Catharanthus roseus (Apocynaceae) naturally infected with diverse phytoplasmas in Costa Rica William Villalobos1, Kristi Bottner-Parker2 , Ing-Ming Lee2, Mauricio Montero-Astúa1, 3, Federico J. Albertazzi1,4, Teresita Coto-Morales1, Izayana Sandoval-Carvajal1, Laura Garita1 & Lisela Moreira1,3 1. Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica (UCR), San José 11501-2060, Costa Rica; william.villalobos@ucr.ac.cr, teresita.coto@ucr.ac.cr, izayana.sandoval@ucr.ac.cr, laura.garita@ucr.ac.cr 2. Molecular Plant Pathology Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705, USA; kristi.bottner@ars.usda.gov, ingming.lee@ars.usda.gov 3. Escuela de Agronomía, UCR, San José 11501-2060, Costa Rica; mauricio.monteroastua@ucr.ac.cr, lisela.moreira@ucr.ac.cr 4. Escuela de Biología, UCR, San José 11501-2060, Costa Rica; federico.albertazzi@ucr.ac.cr Received 08-VIII-2018. Corrected 03-X-2018. Accepted 10-XII-2018. Abstract: Phytoplasmas (class Mollicutes) are causal agents of plant diseases with an economic impact on crops or threatening local biodiversity. A survey was conducted from 2012 to 2016 on infected Catharanthus roseus plants that exhibited symptoms reminiscent of phytoplasma infection throughout Costa Rica. A total of 73 plants were collected exhibiting symptoms such as virescence, phyllody, axillary proliferation, little leaf, leaf malformation, chlorosis, or yellowing. All samples were tested by nested PCR using phytoplasma universal and specific primer pairs. Phytoplasma infection was detected in 52 (71.2 %) of the plants collected. Phytoplasmas of six subgroups belonging to 16Sr groups I, III, IX, XIII and XV were identified based on sequencing and in silico RFLP analyses. ‘Candidatus Phytoplasma asteris’ (16SrI) was the predominant group among the positive samples (n = 30) showing variety of symptoms and wide distribution from sea level to ca. 1 400 m.a.s.l. in six of the seven Costa Rican provinces. Group 16SrIII was the second most abundant (14 samples); and the remaining three groups were seldom found in C. roseus (8 samples). Moreover, group 16SrXIII phytoplasma was detected for the first time in the country. To the best of our knowledge, this is the first report of natural infection of C. roseus with phytoplasma subgroups 16SrI-B, 16SrI-P, 16SrIII-F, 16SrIX-F, 16SrXIII-A, and 16SrXV-B in Costa Rica and Central America. Key words: periwinkle; nested PCR; 16SrI; 16SrIII; 16SrIX; 16SrXIII; 16SrXV. Villalobos, W., Bottner-Parker, K., Lee, I. M., Montero-Astúa, M., Albertazzi, F. J., Coto- Morales, T., Sandoval-Carvajal, I., Garita, L., & Moreira, L. (2019). Catharanthus roseus (Apocynaceae) naturally infected with diverse phytoplasmas in Costa Rica. Revista de Biología Tropical, 67(1), 321-336. Phytoplasmas, previously termed myco- 2000; Hogenhout, et al., 2008). They were plasma-like organisms, are small, cell wall- first discovered, through electron microscopy less, phloem-limited prokaryotes that have been studies, by a group of Japanese scientists reported to be associated with diseases in sev- in 1967 (Doi, Teranaka, Yora, & Asuyama, eral hundred to more than 1 000 plant species 1967). Based on phylogenetic analyses using worldwide (Lee, Davis, & Gundersen-Rindal, 16S rRNA and/or ribosomal protein gene Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 321 sequences, phytoplasmas have been placed in on molecular classification and the guidelines the class Mollicutes (Gundersen, Lee, Rehner, established by the IRPCM Phytoplasma/Spiro- Davis, & Kingsbury, 1994). Phytoplasma plasma Working Team-Phytoplasma Taxonomy infection interferes with plant development Group (IRPCM, 2004), phytoplasmas have and induces morphological and physiological been classified into 43 ‘Candidatus Phyto- changes, including: witches’-broom, phyllody, plasma’ species (Arneodo, et al., 2007; Fernán- virescence, bolting, reddening of leaves and dez, Galdeano, Kornowski, Arneado, & Conci, stems, generalized yellowing, stunting and 2016; Zhao & Davis, 2016; Miyazaki et al., decline (Hogenhout et al., 2008). These mol- 2017; Naderali et al., 2017). licutes are responsible for devastating dam- Phytoplasma infection with one of four age to many economically important crops, 16Sr groups (16SrI, 16SrIII, 16SrIX, and 16Sr fruit trees, woody trees and ornamental plants XV) has been reported in various plant spe- worldwide. Some of these diseases can cause cies in Costa Rica (Gámez & León, 1985; death of the plant host resulting in serious eco- Kenyon, Harrison, & Richardson, 1999; Vil- nomic impact and may affect local biodiversity lalobos, Moreira, Bottner, Lee, & Rivera, 2002; (Maejima, Kenro Oshima, & Namda, 2014; Villalobos et al., 2011; Pardo, Truke, Cardozo, Marcone, 2014). Varela, & Alvarez, 2014). Although natural In nature, phytoplasmas live and reproduce phytoplasma infection in Catharanthus roseus in the phloem tissue of plants, as well as in G. Don (Apocynaceae) has been reported else- the salivary glands and other tissues of some where (Pérez-López, Olivier, Luna-Rodríguez, phloem feeding Hemiptera of the families Adame-García, & Dumonceaux, 2016b), to our