Fringed Spiderflower (Cleome rutidosperma) Is a New Host for Purple Coneflower Witches’ Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan

Abstract

Fringed spiderflower (Cleome rutidosperma) is an annual herbaceous plant that grows as a weed. Originally from tropical Africa, fringed spiderflower has been introduced and is becoming widely distributed at low altitudes in Taiwan. In June 2019, symptoms such as small leaves, witches’ broom, floral virescence, and phyllody were observed on fringed spiderflowers grown at the agricultural experiment station in Wufeng, Taichung City, Taiwan. These disease symptoms are similar to those associated with purple coneflower (Echinacea purpurea) witches’ broom disease caused by a 16SrII-A subgroup phytoplasma identified in the same area (Tseng et al. 2012; Chang et al. 2015). Leaf samples of spiderflowers exhibiting disease symptoms were collected for western blotting and PCR assays. Total proteins were extracted from plant samples and examined by western blotting using the polyclonal antibody raised against immunodominant membrane protein (Imp, accession no. KPN61379) of Purple Coneflower Witches’ Broom Phytoplasma. A specific signal of expected size (19 kDa) for Imp was detected in the symptomatic leaf samples, but not in healthy leaf samples. For PCR analysis, the total DNA of each sample was extracted using the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) and used as template in nested PCR with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). DNA extracted from leaves sampled on healthy plants, were used as negative control. An amplified DNA fragment of 1.2 kb specific for 16S ribosomal RNA (16S rRNA) obtained from symptomatic leaf samples was gel purified and sequenced. The nucleotide sequence was deposited into the GenBank (accession no. MN213634). Blast analysis revealed that MN213634 is identical to the nucleotide sequence of GenBank accession LKAC01000001.1 [complement (31093..32611)] of phytoplasma associated with purple coneflower witches’ broom disease. Using iPhyClassifier, the virtual RFLP pattern of 16S rDNA fragment (accession no. MN213634) confirmed that the phytoplasma detected in symptomatic fringed spiderflower belongs to the 16SrII-V subgroup. Subsequent PCR and DNA sequencing assays further confirmed that the genes encoding the putative secreted proteins (accession no. WP_054598519 and KPN61138) identified in Purple Coneflower Witches’ Broom Phytoplasma were also found in samples of symptomatic fringed spiderflowers. Our findings revealed that the fringed spiderflower is a new weed plant host for Purple Coneflower Witches’ Broom Phytoplasma. This result expands our knowledge of the natural host range of 16SrII-V subgroup strains.