1. Introduction
Xylella fastidiosa is one of the fifteen quarantine bacteria of highest phytopathological interest in the European Union (EU) [
1]. It causes damaging diseases in strategic crops of socio-economic importance, and in ornamental and wild plants of a wide variety of botanical species [
2]; the overall number of host plants in the last update by EFSA reached 595 plant species, 275 genera, and 85 families [
2]. This pathogen is disseminated over long distances by the uncontrolled movement of infected, but frequently symptomless, plant material, and it is naturally transmitted by different species of insect vectors that spread the bacterium locally in a persistent and efficient manner [
3,
4,
5,
6].
X. fastidiosa is currently present in several European countries, mainly in Italy, France, and Spain [
7], where a number of areas are under eradication or containment strategies in order to avoid dissemination of this quarantine organism [
8]. The impact of the diseases caused by
X. fastidiosa is very high, both in terms of production losses and the number of hectares affected [
4]. In fact, in the Mediterranean area there are strategic crops for the various national economies, such as olive and almond trees, and even very important forest species, which have been threatened in the outbreaks reported in southern Italy, France, and Spain (Balearic Islands and Alicante) [
4]. Consequently, the situation is also of great concern to international organizations such as the European and Mediterranean Plant Protection Organization (EPPO), the International Plant Protection Convention of the Food and Agriculture Organization (IPPC-FAO), and the European Food Security Agency (EFSA), which coordinate standards for diagnosis and detection of the pathogen [
6,
9], as well as guides for sampling [
10] and an updated host list that is regularly revised due to the continuous increase of new host species [
2].
The use of accurate and gold standard methods and reagents for detection and diagnosis of
X. fastidiosa is needful to contain the spread and to ensure the commercialization of
Xylella-free plant material, in order to preserve crops and forests of economic and environmental importance in the EU and in many
Xylella-free countries worldwide. The molecular amplification techniques currently available [
6] are very useful and play a key role in the preventive control and management of the disease. Nevertheless, real-time PCR protocols involve a high cost per sample and have some inhibition challenges, especially in certain hosts, and risk of contamination [
11]. In fact, the PCR inhibitors present in some host species may decrease the sensitivity of optimized protocols because they are not always removed during the extraction and purification of nucleic acids.
X. fastidiosa pathosystems can be considered a case study in microbial diagnosis because their large host range advises the use of polyphasic methods for more accurate detection and diagnosis.
In this context, highly specific immunological or serological techniques could be very useful, because their sensitivity is not affected by these inhibitors and are sustainable for large-scale testing, as has been demonstrated in several diseases [
12,
13]. Thus, protocols based on these techniques—that are economical, accurate, reproducible, sensitive, fast, and user-friendly—complete the availability of methods to reliably detect
X. fastidiosa. Moreover, they could be used in public and private laboratories, nurseries, etc., for effective elimination of infected plants, even with no symptoms but with latent infections. Immunological methods and tools based on specific, homogeneous, and well-characterized antibodies must be part of a polyphasic strategy for the detection of
X. fastidiosa, as has already been demonstrated for other pathosystems, which allows a drastic increase in the number of samples analyzed [
11,
14].
According to the IPPC-FAO [
11], a number of serological methods, all of them based on conventional polyclonal antibodies (PAbs), have been developed for the detection of
X. fastidiosa, including ELISA [
15], membrane entrapment immunofluorescence [
16], dot immunobinding assay [
17], western blotting [
18], and indirect immunofluorescence [
19]. More recently, tissue print-ELISA or direct tissue blot immunoassay (DTBIA) has been reported as an alternative for rapid screening of olive samples for
X. fastidiosa in Italy [
20]. Some of these methods are also recommended in the EPPO standard for diagnosis of the bacterium [
6]. However, in general, the immunological detection methods are less used than in the past, partially due to the non-availability of homogeneous and well characterized specific antibodies for some pathogens. The main drawbacks associated to PAbs are: (i) the poor specificity, (ii) the lack of homogeneity among different batches, (iii) the strong dependence on the individual immunized animal, and (iv) that only a maximum of 10–15% of the antibodies developed act against the injected antigen. This poses a serious problem regarding the standardization of commercial lots of antisera of uncertain specificity and cross-reactivity [
12,
13,
21].
Monoclonal antibodies (MAbs) offer numerous advantages, such as specificity, since nearly 100% of immunoglobulins are identical with a predefined specificity, which significantly reduces the number of false positives and cross-reactions in the assays. This confers accuracy and reproducibility to the immunological tests, as a result of the selection and characterization of antibodies of high affinity, avidity, and homogeneity. In addition, MAbs can be obtained in unrestricted amounts in a reproducible manner, which is a safeguard for the standardization of commercial kits. The perpetuity of hybridoma cell lines through culture and freezer storage offers the advantage of the reproducibility of MAbs over time and in different laboratories [
22]. MAbs for
X. fastidiosa were reported long time ago by Garnier et al. [
23], but they were not evaluated or characterized and are not available.