Cicadellidae, Cixiidae, Delphacidae, Derbidae, Flatidae and Psyllidae. Phytoplasmal diseases knowledge, it has not been reported in Costa are transmitted from plant to plant by one Rica. This plant species, a perennial herba- of such insect vectors, as well as by grafting ceous plant native to Madagascar, is commonly and Cuscuta spp. A suitable combination of known as Madagascar periwinkle, vinca, or various plant hosts and insect vector-mediated “mariposa” (butterfly) in Costa Rica. It is transmission are responsible for the horizontal widely cultivated as a popular ornamental plant transmission of phytoplasmas between plants and can be found in gardens and homes across (Weintraub & Beanland, 2006). Some studies the warmer parts of tropical and subtropical suggest the possibility of transovarial transmis- countries. Periwinkle plants are used as an sion of phytoplasmas (Alma et al., 1997; Han- experimental host for the maintenance of phy- boonsong, Choosai, Panyim, & Damak, 2002; toplasma strains, as well as to study phytoplas- Mittelberger et al., 2017). ma-host interactions (Nejat et al., 2013; 2015). Phytoplasmas are difficult to cultivate in We consistently found Madagascar peri- vitro, and are therefore poorly characterized winkle plants exhibiting symptoms reminiscent bacteria. Molecular methods using the highly of phytoplasma infection (Hogenhout et al., conserved 16S rRNA gene as well as other 2008) in gardens, parks, living-fences and conserved biomarkers, including ribosomal along sidewalks throughout Costa Rica. In this protein, tuf, secA, and secY genes, have been study, we examined a sample of 73 symptom- used for the detection, differentiation, and atic C. roseus plants collected from 2012 to classification of phytoplasmas (Lee, Gunders- 2016. The phytoplasmas were detected and en-Rindal, Davis, & Batoszyk, 1998b; Seemül- identified by nested PCR, sequencing, in silico ler, Marcone, Lauer, Ragozzino, & Göschl, RFLP and phylogenetic analyses. This work 1998; Marcone, Lee, Davis, Ragozzino, & reports natural infection of Madagascar peri- Seemüller, 2000; Hodgetts, Boonham, Mum- winkle plants with different phytoplasma 16Sr ford, Harrison, & Dickinson, 2008). Based subgroups in Costa Rica. 322 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 MATERIALS AND METHODS DNA polymerase (Thermo Fisher Scientific Inc. USA), and 2 μl of DNA extracted. Diluted Sampling: A total of 73 C. roseus plants PCR product (1:20) with UltraPure DNase/ exhibiting different symptoms related to phy- RNase free distilled water (Thermo Fisher toplasma infection (witches’-broom, phyllody, Scientific Inc.) from the first amplification was virescense, leaf yellowing, dwarfing, etc.) were used as template in the nested PCR. The PCR collected from home gardens, sidewalks, and thermocycler profile for both amplification parks at different locations in the seven provinc- steps was denaturation at 94 °C for 1 min (2 es of Costa Rica [Alajuela province: Alajuela, min for the first cycle), annealing at 55 °C for Cartago province: Dulce Nombre, Paraíso, 1 min, and extension at 72 °C for 2 min (10 min Turrialba; Guanacaste province: Hojancha, for the last cycle). The amplified products (5 μl Filadelfia, Cañas, Sámara, Santa Cruz; Here- of each PCR reaction) were evaluated by elec- dia province: Santo Domingo; Limón prov- trophoresis through 1 % agarose gels, stained ince: Cahuita; Puntarenas province: Chomes, with GelRedTM (Biotium, California, USA) and Esparza, Paso Canoas, Potrero Grande, Que- visualized with a UV transilluminator. brada Grande, Tárcoles; and San José province: Coronado, Moravia, Pérez Zeledón, San Pedro, Sequencing and phylogenetic analyses: Sabanilla]. Two Gliricidia sepium trees show- All samples (n = 52) that yielded amplicons ing little leaf disease (GLLD) were collected of about 1.2 Kb in the previous nested PCR in La Guácima (Alajuela province) to compare were used to prepare new reactions using only sequences with those from C. roseus samples the internal primer pairs of the nested PCR harboring group 16SrIX phytoplasmas. protocol (Gundersen & Lee, 1996a) and were directly sequenced in both directions by Mac- DNA extraction and amplification: Leaf rogen Inc. (Korea). A contig sequence using the midribs and petiole (100 mg) from each sample forward and reverse sequences was obtained of C. roseus and G. sepium were extracted for each sample using BioEdit software v. 7.2.5 using the DNeasy Plant Mini Kit (Qiagen, (Hall, 1999). Confirmation of phytoplasmas Hilden, Germany) according to the manufac- infection and preliminary identification of the turer’s instruction. Additionally, DNA from phytoplasma group for each sample was done healthy periwinkle plants grown in an insect- using BLAST algorithm (http://blast.ncbi.nlm. proof greenhouse was extracted as negative nih.gov/Blast.cgi). control. DNA extracted from Sechium edule A total of five phytoplasma groups were infected with 16SrI-B phytoplasma (Villalobos identified within those 52 positive samples et al., 2002) was used as a positive control. (Digital Appendix 1). Four representative Detection of phytoplasmas was carried samples for group 16SrI (the most abundant) out by amplification of 16S rDNA in a nested and one each from the other four phyto- polymerase chain reaction (PCR) assay, using plasma groups (16SrIII, 16SrIX, 16SrXIII and universal primer pair P1/P7 (Deng & Hiruki, 16SrXV) were selected for further analyses. 