First report of Diaphorina citri and 'Candidatus Liberibacter asiaticus' associated with the Huanglongbing disease on Citrus spp. in French Guiana

Abstract

Huanglongbing (HLB) is a disease on citrus associated with three unculturable phloem-limited gram-negative bacterial species transmitted through grafting or by two psyllids: Diaphorina citri (ACP) and Trioza erytreae (Bové 2006). HLB is considered the worst constraint on citrus worldwide, where major active epidemics involve 'Candidatus Liberibacter asiaticus' (CLas) and ACP. In the French West Indies, ACP was detected in Guadeloupe (1998) and Martinique (2012), followed by CLas detection in 2012 and 2013, respectively. In French Guiana, under the direction of the French Ministry of Agriculture regional services (DEAAF), FREDON Guyane has been monitoring HLB and its psyllid vectors since 2016. The first detection of D. citri occurred on 22 July 2021 in the Madeleine district of Cayenne within the Murraya paniculata sentinel network and on various Citrus spp. Species identification was confirmed morphologically on 2 August 2021 by the French National Reference Laboratory for Insects (ANSES, Plant Health Laboratory, Entomology and Botany Unit), but no Liberibacter sp. test was done. At this stage, citrus trees did not show clear HLB symptoms (blotchy mottle patterns and yellow islands on leaves). Suspicious plant samples of M. paniculata and Citrus spp. were regularly transmitted to the National Reference Laboratory for bacteria on citrus and tropical plants (ANSES, Plant Health Laboratory, Tropical Pests and Diseases Unit, Reunion Island) for analysis, using the official French method ANSES/LSV/MA 068 based on the Li et al. (2006) real-time PCR. DNA extractions (DNeasy Plant Mini Kit, Qiagen) were performed on leaf midribs, along with a negative control (Citrus paradisi 'Star Rubis'). Amplifications were performed on two wells, giving an average Cq value. In September 2021, typical amplification curves were obtained on one sample of M. paniculata (Cq = 34.36; SD = 0.314). The same scenario occurred in March 2022 on a sample from an old citrus tree (Cq = 31.27; SD = 0.241) collected in a private courtyard in Sinnamary and in July 2023 on a citrus tree (Cq = 31.83; SD = 0.074) from an orchard in Kourou. This last sample's positive status was further confirmed through the real-time PCR protocols from Morgan et al. (2012) (Cq = 33.25; SD = 0.027), Zheng et al. (2016) (Cq = 33.05; SD = 0.237), and de Chaves et al. (2023) (Cq = 34.19; SD = 0.850). Following the impossibility to characterize the species of 'Ca. Liberibacter spp.' detected (40 out of 352 samples since 2022), hampered by its in planta low titer, a field survey was organized in November 2024. CLas was detected on one sample of citrus tree from a private courtyard in Kourou (Cq = 26.31; SD = 0.288), confirmed by the method of de Chaves et al. (2023) (Cq = 27.507; SD = 0.07). Higher in planta titer allowed for the CLas species characterization through conventional PCR (CLas observed band of 703 bp) (Hocquellet et al. 1999; Teixeira et al. 2005). Sanger sequencing (Genewiz U.K., 678 bp) of this amplicon (Genbank no. PV018783) showed 100% identity (BLASTn) with the CLas strain CP131152. To our knowledge, this is the first report of the presence of HLB in French Guiana, involving the CLas species and the vector D. citri. All year long, high heat could explain in planta HLB low titer. The introduction events remain unclear. This report highlights the importance of plant certification, psyllid population control, and surveillance of territories close to French Guiana