Ixeris chinensis Is a New Host for Peanut Witches’ Broom Phytoplasma, a 16SrII-V Subgroup Strain, in Taiwan

Abstract

Ixeris chinensis (Thunb. ex Thunb.) Nakai, a perennial herbaceous plant that belongs to the family of Asteraceae, is widely distributed at mid-low altitude regions in Taiwan. I. chinensis is commonly used as traditional herbal medicine for the treatment of inflammation, bronchitis, pneumonia, and diarrhea. In March 2020, disease symptoms such as shoot proliferation, phyllody, virescence, purple top, and witches' broom were observed on I. chinensis at the sansheng community park in Mailiao, Yunlin County, Taiwan. Totally, eight I. chinensis plants were checked and half of them were symptomatic. These disease symptoms are similar to those associated with peanut witches’ broom (PnWB) disease identified in the same area (Liu et al. 2015). Three samples mixed with leaf, stem, and flower were tested including one healthy and two symptomatic I. chinensis. The total DNA of each sample was extracted and examined by nested PCR for the amplification of 16S rDNA with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). A specific signal of expected size (1.2 kb) for 16S rDNA was only detected in the symptomatic I. chinensis, but not in healthy I. chinensis. The nucleotide sequence (accession no. MT416114) of the amplified DNA fragment using primer pairs P1/P7 from symptomatic I. chinensis is identical to that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Analysis of the virtual RFLP pattern of MT416114 generated by iPhyClassifier revealed that the phytoplasma detected in symptomatic I. chinensis belongs to a 16SrII-V subgroup. The total protein of each sample was also extracted and examined by western blotting using the polyclonal antibody raised against Imp protein of purple coneflower witches’ broom phytoplasma (Chien et al. 2020), which is identical with that (accession no. ADD59806) of PnWB phytoplasma. An expected signal of 19 kDa specific for Imp was detected in symptomatic I. chinensis, but not in healthy I. chinensis. Subsequent PCR, DNA sequencing and western blotting assays further confirmed that the gene encoding a SAP11-like protein was only detected in symptomatic I. chinensis, and shares 100% identity with that (accession no. EMR14684) of PnWB phytoplasma. Our results indicate that PnWB phytoplasma causes disease in I. chinensis, a common weed, which may act as an alternative natural host and facilitate the spreading of phytoplasma disease in Taiwan.