1991; Smart, et al., 1996) in the first reac- Additionally, two samples of G. sepium were tion followed by R16F2n/R16R2 (Gunder- included as control for phytoplasma group sen, Lee, Rehner, Davis, & Kingsbury, 1994; 16SrIX. A semi-nested PCR assay was per- Lee, Gundersen, Hammond, & Davis, 1994) formed using primer pair P1/16S-SR in the in the second reaction. Amplifications were first reaction followed by P1A/16S-SR (Lee, performed with a PCR Gradient Palm Cycler et al., 2004) in the second reaction to amplify (Corbett Research Model CG1-96, Australia) the near full length 16S rRNA gene (about 1.5 in 27 μl reactions containing 200 μM of each Kb). The nested PCR products (a total of eight) of the four dNTPs, 0.4 μM of each primer, were purified using PCR Kleen Spin Col- 1.5 mM MgCl2, 0.625 units of DreamTaq umns (Bio-Rad, Hercules, CA) and cloned into Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 323 Escherichia coli (TOP10) by using the TOPO analysis was inferred with MEGA7 using the TA Cloning Kit (Invitrogen, Carlsbad, CA) Maximum Likelihood method based on the according to the manufacturers’ instructions. General Time Reversible model and the rate of Two clones per sample were sequenced with variation per site was determined by a gamma an automated DNA sequencer (Macrogen Inc., distribution with a proportion of invariable Rockville, MD USA). Sequencing reads were sites (G+I). The estimation of stability and assembled using the SeqMan program from support for the inferred clades were performed the DNAStar LaserGene software package using bootstrap analyses of 2 500 replicates. (DNAStar, Madison, WI) and a final sequence was obtained for each clone. The iPhyClassi- RESULTS fier tool (Zhao, et al., 2009) was then used to generate in silico RFLP profiles and determine A total of 73 C. roseus samples were col- phytoplasma group classifications. A BLAST lected in different regions of the country and search (BLASTn) was utilized to find similar phytoplasma infection was detected in 71.2 % sequences deposited in GenBank. Sequences (52/73) by nested PCR. Ten different symptom from clones obtained from C. roseus have been combinations (Table 1) were observed in the deposited to GenBank under accession num- phytoplasma-positive C. roseus plants (Fig. bers MH428957 to MH428964. Additionally, 1A, Fig. 1B, Fig. 1C, Fig. 1D, Fig. 1E and sequences of Costa Rican GLLD are available Fig. 1F). The most common symptom was as MH428965 and MH428966. virescence (19 plants) or virescence combined The sequences of subgroup representa- with other symptoms (11 plants), represent- tives obtained in this study as well as 30 phy- ing in total 57.7 % of the positive plants. The toplasma strains available from GenBank, and second most abundant symptom was yellow- Acholeplasma palmae J233 (as outgroup) were ing, observed on 19 plants (36.5 %). We were aligned using ClustalW (Thompson, Higgins, & not able to find a pattern/association between Gibson, 1994) in MEGA v. 7.0 (Kumar, Stecher, type of symptoms and other variables, includ- & Tamura, 2016). The final alignment included ing phytoplasma group or geographic altitude a total of 1 401 positions. The phylogenetic where the plant was collected. Nonetheless, TABLE 1 Number of samples in each symptom category associated with each of five phytoplasma 16Sr groups identified in Catharanthus roseus collected in Costa Rica from 2012-2016 CUADRO 1 Número de muestras según síntoma observado en Catharanthus roseus infectados con fitoplasmas, recolectadas en Costa Rica, 2012-2016 Phytoplasma group/ Grupo de fitoplasma Symptoms / Síntomas Total 16SrI 16SrIII 16SrIX 16SrXIII 16SrXV Bolting 1 – – – – 1 Phyllody + little leaf + proliferation 2 – – – – 2 Virescence 14 2 1 1 1 19 Virescence + big bud – – – – 1 1 Virescence + phyllody 1 – – – 1 2 Virescence + proliferation 1 – – – – 1 Virescence + proliferation + little leaf 2 – – 1 – 3 Virescence + little leaf + stunting + short internodes – 3 – – – 3 Virescence + yellowing 1 – – – – 1 Yellowing 8 9 2 – – 19 324 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 Fig. 1. Symptoms exhibited by periwinkle plants infected with phytoplasmas including A) yellowing, dwarfing, little leaf and axilar proliferation, B) virescence, C) floral abortion and malformation, D) stalk elongation (bolting), E) big bud, F) phyllody. Fig. 1. Síntomas presentados por plantas de vinca infectadas con fitoplasmas incluyeron A) amarillamiento, enanismo, hoja pequeña y brotación múltiple axilar, B) enverdecimiento floral, C) aborto y deformación floral, D) tallos elongados, E) base floral engrosada, F) filodia. perhaps due to a greater number of samples, 16SrIX, 16SrXIII and 16SrXV were detected the 16SrI group showed variation regarding in 30, 14, 3, 2 and 3 symptomatic C. roseus symptom