Introduction

Tomato (Solanum lycopersicum L.) is by far the most important vegetable crop representing 72 % of the value of fresh vegetables produced worldwide. Tomato crops are susceptible to a wide range of pathogens including fungi, bacteria, viruses and viroids. Economically relevant pathogens infecting tomato include Clavibacter michiganensis subsp. michiganensis, Pepino mosaic virus (PepMV) and Mexican papita viroid (MPVd). C. michiganensis subsp. michiganensis is a gram-positive bacterium that causes bacterial canker of tomato and represents a serious problem in global main production areas of this crop. This bacterium has been included by the European Plant Protection Organization (EPPO) in the list of quarantine organisms for all member states (Anonymous). The pathogen can infect systemically causing failure of fruit development or uneven ripening; under - favorable conditions, plants become totally dehydrated (OEPP/EPPO 2005). C. michiganensis subsp. michiganensis is a seed borne pathogen; its spread over long distances is facilitated by seeds trade and commerce, but latently infected young plants also play an important role in long distance spread (OEPP/EPPO 2005). PepMV is a member of genus Potexvirus (Jones et al. 1980, also recently included on EPPO A2 list (http://www.eppo.int/QUARANTINE/listA2.htm). The severity of the disease induced by the virus depends on the environmental conditions. Severely affected plants become stunted and distorted with significantly reduction of fruit quality (Spence et al. 2006). The virus is transmitted mechanically by contaminated tools, direct plant-to-plant contact, grafting and by seeds obtained from infected tomato plants, although at very low rate (Jorda et al. 2001; Ling and Carpenter 2005; Córdoba-Sellės et al. 2007; Ling 2008). MPVd is a member of the family Pospiviroidae (Di Serio et al. 2014); so far, it has only been identified in tomato crops in Mexico and Canada. Infected plants show general stunting and reduced fruit size (Ling and Zhang 2009).

Many pathogens cause substantial economic losses, so resistant crops are a good management strategy to reduce them; however, such resistant varieties are not always available. In these cases, the availability of simple and reliable detection procedure is a requisite to ensure the identification of plant material free of pathogens. In addition, since a plant could be infected with more than one pathogen, multiple detection assays may have to be conducted, increasing the time and the associated costs of insuring plant material free of pathogens. An alternative is the simultaneous detection of several pathogens in a single reaction (multiplex detection). During the last two decades, multiplex detection of several pathogens by non-isotopic molecular hybridization (NiMH) using digoxigenin-labelled probes has proved to be an appealing detection procedure (reviewed in James et al. 2010). This technique has mainly been applied for the simultaneous detection of viruses and/or viroids affecting a crop. In one approach individual specific digoxigenin-labelled probes are mixed in the hybridization solution (e.g. Saldarelli et al. 1996; Sanchez-Navarro et al. 1999; Saade et al. 2000; Ivars et al. 2004; Minutillo et al. 2012). Another approach uses a unique riboprobe, called polyprobe, which contains fused-in-tandem partial nucleic acid sequences of the target pathogens (Herranz et al. 2005). Polyvalent detection using a unique polyprobe has been successfully applied to the detection of different plant viruses infecting stone fruits (Herranz et al. 2005; Peiró et al. 2012; Fajardo and Nickel 2014), tomato (Aparicio et al. 2009) and different viroids that infect citrus (Cohen et al. 2006) pome and stone fruit trees (Lin et al. 2011), grapevine (Zhang et al. 2012) and ornamentals and vegetables (Torchetti et al. 2012). Moreover, the use of the polyprobe technology by NiMH has been proven to allow the detection of virus and viroids in a single assay. Recently, a polyprobe was developed to detect eight viruses and two viroids commonly found in cultivated stone fruit trees (Peiró et al. 2012). Although NiHM was used to detect Pseudomonas syringae pv tomato from both pure bacterial cultures or artificially contaminated tomato seeds (Fanelli et al. 2007), its use with polyprobes has not been reported to detect pathogens other than viruses and viroids in naturally infected plants.

