First Report of a ‘Candidatus Phytoplasma aurantifolia’ Strain Associated with Virescence, Floral Proliferation, and Dwarf Symptoms on Indigofera suffruticosa in Taiwan

Abstract

Indigofera suffruticosa Mill., generally known as anil or Guatemalan indigo, is a perennial shrub planting in tropical and subtropical regions, including Taiwan; it was commonly used to produce the indigo dye, which is still being used in many personal workshops in Taiwan. There were a few reported cases of the association of phytoplasma with Indigofera spp. In 1983, I. suffruticosa showing phyllody and small leaf symptoms was first described, but no molecular identification was conducted (Albanese et al. 1995). Indigofera linifolia with little leaf, phyllody, and proliferation symptoms and Indigofera colutea showing little leaf symptoms in Australia caused by phytoplasma strains were reportedly associated with papaya yellow crinkle disease (Padovan and Gibb 2001). Affected Indigofera juncea and Indigofera hirsuta exhibiting witches’-broom symptoms were reported to be the same as the sesame phyllody phytoplasma isolate in Thailand (Nakashima et al. 1995). In February 2016, I. suffruticosa plants showing virescence, floral proliferation, and dwarf symptoms were collected from commercial fields of Mingjian Township, Nantou County, Taiwan. Two each of symptomatic and asymptomatic samples were collected from the field for further study. Transmission electron microscopic observation revealed the presence of the pleomorphic phytoplasma cells only in the phloem sieve cells of the two symptomatic I. suffruticosa plants. The two asymptomatic and two symptomatic samples were used for total DNA extraction with the modified CTAB method, and the amplicons of the nested PCR with universal primer pairs for nearly full-length 16S rDNA amplification (P1/P7 followed by R16F2n/R16R2) were electrophoresed in 0.8% agarose gel (Fulton et al. 1995; Lee et al. 1998). A 1.2-kb fragment (GenBank accession no. KX268346) was identified only from the two symptomatic I. suffruticosa plants. Analysis of 16S rDNA sequences by iPhyClassifier indicated that the phytoplasma associated with virescence symptoms in I. suffruticosa shared a 98.2% identity with that of the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession no. U15442). Virtual restriction fragment length polymorphism patterns were identical to those of the 16SrII-V subgroup (Zhao et al. 2009). Taken together, the results indicated that the phytoplasma associated with I. suffruticosa in Taiwan is a ‘Ca. P. aurantifolia’-related strain. The occurrence of phytoplasma in I. suffruticosa could have direct implication on economic profit for growers and the handicraft industry in Taiwan. To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma causing virescence, floral proliferation, and dwarf symptoms on I. suffruticosa in Taiwan.