expression in the plant host C. roseus. samples (Table 2), respectively. No mixed According to BLAST search results, phy- infections were found, despite the presence of toplasmas belonging to groups: 16SrI, 16SrIII, two or three different phytoplasmas in some Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 325 TABLE 2 Number of infected Catharanthus roseus samples associated with each of five phytoplasma 16Sr groups identified and province of collection in Costa Rica CUADRO 2 Número de muestras de Catharanthus roseus según grupo de fitoplasma detectado por provincia de recolección en Costa Rica Phytoplasma group / Grupo de fitoplasma Province / Provincia Total 16SrI 16SrIII 16SrIX 16SrXIII 16SrXV Cartago 7 1 – 1 – 9 Guanacaste 6 – – 1 3 10 Heredia 1 1 – – – 2 Limón 1 – – – – 1 Puntarenas 4 – 1 – – 5 San José 11 12 2 – – 25 Total 30 14 3 2 3 52 locations in the same province (Table 2). The were found in six out of seven provinces in altitudinal distribution of phytoplasmas sug- the country (Table 2). Only one infected C. gested that 16SrI group has a wide distribution, roseus (CR02) expressing bolting symptoms while groups 16SrIII and 16SrXV showed (GenBank accession no. MH428961), detected presence above 1 200 masl or below 500 masl, in Turrialba city (Cartago province), showed respectively (Fig. 2). 97 % similarity to subgroup 16SrI-P (GenBank The majority of nucleotide sequenc- accession no. AF503568). es obtained from positive plants (30 of 52 A summary of data obtained using the sequences) indicated infection with ‘Ca. Phy- iPhyClassifier tool to analyze cloned phyto- toplasma asteris’, with similarities between plasma 16S rRNA gene sequences obtained in 98-99 % with subgroup 16SrI-B represen- this study is presented in Table 3. The virtual tatives. Plants infected with this subgroup RFLPs patterns of three sequences representa- tive of subgroup 16SrI-B (GenBank accession nos. MH428957, MH428960 and MH428964) were identical to the reference strain of this subgroup. However, the sequence of a sample from Turrialba identified as subgroup 16SrI-P (GenBank accession no. MH428961) showed identical virtual RFLP patterns to this subgroup with 16 out of 17 enzymes used, but the pattern resolved using HhaI was like other strains in the 16SrI group although dissimilar to sub- group 16SrI-P (Fig. 3A), suggesting a possible new subgroup within the ‘Ca. Phytoplasma asteris’ group in Costa Rica. Fig. 2. Altitudinal distribution (meters above sea level, The second group in importance, by num- m.a.s.l.) of Catharantus roseus samples per phytoplasma ber of detections, was group 16SrIII (X-disease group collected in this study in Costa Rica. group or ‘Ca. Phytoplasma pruni’). Eleven Fig. 2. Distribución altitudinal (metros sobre el nivel del mar, m.s.n.m.) de muestras de Catharantus roseus según samples collected from gardens in three differ- grupo de fitoplasmas detectado en este estudio realizado ent provinces (Heredia, Cartago and San José) en Costa Rica. were infected with group 16SrIII phytoplasma 326 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 327 TABLE 3 Summary of data obtained using the iPhyClassifier tool to analyze cloned phytoplasma 16S rRNA gene sequences obtained in this study CUADRO 3 Resumen del análisis de datos para las secuencias de fitoplasmas obtenidos en este estudio usando la herramienta iPhyClassifier GenBank ‘Candidatus Phytoplasma sp.’ Sequence similarity Similarity coefficient to the Sample ID Accession assignment using reference strains. to the assigned ‘Ca. reference pattern of phytoplasma Relation to 16Sr Number GenBank accession numbers Phytoplasma sp.’ 16Sr group/subgroup (GenBank subgroup/group classified Delineating restriction enzyme indicated in parentheses. reference strain (%) accession number) CR078 MH428957 99.5 1.00, 16Sr I-B (AP006628) 16SrI-B member Virtual RFLP patterns CR41 MH428960 ‘Ca. Phytoplasma asteris’ identical to 16SrI-B CR47 MH428964 (M30790) 99.6 0.97, 16SrI-P (AF503568) tentative 16SrI HhaI, pattern correspond to CR02 MH428961 new subgroup 16SrI-B instead of 16SrI-P CR33 MH428959 ‘Ca. Phytoplasma pruni' rrnA’ 99.1 0.98, 16SrIII-F (AF510724) 16SrIII-F variant BstUI, pattern correspond to (JQ044393) 16SrIII-D instead of 16SrIII-F CR06 MH428962 98.4 0.97, 16SrIX-A (AF248957) tentative 16SrIX RsaI, new pattern ‘Ca. Phytoplasma phoenicium’ new subgroup GLLDCR1 MH428965 (AF515636) 98.3 1.00, 16SrIX-F (AF361017) 16SrIX-F member Virtual RFLP patterns GLLDCR2 MH428966 identical to 16SrIX-F CR25 MH428958 ‘Ca. Phytoplasma hispanicum’ 99.5 0.98, 16SrXIII-H (JX626329) 16SrXIII-H variant MseI, pattern correspond to (AF248960) 16SrXIII-A instead of 16SrXIII-H CR14 MH428963 ‘Ca. Phytoplasma brasiliense’ 99.2 0.96, 16SrXV-A (AF147708). tentative 16SrXV HaeIII, new pattern (AF147708) new subgroup Fig. 3. Computer-simulated virtual RFLP patterns generated from in silico digestions of phytoplasma 16S rRNA genes from strains detected in this study belonging to groups: (A) 16SrI using HhaI (MH428961: 16SrI-P*); (B) 16SrIII using BstUI (MH428959: 16SrIII-F*); (C) 16SrIX using RsaI (MH428962:16SrIX-A∞); (D) 16SrXIII