First Report of "Candidatus Liberibacter asiaticus" and Its Vector Diaphorina citri in Murraya paniculata in Santiago del Estero Province, Argentina

Abstract

Candidatus Liberibacter asiaticus is one of the bacterial species associated with Huanglongbing (HLB), a severe citrus disease that causes major losses worldwide. It is transmitted by the psyllid Diaphorina citri. HLB can also affect Murraya paniculata, an ornamental plant common in sidewalks and home yards. In Argentina, the first detection of HLB was on citrus plants in Misiones province in 2012 and has since been detected in other provinces of northeastern Argentina (Badaracco et al. 2017). HLB also represents a menace to the other important citrus growing region of Argentina, the Northwest (NWA), which produces 56% of the citrus of the country in the provinces of Tucumán (1,400,000 t per year, mainly lemon), Salta (323,000 t), Jujuy (257,000 t), Catamarca (9,000 t), and Santiago del Estero (only in backyard and urban areas). In 2005, the Estación Experimental Agroindustrial Obispo Colombres (EEAOC) started monitoring and analyzing samples from commercial citrus fields, urban areas and nurseries from NWA. Since 2009, the EEAOC participates in the National HLB Prevention Program, coordinated by the federal phytosanitary agency Servicio Nacional de Sanidad y Calidad Agroalimentaria (Senasa). To date, 48 locations in NWA have been visually inspected and leaf samples displaying HLB-type symptoms and psyllid samples were collected. A total of 12,462 samples were analyzed by real-time PCR (Li et al. 2006); 51% corresponded to citrus and 4% to M. paniculata from the five provinces of NWA, and 45% to D. citri from Salta and Jujuy, all with negative results. In July 2017, Senasa personnel detected in the urban area of La Banda city in Santiago del Estero, a M. paniculata tree with chlorotic branches that presented only a few leaves showing blotchy mottle and yellow midrib. During this inspection, the psyllid was also detected for the first time in Santiago del Estero province. Immediately, insect and leaf samples were submitted to the EEAOC laboratory for analysis. Total DNA was extracted from 500 mg of petiole and midrib tissue from leaves and from five adult insects using a modified CTAB method and analyzed with real-time PCR. The plant sample analyzed produced a threshold cycle (Ct) value of 26.31 (positive range). The presence of Ca. L. asiaticus in M. paniculata was confirmed by Nested real-time PCR (Lin et al. 2010) and conventional PCR, as well as by the sequence of its partial 16S rRNA and ribosomal protein genes. Sequences were PCR-amplified using specific primers OI1 and OI2c for the Asian form (Jagoueix et al. 1996). Blast analysis of the sequence obtained showed a 99% identity with the sequence of the organism Ca. L. asiaticus (GenBank Accession No. CP019958.1). The positive plant was eradicated and additional actions, included in the Program to prevent the spread of HLB in NWA, were taken. Furthermore, actions are being implemented to avoid entry of the psyllid from Salta, Jujuy and Santiago del Estero into Tucumán and Catamarca, where the insect has not yet been detected. To date, there have been no additional positive detections in plant or insect samples from NWA. Symptoms observed in negative samples could be attributed to other causes such as nutritional deficiencies or other pests. To our knowledge, this is the first report of Ca. L. asiaticus in Santiago del Estero province, in an urban area located approximately 150 km from the closest citrus growing areas in Tucumán province, the world main producer and processor of lemon and one of the main exporters of lemon fresh fruits.