First Report of 'Candidatus Phytoplasma aurantifolia' Associated with the Invasive Weed Eclipta prostrata (L.) in Taiwan.

Eclipta prostrata (L.), commonly known as false daisy of the family Asteraceae, is an erect or prostrate annual herb that grows 5 to 45 cm tall. It is widespread mainly in tropical and subtropical regions like India, China, Taiwan, Thailand, and Brazil (Chung et al., 2017). E. prostrata has very wide medicinal properties accounted by several phytochemicals like thiophene derivatives, steroids, flavonoids, and polypeptides (Feng et. al., 2019). It is also used as a traditional herbal medicine for the treatment of bleeding, hemoptysis and itching, hepatitis diarrhea, and even hair loss (Timalsina et al., 2021). In September 2021, E. prostrata displaying branch proliferation and phyllody symptoms with about 30% (6 were symptomatic and 14 were healthy) incidence rate was observed in Mailiao, Yunlin, Taiwan where phytoplasma disease is permeating and has affected many crops and non-crop species including peanut, mungbean, curl-leaved tobacco, false amaranth, etc. Compared to healthy E. prostrata bearing white ray florets and cream or dull white disk florets, symptomatic ones developed phyllody which is more pronounced on the severely infected ones. Further examination by transmission electron microscope revealed a pleomorphic (circular, elliptical, and bell-shaped) phytoplasma-like organisms accumulated in the sieve elements of the symptomatic leaves. Phytoplasma infection was further confirmed by nested polymerase chain reaction using universal primers P1/P7 (carried out for 12 cycles), followed by R16F2n/R16R2 (carried out for 35 cycles) on the genomic DNA extracted by Plant Genomic DNA Purification Kit (DP022-150, GeneMark) (Lee et al. 1993). Results revealed that the conserved 16S rRNA gene with a 1.2 kb fragment size was amplified only by the symptomatic samples. Furthermore, western blotting was done using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of peanut witches'-broom (PnWB) phytoplasma, a 'Candidatus Phytoplasma aurantifolia' in Taiwan that belongs group to 16SrII (Chen et al. 2021). Consistent with the nested PCR, only the symptomatic samples revealed a specific Imp signal with a size of 19 kDa. To classify the phytoplasma associated with the symptomatic E. prostrata, the DNA sequence (No. OM397418) of the P1/P7 primer pair-amplified DNA fragment was obtained using P1 and a nested primer (5'-GGGTCTTTACTGACGCTGAGG-3'), which shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of PnWB phytoplasma. Further analysis of the virtual RFLP pattern of OM397418 by iPhyClassifier confirmed that the phytoplasma identified in the symptomatic E. prostrata belongs 16SrII-V subgroup. To the best of our knowledge, this is the first report of phytoplasma disease in E. prostrata associated with the 'Ca. P. aurantifolia' in Taiwan.

Detection, Identification, and Molecular Characterization of the 16SrII-V Subgroup Phytoplasma Strain Associated with Digera muricata in Taiwan.

Digera muricata (L.) Mart. is a pantropical annual herb belonging to the Amaranthaceae family. In August 2021, D. muricata with indicative phytoplasma symptoms of phyllody, witches'-broom, and virescence was discovered adjacent to a peanut field in Mailiao, Yunlin, Taiwan. The causal agent of the observed symptoms was detected and identified by a series of molecular characterizations. Sieve elements of the phloem tissue were perused under the transmission electron microscope and revealed the presence of pleomorphic phytoplasma-like organisms. Nested PCR using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 was able to amplify a 1.2-kb DNA fragment for the 16S rRNA gene only from the symptomatic D. muricata. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP further affirmed that the phytoplasma associated with the diseased D. muricata can be classified into the 16SrII-V subgroup. Moreover, displayed evident symptoms were explained by the concomitant detection of PHYL1 and SAP11, the virulence genes responsible for the development of leaf-like flowers and shoot proliferation, respectively. Although phytoplasma infection on the noncrop species does not have a direct economic impact, its role in disease spread and perpetuation is indubitable.

Detection, Identification, and Molecular Characterization of a 16SrII-V Subgroup Phytoplasma Associated with Nicotiana plumbaginifolia.

Nicotiana plumbaginifolia Viviani, commonly known as curl-leaved tobacco, is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani, found in Yunlin County, Taiwan, was observed to have phyllody, virescence, and witches'-broom, which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S ribosomal RNA (rRNA)-based phylogenetic analysis and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches'-broom phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain. Further identification of SAP54/PHYL1 and SAP11 homologs in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches'-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease, which poses a threat to the agricultural sector and food security in Taiwan.

Detection, Identification and Molecular Characterization of the 16SrII-V Subgroup Phytoplasma Strain Associated with Pisum sativum and Parthenium hysterophorus L.

Two unrelated plant species, green pea and parthenium weed, harboring typical phytoplasma symptoms, were discovered in Yunlin, Taiwan. Green pea (Pisum sativum.) and parthenium weed (Parthenium hysterophorus L.) are both herbaceous annual plants belonging to the Fabaceae and Asteraceae families, respectively. Displayed symptoms were witches' broom, phyllody and virescence, which are typical indications of phytoplasma infection. Pleomorphic phytoplasma-like bodies were observed under the transmission electron microscope in the sieve elements of symptomatic green pea and parthenium weed. The iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma associated with the diseased plants belongs to the 16SrII-V subgroup. The disease symptoms of both plants can be explained by the identification of PHYL1 and SAP11 effectors, identical to those of peanut witches' broom phytoplasma. The phytoplasma strains identified in this study present a very close phylogenetic relationship with other 16SrII-V subgroup phytoplasma strains discovered in Taiwan. These results not only convey the local status of the 16SrII-V subgroup phytoplasma strains but also encourage attention to be given to preventing the spread of this threat before it becomes pervasive.