Fine Tuning of Real Time PCR as a First Tool for the Detection of G143A Substitution in Venturia inaequalis Samples

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple trees worldwide and requires a high number of fungicide applications. The G143A substitution in the inhibitor binding site of cytochrome b of V. inaequalis confers a high level of resistance to strobilurins targeting the bc 1 complex. The aim of this work was to substitute the labor intensive in vitro assays, with the faster quantitative PCR. An allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis. To be able to suggest that the molecular method could be applied as unique and robust technique, we carried out in vitro sensitivity test to trifloxystrobin; first testing the relative germination and subsequently confirmed with the quantification of mutated allele frequencies by qPCR on forty-nine Italian V. inaequalis populations. qPCR gave a similar pattern to that obtained using in vitro conidial germination test in predominantly sensitive and resistant populations, the variability between these two tests was observed in some heterogeneous populations. The qPCR assay developed in this study efficiently quantifies the A143 allele and we can conclude that this method could be use-ful for the study of the fungicide resistance at population level in the fields, giving a quick response also with a large amount of samples.

in almost all apple growing areas in the world, and in Italy requires numerous fungicide applications (from 10 to 20 and more) especially in Northern areas because of frequent rain and fairly high temperatures in the early growth stages.
Old protectant fungicides (dithiocarbamates, dithianon, captan and dodine) have an important role in apple scab control. In the early 1970s, the most important fungicides for scab control were the benzimidazoles, but in a few years they were dramatically affected by resistance development. This led to a return to the use of dodine until the late 1970s, when fenarimol's registration started the Sterol Biosynthesis Inhibiting (SBI) fungicide period. These fungicides have been used for many years with triazoles but they were progressively affected by resistance from the middle of the 1980s to nowadays [1].
An improvement of control was made possible by anilinopyrimidines introduced in the middle of the 1990s and by strobilurins in the late 1990s. Strobilurin fungicides, belonging to the group of Quinone outside Inhibitors (QoIs), are highly effective against a wide range of fungal pathogens. The mode of action of QoIs consists in inhibiting mitochondrial respiration by binding at the so-called Qo site of cytochrome b. Cytochrome b is part of the cytochrome bc1 complex (or complex III), located in the inner mitochondrial membrane of fungi and other eukaryotes [2] [3]. Strobilurins, first as kresoxim-methyl and later as trifloxystrobin, permitted improvements in apple scab control because they made it possible to extend spray intervals between treatments even up to ten days [4].
The first occurrences of resistance to QoIs, also with a reduction in performance, were reported for several pathogens, including V. inaequalis [5] [6] [7]. The first mechanism of resistance observed in many phytopathogenic fungi was a single-nucleotide mutation in the cytochrome b (cytb) gene, leading to amino acid exchange [8]. Three of the most common amino acid substitutions were detected in several phytopathogenic fungi and Oomycetes that are resistant to QoIs: from glycine to alanine at position 143 (G143A), from phenylalanine to leucine at position 129 (F129L) and from glycine to arginine at position 137 (G137R) [9].
Sierotzki an collaborators detected for the first time the G143A substitution in cytochrome b gene in QoI-resistant field isolates of plant pathogens [10]. This target site mutation has been identified in more than twenty species, including phytopathogenic Ascomycetes such as V. inaequalis [11], several powdery mildews [10] [12] and Alternaria species [6] [13] [14] [15]. This mutation was able to cause severe decreases of sensitivity (Resistance factor [RF] often higher than 100) and field control failure, while the other detected mutation, F129L, determined moderate effects on sensitivity and field performances. The V. inaequalis situation was more complex because the high reduction of V. inaequalis population sensitivity was not always related to the detected mutations or the field control reductions [16] [17] [18]. In these situation, the alternative respiration pathway was assumed to have an important function, however its in planta activity has not been clarified [19]. Consequently, the results on V. inaequalis were  [20].
Another resistance mechanism; an external esterase able to metabolise the fungicide conferring a partial loss of control of kresoxim-methyl was reported [21].
However, studies proved that it was much less effective in the case of other strobilurins [18].
In Italian apple areas, a reduced activity of QoIs on apple scab has been suspected since 2000 and 2001 [22]. The field control complaints made by farmers in the last few years in Italy led to reconsider the application of these fungicides.
Strobilurins were abandoned in some areas, especially where control failures were rather frequent, or utilized only in low risk periods, while in other areas they continue to be applied with the usual anti resistance recommendations. Some authors reported the presence of resistant populations of V. inaequalis [23] [24] and they defined in vitro values for resistant populations similar to the one defined in our previous work [22]. Moreover, Fontaine and collabrators [24] were in agreement with our previous study supporting the concept that the G143A substitution was almost always present in resistant populations and sometimes also in sensitive ones [25]. Even when it is present at very low frequencies, the mutation can be identified utilizing quantitative Real-Time PCR [26]. Once the resistance mechanism is known, it is often faster to use molecular techniques compared to biological assays. The in vitro methods are widely used for sensitivity screening among fungal populations. However, they are labor-intensive and time-consuming; moreover, only a part of the samples from orchards (generally no more than 50% -60%) have conidia with a sufficient viability to be multiplied in order to carry out the in vitro assays. The main advantage of qPCR methods is their high sensitivity and cultivation-independency, in fact, qPCR allows the detection of small amounts of the desired allele in bulk samples, thus it eliminates the need to maintain pure cultures of the pathogens.

