Enquête préliminaire sur la présence de Xylella fastidiosa sur l'olivier, les agrumes et la vigne au Maroc
Xylella fastidiosa, bactérie gram-négative, confinée au xylème, très polyphage, provoque diverses maladies sur plus de 300 plantes hôtes. Compte tenu de la récente détection confirmée de X. fastidiosa dans l'Union Européenne, cette bactérie est en train de devenir une menace sérieuse pour le secteur agricole marocain. Une enquête a été menée durant Mai-Septembre 2015 sur la présence de X. fastidiosa dans plusieurs plantations de l'olivier, des agrumes et de la vigne à travers le pays. Des symptômes sévères qui pourraient être associés à la bactérie ont été relevés. Un total de 900 échantillons de différentes cultures à partir de différentes régions ont été collectés au hasard: 220 oliviers (cv. Picholine Marocaine) de deux régions, 410 arbres d'agrumes appartenant à 7 cultivars collectés dans 4 régions et 270 plants de vigne appartenant à 6 cultivars de 3 régions; tous ces échantillons ont été testés pour la présence de X. fastidiosa en utilisant un kit commercial ELISA. Les résultats obtenus ne montrent aucun échantillon positif. Ces résultats préliminaires sont considérés comme une bonne première indication, étant donné que X. fastidiosa n'a pas été détectée au Maroc. Cependant, des enquêtes continues à grande échelle sont nécessaires pour empêcher son entrée dans le pays.
Mots-clés: Maroc, Xylella fastidiosa, enquête, Olivier, agrumes, vigne, ELISA.
Xylella fastidiosa, a gram negative, devastating plant pathogenic bacterium and the causal agent of a number of severe diseases, among which olive quick decline syndrome, Pierce’s disease of grapevine, leaf scorch of almond, oleander and coffee, citrus variegated chlorosis, and other disorders of perennial crops and landscape plants (Hopkins and Purcell, 2002; Janse and Obradovic, 2010; Purcell, 2013;Saponari et al., 2013). Many wild plants may carry the pathogen with, but more often without showing symptoms, such as grasses, sedges and trees (Janse and Obradovic, 2010). Four subspecies of the bacterium have been discriminated so far: ssp. fastidiosa, ssp. pauca, ssp. multiplex and ssp. Sandyi (Schaad et al., 2004; Schuenzel et al., 2005). Randal et al., (2009) proposed a fifth one (ssp. tashke), that can be differentiated by DNA: DNA hybridization (Schaad et al., 2004) and multi-locus sequence typing (Scally et al., 2005). X. fastidiosa causes various diseases, on more than 300 plant hosts, inducing various symptoms: marginal leaf scorching, wilting of foliage and withering of branches, dieback and stunting with eventual plant death in susceptible host plants (Djelouah et al., 2014). Xylela fastidiosa is transmitted by several species of sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and spittlebugs or froghoppers (Hemiptera: Cercopoidea), which are xylem-fluid feeders (Redak et al., 2004). There is also evidence that cicadas (Hemiptera: Cicadoidea), another group of xylem-fluid feeders, transmit X. fastidiosa in grape (Krell et al., 2007).
X. fastidiosa is a slow-growing (fastidious) bacterium that does not grow on many common culture media, but some good selective media are available (Schaad et al., 2001). In addition to symptom observation and isolation and culturing, detection and identification of the bacterium relies on several laboratory assays. Serological tests include enzyme linked immunosorbent assay (ELISA) (Sherald and Lei, 1991), dot immunobinding assay (DIBA), western blotting (Lee et al., 1992; Chang et al., 1993), immunofluorescence (Carbajal et al., 2004) and direct tissue blot immunoassay (Djelouah et al., 2014). Polymerase chain reaction (PCR)-based assays (Minsavage et al., 1994; Rodriguez et al., 2003; Huang, 2009), as well as real-time and loop-mediated isothermal amplification (LAMP) PCR (Oliveira et al., 2002; Francis et al., 2006; Harper et al., 2010; Li et al., 2013; Guan et al., 2013), have been used to detect the bacterium in grapevine, citrus, almond and other hosts. Although culturing remains the fundamental procedure to unequivocally confirm the presence of the bacterium, serological and molecular techniques are more suitable methods for screening a large number of samples (Loconsole et al., 2014).
In October 2013, an outbreak of X. fastidiosa was found in Apulia (south-eastern Italy) in olive trees affected by a disease denoted «Olive Quick Decline Syndrome» (Saponari et al., 2013). In July 2015, the bacteria X. fastidiosa was detected in the island of Corsica (France) affecting ornamental plants of Polygala myrtifolia (EPPO, 2015a). Later, in October 2015, it was detected for the first time in Alpes-Maritimes department (Southern France) (EPPO, 2015b). In July 2016, the bacterium was reported for the first time in Germany on a single potted plant of Nerium oleander in a small nursery producing young vegetable and ornamental plants in Saxony (EPPO, 2016a). In November 2016, the presence of X. fastidiosa was confirmed in Islas Baleares. The bacterium was detected in sweet cherry (Prunus avium) in a garden centre in Porto Cristo, municipality of Manacor, on the island of Mallorca (EPPO, 2016b). More recently, in May 2017, X. fastidiosa was detected in Mallorca, Ibiza and Menorca on 12 plant species including citrus and grapevine (EPPO, 2017).
Following these confirmed findings of the bacterium in the European Union, it was decided to conduct a preliminary survey on the presence of the bacterium in olive, citrus and grapevine groves in Morocco.