In the case of C. michiganensis subsp. michiganensis, seed testing is essential for pathogen control and the EPPO standard protocols are based on the isolation and the posterior identification of the pathogen. Thus, seed extracts are grown on semi-selective media or directly tested by serological or PCR based methods. Negative samples on selective media, but positive by one of the molecular techniques, are analyzed in tomato seedlings. After bacteria multiplication in planta, C. michiganensis subsp. michiganensis identity has to be confirmed by pathogenicity, biochemical, ELISA and PCR-based tests (OEPP/EPPO 2005; De León et al. 2011). Immunofluorescence (IF) and ELISA, using commercially available antibodies, and PCR based methods are the techniques most frequently used for either the detection in seed extracts and for the confirmation assays in field surveys (reviewed in De León et al. 2011). However, these technologies are expensive and have several limitations including low sensitivity and false positives caused by cross-reactivity. In the case of the PCR methodologies, two main limitations should be considered: i) the requirement for specialized laboratories and equipment, and ii) the observation that the reaction can be inhibited by contaminants present in plant extracts (Kokosková et al. 2010; Olivier et al. 2010; de León et al. 2008; Dreier et al. 1995). Related to simultaneous detection procedures, a multiplex PCR was developed to detect C. michiganensis subsp. michiganensis and two other tomato-infecting bacteria, Pesudomonas syringae pv. tomato and Xanthomonas axonopodis pv. vesicatoria (Özdemir 2009). More recently, Johnson and Walcott (2012) carried out a magnetic capture hybridization procedure to concentrate and purify target nucleic acids coupled to a multiplex real-time PCR assay that permits the simultaneous detection of C. michiganensis subsp. michiganensis and PepMV. However, both protocols used bacterial cultures and no results were presented from naturally infected seeds or tomato plants.

We have analyzed the versatility of polyprobe technology to detect three different pathogens by creating a poly-3 riboprobe to detect C. michiganensis subsp. michiganensis, PepMV and MPVd. We have also evaluated the capability of the NiMH technique as alternative method to serological and PCR tests for the specific detection of this bacterium. Our results showed that this approach allowed the specific detection and differentiation of C. michiganensis subsp. michiganensis from other pathogenic bacteria in pure cultures. Furthermore, the poly-3 technology was able to detect specifically all three pathogens from naturally infected tomato plants in a single assay, demonstrating the capacity of this technology to simultaneously detect viruses, viroids and a bacterium. The use of this technology for the routine analysis of tomato crops is discussed.

Material and methods

Plant material and pathogenic bacteria

Tomato plants were randomly collected in greenhouses located in the central and eastern regions of Mexico (Montecillo, Yecapixtla, and Zacualpan) during the month of July 2014. The C. michiganensis subsp. michiganensis strain was isolated from infected plant tissue and purified in nutritive agar medium. The plant pathogenic bacteria Agrobacterium tumefasciencies, Erwinia caticida, Pectovacterium carotovorum, Pseudomonas syringae pv. tagetis, Pseudomonas marginalis, and Pseudomonas helianthi were obtained from the Colegio de Postgraduados Bacteria Collection at the Campus Montecillo, Texcoco in the Estado de México, México..

Single and poly-3 probes construction and synthesis of digoxigenin-labelled probes

Total RNA was extracted using Trizol Reagent (Sigma) from infected plants. Individual and poly-3 probes were generated using the pSK+ Bluescript plasmid following the methodology previously described by Peiró et al. (2012). Briefly, reverse transcription and PCR reactions were carried out using specific primers that contain the XhoI and SalI restriction sites (Table 1) that amplified the full-length MPVd (360 nt), a fragment corresponding to part of the coat protein gene (CP) of PepMV (200 bp) and to the putative serine protease gene (pat-1) (370 bp) encoded in the pCM2 plasmid of C. michiganensis subsp. michiganensis. pat-1 is an essential gene for bacterial virulence which is used as target for the PCR identification of C. michiganensis subsp. michiganensis (Meletzus et al. 1993; Dreier et al. 1995; Chalupowicz et al. 2010; De León et al. 2011). The amplicons were digested with the XhoI and SalI restrictions enzymes, purified from agarose gels and cloned in the pSK+ plasmid, previously digested with XhoI and dephosphorylated, to generate the pSK/Cmm, pSK/PepMV and pSK/MPVd constructs. To generate the pSK/poly-3 construct, the PepMV and MPVd PCR fragments were sequentially incorporated into the pSK/Cmm, previously digested with XhoI and dephosphorylated. The introduction of the amplicon in the right orientation permits the inactivation of the original XhoI site present in the pKS/Cmm plasmid, by the ligation of the compatible SalI/XhoI restriction sites. The nucleotide sequences of all constructs were corroborated by nucleotide sequencing.

Table 1 Primers used in this study
Full size table

Single and poly-3 plasmids were linearized with XhoI restriction enzyme and the corresponding digoxigenin- labeled riboprobes were synthesized using the T7 RNA polymerase following the manufacturer’s instructions (TaKaRa).

