Detection of Parietaria Mottle Virus by RT-qPCR: An Emerging Virus Native of Mediterranean Area That Undermine Tomato and Pepper Production in Southern Italy

Parietaria mottle virus (PMoV) is considered an emerging virus in many countries of the Mediterranean basin, especially on tomato and pepper crops. Symptoms on tomato leaves and fruits can be easily confused with those induced by cucumber mosaic virus (CMV) with necrogenic satellite RNA (CMV-satRNA), tomato spotted wilt virus (TSWV) or tomato mosaic virus (ToMV). Mixed infection of these viruses has been also reported in some tomato cultivars, with an increase in the complexity of the symptoms and severity of the disease. Although a specific serum and riboprobes have been produced, nowadays no sensitive diagnostic methods are available for the rapid PMoV detection. Here, we have developed a RT-qPCR assay with the aim to establish a more sensitive and specific method for PMoV detection. Specific primers and TaqMan probe were designed and in silico tested with all PMoV isolates available in GenBank. Moreover, this method was evaluated on tomato naturally infected samples from Sicily region (Italy). Results obtained showed that the RT-qPCR assay developed in this work is extremely sensitive, in fact, it is able to detect as few as 10 PMoV RNA copies in tomato total RNA; moreover, it will be a particularly valuable tool for early detection of PMoV. Furthermore, the analyzes on field samples show how this pathogen is increasingly present in tomato crops in the last years, helping to undermine the Italian horticultural sector.


INTRODUCTION
Vegetable crops represent one of the main sources of income and food worldwide. Among these crops of particular importance are tomatoes and peppers. Tomato (Solanum lycopersicum L., family Solanaceae) is a very important vegetable crop cultivated worldwide, with an area harvested of over five million hectares and a worldwide production of over 180 million tons (Food and Agriculture Organization of the United Nations (FAO), 2021). In the 2009-2019 decade, the global tomato production increased by more than 25 million tons (Food and Agriculture Organization of the United Nations (FAO), 2021). Another important horticultural crop is the pepper (Capsicum annuum L., family Solanaceae), that is cultivated worldwide in almost two million hectares, with a total production of 38 million tons.
In Italy, tomato production in open field is mainly located in Apulia and Emilia-Romagna regions, whereas tomato production in protected conditions is primarily based in Sicily (Agri ISTAT, 2020).
In the Mediterranean basin, Italy represents the main access point for plant material to European countries, due to its central geographical position; this significantly increases the danger of introducing new pathogens, posing a serious risk to agriculture, biosecurity, and food production, jeopardizing the future of Italian horticulture (Panno et al., 2019c).
PMoV, genus Ilarvirus (subgroup 1), family Bromoviridae, was identified and characterized for the first time in 1987 in Turin province (northern Italy) in Parietaria officinalis, causing a typical mottling in leaves (Caciagli et al., 1989) and subsequently in tomato (Lisa et al., 1998) and pepper (Janssen et al., 2005) plants. PMoV belongs to the same subgroup of tobacco streak virus (TSV), the type member of the genus Ilarvirus, but these two viruses are not serologically correlated.
To date, PMoV has only been reported in a few countries of the Mediterranean basin, in particular Italy (Caciagli et al., 1989;Parrella et al., 2021), France (Marchoux et al., 1999), Greece (Roggero et al., 2000), and Spain (Aramburu, 2001). The real incidence of PMoV was often underestimated in the past, due to the confusion generated by similar symptoms, caused by TSWV, tomato mosaic virus (ToMV), cucumber mosaic virus (CMV) with the necrogenic satellite (satRNA), and to the lack of knowledge about this virus.
The symptomatology on tomato leaves consists of irregular necrotic lesions, coalescent and usually concentrated in the basal area; in addition, the necrotic lesions can perforate the leaf, which often appears deformed and curved ( Figure 1A). The necrosis can progress to the leaf petiole and the stem, that often presents longitudinal necrotic stripes; in the worst cases, the stem apex necrotises and folds upon itself (Aramburu, 2001; Figure 1A). In tomato fruits the symptom consists in the pigmentation alteration: at the initial stage of infection, the fruits show greenish translucent rings, which become brownish, generally in relief, more or less corky and coalescent, causing fruit deformations (Galipienso et al., 2005; Figure 1B). On ripe and mature fruits, chlorotic rings associated to PMoV infection, have been also observed.
The symptoms on PMoV-infected pepper plants are very similar to symptoms described on tomato, consisting in corky necrotic stripes on the stems, corky rings and deformations on the fruits (Figure 1C), mosaic and basal necrosis on the leaves, causing in some cases the leaves perforation (Janssen et al., 2005).
PMoV can be passively transmitted by insects, such as thrips through the infected pollen (Aramburu et al., 2010).
Currently, the containment of PMoV dispersion can be implemented by early diagnosis and the application of preventive measures in crop management, reducing the introduction and subsequent spread in other countries. To date, there are no commercially available specific antibodies for the serological diagnosis of PMoV; the only currently available tool for PMoV identification is based on the end-point reverse transcriptionpolymerase chain reaction (RT-PCR) (Galipienso et al., 2008) and molecular hybridization techniques (Parrella et al., 2001Galipienso et al., 2005). Consequently, it is necessary to develop In bold the primer pair with the lowest Ct value.
an alternative and more sensitive diagnostic methods for the PMoV early diagnosis. For this reason, the aim of the present work was to develop a sensitive and specific molecular method to detect PMoV in infected tomato and pepper plants, based on reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with TaqMan R probe technique and estimate the dispersion of this pathogen in Southern Italy (Sicily).

