Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

Strains of Ceratocystis platani used in this study (Table 1) were obtained from the Central Bureau Voor Schimmelcaltures (CBS, The Netherlands) and from the Istituto per la Protezione delle Piante-Consiglio Nazionale delle Ricerche (IPP-CNR, Italy) collections. Fungal isolates were grown on 300PT cellophane discs (Celsa, Varese, Italy) on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI) in 90-mm-diameter petri dishes and incubated in the dark at 20°C. After 7 days, the mycelium was scraped from the surface of the cellophane and stored in 1.5-ml microcentrifuge tubes (Sarstedt, Verona, Italy) at −20°C. DNA extraction was performed from mycelium using the E.Z.N.A. plant DNA minikit (Omega Bio-tek, Norcross, GA), following the manufacturer's instructions. The concentration of extracted DNA was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). To elucidate the phylogenetic relationships between C. platani and the other Ceratocystis species used (Table 1) and to test the specificity of the qPCR assay, sequence alignment of the internal transcribed spacer (ITS), β-tubulin, and cerato-platanin (CP) genes, was conducted. Cerato-platanin is a small hydrophobic protein from C. platani (24). The ITS2 region was amplified with the general primers ITS1 and ITS4 according to White et al. (25), while the β-tubulin gene was amplified using primers Bt2a and Bt2b, as described by Glass and Donaldson (26). Cerato-platanin was amplified with primer set CeraF (5′-ATGAAGTTCTCTATCCTACCC-3′) and CeraR (5′-AGGAGCTTCCGGAGAAGTCAC-3′) (this study) with the following cycling conditions: initial denaturation of 95°C for 2 min, followed by 40 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 90 s, with a final elongation step at 72°C for 9 min. PCR products were purified and sequenced directly using a BigDye197 terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) and analyzed with an ABI-3130xl 48-capillary DNA sequencer (Applied Biosystems). Maximum likelihood (ML) phylogenetic trees were produced for ITS and for concatenated β-tubulin and CP sequences using the program RAxML (27) with the GTR model (28, 29). Bootstrap values were estimated from 1,000 replicates. Computations were performed at the Vital-IT Center (http://www.vital-it.ch) for high-performance computing of the Swiss Institute of Bioinformatics (SIB). Petrellia setifera (AF043596) from Witthuhn et al. (30) was used as an outgroup for the ITS tree, and Ceratocystis albifundus (EU244986) from Heath et al. (31) was used as the outgroup for the concatenated tree.

In order to obtain markers specific for C. platani, two sets of primers and TaqMan minor groove binding (MGB) probes were designed to amplify the CP single-copy and the ITS multiple-copy genes. Primers and probes were designed using Primer Express Software 3.0 (Applied Biosystems) on the basis of consensus sequences of ITS2 (EU426554) and CP (EF017218) derived from the NCBI database (www.ncbi.nlm.nih.gov). TaqMan MGB probes were labeled with 6-carboxyfluorescein (FAM) at the 5′ end and a nonfluorescent quencher (NFQ) with minor groove binder (MGB) ligands as the quencher at the 3′ end. The specificity of newly designed primers and probes was further tested with BLAST (32) using published sequences in GenBank. The specificity of the primers and probes was also tested by qPCR on DNA from axenic cultures of (i) Ceratocystis species strictly related to C. platani, (ii) common airborne fungi, and (iii) fungal species inhabiting plane trees (Table 1).

A 25-μl PCR mixture containing 12.5 μl TaqMan Universal master mix (Applied Biosystems) was performed on an ABI Prism 7300 sequence detector (Applied Biosystems). The primer and probe final concentrations used were as follows: 300 nM forward primer (Eurofins MWG Operon, Ebersberg, Germany), 300 nM reverse primer (Eurofins MWG Operon), and 200 nM TaqMan MGB probe (Applied Biosystems) designed for amplification of ITS and CP genes. For each tube, 5 μl sample DNA was used.

All DNA samples were assayed in MicroAmp optical 96-well plates (Applied Biosystems), closed with MicroAmp optical caps, using the ABI Prism 7300 sequence detector (Applied Biosystems). Each DNA sample was assayed in three replicates. Four wells, each containing 5 μl sterile water, were used as the no-template control (NTC). The PCR protocol was 50°C (2 min), 95°C (10 min), 50 cycles of 95°C (30 s), and 60°C (1 min).

