This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Deliverable Title Report on the phytoplasma detection and identification in Haplaxius crudus nymphs and other potential cixiid lethal yellowing (LY) vector identification. Deliverable Number Work Package D2.2 WP2 Lead Beneficiary Deliverable Author(S) COLPO Carlos Fredy Ortiz Beneficiaries Deliverable Co-Author (S) COLPO Carlos Fredy Ortiz UCHIL Nicola Fiore UNIBO Assunta Bertaccini Planned Delivery Date Actual Delivery Date 31/10/2019 28/10/2019 R Document, report (excluding periodic and final X reports) Type of deliverable DEC Websites, patents filing, press & media actions, videos E Ethycs PU Public X Dissemination Level CO Confidential, only for members of the consortium 1 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Table of contents List of figures 5 List of tables 6 List of acronyms and abbreviations 7 Executive summary 8 1. Introduction 9 2. Reproduction in captivity of Haplaxius crudus 10 2.1. Material and methods 10 Preliminary assays for reproduction in captivity of H. crudus 11 Assays for reproduction in captivity of H. crudus 12 2.2. Results and discussion 13 Preliminary assays for reproduction in captivity of H. crudus 13 Assays for reproduction in captivity of H. crudus 14 2.3. Conclusions 14 3. Detection and identification of phytoplasmas in nymphs of cixiids 15 3.1. Material and methods 15 DNA extraction 15 Nested Polymerase Chain Reaction (nested PCR) 15 PCR products purification and sequencing 16 3.2. Results and discussion 16 3.3 Conclusions 18 4. Molecular identification of H. crudus and H. caldwelli nymphs 18 4.1. Material and methods 18 Insect collection and DNA extraction 18 PCR amplification of the cytochrome C oxidase subunit I region in the mtDNA 18 (mitochondrial DNA) (mitochondrial DNA) PCR products purification and sequencing 18 Sequencing and restriction fragment length polymorphism (RFLP) analyses 20 of amplified DNA obtained by the primers C1-J-2183 and UEA8 4.2. Results and discussion 20 2 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 PCR amplification of the cytochrome C oxidase subunit I region in the mtDNA 20 (mitochondrial DNA) Sequencing and restriction fragment length polymorphism (RFLP) analyses 20 of amplified DNA obtained by the primers C1-J-2183 and UEA8 4.3. Conclusions 21 5. References 22 3 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 List of figures Figure 1: Sampling during 2019 in the locality of Pailebot 11 Figure 2: Preliminary assays for reproduction in captivity of H. crudus 12 Figure 3: Assays for reproduction in captivity of H. crudus with pot-cages and P. 13 laxum grass Figure 4: Cixiid nymph in the 4th instar, associated to the rhizosphere of Eustachys 17 petreae Figure 5: Phylogenetic tree derived by the sequence analysis of the primers 17 503F/LY16Sr Figure 6: Phylogenetic tree derived by the sequence analysis of the primers C1-J- 21 2183/UEA8 4 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 List of tables Table 1: Alternative host families and species found in the coconut localities 10 visited. Soil parameters measured Table 2: Number of adult individuals of H. crudus emerged in the pot-cages used 13 in the preliminary assay for reproduction in captivity Table 3: Adult insects of H. crudus born in captivity 14 Table 4: Sequences of primers used for the PCR amplification of a fragment of the 19 COI gene of H. crudus and H. caldwelli Table 5: Virtual RFLP analyses of DNA from H. crudus and H. caldwelli with AluI, 21 SspI, ClaI, RsaI and TaqI on C1-J-2183/UEA8 amplicons 5 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 List of acronyms and abbreviations 16S rDNA - 16S ribosomal deoxyribonucleic acid 16S rRNA - 16S ribosomal ribonucleic acid 18S rDNA - 18S ribosomal deoxyribonucleic acid μM - micromolar bp - base pair BSA - bovine serum albumin CICY - Centro de Investigación Científica de Yucatán C. nucifera - Cocos nucifera COLPO - Colegio de Postgraduados CTAB - cetyl trimethylammonium bromide DNA - deoxyribonucleic acid dNTPs - deoxynucleotides EDTA - ethylenediaminetetraacetic acid E. petreae - Eustachys petreae H2O - water H. caldwelli - Haplaxius caldwelli H. crudus - Haplaxius crudus H. skarphion - Haplaxius skarphion LY - lethal yellowing M - molar ml - milliliter mtCOI DNA - mitochondrial cytochrome C oxidase I deoxyribonucleic acid mtDNA - mitochondrial deoxyribonucleic acid