Source of Viral Material
One lyophilized PMoV isolate from tomato plant, named ST-1 (GenBank Acc. No. KP234349, Galipienso et al., 2015), stored in the plant virology laboratory of the University of Palermo, was used as source material for subsequent experiments. Total RNA from 100 mg of previously re-hydrated ST-1 isolate was extracted using a Plant RNA/DNA Purification Kit (Norgen Biotek Corp., ON, Canada), following the manufacturer's instructions. Total RNA concentration was determined in duplicate with NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, United States) and used for cDNA synthesis. In the reverse transcription step (RT), the cDNA was obtained using the M-MLV Reverse Transcriptase (Thermo Fisher Scientific, MA, United States) in a 20 µL reaction volume containing 2 µL of total RNA and 100 pmol of the reverse primer PMoV-3R (5 -CACTCTTTACGTTGGCATCG-3 , position 1991-2010, Galipienso et al., 2008), according to the following cycling conditions: 42 • C for 45 min and 95 • C for 10 min. PCR was performed in 25 µL (final volume) containing 2 µL of the obtained cDNA, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl 2 , 0.4 mM dNTPs, 100 pmol of the forward primer PMoV-2F (5 -AATCGAGACTTTCGCCAGGA-3 , position 865-884) and 100 pmol of the reverse primer PMoV-3R (Galipienso et al., 2008), 2U of Taq DNA polymerase (Thermo Fisher Scientific, MA, United States) and RNase free water to reach the final volume, according to the following cycling conditions: 95 • C for 3 min; 40 cycles of 30 s at 95 • C, 45 s at 55 • C, 90 s at 72 • C and a final elongation of 10 min at 72 • C. The PCR was carried out in a MultiGene OptiMax thermal cycler (Labnet International Inc., NJ, United States). The obtained DNA products of expected size (1,146 nt) were confirmed by electrophoretic separation in a 1.5% agarose gel stained with SybrSafe (Thermo Fisher Scientific, MA, United States). The obtained cDNA product using the PMoV-2F/PMoV-3R primer pair was cloned in the pGEM-T vector (Promega, WI, United States) following the manufacturer's instructions, and subsequently sequenced in both directions using an ABI PRISM 3100 DNA sequence analyzer (Applied Biosystems, CA, United States).