Origin and Maintenance of Venturia inaequalis Populations
Forty-nine bulk V. inaequalis populations were sampled in Northern Italy orchards during 2002-2010. V. inaequalis populations were collected in apple trees with different scab management: wild-type trees (WT), which have never been treated with fungicides and located in uncultivated areas of Northern Italy; untreated trees (UNT), which have never been exposed to QoIs; commercial orchards where several groups of fungicides, often including strobilurins, have been applied with good disease control (GC) or poor disease control (PC) (i.e. with practical resistance to QoI fungicides). Samples consisted of 40 -50 scabbed leaves randomly collected in each orchard from May to July.
In order to obtain populations with high viability, a drop of sterile water was put on 30 -40 scab lesions from each sample and conidia were harvested by rinsing with sterile water. The conidial concentration was adjusted to (1 − 3) × 10 5 spores/mL and inoculated on potted apple seedlings. Inoculated seedlings were incubated for 15 -20 days at 21˚C in a greenhouse. All scabbed seedling leaves were then conserved in silica gel at 4˚C for fungicide sensitivity assays and molecular analysis. In addition, scabbed-infected leaf discs obtained by a sterile cork borer (9 mm ∅) from 15 -20 leaves for each population were stored at −20˚C until DNA extraction. The resistance response for each population was expressed as mean percent relative germination "RG%" (percentage of germination at 2 mg/L/percentage of germination in untreated × 100), as previously described [39].

DNA Extraction
The fungal genomic DNA was extracted directly from lyophilised scabbed leaves which were ground in liquid nitrogen by a CTAB-based method and then puri-

Primers Set-Up
The G143A substitution in V. inaequalis population was detected using the qPCR method. A specific primers set was designed. The forward primers FwS5418 and FwR5418 were used respectively for sensitive (S) and resistant (R) alleles, while the reverse common primer, R5548U, was used (Table 1). With the aim of verifying the qPCR efficiency (E) and specificity of each primer pair, different percentages of reference sensitive and resistant plasmidial DNA were analyzed: 100% S, 1% S + 99% R, 5% S + 95% R, 10% S + 90% R, 50% S + 50% R, 99% S + 1% R, 100% R ( Figure 1). Even when it is present at low frequencies, the mutation can be identified utilizing quantitative Real-Time PCR. The ratio (%) of the two alleles present in samples was calculated using the ΔC q method according to the equation [40] 10^ (Cq S allele − Cq R allele)/-slope); R (R/1 + R) × 100 = R allele; where Cq is the quantification cycle and reflects the cycle number at which the fluorescence generated within the reaction of each sample crosses the threshold and the slope was calculated according to the standard curves, which were specific for each allele. The data are expressed as percentages of mutated allele.

Standard Curves Development
Standard curves were obtained mixing S and R alleles strains (50% S and 50% R) 10-fold serial dilutions ranging from 20 ng × 10 −3 to 20 ng × 10 −6 of plasmid DNA.
The standard curve efficiencies were: S allele, E = 101.9%, R allele, E = 100.1%. The high efficiency for each gene allowed the assumption that the genes are amplified with the same efficiency, and an average slope of −3.329 was used in the equation.

Quantitative Real-Time PCR Conditions
The qPCR analysis of the forty-nine V. inaequalis populations was performed To verify the specificity of the product obtained, a melting curve was performed at the end of the PCR reaction with an increase of the temperature specificity of 0.05˚C/s, from 55˚C to 95˚C.
For each population, the specific primers for both QoI R and S alleles were analysed in each plate. The assays were carried out in duplicate, each experiment was repeated three times and the means comparisons was conducted. Data were calculated using the supplied Real-Time Detection System software version 3.0.
for Windows.

