Disease Note Diseases Caused by Bacteria and Phytoplasmas First Report of ‘Candidatus Liberibacter africanus’ Associated with Citrus Greening Disease in Nigeria Inusa Jacob Ajene, 1,2 Fathiya Khamis, 1,† Samira Mohammed, 1 Ade- dapo Olutola Adediji, 3 Gabriel Ifo Atiri, 3 Shakiru Adewale Kazeem, 4 and Sunday Ekesi 1 1 International Center of Insect Physiology and Ecology, Nairobi, Kenya 2 Department of Crop Protection, Ahmadu Bello University, Zaria, Nigeria 3 Department of Crop Protection and Environmental Biology, Faculty of Agriculture, University of Ibadan, Ibadan, Nigeria 4 Nigeria Agricultural Quarantine Service, Post-Entry Quarantine, Surveil- lance and Diagnostic Station, Ibadan, Nigeria Funding: The authors gratefully acknowledge the support for this research by the following organizations and agencies: German Ministry for Economic Cooperation and Development (BMZ) through GIZ to the project ‘Strengthening Citrus Production Systems through the Introduction of Integrated Pest Management (IPM) Measures for Pests and Diseases in Kenya and Tanzania (SCIPM)’ (Deutsche Gesellschaft f¨ ur Internationale Zusammenarbeit, Project no.:14.1432.5‐001.00/Contract no.: SCIPM), UK Aid from the U.K. Government, Swedish International Development Cooperation Agency (Sida), Swiss Agency for Development and Co- operation (SDC), and Kenyan Government. Plant Dis. 0:1, 2020; published online as https://doi.org/10.1094/PDIS-11-19-2380-PDN. Accepted for publication 17 December 2019. Citrus greening is a serious disease of Citrus sp. and has been associated with the collapse of several citrus industries in Asia and America (Bov´ e 2006). The disease is associated with the presence of a phloem-limited bacterium, of the ‘Candidatus Liberibacter’ genus. ‘Candidatus Liberi- bacter 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 Bov´ e 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 align- ments of sequences were assembled using MAFFT version 7, whereas phy- logenetic 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 in- cluded 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. References: Altschul, S. F., et al. 1990. J. Mol. Biol. 215:403. Aubert, B., et al. 1988. Page 231 in: Proc. 10th Conf. IOCV. IOCV, Riverside, CA. Bov´ e, J. M. 2006. J. Plant Pathol. 88:7. Garnier, M., and Bov´ e, J. M. 1996. Page 388 in: Proc. 13th Conf. IOCV. IOCV, Riverside, CA. Hall, T. A. 1999. Nucleic Acids Symp. Ser. 41:95. Hocquellet, A., et al. 1999. Mol. Cell. Probes 13:373. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Roberts, R., et al. 2015. Int. J. Syst. Evol. Microbiol. 65:723. Tamura, K., and Nei, M. 1993. Mol. Biol. Evol. 10:512. Varma, A., and Atiri, G.I. 1993. Page 462 in: Proc. 12th Conf. IOCV. IOCV, Riverside, CA. The author(s) declare no conflict of interest. e-Xtra Keywords: prokaryotes Q: 1 , fruit, tree fruits, epidemiology, disease development and spread Plant Disease / nnn 2020 1