First Report of Field Population of Trioza erytreae Carrying the Huanglongbing-Associated Pathogen, ‘Candidatus Liberibacter asiaticus’, in Ethiopia

African citrus greening is a destructive disease of citrus that has been reported in South Africa since the 1920s. The disease is associated with ‘Candidatus Liberibacter africanus’ (Laf) and is transmitted by Trioza erytreae (Cook et al. 2014; McClean 1974). The related bacteria ‘Candidatus Liberibacter asiaticus’ (Las), which is associated with the much more severe Huanglongbing disease and is transmitted by Diaphorina citri, was recently reported in Ethiopia (Saponari et al. 2010). Experimentally, T. erytreae has been proven to transmit Las (Massonie et al. 1976), but natural occurrence of Las in field populations of this psyllid has not been reported. A survey was conducted for the citrus greening vector T. erytreae in the Amhara region of Ethiopia in November 2017. T. erytreae adults were identified as per the descriptions of OEPP/EPPO (2005). Sampling sites included large- and small-scale citrus orchards (in both highland and lowland areas) and citrus trees grown in backyard gardens. T. erytreae were found and collected from sweet orange, lemon, and tangerine trees in backyard gardens and a small-scale commercial orchard. Voucher specimens were deposited at the International Centre of Insect Physiology and Ecology repository. Adult T. erytreae collected were screened for the presence of various strains of Liberibacter bacteria. The samples were surface sterilized using 3% sodium hypochlorite and rinsed with distilled water. Genomic DNA was extracted from individual insects using the Isolate II Genomic DNA Kit (Bioline, United Kingdom), following the manufacturer’s instructions. Leaf samples from citrus trees on which the psyllids were collected were also tested, and plant total DNA was extracted from individual petioles. DNA quality and quantity checks were performed using a Nanodrop 2000/2000c Spectrophotometer (Thermo Fischer Scientific, U.S.A.). Conventional polymerase chain reaction assays were done to amplify the 50s ribosomal protein L10 gene region (rplA-rplJ) of Las and Laf using primers A2 and J5 (Hocquellet et al. 1999), generating the expected 650-bp product from 70 insect samples and the plant samples from each site. Amplicons were purified and bidirectionally sequenced. The rplA-rplJ sequences were aligned with reference Las sequences retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) using the MUSCLE tool in MEGA X (Kumar et al. 2018). Sequences from this study (GenBank accession no. MH809485) had a 100% base-pair match with Las (GenBank accession no. MG418842.1). A maximum-likelihood phylogenetic tree constructed for the ribosomal protein gene sequences revealed that the Liberibacter obtained from T. erytreae clustered with Las and clustered separately from Laf and ‘Candidatus Liberibacter solanacearum’ species. To the best of our knowledge, this is the first report of field populations of T. erytreae carrying Las in Ethiopia. Furthermore, the detection of Las (initially reported to be solely transmitted by D. citri) in sweet orange, lemon, and tangerine trees in an area with the presence of T. erytreae highlights the potential of this psyllid to transmit Las. Therefore, this study provides new insight into a possible alternate route of proliferation of Las in the absence of D. citri, and it raises the need to determine the transmission efficiency, vector competency, and the vector–pathogen relationships in field populations of the psyllid (T. erytreae).