Sample analisis, total RNA extraction and hybridization procedures

Total nucleic acids were extracted from 0.1 g of fresh tomato tissues using a silica extraction procedure (Thompson et al. 2003). In the case of plants infected with C. michiganensis subsp. michiganensis and pure cell cultures, sap extracts were boiled for 5 min prior to the extraction procedure. Pure bacteria cultures cells were growth in selective medium until the indicated CFU/ml measured as viable plate colonies and subsequently sedimented by centrifugation 5 min at 12.000 rpm. Pellets were resuspended with the silica buffer and boiled for 5 min prior the total nucleic acid extraction procedure.

To analyze the distribution of C. michiganensis subsp. michiganensis, PepMV and MPVd in infected tomato plants and for field analysis, total nucleic acids equivalent to 5 mg of fresh tissue were applied onto positively charged nylon membranes (ROCHE) as previously described (Astruc et al. 1996). When required the corresponding serial dilutions of total nucleic acids were made with extraction buffer. Membranes were air-dried and the nucleic acids were covalently fused by UV cross-linking (700 × 100 J/cm2). Pre-hybridization and hybridization with the single or the poly-3 riboprobes were conducted as described previously (Pallás et al. 1998). Chemiluminescent detection with CSPD substrate (ROCHE) was performed as recommended by the manufacturer.

RT-PCR reactions

The RT-PCR reactions were carried out using the primers listed in Table 1. The reaction mixture for the reverse transcription consisted of 30 ng of total nucleic acids, 1 mM dNTPs, 100 mM antisense primer, 40 U of ribonuclease inhibitor and 10 U of the Reverse Transcriptase RevertAid M-MuLV (Thermo Scientific) in a final volume of 20 μl. The reaction was carried out following the manufacturer’s recommendations. PCR was done in a Techne TC-512 thermocycler using 2 μL cDNA, 2 mM MgCl2, 0.2 μM dNTPs, 0.2 μM of each primer and 1 U TaqPol (Promega®) in a 20 μl final reaction volume. Thermal cycling conditions included a denaturation step at 94 ° C for 3 min and 35 cycles of denaturing at 94 °C for 20 s, annealing at 55 °C for 20 s, and extension at 72 ° C for 60 . Finally, an incubation of 10 min at 72 °C to finish the incomplete PCR fragments was performed.

Results

Many studies have demonstrated the usefulness of the NiMH to simultaneously detecting viruses and/or viroids. Although this technology was also used to detect DNA sequences specific for plant pathogenic bacteria (Dessaux et al. 1995) its use for the simultaneous detection with other pathogens in naturally infected plants has not been reported to date. The goal of this work was to evaluate the use of a digoxigenin-labelled poly-3 polyprobe to detect simultaneously three different pathogens, C. michiganensis subsp. Michiganensis, PepMV and MPVd that significantly affect tomato plants.

The pSK/poly-3 plasmid generated in this work contains the 5′ and 3′ terminal regions complementary to the pat-1 gene of C. michiganiensis subsp. michiganensis and the full-lenght MPVd genome, respectively meanwhile the central region is complementary to the CP of PepMV (Fig. 1a). This plasmid was used to synthesize the digoxigenin-labelled poly-3. The first step was to adjust the NiMH procedure for the specific detection of C. michiganensis subsp. michiganensis. One important question to solve was whether, in spite that the pat-1 gene is only present in the pathogenic C. michiganensis subsp. michiganensis strains our poly-3 would give some cross-reaction with other bacteria. To address this question, total nucleic acids from six species of plant pathogenic bacteria were hybridized with the poly-3. The results showed specific hybridization signal only with C. michiganensis subsp. michiganensis (Fig. 1b, panels 7 and 8), and no signal was observed in the rest of the analyzed bacteria (Fig. 1b, panels 1 to 6, A. tumesfaciencies, E. caticida, P. carotovorum, P. syringae pv. tagetis, P. marginalis and P. helianthi, respectively). After that, we determined the endpoint limit of the poly-3 to detect C. michiganensis subsp. michiganensis by using pure cell cultures. As shown in Fig. 1c, the endpoint detection limit was 106 CFU/ml. The same extracts were used to determine the limit of detection by RT-PCR, where the endpoint corresponded to 104 CFU/ml (Fig. 1d), similar to that previously reported using Real-time PCR (Johnson and Walcott 2012; Bach et al. 2003). The RT-PCR detection limit was 100 times higher than that observed for NiMH, a difference that was in the same order of magnitude reported for the detection of other pathogens (Sanchez-Navarro et al. 1998).

