Detection of Phytoplasmas Associated with Clover Proliferation (16SRVI-A) and Pigeon Pea Witches’ Broom (16SRIX-C) Group in Periwinkle (Catharanthus roseus) Plants in Recreation Areas in Hatay Province of Türkiye

Year 2025, Volume: 8 Issue: 6, 1890 - 1894, 15.11.2025

Hakan Çarpar

https://doi.org/10.34248/bsengineering.1720137

Abstract

Periwinkle (Catharanthus roseus), also known as the Madagascar flower, is an important ornamental plant used in recreational areas as well as being a medicinal plant. In this study, conducted between 2021 and 2022 in Hatay province of Türkiye, shoot samples were collected from 25 plants exhibiting phyllody, virescence, yellowing, little leaf, dwarfing and witches' broom symptoms related to phytoplasmas in recreation areas. Leaf and stem samples from suspected periwinkle plants were tested by biological indexing and molecular methods. Total nucleic acid in symptomatic plants was isolated by CTAB method. All symptomatic plants were tested by direct and nested PCR (nPCR) using universal primer pairs (R16F1/R0 and R16F2n/R2). Phytoplasma infections were detected in all symptomatic plants collected. Four selected samples from Arsuz (2 samples) and Antakya (2 samples) districts of Hatay, representing the geographical variations, were sequenced. One from each district belonging to different groups deposited to the GenBank (Accession No: PP763874 and PP763741). Sequenced samples were verified by BLAST, iPhyClassifier (virtual RFLP) and phylogenetic analysis. Two subgroup phytoplasmas belonging to 16Sr group VI and IX were detected by sequencing and virtual RFLP. While Clover Proliferation Group, subgroup 16SrVI-A, was detected in selected 2 samples taken from Antakya, phytoplasma associated with Pigeon Pea Witches’ Broom, subgroup 16SrIX-C, was detected in 2 samples taken from Arsuz. This is the first report of natural infection of periwinkle infecting by phytoplasma belonging to subgroup 16SrIX-C in Türkiye.

Keywords

BLAST , iPhyClassifier (virtual RFLP) , Natural infection , Periwinkle (Catharanthus roseus) , PCR

Ethical Statement

Ethics committee approval was not required for this study because of there was no study on animals or humans.