In summary, integrated protocols for the detection of bacteria are advised, which include the use of molecular and serological techniques as screening methods, followed by confirmation using techniques supported by different biological principles [
24]. The choice of the most accurate and suitable detection method is crucial and should be related to the final purpose of the analysis, especially in areas with the presence of
X. fastidiosa, where extensive surveys and testing are required. It would be very useful to be able to include in the flowchart for the diagnosis of
X. fastidiosa of the EPPO standard [
6] the use of immunological methods based on MAbs.
The purpose of this research was to develop a useful specific tool and immunological methods for the detection of X. fastidiosa in large-scale programs that meet the requirements of speed, simplicity, low price, and accuracy desirable in a diagnostic technique intended for this purpose. This work demonstrates that the selected MAb2G1/PPDis a very promising tool that can be used for an accurate detection of X. fastidiosa by DAS-ELISA, tissue print-ELISA or by other sustainable serological techniques in massive monitoring programs, essential for the development of appropriate integrated management approaches to prevent the spread and establishment of this quarantine pathogen into new areas in the EU members and other countries.
4. Discussion
Immunological methods based on specific, homogeneous and well characterized antibodies are among the tools within a polyphasic strategy for the detection of
X. fastidiosa. These methods can drastically increase the number of analyzed samples with good accuracy when based on specific MAbs [
9,
11,
14]. The successful use of specific MAbs produced in this work by different direct and indirect serological techniques, such as DAS and DASI-ELISA, and tissue print-ELISA, show the ability of these antibodies to recognize
X. fastidiosa. The intensity of the serological reaction suggests a high affinity and avidity of the selected MAbs to the antigenic determinants or detectable antigens of the target. These MAbs against
X. fastidiosa, available for the first time, showed their usefulness and have been validated in this work as a reliable tool for the detection, identification, and diagnosis of this important phytopathogenic bacterium.
Interestingly, the selected MAbs, in particular 2G1/PPD (isotype IgG
1), proved to be highly specific for
X. fastidiosa, detecting all the strains analyzed representing different subspecies, STs and hosts. The high serological relationship observed by both DASI-ELISA and DAS-ELISA among the different
X. fastidiosa strains tested, mainly with the MAb2G1/PPD, suggests that it recognizes a widespreadand well conserved
X. fastidiosa epitope or antigenic determinant shared among the different strains. In the development of a rapid and large-scale diagnostic tool, species level recognition is the most suitable strategy, so that none of the variants that may be involved are left out, since co-infection of
X. fastidiosa strains from different subspecies have been found in individual samples [
38]. Furthermore, the MAbs obtained were highly specific because no cross reactions were observed against other phytopathogenic bacteria tested, not even with phylogenetically related strains of the genus
Xanthomonas, nor with the usual microbiota of the host plants routinely analyzed.
When comparing the developed DAS-ELISA based on MAb2G1/PPD with the commercially available kit of Loewe based on polyclonal antibodies from antisera, both systems achieved the same sensitivity in both pure cultures and spiked samples, but the MAbs showed less background noise. In fact, leaf extracts of different plant species were tested and no significant differences in sensitivity were found between them, which demonstrates the general usefulness of DAS-ELISA MAb2G1/PPD for
X. fastidiosa detection in a wide range of hosts, suggesting that there is not a remarkable influence of the plant material in its sensitivity. This may indicate that DAS-ELISA MAb2G1/PPD is not affected by the presence of potential inhibitors, like phenolic compounds, which is an advantage over molecular detection methodologies [
26]. In the naturally infected samples analyzed, those that gave negative results by DAS-ELISA MAb2G1/PPD (n = 15 respect to Harper’s real-time PCR and
n = 12 respect to Francis’ real-time PCR) belonged to different plant species, so no correlation was found between type of host and the negative result attributable to inhibition. What probably happens is that in trees with recent infections, the load of
X. fastidiosa is very low, below the detection limit. Nevertheless, the overall capacity of DAS-ELISA MAb2G1/PPD for the detection of
X. fastidiosa in different plant matrices can be considered excellent, since the pathogen was detected by the MAb in almond tree samples showing or not leaf scorch symptoms.