using MseI (MH428958: 16SrXIII-H*); (E, F) 16SrXV using HaeIII and HpaII (MH428963: 16SrXV-A∞), obtained using iPhyClassifier tool. GenBank accession numbers are shown in parenthesis after each subgroup. Phytoplasma subgroup variant are indicated by an asterisk and tentative new subgroup by ∞ symbol after subgroup number. GenBank acc.nos. KX810335 and HQ230579 in E and F correspond to other variants to subgroups 16SrXV-A and 16SrXV-B reported previously. Lanes MW, HaeIII digest of ΦX174 RFI DNA; fragment sizes (bp) from top to bottom: 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72. Figure edited with Paint.net software. Fig. 3. Patrones RFLPs virtuales para los fitoplasmas detectados en el estudio respecto a los patrones mostrados en el grupo asignado: (A) 16SrI usando HhaI (MH428961: 16SrI-P*); (B) 16SrIII con BstUI (MH428959: 16SrIII-F*); (C) 16SrIX usando RsaI (MH428962:16SrIX-A∞); (D) 16SrXIII usando MseI (MH428958: 16SrXIII-H*); (E, F) 16SrXV con HaeIII y HpaII (MH428963: 16SrXV-A∞), obtenidos con el programa iPhyClassifier. Los números de registro del GenBank para cada subgrupo se muestran en paréntesis. Los variantes de subgrupos se indican con un asterisco mientras que los potenciales nuevos subgrupos con el símbolo ∞. Los números de registro GenBank: KX810335 y HQ230579 en E y F corresponden a otras variantes en 16SrXV reportadas. Los carriles del marcador de peso molecular (MW) corresponden a fragmentos de ADN del bacteriófago ΦX174 RFI digerido con HaeIII, el tamaño de los fragmentos de arriba hacia abajo es: 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72 pares de bases. Figura editada con Paint.net. strains. The search for similar sequences in in a limited number of samples. Only eight public databases showed 99 % identity to Mex- plants in total from three provinces (Cartago, ican Xalapa periwinkle virescence phytoplas- Guanacaste, and San José) carried one of these ma (GenBank accession no. KY778009). The groups. Three C. roseus samples infected with virtual RFLP pattern derived from one of our 16SrIX group phytoplasmas were detected. samples (GenBank accession no. MH428959) A sample collected in Potrero Grande (Pun- was most similar to the reference pattern of tarenas), GenBank accession no. MH428962, the 16SrIII-F subgroup (GenBank accession showed 99 % similarity to pigeon pea witches’- no. AF510724), with a similarity coefficient of broom (GenBank accession no. KJ817866), 0.98. However, the MH428959 phytoplasma Crotalaria juncea witches’-broom (GenBank is a variant of subgroup 16SrIII-F, because the accession no. KF941131), and other phytoplas- virtual RFLP pattern resolved by BstUI cor- mal strains belonging to that group. Results responded to subgroup 16SrIII-D instead of from the iPhyclassifier tool for this sample 16SrIII-F (Fig. 3B). classified it (similarity coefficient = 0.97) as The presence of three other groups: 16SrIX belonging to subgroup 16SrIX-A (PPWB, Gen- (‘Ca. Phytoplasma phoenicium’), 16Sr-XIII Bank accession: AF248957). However, the vir- (‘Ca. Phytoplasma hispanicum’) and 16Sr-XV tual RFLPs obtained for this sample displayed (‘Ca. Phytoplasma brasiliense’) was detected a unique RFLP pattern of three bands with 328 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 enzyme RsaI (Fig. 3C). When this sample was respectively. However, an in silico RFLP com- compared to sequences obtained in Costa Rica parison using the iPhyClassifier showed that during this research from GLLD, GenBank the phytoplasma found in C. roseus is not the accession nos. MH428965 and MH428966, same as the 16SrXV-B subgroup previously results showed that the phytoplasmas from reported in Costa Rica infecting G. ulmifolia. group 16SrIX detected in periwinkle and Gliri- According to the virtual RFLP pattern obtained cidia in Costa Rica are different from each for the sequence of GenBank accession no. other, and the strain found in periwinkle may MH428963, the most similar to it was the refer- represent a variant of subgroup 16SrIX-A, ence pattern of subgroup 16SrXV-A (GenBank meanwhile Costa Rican GLLD strains have accession no. AF147708), with a similarity 100 % identity to those from Honduran GLLD. coefficient of 0.96, suggesting this strain may The presence of group 16SrXIII, ‘Ca. represent a tentative new 16SrXV subgroup. Phytoplasma hispanicum’, in Costa Rica was The virtual RFLP analysis displayed a unique first detected in this survey. Two plants show- and different RFLP pattern with HaeIII to ing virescence and harboring this phytoplasma previously reported strains (Fig. 3E). The 16 group were collected in Dulce Nombre (Car- remaining virtual RFLPs showed exactly the tago) and Hojancha (Guanacaste, GenBank same patterns to those of subgroup 16SrXV-A, accession no. MH428958). The 16S rRNA for example with enzyme HpaII (Fig. 3F). gene sequences obtained and compared by the Phylogenetic analysis comparison based BLAST algorithm showed 99 % identity with on 16S rDNA sequences of phytoplasma sub- Mexican periwinkle virescence phytoplasma groups found in this study showed 16Sr group (MPVP, GenBank accession no. AF248960). clustering