Results were analyzed using an SDS 1.9 sequence detection system (Applied Biosystems) after manual adjustment of the baseline and fluorescence threshold. Measurements of C. platani DNA in unknown samples were made by interpolation from a standard curve generated with a C. platani DNA standard, which was amplified in the same PCR run. The standard curve was generated from 5-fold serial dilutions (ranging from 100 ng/tube to 0.25 pg/tube) of a known concentration of C. platani DNA (strain Cp5) and analyzed in triplicate in seven independent assays. The amount of C. platani DNA was expressed as pg/μl.

The effectiveness of the airborne inoculum trap (AIT) system was evaluated by inoculation of Whatman filter papers with a conidial suspension of C. platani, followed by molecular detection. AITs were prepared using 90-mm-diameter petri dishes (Sarstedt, Verona, Italy) containing a Whatman no. 1 filter paper (Sigma-Aldrich, Milan, Italy), moistened with 4× TE buffer (40 mM Tris HCl, 4 mM EDTA [pH 8.0]) (20). Each petri dish was attached to the base of a small aluminum tray (500 ml) using double-sided adhesive tape. The tray was covered with a plastic net (6- by 6-mm mesh). A conidial suspension (1 ml in sterile water) was obtained from C. platani isolate CF0 (Table 1), following incubation on 1.5% PDA for 10 days. The number of conidia was determined in a Burker chamber, and five 1:10 serial dilutions ranging from 10 to 10⁵ conidia/ml were prepared. For each conidial dilution, a pellet was obtained after centrifugation at 16,100 × g for 5 min and used for DNA extraction. Two Whatman no. 1 filter papers were inoculated with each of the conidial dilutions. Filters were then washed in a 50-ml centrifuge tube (Sarstedt) containing 20 ml TE buffer (4×) at 65°C. Each tube was centrifuged at 289 × g for 90 min to precipitate the pellet. One milliliter of the suspension containing the pellet and TE buffer was then transferred to a 2-ml microcentrifuge tube (Sarstedt), and following centrifugation at 16,100 × g for 5 min, the pellet was used for DNA extraction using the E.Z.N.A. plant DNA kit, as previously described. Both the DNA from the conidial suspension and the inoculated filter papers were tested by qPCR, as described above.

The environmental survey was conducted in October 2011 on street plane trees in Florence, Italy (43°47′59″N, 11°13′94″E; 51 m above sea level), to assess the presence of the C. platani inoculum under urban conditions using AITs. The presence of the pathogen was determined in two sampling areas: (i) an infection area with Platanus × acerifolia showing symptoms of canker stain and (ii) the surrounding area, with healthy plane trees. Sampling was conducted in areas at least 50 m apart from each other. In the infection area, a sanitation cutting had been planned to remove infected plane trees. The presence of C. platani in the infection area on symptomatic planes was confirmed by isolations onto PDA (33). Briefly, isolations were carried out from samples collected from the boundary between necrotic and healthy tissues. Small fragments were collected from the margin of the necrotic tissue and placed in 90-mm-diameter petri dishes on 1.5% PDA and incubated at 20°C for 10 days. In addition, DNA was extracted from both necrotic and healthy tissues and analyzed by qPCR, as described above. In total, 25 AITs were prepared: 11 were placed at the infected site, and the remaining 14 were placed in the surrounding area. Each AIT was placed 2 m above the soil level at a 10-m distance from each other, on each streetlight. In the surrounding area, traps were placed 7 days before the sanitation fellings and wetted daily with 5 ml of 4× TE buffer (pH 8.0) on to the filter. To avoid contamination of the traps by C. platani before felling began, traps were placed in the infection site in the early morning and removed on the same day, once the felling operation concluded. AITs were transported to the laboratory, DNA was extracted from the filters as described above, and qPCR was carried out to determine the presence of C. platani.

To assess the minimal amount of C. platani able to cause damage on plane trees, inoculation tests were carried out on 18 5-year-old potted Platanus × acerifolia ramets obtained by rooted cuttings of an individual plane tree. (Ramets are vegetatively reproduced copies of a plant; each ramet has the same genotype as the original parent tree.) Inoculation was performed in May 2012 by wounding 40 cm above the soil (stem diameter, 1 cm), with a single wound per plant, using a knife blade carrying a drop of a 0.2 ml of five 1:10 serial dilutions, previously used for AIT inoculations (conidial concentrations ranging from 10 to 10⁵ conidia/ml). Three ramets for each conidial concentration were inoculated, and three wounded noninoculated ramets were used as control plants. Ramets were wrapped in Parafilm at the inoculation sites to reduce contamination and prevent excessive drying. After 4 months, C. platani was detected by the presence of specific symptoms and mycelium reisolation from inoculated plane tissue on PDA (33).