MATERIAL AND METHODS
Considering the potential risk presented by X. fastidiosa on several crops in Morocco, a preliminary survey was conducted across the country, covering olive, citrus and grapevine growing areas. Field surveys were conducted from May to September 2015 in six different regions. A total of 43 commercial groves were visited and their trees inspected for the presence of typical symptoms of X. fastidiosa. In a few trees, severe symptoms which could be associated to the bacterium were observed. A total of 900 samples of different crops from different regions were randomly collected: 220 olive trees (cv. Picholine Marocaine) from two regions (Azilal and Meknès), 410 citrus trees belonging to 7 different cultivars collected in 4 regions (Gharb, Haouz, Loukkos and Souss) and 270 grapevine plants belonging to 6 different cultivars (4 table and 2 wine grape cultivars) from 3 regions (Gharb, Haouz and Meknès) (Table 1). A detailed map was drawn up for each surveyed site, where information about position, cultivar, age, origin and location of each sampled tree were recorded. Any suspicious symptom and information potentially helpful for further sampling and investigation were recorded, and representative photos were also taken. Collected samples were stored in closed plastic bags in a cooling box during the delivery to the laboratory for analysis with ELISA and PCR diagnostic methods.
Double antibody sandwich-ELISA
All the collected samples from olive, citrus and grapevine trees were tested by ELISA, using specific polyclonal antibodies to X. fastidiosa (Loewe Biochemica, Germany), according to the manufacturer’s instructions. In the presence of PBS-buffer, extracts were obtained from leaf petioles and midveins, using mortars and pestles. Absorbance was measured after 30, 60, 120 and 180 minutes (min) with a microplate reader (ELX800, BioTek) at 405 nm.
Polymerase chain reaction
A total of 10 samples per each tree crop (olive, citrus and grapevine) were randomly selected and tested by PCR for the detection of X. fastidiosa. Total nucleic acids were extracted from leaf petioles and midveins using a CTAB-based extraction buffer (Rodrigues et al., 2003; Djelouah et al., 2014). For PCR, the RST31/RST33 set of primers targeting the 16S rDNA gene was used (Minsavage et al., 1994). Reactions were conducted in a final volume of 25 μL, using 5 μL of 5× GoTaq polymerase (Promega), 250 nM each of forward and reverse primer, and 1 μL of total DNA template (50 ng.μL-1). Thermocycling conditions were as follows: 94°C for 3 min, 35 cycles of 94°C for 30 seconds (s), 50-55°C for 30-45 sec and 72°C for 30 sec, and a final extension of 5 min at 72°C (Djelouah et al., 2014). PCR products were analyzed by electrophoresis in 1% Tris-Acetate-EDTA (TAE)-agarose gels and DNA bands visualized by staining the gel in ethidium bromide.
RESULTS AND DISCUSSION
All the collected samples were tested for the presence of X. fastidiosa by using an ELISA commercial kit (Loewe Biochemica, GmbH). The obtained results did not show any positive sample. The ELISA test worked properly: the positive control of the kit reacted positively, whereas no color change was observed with the negative control of the kit. Loconsole et al. (2014) performed several laboratory tests, in which the reactivity of different commercially available ELISA kits was compared, and showed that a kit from Loewe (Biochemica GmbH, Germany) detected a higher number of known positive samples with reactions occurring within 2 hours, following manufacturer’s instructions and using the controls supplied with the kit. Thus, this kit was used for this preliminary survey on the presence of X. fastidiosa in Morocco.
Moreover, no amplified DNA was obtained from any of the tested samples using PCR, confirming the absence of the bacterium in these samples. The used primers (RST31/33) are widely accepted for the detection of the bacterium in quarantine programs (EPPO, 2004), as well as primers targeting the 16S rDNA genomic region (Firrao and Bazzi, 1994; Rodriguez et al., 2003), which are more suitable for accurate detection of a wider number of genetically diverse strains of X. fastidiosa (Harper et al., 2010).
These obtained preliminary results are taken as an encouraging indication, considering that X. fastidiosa was not found in Morocco, at least in the surveyed tree crops. However, frequent extensive surveys in different regions and on different potential host plants are needed to prevent its entrance into the country. It is noted that the presence of leaf scorch symptoms which were frequently observed during the survey may have multiple origins, abiotic or biotic (e.g. drought, salty winds, nutrient deficiency/toxicity, frost damage, fungal pathogens…). While the hazard presented by X. fastidiosa in other hosts (oak, plane, maple and others) still has to be evaluated, inspection services should be aware that these hosts also present a certain risk.
X. fastidiosa is a regulated quarantine pest whose introduction and spread in Morocco is banned. Following the recent confirmed finding of X. fastidiosa in the European Union (Italy and France); this bacterium is becoming a serious threat to the Moroccan agricultural sector. In this context, the National Food Safety Office (ONSSA) adopted measures to prevent the introduction of X. fastidiosa into the national territory by deciding to suspend importation of host plant species to Pierce’s disease from Italy. Results obtained in this study clearly indicated that X. fastidiosa was not found in Morocco. However, frequent extensive surveys in different regions are needed to prevent its entrance into the country.
For any effective control and containment strategy of X. fastidiosa, the correct identification of the pathogen by quick, sensitive and reliable laboratory tests is crucial. In routine monitoring and surveying, culturing X. fastidiosa is time consuming (with some subspecies it takes up to three weeks for the colonies to grow) and is labor-intensive, particularly when a large number of samples are processed. Therefore, the use of serological and molecular diagnostic tools is of utmost importance for the detection of the bacterium in plant tissues and in potential insect vectors.
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