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1 Development of Direct Tissue Blot Immunoassay (DTBIA) as an efficient tool for the on- site detection of Spiroplasma citri, causal agent of Citrus stubborn disease Mounira Inas Drais 1, 2*, Leonardo Varvaro 2, Khaled Djelouah 1 1.Mediterranean Agronomic Institute of Bari (IAMB) Via Ceglie 9, Valenzano, BA - Italy. 2. Università degli studi della Tuscia [Viterbo]Via S. Camillo de Lellis, Viterbo VT - Italy 1

2 INTRODUCTION Spiroplasma citri is the causal agent of Citrus Stubborn disease (CSD), affecting growth, yield and fruit quality of the citrus trees. The stubborn-affected trees produce small fruit that are lop-sided or acornshaped. Stunted trees This pathogen is a phloem-inhabiting bacterium, belonging to the class of Mollicutes, its Identification and detection is challenging due to its fluctuating titer and sporadic distribution in infected citrus trees. In order to allow the mass detection of S. citri, Direct Tissue Blot ImmunoAssay (DTBIA) technique, a process of transferring protein antigens from a freshly cut plant tissue to nitrocellulose membrane and Small, mottled leaves detected with enzyme-labelled antigen-specific antibodies, has been developed Lopsided fruits 2

3 Materials and Methods Based on similar approach applied recently for the mass detection of Xylella fastidiosa in olive trees (Djelouah et al., 2014), Several parameters were compared; thus, including the 0.20 and 0.45μm pore size of nitrocellulose membranes; the bovine serum albumin (BSA), fat milk, gelatin and polivynyl alcohol solutions for the saturation of protein-binding sites. The blotted membranes were exposed for 2h to alkaline phosphataseconjugated polyclonal or monoclonal antibodies specific to S.citri at different dilution The developed protocol was evaluated and validated during a wide survey in Algerian Citrus orchards In this context, 112 trees were sampled, printed on the nitrocellulose membranes and tested. The results obtained from the DTBIA were compared with PCR assays using SC1 primer pairs (Saillard, not published) targeting the Spiralin gene, the most abundant membrane proteins in S. citri. 5. Addition of S.citri specific antibodies 6. Membrane staining SigmaFastTM BCIP- NBT solution 1. citrus twigs 2.Membrane printing 3. Blocking step 4. Membrane washing 7. Membrane observation 3

4 (+) control 4 Results and discussion Following all the trials, the appropriate DTBIA protocol was developed evidencing that the 0.45µm pore size nitrocellulose membrane, the twigs, the polyvinyl alcool blocking solution for 2 minutes and the alkaline phosphatase conjugated polyclonal antibodies (Sediag, France) specific to S.citri performed better than the other reagents tested. During the validation of the protocol in Algeria, two samples out of 112 tested trees reacted positively with the DTBIA assay, showing purple-stained area in the S. citri -infected imprints on blotted membranes. All the obtained results through the developed protocol were confirmed by PCR assays targeting the spiralin, gene evidencing bands of the expected size 336 bp. M M Isolate 34 Isolate G Amplicons produced using spiralin-f/r primers with S.citri DNA extracted from plant tissue from the field plants N. Membrane Explant Blocking solution Antisera 0.45 µm Pore size 0.20 µm Pore size Petiole Twig Gelatin Fat milk BSA PVA (2 min) 1/50 S.c kit /250 Mab Blue staining area around the imprint

5 Conclusions This study indicate that Direct Tissue Blot Immunosorbent Assay can be a reliable technique for detecting S. citri at large scale in Citrus trees and doesn t require sophisticated equipment or highly skilled operators; thus, reducing time processing, costs, number of daily analyzed samples per working unit compared to other detection techniques. The developed (DTBIA) protocol, represents an important contribution to further improvement of the serological detection at large scale of S.citri. It can prevent the propagation of the disease in the area and contributes in reducing its incidence This investigation consented to detect for the first time two S. citri infected trees among the tested samples by the newly developed DTBIA protocol. The obtained results and the efficiency of the test were confirmed by molecular assays. Interestingly, the molecular characterization of the newly detected Algerian S. citri isolate, evidenced 99% nucleotide homology of the spiralin gene with the Iranian fasa I, isolated from a leafhopper vector. In addition, the Algerian isolate also reacted positively with the primer pairs, targeting the TraG gene, which is essential for insect transmission and predicts a natural diffusion of the pathogen in the case of the presence of insect vectors. 5

6 Thank You Get in Touch Mounira Inas Drais Via Ceglie 9, Valenzano, BARI (+39)