PCR efficiency was calculated from the slope of the standard curve: efficiency = 10^(−1/slope) − 1 (34). Reproducibility of the qPCR assay was assessed by computing the coefficient of variation (%CV) among the mean values in seven independent assays. The threshold value was 0.15, and the baseline emission was calculated from cycles 3 to 15. The accuracy of the AITs in intercepting the aerial inoculum of C. platani at various conidial concentrations was tested by quantifying the ITS region by qPCR, with the DNA extracted from a large series of dilutions of fungal conidia, obtained either from inoculated Whatman filter papers or directly from distilled water. A log-log exponential function on the form y = a + be^x was fitted to the data to describe the relationship between conidial dilution (x) and quantity of fungal DNA (y) in both sample types, and the parameter estimates were compared. Model comparison involved testing the hypothesis that both data sets (extraction from conidia in water or from conidia on Whatman filter papers) shared the same curve. Parameter estimates obtained by separately applying the model to two data sets were tested (F test) against estimates obtained by global fitting of the same model to both data sets. Factorial analysis of variance (ANOVA) (substrate type × conidial concentration interaction) was applied to further test for differences between the two techniques. The logit model (generalized linear model [GLZ]) was applied to test the diffusion of the inoculum (both its presence and quantity) as a function of the distance from the closest symptomatic tree and of the number of symptomatic trees in the surroundings of the traps. The relationships between presence or absence or quantity of the inoculum in AITs and distance from symptomatic trees was described by fitting an inverse exponential model shown by the equation (y = a + be^x) to these data. GLZ analyses, nonlinear estimation (least-square curve-fitting procedure, Levenberg-Marquardt algorithm), and ANOVA were performed in Statistica 6.0 (35).

Newly determined sequences (for ITS1-5.8S-ITS2, CP, and β-tubulin DNA sequences) have been deposited in GenBank under accession numbers KC493159 to KC493171, KF302676 to KF302687, and KF302688 to KF302703 (for more details, see Table S1 in the supplemental material).

Phylogenetic topology, reconstructed with ITS sequences and from concatenated β-tubulin and CP sequences, showed that C. platani occurred in the main cluster of C. fimbriata isolates (see Fig. S2 and S3 in the supplemental material). Classification of Ceratocystis cacaofunesta in the same cluster confirmed a close phylogenetic relationship between these strains. In contrast, C. variospora and C. populicola differentiated into a separate clade (see Fig. S2 and S3).

For each target gene (ITS and CP) the positions of the upstream primer, reverse primer, and the internal probe are shown in Fig. 1. The total lengths of the amplicons were 55 bp and 59 bp for the ITS and CP target genes, respectively. Characteristics of the primers and probe are given in Table 2. The nucleotide-nucleotide BLAST search showed complete homology (100%) between the CP amplicon sequence designed for both C. platani target genes (CP and ITS). Only C. platani isolates were positively amplified by qPCR on fungal DNA, using both primer-probe combinations designed for multicopy (ITS) and single-copy (CP) genes. DNAs from isolates of other Ceratocystis species and fungi in other genera were not amplified (Table 3).

Quality of the standard curves was evaluated by comparison of the cycle thresholds (C_T) for each point of the curve, since each value should hypothetically be the same in replicate samples run under the same conditions. Differences were observed between the standard curves constructed with the CP and ITS genes (Table 4, Fig. 2). The reproducibility of the standard curves, however, was high as the variation coefficient for each point was below 5% for both C. platani target genes (Table 4). For each target gene, measurements of linearity such as slope, y intercept, and correlation coefficient (r²) did not vary significantly among the seven assays (%CV < 2%). Comparison between the two CP and ITS primer-probe combinations showed differences in sensitivity: C_T values of molecular markers designed for the CP gene were higher than those obtained for the ITS genes (Fig. 2). The difference (ΔC_T) between these two target genes was 6.22 ± 0.2 (mean ± standard deviation [SD]). The detection limits of the qPCR assay were 0.05 pg/μl and 2 fg/μl DNA for the CP and ITS primer-probe combinations, respectively. Due to the higher sensitivity, the ITS primer-probe combination sets were used subsequently to detect and quantify C. platani from environmental AIT samples.

