Eur J Plant Pathol https://doi.org/10.1007/s10658-021-02234-7 Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean Maritza Luis-Pantoja & Camilo Paredes-Tomás & Youri Uneau & Wayne Myrie & Raphael Morillon & Eleonora Satta & Nicoletta Contaldo & Francesco Pacini & Assunta Bertaccini Accepted: 8 February 2021 # The Author(s) 2021 Abstract “Huanglongbing” (HLB) is one of the most phytoplasma infection was detected. Moreover the de- devastating diseases of citrus orchards worldwide. Sam- tection of only phytoplasmas in 11 symptomatic citrus ples from 183 citrus plants of different cultivars and samples collected from Cuba and Guadeloupe without rootstock/cultivar combinations, showing HLB symp- ‘Ca.Liberibacter’ detection, confirmed that the symp- toms in three Caribbean countries (Cuba, Jamaica, and tomatology cannot be the sole criterium to discriminate Guadeloupe-France), were collected to verify the possi- between the presence of the two pathogens, and molec- ble co-infection of ‘Candidatus Phytoplasma’ and ular detection is necessary to identify single or mixed ‘Candidatus Liberibacter’ species. The 64% of the sam- infections. Diaphorina citri insects collected from Cuba ples resulted positive to the ‘Ca. L. asiaticus’ and the andGuadelouperesultedinfectedwith‘Ca.L.asiaticus’ 27% to diverse ‘Ca.Phytoplasma’-related species, confirming its active role in the dissemination of the moreover about the 14% of the samples infected with pathogen. Only one insect of the Cicadidae family, ‘Ca. Liberibacter’ were also found positive to collected in Guadeloupe, was found positive for phyto- phytoplasmas, indicating the presence of mixed infec- plasma presence. Considering that the phytoplasmas tion especially in the orchards located in Cuba. More- belonging to some ‘Candidatus species’ were detected over, in one of the samples from Jamaica mixed in the three countries in different citrus varieties, a relevant role as phytoplasma reservoir can be attribute M. Luis-Pantoja : C. Paredes-Tomás to citrus orchards. Research Institute of Tropical Fruit Crops, La Habana, Cuba Keywords Citrus greening . Phloematic bacteria . Y. Uneau Molecular detection . Mixed infection . Insect vectors ASSOFWI, Vieux-Habitants, Guadeloupe, France W. Myrie Introduction Coconut Industry Board, Kingston, Jamaica “Huanglongbing” (HLB) is one of the most devastating R. Morillon citrus diseases in the world (Bové 2006), mainly present CIRAD, Station de Roujol, Guadeloupe, Petit Bourg, France with severe outbreaks in Asian and American continents (CABI 2019). It was detected also in several areas of the Caribbean, and, in particular, Cuba at the end of 2006 E. Satta : N. Contaldo : F. Pacini : A. Bertaccini (*) Department of Agricultural and Food Sciences, Alma Mater (Luis et al. 2009), Jamaica in 2009 (Brown et al. 2011), Studiorum - University of Bologna, Bologna, Italy and Guadeloupe in 2012 (Cellier et al. 2014). Citrus e-mail: [email protected] industry is present in 140 countries worldwide covering Eur J Plant Pathol an area of 10.3 million hectares, with a production of morphological identification, tested to verify ‘Ca. about 167 million tonnes in 2016 (FAOSTAT n.d.). In Liberibacter’ and phytoplasma presence as described the Caribbean countries the impact of HLB on the citrus below. industry is so relevant that both production and export of Cuba and Guadeloupe economy are severely affected. DNA extraction Despite the lack of fulfilment of Koch’spostulates, HLB occurrence has been primarily associated with DNA extraction was performed from 1 g of leaf midribs ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’ using CTAB (Murray and Thompson 1980) and phenol- and ‘Ca. L. americanus’ (Jagoueix et al. 1994; Teixeira chloroform (Prince et al. 1993) based methods. The et al. 2005) presence in symptomatic citrus plants. How- CTAB method was also applied to extract the DNA ever, phytoplasmas belonging to diverse ribosomal from the specimens of insect vectors or potential vectors groups were also reported in several citrus species show- collected in and near citrus orchards in Cuba (extracted ing HLB-like symptoms with or without ‘Ca. in batches of 5) and Guadeloupe (extracted as single Liberibacter’ detection (Martinello-Sanches et al. specimen). 