Primer and TaqMan R Probe Design
Multiple alignments using the ClustalX2 program (Larkin et al., 2007)  Nucleotide identities between sequence pairs were estimated with the MEGA X program (Kumar et al., 2018), and two oligonucleotide primer pairs for RT-qPCR within the CP gene were designed using Vector NTI 11.5 (Thermo Fisher Scientific, MA, United States) ( Table 1). Both sets of primers were tested, using the total RNA of ST-1 isolate with the concentration of ∼10 ng RNA/µL, by RT-qPCR with SYBR Green, in order to understand which primer pair has the lowest threshold cycle (Ct) value. The RT-qPCR was performed in a Rotor-Gene Q2plex HRM Platform Thermal Cycler (Qiagen, Hilden, Germany). The mixture consists in a 20 µL final volume, containing 1 µL of total RNA extract (ST-1), 1 µL of 0.5 mM of each primer, 10 µL of Master mix QuantiNova SYBR R Green PCR Kit (Qiagen, Hilden, Germany) and H 2 O DEPC to reach the final volume.
Total RNA from a healthy tomato plant and water were used as negative control samples. Each sample was analyzed twice. Cycling conditions included reverse transcription at 45 • C for 10 min, incubation at 95 • C for 10 min, 40 cycles of 95 • C for 5 s and 60 • C for 10 s; the fluorescence was measured at the end of each cycle. Melting curve steps were added at the end of RT-qPCR as following: 95 • C for 1 min, 40 • C for 1 min, 70 • C for 1 min and a temperature increase to 95 • C at 0.5 • C/s to record the fluorescence.
A specific PMoV TaqMan R probe (Eurofins Genomics, Luxembourg) was designed in a conserved domain within the region encompassed by the primers. The probe, with a length of 22 nucleotides, was 5 -labeled with the reporter dye FAM (6-carboxyfluorescein) and 3 -labeled with a non-fluorescent quencher (NFQ) TAMRA ( Table 1). The predicted Tm values for PMoV primers and probe were 58 and 68 • C, respectively, calculated with the prediction tool provided by Primer Express Software v3.0.1 (Thermo Fisher Scientific, MA, United States). RT-qPCR was performed in a Rotor-Gene Q2plex HRM Platform Thermal Cycler in a reaction mix of 25 µL final volume, containing 5 µL of total RNA extract with the concentration of ∼10 ng RNA/µL, 0.5 µM of the forward primer PMoV21F and the reverse primer PMoV115R (the primer set that yielded the most sensitive detection for all PMoV isolates providing the lowest Ct values), 0.25 µM of TaqMan PMoV43 probe, 0.5 µL of RNase Inhibitor (Applied Biosystems, CA, United States), 12.5 µL of 2X QuantiNova Probe RT-PCR Master Mix, 0.25 µL of QN Probe RT-Mix, and H 2 O DEPC water to reach final volume.
Two independent RT-qPCR assays were carried out. Each sample was analyzed in duplicate, moreover, the control samples in each test included total RNA purified from a healthy tomato plant, water instead of sample and at least two RNA transcripts (see below). The probe annealed specifically in an internal region of the PCR product amplified with primers PMoV21F and PMoV115R (primer pair that showed the lowest Ct value). The RT-qPCR conditions consisted in reverse transcription at 45 • C for 10 min, enzyme denaturation at 95 • C for 10 min, and 40 cycles of 95 • C for 10 s and 60 • C for 60 s; the fluorescence was measured at the end of each cycle. The mean (X) Ct value and the standard deviation (SD) for each sample were calculated from the four Ct values obtained.

Standard Curve
An external standard curve was obtained in order to determine the sensitivity of the RT-qPCR protocol. Serial dilutions of an in vitro synthesized positive-sense RNA transcript of the selected gRNA region were amplified using the RT-qPCR TaqMan assay. The template for the in vitro transcription was obtained using one ST-1 clone by conventional PCR amplification with the PMoV-3R primer and a modified PMoV-2F primer with the T7 promoter (Galipienso et al., 2008).
The obtained PCR product was transcribed in vitro with the HiScribe R T7 High Yield RNA Synthesis Kit (New England Biolabs, MA, United States) following the manufacturer's instructions.
Transcripts were purified with RNaid Spin kit (Bio101, CA, United States), treated twice with RNase free DNase (Turbo DNA-free from Ambion, TE, United States); their concentration was determined in duplicate with NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, United States).
Ten-fold serial dilutions of the transcript in healthy tomato total RNA extract (10 ng/µL) containing 10 10 -10 1 copies were used in the RT-qPCR TaqMan assay. To ensure the absence of DNA template in transcript preparations, the assay was performed with and without the reverse transcriptase.
The RNA transcript concentration (pmol) in each dilution was calculated with the formula: micrograms of transcript RNA × (106 pg/1 µg) × (1 pmol/340 pg) × (1/number of bases of the transcript), and the number of RNA copies was calculated using this concentration value and Avogadro's constant. The standard curve was obtained plotting the Ct values from two independent assays with four replicates per standard dilution vs. the logarithm of the RNA concentration dilution. The amplification efficiency was calculated from the slope of the corresponding curve using the formula 10 (−1/slope of the standard curve) , or the same formula × 100 (when given as a percentage value). The sampling areas selected were marked by GPS using the Planthology mobile application (Davino et al., 2017a). Each sample was placed in an extraction plastic bag (Bioreba, Kanton Reinach, Switzerland) containing 1 mL of extraction buffer (50 mM Tris pH 9.0, 150 mM LiCl, 5 mM EDTA), and homogenized with a HOMEX 6 homogenizer (Bioreba, Kanton Reinach, Switzerland). Healthy tomato plants used as negative controls were grown in a phytotron with a 16/8 hrs light/dark photoperiod, 27 • C and 70-90% relative humidity. Total RNA from each sample was extracted by using a Plant RNA/DNA Purification Kit (Norgen Biotek Corp., ON, Canada), following the manufacturer's instructions, and the concentration was determined in duplicate with NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, United States). In order to assess the presence/absence of PMoV, the collected samples were analyzed by conventional end-point RT-PCR (Galipienso et al., 2008). Subsequently, the same samples were analyzed in duplicate by RT-qPCR, following the protocol described previously, in order to confirm the validity of the technique developed in this work and to compare the results of the two diagnostic methods.

