First report of field population of Trioza erytreae carrying the Huanglongbing associated pathogen, Candidatus Liberibacter asiaticus, in Ethiopia

Abstract

Citrus greening is a serious disease of Citrus sp. and has been associated with the collapse of several citrus industries in Asia and America (Bové 2006). The disease is associated with the presence of a phloem-limited bacterium, of the ‘Candidatus Liberibacter’ genus. ‘Candidatus Liberibacter africanus’, the principal agent of African citrus greening disease (ACG) has been reported in South, East, and Central Africa (Aubert et al. 1988; Garnier and Bové 1996), but its presence in Nigeria is unverified. This study, therefore, sought to identify the status of ACG in Nigeria and its associated ‘Ca. Liberibacter’ species. A survey was carried out for greening in citrus orchards in April 2018 in Benue and Nasarawa states in Nigeria. Twenty sites were surveyed, and symptomatic trees were observed in 11 sites. Five symptomatic trees per site were randomly selected, and leaf samples were collected from sweet orange (Citrus sinensis) plants exhibiting typical citrus greening symptoms, including mild to severe yellowing on the shoots and mottling and chlorosis on the leaves. Plant total DNA was extracted from individual petioles using the Bioline Isolate II Plant DNA Kit (Bioline, U.K.). DNA quality and quantity checks were performed using a Nanodrop 2000/2000c Spectrophotometer (Thermo Fischer Scientific, U.S.A.). Liberibacter presence in DNA extracts was detected using quantitative polymerase chain reaction (PCR) with primer pairs LibUF/HLBR (Roberts et al. 2015) targeting the 16S rRNA and PCR to amplify the 50S ribosomal protein L10 gene region (rplA-rplJ) of ‘Ca. L. africanus’ using primers A2 and J5 (Hocquellet et al. 1999). The presence of ‘Ca. Liberibacter’ was assessed in 11 of the 20 sites surveyed, and five leaf samples per tree were tested from each site. All tested leaf samples from four sites in Benue state were positive, whereas there were no positive detections from samples from Nasarawa state. The amplification of the rplJ gene region generated the expected 650-bp product from all plant samples obtained from the four sites. One amplicon per site was purified and bidirectionally sequenced. Sequences were assembled using BioEdit version 7.2.5 (Hall 1999) and identities determined using Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). Multiple alignments of sequences were assembled using MAFFT version 7, whereas phylogenetic analyses were done using MEGA X (Kumar et al. 2018). The maximum likelihood method was applied as the tree-building algorithm to visualize patterns of divergence among the Liberibacter sequences from the positive samples (Tamura and Nei 1993). ‘Ca. L. solanacearum’ was included as an outgroup. Sequences from this study (GenBank accession nos. MN205932 to MN205935) had a 100% base-pair identity with ‘Ca. L. africanus’ (GenBank accession no. MK542518). Phylogenetic analysis of the rplJ gene region indicated the clustering of the Nigerian sequences within a distinct ‘Ca. L. africanus’ clade. This study provided the first verified report on ACG presence in Nigeria. A previous study by Varma and Atiri (1993) listed greening as one of the diseases affecting citrus in Nigeria, but the identity of the associated pathogen was not determined. This detection will guide in the development of an integrated pest management approach to ACG disease management in the country.