consistent with the group identifica- According to virtual RFLP analysis, it was tion by BLAST analysis. The same consis- most similar to the reference pattern of sub- tent phylogenetic clustering was found with group 16SrXIII-H (GenBank accession no. representatives of groups 16SrIII, 16SrIX and JX626329), with a similarity coefficient of 16SrXIII. However, representatives of sub- 0.98, suggesting this may be a variant of groups 16SrI-B and 16SrI-P, obtained in this 16SrXIII-H. The virtual RFLPs displayed pat- study, clustered together instead of with related terns like those of subgroup 16SrXIII-H, except sequences obtained from GenBank, suggesting for a unique pattern for enzyme MseI (Fig. 3D). a possible relationship due to geographic ori- The fifth phytoplasmas group detected gin. Also, contrary to the iPhyClassifier results, during this work was ‘Ca. Phytoplasma brasil- the representative strain of group 16SrXV from iense’ (16SrXV). Infection of C. roseus by C. roseus, obtained in this study, clustered this group was confined to Guanacaste prov- more closely to subgroup 16SrXV-B strains ince. All three plants harboring group 16SrXV than subgroup 16SrXV-A (Fig. 4). showed virescence; with one plant (CR03) collected in Samara (Guanacaste) also exhibit- DISCUSSION ing phyllody and another one (CR14, Santa Cruz, Guanacaste) also exhibiting big bud (Fig. In this study, phytoplasmas were detected 1E). The 16S rRNA gene sequences obtained in naturally infected C. roseus, in six of seven showed 99 % similarity with sequences cor- provinces in Costa Rica (excluding Alajuela), responding to this phytoplasma infecting Vitis 16SrRNA gene sequences indicated that they vinifera, Carica papaya, Crotalaria juncea, belonged to five groups: 16SrI, 16SrIII, 16SrIX, Hibiscus sp. and Guazuma ulmifolia from 16SrXV, and 16SrXIII. The first four groups Peru (GenBank accession nos. KX670807- were previously identified in other plant hosts 9, KX810334-6), Brazil (GenBank accession in Costa Rica (Kenyon et al., 1999; Villalobos nos. KF878382 and AF147708), and Costa et al., 2002; 2011; Pardo et al., 2014). Group Rica (GenBank accession nos. HQ258882-3), 16SrXIII was detected in the country for the Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 329 Fig. 4. Phylogenetic tree inferred from analysis of 16S rRNA gene sequences by Maximum Likelihood method based on the General Time Reversible model. A bootstrap test with 2500 replicates was done to support reliability of the analysis. The analysis involved 41 nucleotide sequences, included reference strains of 30 previously described Ca. Phytoplasma species and subgroup strains obtained from GenBank and Acholeplasma palmae J233 (NR_029152), as outgroup to root the phylogenetic tree. GenBank accession numbers of all sequences are showed in parenthesis and sequences obtained in this study are indicated in bold font. Bar = 0.02 nucleotide substitutions per site. Fig. 4. Árbol filogenético inferido del análisis de las secuencias del gen 16S rRNA por el método de Máxima Verosimilitud empleando el modelo “General Time Reversible”, empleando 2500 iteraciones para respaldar la confiabilidad del análisis. El análisis incluyó 41 secuencias nucleotídicas, entre ellas 30 de Ca. Phytoplasma spp. y de los subgrupos de referencia previamente descritas, disponibles en el GenBank, además del aislamiento J233 de Acholeplasma palmae (NR_029152), como grupo externo, empleado para enraizar el árbol filogenético. Los números de registro en el GenBank para las secuencias empleadas se muestran en paréntesis y las obtenidas en este estudio se resaltan en negrita. Bar = 0.02 sustituciones nucleotídicas por sitio. first time. Moreover, three new tentative sub- are more diverse and complex than previously groups into 16SrI, 16SrIX and 16SrXV groups known, and these rise potential risk of phyto- were also described. These findings suggested plasmal diseases to economically crops as well that the phytoplasmas present in Costa Rica as wild biodiversity. Despite the presence of 330 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 two different phytoplasmas in some locations wild plant species belonging to Anacardiaceae in the same province (Table 2), no mixed infec- (Villalobos, unpublished), Asteraceae / Com- tions were found in this study probably because positae and Rubiaceae (Villalobos, Montero- few clones per plant were analyzed. Astúa, Coto, Sandoval, & Moreira, 2018) have Catharanthus roseus has been reported been detected as new hosts for this group. as a natural host for phytoplasmas groups The phytoplasma strain variant of sub- 16SrI, 16SrIII, 16SrIX, 16SrXV, 16SrXIII, group 16SrI-P found in Turrialba (Cartago in North and South America, and the Carib- province, GenBank accession no. MH428961) bean islands, but not from the Central Ameri- has not been previously reported in Costa can region (Lee et al., 1998b; Lee, Davis, Rica or Central America. This subgroup was & Gundersen-Rindall, 2000; Montano, Dally, reported by Šeruga et al. (2003) infecting Davis, Pimentel, & Brioso, 2001b; Torres, Populus nigra trees in