NaCl - sodium chloride NJ - Neighbor Joining PCR - polymerase chain reaction P. laxum - Panicum laxum 6 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 PVP-40 - polyvinylpyrrolidone 40 RFLP - restriction fragment length polymorphism rDNA - ribosomal deoxyribonucleic acid RNA - ribonucleic acid rpm - revolution per minute Tris - tris(hydroxymethyl) aminomethane Tris-HCl - tris(hydroxymethyl) aminomethane chloridrate U - unit UCHIL - Universidad de Chile UNIBO - Alma Mater Studiorum - University of Bologna UV - ultraviolet 7 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Executive summary Nowadays the only species that has been confirmed as vector of the phytoplasmas 16SrIV- A and -D in coconut palms in Mexico, is Haplaxius crudus. However, other species of Haplaxius, such as H. skarphion, H. caldwelli, among others, captured in coconut palm orchards in Mexico, have been found positive for the presence of these phytoplasmas, but it has not yet been possible to confirm their role as insect vectors. To generate information in this regard, the focus of the present work was to develop the rearing technique for Haplaxius species, detect phytoplasmas in nymphs of cixiids and identify molecularly the Haplaxius species when the insects are still in the nymph stage. For the first time, the rearing of H. crudus in captivity was obtained and the first report about the detection of the phytoplasma 16SrIV-A in nymphs of the Cixiidae is presented here. Moreover, by using PCR and RFLP analyses, it was possible identify rapidly and specifically H. crudus and H. caldwelli in their nymph stages. 8 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 1. Introduction The genus Haplaxius Fowler has a distribution in the New World (Ferreira et al., 2010), which includes 34 species from North America and Northern Mexico; in addition, there are 31 species in the Neotropic (Bartlett et al., 2011). However, currently the only species that has been confirmed as vector of the phytoplasmas belonging to the ribosomal subgroups 16SrIV- A and -D, is Haplaxius (Myndus) crudus (Narvaez et al., 2018), however other species are suspected to play this role in areas with presence of lethal palms decay (Dollet et al., 2010; Halbert et al., 2014). In some coconut plantations, it is reasonable to think that other Haplaxius species, such as H. skarphion, H. caldwelli, among the others, may be participating in the transmission of phytoplasmas due to their phylogenetic proximity to the main vector (Bertin et al., 2010a), also having overlapping ecological niches in coconut plantations (Ramos et al., 2018). Certainly, H. skarphion and H. caldwelli have already been found to be positive for the phytoplasma of group 16SrIV (Ramos, 2018), but it is necessary to identify the ribosomal subgroup and which are the vectors involved in the transmission. Moreover, in some exploratory surveys in these plantations, phytoplasma-positive cixiids nymphs have been found which have not been identified in either genus or species. Bertin et al. (2010b), mention that the modern identification of cixiids is based on morphological characteristics and is restricted to a small number of specialist entomologists with extensive experience about the families belonging to the Cixiidae genus. Even for experts, the morphological distinction of closely related species is difficult. The morphological identification of H. crudus, H. skarphion and H. caldwelli is possible for adults with dichotomous clues such as those of Kramer (1983), but not for their nymph stages. These nymph stages of cixiids species are difficult to observe in the field, because they feed on roots of their host plants (Sforza et al., 1999) and have a low mobility. The molecular identification of insects provides a fast and reliable key tool that can contribute to expand the knowledge about the identifications of cixiids. In addition, these markers can also be applied successfully to nymphs, whose morphological characteristic is limited to a few species of the Cixiidae family. Ceotto et al. (2008) demonstrated that the phylogenetic trees obtained using the mitochondrial cytochrome C oxidase I (mtCOI DNA) and the 18S rDNA nucleotide sequences were congruent, and that the trees were consistent with the morphological classification. Nuclear and mitochondrial genomes have different modes of inheritance, and the effectiveness of nuclear