C. platani isolates (Cp3, Cp5, Cp6, Cp8, and Cp9) (Table 1) obtained from symptomatic plane trees were identified on the basis of morphological characteristics and by using qPCR tested on DNA extracted from mycelium.

qPCR was also used to identify and quantify C. platani in DNA extracted from symptomatic plane tissues (Table 3). The quantity of C. platani DNA detected in these samples using both primer and probes (sets for CP and ITS) ranged between 2.0 × 10³ and 5.0 × 10³ pg DNA per μg of total DNA extracted. C. platani DNA was absent in asymptomatic plane tissues, used as negative controls (Table 3). Selected amplification plots of PCRs obtained from the samples tested are shown in Fig. 3.

Using the ITS primer-probe combination, the amount of C. platani DNA present in conidial suspensions was between 0.5 × 10⁻² and 1.8 × 10² pg/μl total DNA (∼10 to 10⁵conidia/ml, respectively). Detection of the pathogen from AITs differed according to the molecular marker used. When the ITS primer-probe combination was used, all the 11 AITs from the infection site included C. platani, whereas 4 out of 14 were positive in the surrounding area. The quantity of fungal DNA on these AITs was between 1.2 × 10⁻² and 5.3 pg/μl. In contrast, the CP primer-probe combination gave positive results for only 3 out of 11 AITs from the infected site, while traps located from the surrounding area were all negative. The quantity of C. platani DNA ranged between 4.5 × 10⁻² and 1.1 pg/μl.

With the exception of the highest concentration of C. platani conidia, the quantity of DNA obtained did not vary between extraction substrates, i.e., conidial suspensions in water or suspension applied to filter papers (ANOVA, significant substrate × concentration interaction, P < 0.001; honestly significant difference [HSD] post hoc test, P > 0.1). At 10⁵ conidia per ml, a significantly greater quantity of DNA was obtained from the conidia suspension in water (HSD post hoc test, P < 0.001) than from the AITs. The relationship between conidial dilution (x) and the quantity of C. platani DNA detected by qPCR using the ITS primer-probe combination (y) produced an exponential curve (as shown in the equation y = a + be^x). Curves obtained by fitting the model to the quantities of DNA obtained from inoculated filter papers (y = −3.97 + 0.078e^x) and from conidial suspensions in water (y = −4.22 + 0.093e^x) were similar (Fig. 4). Estimates for both a and b in the equation y = a + be^x had overlapping confidence intervals (Fig. 4, insert) and did not differ significantly between the two curves (a F value = 0.41; P = 0.532; b F value = 4.21, P = 0.063).

C. platani was detected in AITs within 200 m of the closest symptomatic tree. Compared to negative AITs, positive AITs were surrounded by a higher number of symptomatic trees in a 100-m radius and were significantly closer to infected trees. The frequency of positive AITs (F_S) increased significantly with higher numbers of symptomatic trees (N_ST) located within a 100-m radius (Fig. 5A), and decreased with increasing distance (D) from the closest symptomatic tree (Fig. 5B). Logit regression showed that both N_ST [χ²₍₁₎ = 8.26, P = 0.004] and D [χ²₍₁₎ = 10.33, P = 0.001] had a significant effect on the detection of the fungus on AITs. The dependence of successful detection on D was described by an inverse exponential function in the form F_S = a + e^bD (Fig. 5C) (a = 1.002, b = 0.008, P ≤ 0.05; explained variance = 81.6%). A similar curve efficiently described the reduction of the quantity of the DNA (Q_DNA) of C. platani retrieved from AITs when the distance between the trap and the closest symptomatic tree increased (Fig. 5D) (Q_DNA = a + e^bD; a = 4.612, b = 0.016, P ≤ 0.05; explained variance = 95.3%).

After 4 months, all ramets inoculated with a conidial concentration ranging from 10² to 10⁵ conidia/ml were dead. These plants showed typical symptoms of blue stain canker consisting of stem cankers around the infection site and wood discolorations. Isolation on PDA medium showed the presence of fungal mycelium that was identified as C. platani. No symptoms were observed, and no successful pathogen isolation was possible from control plants and from plants inoculated with 10 conidia/ml.