Occurrence of a Clover Proliferation (16SrVI) Group Phytoplasma Associated with Little Leaf Disease of Portulaca grandiflora in India

Abstract

Portulaca grandiflora (family Portulacaceae), commonly known as moss rose purslane, is a popular ornamental plant widely grown in temperate climates because it blooms all summer. Portulaca is also used for medicinal purposes since it is rich in vitamins A, B1, and C and has antimicrobial and cytotoxic activity. Since March 2005, 30 to 50% of P. grandiflora plants in the ornamental gardens as well as in pots at the Central Institute of Medicinal and Aromatic Plants, Lucknow, India have displayed symptoms resembling phytoplasma infection. Disease symptoms start as a typical bud proliferation, downward curling, and diminishing size of leaves, followed by overall stunted growth and yellowing of the whole plant from April to June. Some plants also formed rosettes and a proliferation of axillary shoots resulting in a witches'-broom appearance. Typical pleomorphic bodies, mostly spherical to oval, ranging from 340 to 1,100 nm were observed only in sieve elements of infected plants by transmission electron microscopy (TEM). On the basis of symptoms, TEM observations, PCR, and response to antibiotic treatment, the causal organism was identified as phytoplasma (1). Total genomic DNA from healthy and infected plants was extracted with the CTAB buffer method (2). Of 27 suspected samples screened by PCR, 23 were phytoplasma positive. Presence of phytoplasmas in plants was demonstrated by a nested PCR assay employing primer pair P1/P6 followed by R16F2n/R16R2 that generated rDNA products of 1.5 and 1.2 kb, respectively, only from symptomatic plants. No differences among phytoplasmas in Portulaca plants were detected by restriction fragment length polymorphism (RFLP) analysis of nested rDNA (1.2 kb) products using endonucleases BamHI, RsaI, AluI, HpaII, and EcoRI. Comparative analysis of RFLP patterns with those derived from reference phytoplasmas tentatively identified the Portulaca little leaf (PLL) phytoplasma as a member of 16S rDNA RFLP group 16SrVI (3). A nested PCR product (1.25 kb) was cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. The sequence was deposited in the GenBank database (Accession No. EF651786). Sequence analysis revealed the PLL phytoplasma to be most similar (98%) to Indian brinjal little leaf (Accession No. EF186820) and ‘Candidatus Phytoplasma trifolii’ (Accession No. AY390261), two 16SrVI group phytoplasmas previously reported from India and Canada, respectively. The status of PLL (EF651786) was also verified by in silico RFLP analysis (4) of the F2n/R2 sequence of six closely related strains (Accession Nos. AF228052, AY390261, AY270156, AY409070, AY409069, and EF186820) of the 16SrVI group using 17 restriction enzymes (AluI, BamHI, BfaI, BsfUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, Sau3AI, RsaI, SspI, and TaqI). In silico restriction digestion and virtual gel plotting showed similar patterns for all enzymes. To our knowledge, this is the first report of a 16SrVI group phytoplasma infecting Portulaca plants in India. References: (1) P. V. Ajayakumar et al. Aust. Plant Dis. Notes 2:67, 2007. (2) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:74, 1999. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) W. Wei et al. Int. J. Syst. Evol. Mic. 57:1855, 2007.

Phytoplasmas and Phytoplasma Diseases: A Severe Threat to Agriculture

“…VI-B Strawberry multiplier disease (MC) AF190224 [147] VI-C Illinois elm yellows (EY-IL1) AF409069/AF409070 [175] VI-D Periwinkle little leaf (PLL-Bd) AF228053 [176] VI-E Centarurea solstitialis virescence (CSVI) AY270156 [177] VI-F Catharanthus phyllody phytoplasma (CPS) EF186819 [25] VI-H Portulaca little leaf phytoplasma (PLL-Ind) EF651786 [178] VI-I Passionfruit (WB-Br4) 'Ca. P. sudamericanum' GU292081 [165] Continued 16SrVII: Ash yellows (America, Europe)…”

Molecular characterization of phytoplasmas of ‘Clover proliferation’ group associated with three ornamental plant species in India

“…In India, 16SrVI group of phytoplasmas was earlier reported to be associated with several diseases of plants, viz. Araucaria little leaf (Gupta et al 2009), brinjal little leaf (Azadvar and Baranwal 2012), Withania little leaf (Zaim and Samad 1995; Samad et al 2006), Portulaca little leaf (Samad et al 2008), Datura little leaf (Raj et al 2009; Singh et al 2012), Croton leaf yellows (Madhupriya et al 2016), and Calotropis gigantea leaf yellows (Madhupriya et al 2010). However, 16SrVI-D subgroup of phytoplasma was only reported with brinjal little leaf (Azadvar and Baranwal 2012) and Catharanthus roseus little leaf (Bertaccini 2015).…”

Plant, Soil and Microbes

“…However, Hibben and Franzen ( 1989 ) reported susceptibility of Syringa to these prokaryotes in the United States. Ajayakumar et al ( 2007 ) and Samad et al ( 2008 ) described a phytoplasma disease associated with little leaf of Portulaca grandifl ora in Lucknow, India. Kaminska and Sliwa ( 2008 ) reported mixed infection of apple proliferation and aster yellows phytoplasmas in dahlia plants.…”