Croatia. However, Galdeano, Docampo, & Conci, 2004; Duduk, BLASTn top hit results showed 99 % similar- Mejia, Calari, & Bertaccini, 2008; Barbosa, ity of the Turrialba’s periwinkle sample with Eckstein, Bergamin Filho, Bedendo, & Kita- other phytoplasmas found in North America, jima, 2012; Galdeano, Guzmán, Fernández, & including a periwinkle with virescence col- Conci, 2013; Dumonceaux, Green, Hammond, lected in Yucatan, Mexico (GenBank accession Pérez, & Olivier, 2014; Caicedo, Rivera-Var- no. EF050085). gas, Segarra, & Davis, 2015; Davis, Harrison, Likewise, the detection of phytoplasmas Zhao, Wei, & Dally, 2016; Pérez-López, Luna- belonging to the 16SrIII-F subgroup (‘Ca. Rodríguez, Olivier, & Dumonceaux, 2016a). Phytoplasma pruni’ related strain) is a new According to Montano et al. (2001b), C. roseus report for Costa Rica. This subgroup was also is a well-known experimental host for phyto- plasmas, however few incidences of natural detected in a different host in Cartago province infections have been reported and its role relat- (Villalobos, unpublished data). Pardo et al. ed to natural dissemination and disease spread (2014) reported subgroup 16SrIII-L infecting is uncertain. Nevertheless, considering a differ- cassava crop associated with frog-skin disease ent perspective, C. roseus may be deployed as in the country. The 16SrIII group has a large a sentinel plant for phytoplasma occurrence in number of subgroups and diversity of host spe- a geographical area, as this study is showing. cies, as well as a wide geographic distribution Subgroup 16SrI-B phytoplasma strains (Zhao et al., 2009). For example, subgroup (‘Ca. Phytoplasma asteris’) have been detected 16SrIII-J is notable for its presence in several in Costa Rica associated with diseases affecting hosts and several countries mainly in South crops, landscape trees, and wild shrubs (Gámez America (Galdeano et al., 2013, Pérez-López & León, 1985; Villalobos et al., 2002, Saborío- et al., 2016a). Rodríguez, Villalobos, & Rivera, 2007). Two It is highlighted that natural infections epidemics associated with this phytoplasma with 16SrIX-A are reported for the first time subgroup caused severe economic losses to in Costa Rica. Its presence in at least two geo- chayote (Sechium edule) growers during 2001- graphically distant sites, Southern area (Potrero 2002, and to coffee growers during 2006-2007 Grande, Puntarenas) and Central Valley (San due the infection of Erythrina trees, which are Pedro, San José), may suggest a high prob- used as shade trees for coffee crop production ability of being found in other areas. The in Costa Rica (Saborío-Rodríguez et al., 2007). symptoms in both geographic locations are In this study, this subgroup was also the most consistent with those previously reported for widespread in the country. We hypothesize the host and subgroup 16SrIX-A, such as vires- that 16SrI group has been present longer in cence and yellowing. Natural infection of this the country; therefore, allowing the emergence plant species by group 16SrIX-A was reported of new host-vector interactions, recently three previously in Colombia (Duduk et al., 2008), Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 331 Brazil (Barbosa et al., 2012) and Puerto Rico KX670807-9, KX810334-6), rather than to the (Caicedo et al., 2015). surrounding Guazuma phytoplasma. Symp- Reports of phytoplasmas belonging to toms associated to natural infection of C. group 16SrXIII have been concentrated in roseus by this phytoplasmas in Brazil (Monta- relatively few hosts and restricted geographic no et al., 2001b) were yellowing and witches’- locations inside North and South America, broom, while in Costa Rica the main symptom infecting C. roseus, Solanum tuberosum, C. was virescence. papaya, S. lycopersicum, Fragaria sp., Melia As previously reported by Gundersen et al. azedarach, Turnera ulmifolia, Dimorphandra (1996b) and Lee et al. (1998a, 2004), genetic spp. and Thunbergia erecta (Lee et al., 1998b; variation in some phytoplasma strains appears Harrison, Boa, & Carpio, 2003; Arneado et al., to be associated with ecological isolation, and 2007; Montano et al., 2011; Melo et al., 2013; distinct phytoplasma strains may result from 2018; Montano, Bertaccini, Guthelle, Paltrin- new epidemiological cycles from an original ieri, & Contaldo, 2015; Fernández et al., 2015; organism (Lee et al., 1998b). The tentative Alves, Souza, Ribeiro, da Silva Xavier, & variants and or tentative subgroups observed Carvalho, 2016). The findings reported in this in phytoplasmas sequenced in this study may study, expand the geographical range of ‘Ca. be the result of ecological isolation in the C. Phytoplasma hispanicum’ (Davis et al., 2016). roseus host. It is worth noting that we observed In this survey, plants infected with subgroup in this study a case where the host species and/ 16SrXIII-A were collected in Cartago province or the putative insect vector associated with and Guanacaste, and no other hosts have been the plant host may represent the ecological detected in the country for this phytoplasma differentiator for a phytoplasma strain, rather