Fig. 1
figure 1

Specificity and detection limit of the pat-1 riboprobe against C. michiganensis subsp. michiganensis. a Schematic representation of the pSK/poly-3 plasmid, indicating the localization of the pat-1 gene of C. michiganensis subsp. michiganensis (Cmm), Pepino mosaic virus (PepMV) and Mexican papita viroid (MPVd) fragments. b Total nucleic acids extracted from different plant pathogenic bacteria suspensions at 109 UFC/ml, were dotted onto nylon membrane and hybridized with the ribopoly-3. Numbers correspond to Agrobacterium tumesfaciencies (1), E. caticida (2), P. carotovorum (3), P. syringae pv. tagetis (4), P. marginalis (5), P. helianthi (6) and C. michiganensis subsp. michiganensis at 109 (7) and 107 UFC/ml (8). c and d Total nucleic acids extracted from C. michiganensis subsp. michiganensis cell cultures at different dilutions (108 to 103 UFC/ml; indicated on top of panels), were dotted onto a nylon membrane and hybridized with the ribopoly-3 (c) or used for RT-PCR reactions to amplify pat-1 gene fragment (d). Lanes a and b correspond to negative and positive controls, respectively

Full size image

The specificity and the sensitivity of the poly-3 was compared to that of the individual riboprobes. Thus, membrane replicates containing total nucleic acids from tomato plants individually infected with each pathogen were hybridized with the individual or poly-3 riboprobes. The endpoint detection limit for both individual and poly-3 riboprobes corresponded to the total nucleic acids extracted from 1 ng of both PepMV and MPVd or 100 ng of C. michiganensis subsp. michiganensis infected tissues (Fig. 2). These results demonstrate that the poly-3 allows the simultaneous detection of the three pathogens with no significant difference in sensitivity respect to the individual riboprobes. In addition, the observation that the individual and poly-3 probes have similar detection limit using complementary synthesized RNA transcripts (pg/μl; data not shown), suggest that the different endpoint detection limits observed in infected tissue among virus/viroids and bacteria could reflect a different pathogen concentration.

Fig. 2
figure 2

Comparison of the sensitivity between single and the poly-3 riboprobes. The end detection limit for each probe was determined by using dilutions of total nucleic acid extract from individually infected plants. Numbers above panels indicate the nanograms of infected tissue equivalent to the total nucleic acids applied onto the nylon membranes. Each replica was hybridized using the indicated riboprobe. Cmm: C. michiganensis subsp. michiganensis

Full size image

A prerequisite to using the polyprobe technology in routine analysis of field samples was to analyze the accumulation of the three pathogens in different tomato tissues. To investigate this, total nucleic acids extracted from petiole, apex, leaf and stem were hybridized with the corresponding individual or poly-3 riboprobes. As shown in Fig. 3, all pathogens accumulated in the analyzed tissues. The individual riboprobes were specific for its corresponding pathogen and the poly-3 results were in concordance with those from the individual probes (Fig. 3).

Fig. 3
figure 3

Distribution of C. michiganensis subsp. michiganensis, PepMV and MPVd in infected tomato plants. Total nucleic acids equivalent to 5 mg of tissue, extracted from petiole, apex, leaf and stem of individually infected tomato plants were applied onto four nylon membranes. Each replica was hybridized using the riboprobe indicated on the right of each membrane. Cmm: C. michiganensis subsp. michiganensis

Full size image

Finally, we analyzed the capability of the poly-3 for routine analysis of tomato samples under field conditions. Thus, 80 samples collected from three tomato producer locations in the center and eastern areas of Mexico, were analyzed with the three single or the poly-3 riboprobes. Field tomato samples, previously identified as individually infected with each pathogen, were used as positive samples. Six samples, including the positive tomato control, rendered positive hybridization signals using the C. michiganensis subsp. michiganensis probe (Fig. 4a, samples A8, B7, G8, H7 and H8 and the positive control I1) whereas only the PepMV and MPVd infected tomato controls were positive using the corresponding individual probes (not shown). All positive samples detected with the single probes were also detected using the poly-3, indicating the capacity of this polyprobe to detect simultaneously the three pathogens in tomato samples (Fig. 4b). The same results were obtained when the samples were analyzed by RT-PCR, using specific primers for each pathogen (Fig. 4c, only 13 representative samples are shown). Taken together, the results suggest that NiMH using the poly-3 is a good detection technique for these three pathogens in tomato plants.