The sensitivity of 10
4–10
5 CFU mL
−1 is similar to that reported with other DAS-ELISA for this pathogen [
39] and better (one order of magnitude lower) than that of the commercially available DAS-ELISA from Agdia, as reported in an interesting comparison between serological and molecular methods [
40]. The DAS-ELISA sensitivity of 10
4 CFU mL
−1 is enough for
X. fastidiosa detection not only in symptomatic samples from areas with high pathogen prevalence, but also in samples from areas with low presence of the pathogen, even if they remain asymptomatic for long periods. In fact, in the area of olive epidemics in Puglia (Italy), conventional PCR and ELISA proved to be equally effective, but ELISA was chosen for the large-scale monitoring programs in the demarcated area due to the simplicity of sample preparation that allows to process a higher number of samples [
41]. Diagnostic specificity of the DAS-ELISA MAb 2G1/PPD was the same (100%) when compared to both real-time PCR techniques, and diagnostic sensitivity was only slightly lower compared to Harper’s real-time PCR (88.5%) than to Francis’ real-time PCR (89.9%).
Naturally infected samples used in this study included different plant species and several plant tissues, i.e., leaves of different ages and, in some cases, wood. Remarkably, there were no false positives by DAS-ELISA MAb 2G1/PPD. This is particularly interesting because the occurrence of false positives in ELISA for the diagnosis of
X. fastidiosa by polyclonal antibodies had been reported [
19], not only in relation to the need to block non-specific binding sites [
42], but also in relation to the potential activity of plant peroxidases [
43]. It is also worth mentioning that some plant tissues did not seem more suitable than others for the detection of
X. fastidiosa by DAS-ELISA MAb 2G1/PPD. This is important because, depending on the season, the use of one plant tissue or another may be more convenient [
6], and this fact would not influence the detection efficiency by DAS-ELISA based on the produced MAbs, at least in almond and olive tree samples.
A comparison of the relative accuracy obtained between DAS-ELISA MAb2G1/PPD and the gold standard real-time PCR (93.5–94.4%) demonstrates a strong correlation between the assayed techniques. In the analysis of almond tree samples from the demarcated area of Alicante (Spain), our results show a substantial or almost perfect agreement [
35] between the DAS-ELISA MAb2G1/PPD and the two real-time protocols validated by EPPO [
6]. This indicates the suitability of the serological techniques developed for
X. fastidiosa diagnosis and detection. Cohen’s kappa coefficient [
34] constitutes a recognized method for evaluating agreement between two diagnostic techniques. This coefficient does not reveal which technique is better, but indicates how often they give the same results. In this work the detection results obtained by two different protocols of real-time PCR, considered gold standard, were compared with those obtained with the same field samples by DAS-ELISA based on the selected MAb 2G1/PPD. The agreement 0.94 was substantial with Harper’s real-time PCR and it was 0.93, almost perfect, with Francis’ real-time PCR, based on kappa index of agreement of Cohen and McNemar test with a highly significant
p value. The McNemar χ2 test indicated that bias was no significant, and Cohen’s kappa index remained strong. In a recent work, Waliullahet al. [
40] also found a high correlation between DAS-ELISA and real-time PCR for field and greenhouse collected blueberry samples infected with
X. fastidiosa. It is known that the sensitivity of real-time PCR is better than that of ELISA, and this, together with the lack of expertise and equipment for serology in many laboratories of EU countries, has probably been the reason why this method has been excluded from the Implementing Regulation of the Commission (EU) 2020/1201 [
8]. However, the results of this work show that serological techniques such as the ELISA developed with the MAbs, which has a high specificity, could be applied to the processing of a large number of samples. Thus, only those samples with negative results could be processed by real-time PCR to overcome false negative results by ELISA due to a low bacterial load.
To avoid the rapid increase of the pathogen population in a given crop, a consistent and early detection protocol is necessary, but, frequently, the use of a single method may not produce 100% diagnostic certainty. In international diagnostic standards it is advisable to use at least two tests based on different biological principles [
6], particularly in areas apparently free of
X. fastidiosa or those under containment strategy. The benefit of any detection technique depends on its simplicity, specificity, sensitivity, robustness, cost-effectiveness, and suitability in all circumstances [
40]. The MAb based DAS-ELISA developed in this study represents a sustainable and low-cost alternative compared to other methods currently used for
X. fastidiosa detection. Like the PCR-based methods, it exhibits high specificity; samples that gave positive results with PCR also reacted by DAS-ELISA MAb2G1/PPD. In contrast to the PCR-based methods, the developed DAS-ELISA can be easily performed in laboratories with a basic structure for microbiology work, even in plant nurseries, and the high performance of these MAbs could also be used for the development of a ‘lab-on-a-chip’device such as the one developed for the CoDiRO strain by Chiriacòet al. [
44], and for other immunological techniques such as tissue print-ELISA, immunofluorescence or lateral flow devices. Especially, tissue print-ELISA is a simple, low-cost, fast, sensitive and accurate way to analyze many samples from nurseries, gardens or large surveys, as clearly showed in other pathosystems [
11] and demonstrated in Italy for olive trees testing [
20]. The specificity would be guaranteed by the use of MAbs, and also speed, simplicity, low analysis cost, and accuracy, which are characteristics desirable in a diagnostic technique [
45].