Sensitivity Assays
Eight WT and one UNT populations were found to be sensitive to trifloxystrobin with RG values from 0 to 9.2% ( Table 2, Column B and Figure 2). Our C. Turan et al.  results concur with the reports of others [17]. Populations obtained from good control orchards had RG ranging from 0 to 14.6% (

Standard Curves and Primer Set up for qPCR
The qPCR analysis showed a specific amplicon using the primer-set for both R and S alleles. The melting curve analyses for all amplicons showed a single peak, and no non-specific products or primer-dimer formation were detected (data not shown). The standard curves obtained using mixed strains (50% S + 50% R) in 10-fold serial dilution showed good efficiency (E) with these primer sets (FwS5418/5548URev), (FwR5418/5548URev) for qPCR ranging from E = 100.1% to E = 101.9%, with a linear correlation coefficient of 0.999 (Figure 3). The high efficiency for each gene allowed the assumption that the genes are amplified with the same efficiency, and an average slope of −3.329 was used in the equation.
Successful allele discrimination was achieved and an excellent reliability related to the ΔC q calculation method was observed.

Relation between R-Allele Frequencies and Relative Germination (RG)
In vitro resistance response to trifloxystrobin was expressed as RG. The populations were classified as sensitive with RG < 14.6% and they were found to contain mutated alleles from 0.015% to 21.32%. Referring to WT and UNT populations, when the relative germination is 0%, the mutated alleles generally ranged from 0% to 2.28%. Sometimes there was no clear relationship between the frequency of A143 allele and RG. For instance, when populations (No 426, 158,

Discussion
In this study, an allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis in order to substitute the biological assays. In order to be able to suggest only this methodology for the monitoring of V. inaequalis we determined how the frequency of mutated alleles relates to the strobilurin resistance, and fundamental was the relationship between in vitro sensitivity tests and molecular assay results.  [42]. This indicates, as suggested by Fraaije et al., [28] that isolates with A143 alleles might play an important role in long-distance dispersal of QoI-resistant genotypes. Moreover, the Fungicide Resistance Action Committee (FRAC) has also shown that it is possible to detect G143A substitutions in fungal populations never exposed to QoI fungicides. An explanation for this phenomenon is, that mutations occur naturally in fungal populations, usually at low frequencies [43]. Mutations can also be found, at low, but variable, frequencies, in situations where the use of QoI based products is providing perfectly accept-  [45].
In this study, quantitative assessments using qPCR followed a similar pattern to that obtained using in vitro conidial germination tests in very sensitive and very resistant populations. In fact, in most cases, it was noted that when RG was <10%, mutated allele frequency was <10% and when RG was >70%, very high mutated allele (>80%) was detected. Similar results were observed in a large number of monoconidial isolates (data not shown). However, some variability between the two tests was observed in heterogeneous populations (presence in the samples of leaf lesions colonised by either mutant or wild type isolates and/or presence of heteroplasmic fungal strains containing mitochondria with or without the G143A substitutions in the leaf samples.
The results demonstrated that a high relative abundance of the A143 allele was associated with high resistance to QoIs. Furthermore, in the current study, the A143 allele was higher than 23% in the orchards, where QoIs failed to control apple scab. Similarly, Villiani et al., [36] reported that in the US, commercial orchards with practical resistance to QoI fungicides had a relative abundance of the A143 allele > 22%.
The qPCR method is able to determine the development of fungicide resistance in apple scab populations and it is a fundamental step in being able to predict possible trends in fungicide resistance on a large scale. More often, it is necessary to detect the presence of the pathogen early and quickly, and to determine the frequencies of resistant and sensitive isolates in one or more regions. The most commonly used test for determining QoI sensitivity of V. inaequalis is a biological test, often conducted at several concentrations and which is very com-American Journal of Plant Sciences plex, this conventional methods to detect resistance are time-consuming and labor-intensive. Additionally, culturing of V. inaequalis is a difficult step because the growth is very slow on culture medium and cross contamination of other fungi occurs very often. The method developed here was designed as a substitution to the traditional method in vitro sensitivity test.

Conclusion
In conclusion, with this work, after processing a large amount of data with different scab management, we can conclude that qPCR analysis could be performed alone without the biological test especially with a high number of samples in order to efficiently and quickly determine the presence of mutated alleles to identify the practical resistance risk in the orchards, with the certainty that the results are consistent and close to the field situation.