Fig. 4
figure 4

Comparative analysis of 80 field tomato samples by NiMH and RT-PCR. Total nucleic acids equivalent to 5 mg of tissue were applied onto nylon membranes that were analyzed using the single Cmm (a) and the poly-3 (b) riboprobes. Healthy samples were applied in A1 and B1 whereas those previously identified as positive samples for MPVd, PepMV and C. michiganensis subsp. michiganensis were applied in boxes D1, F1 and I1, respectively. c Agarose gels showing representative RT-PCR products obtained from 13 tomato samples including the positives by NiMH. Asterisk indicates amplification of unspecific fragments. Lane mk, DNA molecular marker

Full size image

Discussion

Plants can be infected by diverse pathogens (e.g. bacteria, fungi, viruses, viroids, etc.), which may require specific detection procedures for accurate and rapid recognition. The development of techniques to detect simultaneously the most important pathogens threatening a specific crop is desirable, because it reduces time and associated costs of producing pathogen free-material (James et al. 2010). Simultaneous detection of several viruses and/or viroids in a single test using the NiMH technology is a reliable diagnostic procedure (Saldarelli et al. 1996; Sanchez-Navarro et al. 1999; Saade et al. 2000; Ivars et al. 2004; Herranz et al. 2005; Cohen et al. 2006; Aparicio et al. 2009; Lin et al. 2011; Peiró et al. 2012; Fajardo and Nickel 2014). However, as far as we know, no simultaneous detection of a bacterium, virus and viroid by NiMH has been yet reported. In the present work, we evaluated the capacity of the NiMH to detect simultaneously another pathogen different from virus and viroids, as the pathogenic bacteria C. michiganensis subsp.michiganensis.

First we confirmed the capability of the poly-3 to specifically detect C. michiganensis subsp. michiganensis from cell cultures by challenging the riboprobe against other six different pathogenic bacteria. The detection limit in pure cell cultures, was 106 CFU/ml which is comparable to ELISA (105–106 CFU/ml) but 100–1000 higher than serological immunofluorescence technologies (IF) using polyclonal antibodies (103–104 CFU/ml) (Kokosková et al. 2010; reviewed in De León et al. 2011). Despite the lower detection limit of some serological procedures (e.g. IF), false positives may occur (reviewed in De León et al. 2011), an aspect that was not observed in the NiMH procedure presented here. The higher end-point detection limit of the poly-3 with cell cultures compared to the IF, could be explained by the fact that the pat-1 mRNA detected by the poly-3 is mostly induced when the bacterium colonizes the plant. In fact, it has been reported that pat-1 mRNA accumulated between 150 and 50 times more in infected plants than in cell cultures (Chalupowicz et al. 2010). The differences observed between the detection limits of RT-PCR and NiHM, were in the same order of magnitude when the two techniques were used to detect other pathogens (Sanchez-Navarro et al. 1998). However, we observed that NiMH was 1000 times less sensitive than other PCR-based protocols designed to detect the bacteria alone (103 CFU/ml; Kokosková et al. 2010; Zhao et al. 2007; Luo et al. 2008), but only 10 times less sensitive than a multiplex Real-time PCR designed to detect simultaneously C. michiganensis subsp. michiganensis and PepMV (105 CFU/ml; Johnson and Walcott 2012). PCR based techniques to identify a specific pathogen currently present the highest detection limit (Pallas et al. 2009; James et al. 2010; de León et al. 2011), although the presence of reaction inhibitors such as phenols, tannins, and polysaccharides can interfere with the procedure and these techniques are expensive for processing numerous samples.

The results presented here demonstrated that the NiMH polyprobe technology can be used to detect pathogens other than viruses and viroids. There was concordance between the results observed with the NiMH and the RT-PCR to identify C. michiganensis subsp. michiganensis infected tomato samples in the field analysis indicating that NiMH could be used as a routine protocol to detect C. michiganensis subsp. michiganensis in tomato plants with a detection limit comparable to some serological methods.

The NiHM technology using polyprobes has permitted the simultaneous detection of four or six viroids (Cohen et al. 2006; Lin et al. 2011), six viruses (Herranz et al. 2005; Aparicio et al. 2009) or eight viruses plus two viroids (Peiró et al. 2012). Here we present data showing the poly-3 probe allows the simultaneous detection of three pathogens (bacterium, virus and viroid) with the same detection limit and specificity compared to individual riboprobes. This technology could be a very useful tool in both the routine field surveys of tomato crops and to generate plant material pathogen-free stock production. An open question is whether other pathogens from different aetiologies (e.g. fungi, phytoplama, etc.) could be added to polyvalent detection strategies by NiMH.