Source of Viral Material
The RT-PCR product of the ST-1 isolate used in this study showed the expected 1,146-bp size. The obtained sequence after cloning was trimmed in order to remove the external nucleotides, leaving only the coding region of the CP gene which showed a percentage identity of >99% with original ST-1 isolate (Galipienso et al., 2015).

Primer and TaqMan R Probe Design and Protocol Optimization
In Table 1 are reported the two primer pairs and probe designed. The PMoV21F/PMoV115R primer pair showed lower Ct value (8.64 ± 0.2) compared to the PMoV420F/PMoV546R primer pair (9.02 ± 0.2) and were selected chosen as the best candidates together with the TaqMan PMoV43 probe for the subsequent analyses (Figure not shown). The optimal annealing/extension temperature was found at 60 • C and it was selected for the RT-qPCR assay.

PMoV Genotype-Specific RT-qPCR Assay With TaqMan R Probe
As reported in Table 3, the two RNA transcripts, used as positive controls, gave the most sensitive signal with a Ct value ranging from 8.09 ± 0.1 to 8.51 ± 0.2 in the four different assays. The total RNA derived from all tested PMoV isolates (15) gave also positive signal with a Ct value that ranged from 17.18 ± 0.2 to 26.86 ± 0.1, (Supplementary Figure 1), while ToBRFV, ToCV, PepMV, CMV, TSV, TYLCV, TYLCSV, TICV, TSWV, TMV, and ToMV isolates used as outgroups did not give any signal. The qRT-PCR assay confirmed its specificity since it gave no signals from healthy tomato RNA extracts and water as well. As far as the repeatability and reproducibility of the assay is considered, it

Standard Curve
The obtained standard curve covered a wide dynamic range (10 units of concentration) and showed a strong linear relationship, with a correlation coefficient of 0.9996 and 100% amplification efficiency (Figure 2 and Supplementary Figure 2). The high sensitivity of the assay was achieved covering eight orders of magnitude of RNA concentration and enabling the detection of as few as 10 1 PMoV RNA copies in tomato total RNA.

Comparison Between RT-qPCR and Conventional End-Point RT-PCR Techniques on Field Samples
As reported in Table 4 Regarding the difference in score between samples resulted positive at RT-PCR end-point and RT-qPCR, it is important to note that it concerns only asymptomatic samples (Table 5). Probably, this result is due to the low viral titer of asymptomatic samples. In Table 5 are reported and compared the results obtained from the two independent analyses.
Regarding the PMoV spread dynamics in Sicily, during the 3 years of sampling, using the RT-qPCR assay, the incidence showed an upward trend from 22.5% in 2018 to 40% in 2020 (Table 4) in the area object of the survey, demonstrating an increase of PMoV infection of 17.5%.

