is detectable even in asymptomatic leaves, as indeed was expected in light of histological observation on the colonization of the fungus after foliar inoculation (Bassi et al., 1980). Fungal colonization was monitored in root or inoculated seedling stems. In root inoculated one-year-old plants, P. tracheiphila was detected by real-time PCR in different stem portions starting from the collar throughout the apexes and also from the stem sections that were negative by plate isolation. In most samples (7) that were negative by isolation in culture, concentration of DNA was very low ranged between 0.15 and 0.32 pg of DNA per reaction. In some cases the fungus was detected in the plant apexes without being isolated from the intermediate portions of the stems. This suggests that the fungal propagules carried by xylem flow as described by Perrotta et al. (1980) are detectable by real-time PCR. Stem sections sampled from 6 and 24 month-old sour orange and Troyer citrange seedlings revealed different DNA concentrations. Sixty days after inoculations the DNA content was higher (3478.6-4523 pg) in the 6 month-old plants than in those 24 month-old (367-453 pg). Symptoms showed that the latter were more resistant to the disease (data not shown). In both sour orange and Troyer citrange seedlings the highest DNA content was quantified near the apexes.. In field samples we observed that P. tracheiphila DNA decreased from shoot sections sampled near infected tissues towards the shoot base. In these samples we observed DNAs within distinct ranges (Licciardello et al., 2006). Soil Detection A specific protocol was developed to detect DNA of P. tracheiphila from soil. Although P. tracheiphila is a vascular pathogen, and more attention is devoted to epigean infection, radical infections by infected soil occur in the syndromes “mal nero” and “mal fulminante” (Cutuli, 1972; Perrotta and Graniti, 1988). Previous analyses showed that P. tracheiphila DNA is detectable by real-time PCR in both sterile and non-sterile artificially seeded soil without relevant difference in DNA content (Russo et al., 2008). The protocol was evaluated to detect and quantify the fungus in naturally contaminated soil sampled beneath infected lemon trees in 4 different citrus groves between spring and autumn. The results obtained from triplicate sample per plant showed the highest amount of P. tracheiphila DNA in samples taken in March and in April (0.30-0.43 ng DNA g-1 of soil). Fungal DNA was nearly undetectable in July and August (0.05-0.06 ng DNA g-1 of soil) and rose up in September-November (0.10-0.24 ng DNA g-1 of soil) (Table 1). The Real-time PCR represents a promising tool for studying the epidemiology of the fungus in 210 the soil, until now monitored by transplanting healthy plants and checking for symptoms of mal secco (De Cicco et al., 1987) or by a cloned probe (Di Silvestro et al., 1990). Being the detection more sensitive and fast it will contribute to further investigate the role of soil propagule in the mal secco disease cycle. Literature Cited Albanese, G., Grimaldi, V., La Rosa, R., Di Silvestro, I. and Catara, A. 1998. PCR analysis applied to Citrus mal secco diagnosis. J. Plant Path. 80(3):251. 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Okubara, P.A., Schoroeder, K.L. and Paulitz, T.C. 2005. Real-time pcr: applications to studies on soilborne pathogens. Can. J. Plant Pathol. 27:300-313. Oliveri, C., Torta, L. and Catara, V. 2008. A polyphasic approach to the identification of ochratoxin A-producing black Aspergillus isolates from vineyards in Sicily. Int. J. Food Microbiol. 127:147-154. Perrotta, G. and Graniti, A. 1988. Phoma tracheiphila (Petri) Kanchaveli & Gikashvili. p.396-398. In: I.M. Smith, J. Dunez, R.A. Lelliott, D.H. Phillips and S.A. Archer (eds.), European Handbook of Plant Diseases. Blackwell Scientific Publications, Oxford. Perrotta, G., Magnano Di San Lio, G. and Bassi, M. 1980. Some anatomical and morphofunctional aspects of resistance to Phoma tracheiphila in Citrus plants. Phytopath. Z. 98:346-358. Rollo, F., Amici, A., Foresi, F. and Di Silvestro, I. 1987. Construction and characterization of a cloned probe for the detection of Phoma tracheiphila in plant tissues. Appl. Microbiol. 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Month March April May June July August September October November Site Catania Catania Avola (SR) Catania Siracusa Catania Catania Catania Catania Noto (SR) Catania Positive/tot samples 11/11 9/9 10/10 5/10 0/12 4/8 8/11 11/11 12/12 4/18 6/8 Average ng DNA g-1 of soil 0.43 0.30 0.46 0.14 0 0.05 0.06 0.10 0.23 0.02 0.24 Figures A B Fig. 1. Electropherogram of Phoma tracheiphila isolates (A) with the selective primer Mse-sel (+CT) and Eco-sel (+AT) and (B) with Mse-sel (+AA) and Eco-sel (+AT). 213 600 pg DNA 500 y = 137,82x R² = 0,5227 400 300 200 100 0 0 1 2 3 DI Fig. 2. Correlation between disease severity in artificially inoculated leaves (arbitrary scale 0-4) and Phoma tracheiphila DNA concentrations determined by real-time PCR of DNAs from leaf disks sampled at the inoculation sites. 214