2016). Since in Cuba and in the Caribbean only ‘Ca. L. asiaticus’ has been reported associated with HLB PCR, RFLP and sequence analyses presence, the aim of the study was to confirm whether phytoplasmas are also associated with HLB-like symp- The amplifications for the CTAB extraction method toms in single or mixed infection taking into account were carried out using 1 μl of the extracted DNA diluted preliminary investigations carried out in Cuba, Jamaica 1: 30 in sterile distilled water, and 1 μl of a 20 ng/μl and Guadeloupe, (Bertaccini et al. 2019). solution of the chloroform/phenol extracted DNA. The PCR amplification for ‘Ca. Liberibacter’ species detec- tion was performed with primers OA1/OI2c and OI1/ Materials and methods OI2c (Jagoueix et al. 1996), while for the ‘Ca. Phyto- plasma’ species detection the primers R16mF2/ Plant and insect material R16mR1 (Gundersen and Lee 1996), U5/U3 (Lorenz et al. 1995), and M1/M2(=758f/1232r) (Gibb et al. A total of 103 symptomatic citrus samples were collect- 1995) were employed in direct and nested-PCR assays, ed in Cuba (Table 1), six in Jamaica, and 74 in Guade- respectively. Further nested-PCR assays following am- loupe (Table 2) during extensive surveys carried out in plification with primers P1/P7 (Deng and Hiruki 1991; 2017–2019 in diverse growing areas (Fig. 1). The citrus Schneider et al. 1995), 16S503f/LY16Sr (Harrison et al. plants surveyed in Cuba enclosed “Valencia” sweet 1999), and R16(I)F1/R1 (Lee et al. 1994)specificfor orange, grapefruit, “Eureka” lemon, Mexican lime, Per- ribosomal groups 16SrIV and 16SrI, -II and -XII respec- sian lime, and “Nova Tangelo” orange of different ages tively, were also performed. Phytoplasma positive con- (Table 1). In Guadeloupe, the majority of the tested trols from micropropagated collection (Bertaccini 2014) citrus was grafted onto diploid and tetraploid rootstocks, or from the published phytoplasma strain DNAs listed in and the age of the trees varied between 2 and 6 years, Fig. 3, were amplified separately to avoid cross contam- only in a few cases 10 or 20 year-old trees were tested ination of the samples. No positive control was used for (Table 2). In Jamaica the six samples examined were the ‘Ca.Liberibacter’ detection. Negative controls were collectedfromanindustrialorchardof“Valencia” sweet samples from asymptomatic citrus plants, devoid of orange five years old located in the area of Bog Walk nucleic acid template or containing sterile distilled water (Saint Catherine Parish) and from a garden plant of the instead of the DNA template. The ‘Ca. Liberibacter’ and ornamental variety Citrus aurantium in Montego Bay ‘Ca. Phytoplasma’ species identification was achieved (Fig. 1). Leaf samples were collected mainly based on by direct amplicons sequencing and/or RFLP analyses the presence of asymmetric leaf mottling symptoms on 16S rDNA amplicons for the phytoplasma ribosomal (Fig. 2); two additional asymptomatic samples per group/subgroup identification (Lee et al. 1998). Se- Country were also collected as negative controls. Insect quence obtained after alignment were used to verify vectors or putative vectors were collected in the sur- their clustering with reported ‘Candidatus Phytoplasma’ veyed orchards in Cuba and Guadeloupe and, after gross species (Bertaccini and Lee 2018). The evolutionary Eur J Plant Pathol Table 1 Results of the survey for the detection of ‘Ca. Liberibacter’ and ‘Ca. Phytoplasma’ in citrus growing provinces from symptomatic citrus plants in Cuba, in bold samples used also for phytoplasma isolation and results from both plant and colony detection of phytoplasmas Sample Code Location (Province) Scion variety (latin name) ‘Ca. Liberibacter’‘Ca. Phytoplasma’ M9 Jagüey Grande, Matanzas Citrus clementina Hort.exTan.xC. paradisi yes no L3 Jagüey Grande, Matanzas Citrus latifolia Tan. yes no LP1, LP2 Jagüey Grande, Matanzas Citrus latifolia Tan. yes no LP3 Jagüey Grande, Matanzas Citrus latifolia Tan. yes 16SrXII+16SrIV P4 Jagüey Grande, Matanzas Citrus paradisi Macf. yes 16SrIV PC4 Jagüey Grande, Matanzas Citrus paradisi Macf. yes no M23 Jagüey Grande, Matanzas Citrus reticulata Blanco x C. sinensis (L.) Osb. yes 16SrXII+ 16SrXV N2 Jagüey Grande, Matanzas Citrus sinensis (L.) Osb. yes 16SrXI NN Jagüey Grande, Matanzas Citrus sinensis (L.) Osb. no 16SrXII OC2 Jagüey Grande, Matanzas Citrus sinensis (L.) Osb. yes 16SrXII+16SrIV OC5 Jagüey Grande, Matanzas Citrus sinensis (L.) Osb. yes 16SrXII+16SrIV N, N5, OC1, C3, C6 Jagüey Grande, Matanzas Citrus sinensis (L.) Osb. yes no LP4 Jagüey Grande, Matanzas Citrus latifolia Tan. yes no Citrus Habana Miramar, La Habana Citrus aurantifolia (Chrism.) Swing. no 16SrVII 2619, 2623 Sola, Camaguey Citrus latifolia Tan. yes 16SrIV 2616, 2617, 2618, 2622 Sola, Camaguey Citrus latifolia Tan. yes no 2621 Sola, Camaguey Citrus paradisi Macf. yes no 2620 Sola, Camaguey Citrus sinensis (L.) Osb. yes no 2624, 2626, 2627, 2628, 2657, 2660 Ceballos, Ciego de Ávila Citrus latifolia Tan. yes 16SrIV 2625 Ceballos, Ciego de Ávila Citrus latifolia Tan. yes 16SrXII 2644, 2645, 2658, 2659 Ceballos, Ciego de Ávila Citrus latifolia Tan. yes no 2662 Ceballos, Ciego de Ávila Citrus paradisi Macf. yes 16SrIV 2643,