group. Additional analyses must be done to than geographic relationship. Catharanthus determine if this phytoplasma may represent a roseus plants with phytoplasma infection were new subgroup in accordance with differential collected from Guanacaste, where Guazuma virtual RFLP patterns, as mentioned above. trees with subgroup 16SrXV-B phytoplasma Phytoplasmas of group 16SrXV, ‘Ca. Phy- infection are common; nevertheless, contrary toplasma brasiliense’, were reported in Bra- to our preliminary hypothesis, the C. roseus zil infecting Hibiscus spp. (Montano et al., plant growing in Guanacaste, in the same area 2001a), and other hosts including C. roseus, where Guazuma symptomatic trees occur, were Crotalaria juncea Brassica oleracea, and Sida infected with different phytoplasma strains. rhombifolia (Montano et al., 2001b; Eckstein, Including the results from this report, Barboza, Rezende, & Bedendo, 2011; Bianco, phytoplasmas belonging to six groups and et al., 2014; Canale, & Bedendo, 2013). It is nine subgroups have been detected in Costa also present in Costa Rica infecting Guazuma Rica. Moreover, phytoplasma infection has ulmifolia (Villalobos, et al., 2011), and in Peru been detected in more than 17 different plant infecting Vitis vinifera and C. papaya (Wei, et hosts. These findings suggest more diversity al., 2017). The Costa Rican Guazuma strain of phytoplasmas in the country than previously was classified as subgroup 16SrXV-B and the suspected. Attention to their incidence and geo- Peruvian strain was identified as a variant. The graphic spread is important because we hypoth- vinca samples infected with group 16SrXV esize that new host encounters are allowing were found in different places in the Nicoya the development of previously uncharacter- peninsula (Guanacaste), where the presence ized host-phytoplasma associations. On the of Guazuma witches’-broom (GWB) is fre- other hand, accidental or intentional introduc- quent. Nevertheless, sequences showed 99 % tion of phytoplasmas to a new geographical sequence similarity to ‘Ca. Phytoplasma brasil- region must be avoided due to the effects that iense’ representatives of subgroup 16SrXV-B these pathogens may cause to biodiversity. variants from Peru (GenBank accession nos. Examples of this situation increase every year 332 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 67(1): 321-336, March 2019 in scientific reports about new diseases, new fitoplasmas. Infección por fitoplasmas se detectó en 52 hosts, or new geographic range involving these (71.2 %) de las muestras. Fitoplasmas de seis subgrupos plant pathogens. dentro de los grupos 16Sr I, III, IX, XIII y XV fueron identificados basados en secuenciación del ADN y aná- This study extends the knowledge of phy- lisis de polimorfismos de restricción (RFLP) in silico. El toplasmas subgroups in Costa Rica and the grupo predominante encontrado en las muestras positivas Central American region. Here we report for (n = 30) fue el 16SrI (‘Candidatus Phytoplasma asteris’), the first time the detection of natural infection éste mostró variedad de síntomas y amplia distribución of C. roseus with subgroups 16SrI-B, 16SrI-P, desde el nivel del mar hasta casi los 1 400 m.s.n.m. en seis 16SrIII-F, 16SrIX-A, 16SrXIII-A, 16SrXV-A. de las siete provincias de Costa Rica. El grupo 16SrIII fue el segundo más abundante (14 muestras); y los restantes With the exception of 16SrI-B, all of these tres grupos se encontraron en pocas muestras de C. roseus subgroups were previously unknown to occur (8 muestras). Además, fitoplasmas del grupo 16SrXIII se in the Central American region. The findings detectaron por primera vez en el país. De acuerdo a nuestro here reported are not only informative, these conocimiento, este es el primer informe de infección natu- identify C. roseus as a reservoir and potential ral de C. roseus con fitoplasmas de los subgrupos 16SrI-B, 16SrI-P, 16SrIII-F, 16SrIX-F, 16SrXIII-A y 16SrXV-B en inoculum source of phytoplasmas to cultivated Costa Rica y Centroamérica. crops, wild plants, as well as other ornamental plants. Therefore, this plant species may be Palabras clave: vincas; PCR anidado; 16SrI; 16SrIII; considered a sentinel to detect phytoplasmas 16SrIX; 16SrXIII; 16SrXV. within surrounding crops and natural ecosys- tems, as well as their genetic diversity. REFERENCES Ethical statement: authors declare that Alma, A., Bosco, D., Danielli, A., Bertaccini, A., Vibio, M., they all agree with this publication and made & Arzone, A. (1997). Identification of phytoplasmas in eggs, nymphs and adults of Scaphoideus titanus significant contributions; that there is no con- Ball reared on healthy plants. Insect Molecular Bio- flict of interest of any kind; and that we logy, 6(2), 115-121. followed all pertinent ethical and legal proce- Alves, M. S., de Souza, A. N., Ribeiro, G. M., da Silva dures and requirements. A signed document has Xavier, A., & Carvalho, C. M. (2016). A 16SrXIII-E been filed in the journal archives. subgroup phytoplasma is associated with Thunber- gia erecta yellow leaf in Brazil. 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