References

• Barbosa J, Eckstein B, Bergamin A, Bedendo I, Elliot K. 2012. Molecular characterization of a phytoplasma of group 16SrIX related to 'Ca. Phytoplasma phoenicium' in periwinkle in Brazil Trop Plant Pathol, 37: 130-135. https://doi.org/10.1590/S1982-56762012000200005
• Bertaccini A, Duduk B, Paltrinieri S, Contaldo N. 2014. Phytoplasmas and phytoplasma diseases: A severe threat to agriculture. Am J Plant Sci, 5: 1763-1788. https://doi.org/10.4236/ajps.2014.512191
• Bertaccini A. 2007. Phytoplasmas: diversity, taxonomy, and epidemiology. Front Biosci, 12(2): 673-689, https://doi.org/10.2741/2092
• Caicedo JD, Rivera-Vargas LI, Segarra AE, Davis RE. 2015. Detection and molecular characterisation of a group 16SrIX phytoplasma infecting citrus (Citrus sinensis and C. limon), coffee (Coffea arabica), periwinkle (Catharanthus roseus), and tabebuia (Tabebuia heterophylla) in Puerto Rico. Australas Plant Dis, 10(1): 1-8.
• Çarpar H, Sertkaya G. 2022a. Detection and characterization of phytoplasmas in some cucurbits (Cucurbitaceae) and bindweed (Convolvuluceae) in Hatay province of Turkey. MKU J Agric Sci, 27(1): 166-173. https://doi.org/10.37908/mkutbd.1041286
• Çarpar H, Sertkaya G. 2022b. Investigation on phytoplasma diseases, their potential insect vectors and other hosts in pepper (Capsicum annuum L.) growing areas of Hatay-Turkey. MKU J Agric Sci, 27(2): 241-252. https://doi.org/10.37908/mkutbd.1060097
• Çarpar H. 2025. First report of catharanthus roseus phyllody disease associated with candidatus phytoplasma trifolii in recreation areas of Mersin-Türkiye. 11th International Congress on Contemporary Scientific Research, March 12-15, Ho Chi Minh, Vietnam, pp: 585-590.
• Chang HC, Chen JC. 2024. An efficient grafting method for phytoplasma transmission in Catharanthus roseus. Plant Methods, 20(1): 13. https://doi.org/10.1186/s13007-024-01139-w
• Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.
• Duduk B, Mejia JF, Calari A, Bertaccini A. 2008, Identification of 16SrIX group phytoplasmas infecting Colombian periwinkles and molecular characterization on several genes. 17th Congress of the International Organization for Mycoplasmology (IOM), 6-11 July, Tienjin, China, 112: 83.
• Favali MA, Fossati F, Toppi LSD, Musetti R. 2008. Catharanthus roseus phytoplasmas, In: Characterization, diagnosis and management of phytoplasma. NA Harrison, GP Rao, C Marcone, eds. Stadium Press LLC, Texas, US, pp: 195-218.
• Gundersen DE, Lee IM. 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol Mediterr, 35: 144-151.
• Hiruki C, Wang K. 2004. Clover proliferation phytoplasma: 'Candidatus Phytoplasma trifolii'. Int J Syst Evol Microbiol, 54(4): 1349-1353. https://doi.org/10.1099/ijs.0.02842-0
• Hogenhout AS, Music MS. 2010. Phytoplasma genomics, from sequencing to comparative and functional genomics- what have we learnt? Phytoplasmas Genoms, Plant Hosts Vectors, CABI Digital Library, pp: 19-37.
• Lee IM, Gundersen DE, Hammond RW, Davis RE. 1994. Use of mycoplasmalike organism (MLO) groupspecific oligonucleotide primers for nested-PCR assays to detect mixed- MLO infections in a single host plant. Phytopathol, 84(6): 559-566.
• Marcone C, Valiunas D, Mondal S, Sundararaj R. 2021. On some significant phytoplasma diseases of forest trees: An update. Forests, 12(4): 408. https://doi.org/10.3390/f12040408
• Seemüller E, Marcone C, Lauer U, Ragozzino A, Göschl M. 1998. Current status of molecular classification of the phytoplasmas. Plant Pathol J, 80: 3-26.
• Tiwari NN, Jain, RK, Tiwari AK. 2022. Detection of 'Ca. Phytoplasma phoenicium' in periwinkle (Cathranthus roseus) plants in uttar pradesh, India Biosci Res, 38: e38026. https://doi.org/10.14393/BJ-v38n0a2022-56064
• Villalobos W, Bottner-Parker K, Lee IM, Montero-Astúa M, Albertazzi FJ, CotoMorales 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. https://dx.doi.org/10.15517/rbt.v67i1.33972
• Volokhov DV, 2008. Acholeplasma laidlawii strain Haig 179L 16S ribosomal RNA gene and 16S-23S ribosomal RNA intergenic spacer, partial sequence. URL: https://www.ncbi.nlm.nih.gov/nuccore/FJ226570.1?report=GenBank (accessed date: October 29, 2025).
• Wang XY, Zhang RY, Li J, Li YH, Shan HL, Li WF, Huang YK. 2022. The diversity, distribution and status of phytoplasma diseases in China. Front Sustain Food Syst, 6: 943080. https://doi.org/10.3389/fsufs.2022.943080
• Weintraub PG Beanland L. 2006. Insect vectors of phytoplasmas. Ann Rev Entomol, 51: 91-111. https://doi.org/10.1146/annurev.ento.51.110104.151039
• Zhao Y, Wei W, Lee IM, Shao J, Suo X, Davis RE. 2009. Construction of an interactive online Phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease Phytoplasma group (16SrIII). Int J Syst Evol Microbiol, 59: 2582-2593.