DISCUSSION
Climate change, global trade and anthropic activities (and their consequences) seem to be closely related to the emergence of new viruses, triggering an increasing number of outbreaks and epidemics. In fact, emerging and re-emerging virus diseases represent a real threat for horticultural production, as has happened in recent years in southern Italy, with the ToLCNDV  dispersion (Panno et al., 2019b) and, more recently, with the emergence and subsequent rapid ToBRFV spread in Sicily (Panno et al., 2020a) which, in the last 2 years, has been seriously compromising tomato and pepper productions. Numerous works have highlighted that climatic conditions changes are affecting the ecological impact of some plants, including P. officinalis, and thus could be in some extent responsible for increasing the PMoV incidence, substantially by enlarging the flowering period, and thus the pollen production (D'Amato and Cecchi, 2008).
Pathogen detection, identification and quantification are important in plant disease control, and must be accessible in all countries to ensure eco-sustainable crop production and food safety. For these reasons, it is essential to develop, in a plant disease surveillance context, new early diagnosis methods in order to control the pathogens (Ferriol et al., 2015;Puchades et al., 2017;Panno et al., 2020c). For this purpose, distinct parameters including environment conditions, genetic characteristics, transmission methods (Ferriol et al., 2013) and agronomical practices, such as organic amendment should be considered (d 'Errico et al., 2019).
Specifically, as regard virus diseases, the detection is usually performed, when available, by serological tests such as double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal or monoclonal antibodies but, in most cases, the commercial ELISA tests unavailability and/or the low viral titer in nursery plants or during the early stages of virus infection, do not allow the reliable use of these techniques (Jacobi et al., 1998).
Nevertheless, serological methods have some limitations because PMoV is a poorly immunogenic, producing antiserum with a low titer, even against the homologous virus (Caciagli et al., 1989) and currently no commercial kits are available for the specific serological PMoV diagnosis. In addition, TSV and prunus necrotic ringspot virus (PNRSV), which belongs, respectively, to Ilarvirus subgroups I and III, react with PMoV isolates, although very weakly (Lisa et al., 1998).
The one-step RT-PCR end-point has been widely applied for the diagnosis and molecular characterization of PMoV isolates (Galipienso et al., 2005(Galipienso et al., , 2008. The different diagnostic methods described above shows lack of information regarding the sensitivity and specificity, therefore, there is a current need for the development of a specific and sensitive methods for PMoV identification, which could also be usefully applied to epidemiological studies. This study describes the development and evaluation of a RT-qPCR assay for the sensitive and specific PMoV detection which, in recent years, has increased its incidence in tomato and pepper crops in southern Italy. As reported in section "Results", the efficiency of PMoV detection in symptomatic and asymptomatic tomato samples from Sicily was higher with the RT-qPCR developed (52 out of 130 samples) compared to the conventional RT-PCR method (46 of 130 samples) described by Galipienso et al. (2008), with a sensitivity percentage increase of 4.62%. It is possible to note how the RT-qPCR assay developed in this study is more sensitive; this statement is also confirmed by the results of the sensitivity tests (see section "Standard Curve"), which showed that the developed technique is able to detect as few as 10 PMoV RNA copies in tomato total RNA.
Although it is difficult to compare the sensitivity of the PMoV RT-qPCR to the RT-qPCR assays for other ilarvirus species (Osman et al., 2014), as sensitivities are reported rarely in terms of target copy number, but in terms of sample dilution which are not directly comparable, however, a detection limit of approximately ten copies of target is consistent with assays for other, unrelated plant viruses (Agindotan et al., 2007;Gutierrez-Aguirre et al., 2009). In addition, field data demonstrate that the proposed RT-qPCR method being more sensitive, especially in asymptomatic samples, compared to those described so far for the PMoV early diagnosis, and it could represent a good and reliable tool to be applied in low viral titer conditions, during early stage of the disease, and in search for alternative hosts and latent infections.
In conclusion, the results obtained in the present study demonstrate the accuracy, specificity sensitivity, repeatability and reproducibility of the RT-qPCR assay developed for the diagnosis of PMoV in tomato and pepper plants. It is expected that the implementation of this RT-qPCR with TaqMan R probe assay will facilitate efficient and fast phytosanitary certification of nursery stock, when wide samples indexing is required, thereby decreasing the risk of further spread of this dangerous virus. Moreover, the method proposed can also be useful for other application and studies, where sensitivity, accuracy and quantitative date are required, such as field survey, identification of natural virus reservoirs, and screening and characterization of resistances in germplasm.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
SP designed and performed the experiments and bioinformatics analysis, analyzed the data, prepared figures and/or tables, authored the drafts of the manuscript, and approved the final draft. AC performed the experiments and bioinformatics analysis, analyzed the data, prepared figures and/or tables, and approved the final draft. SB performed the experiments and bioinformatics analysis, analyzed the data, and approved the final draft. SM analyzed the data, authored and reviewed drafts of the manuscript, and approved the final draft. SD conceived and designed the experiments, contributed to reagents, materials, and analysis tools, authored or reviewed drafts of the manuscript, and approved the final draft. GP conceived and designed the experiments, authored or reviewed drafts of the manuscript, and approved the final draft. All authors contributed to the article and approved the submitted version.