Phytoplasmas are uncultivated plant-pathogenic bacteria with agricultural importance. Those belonging to the 16SrII group, represented by ‘Candidatus P. aurantifolia’, have a wide range of plant hosts and cause significant yield losses in valuable crops, such as pear, sweet potato, peanut, and soybean. In this study, a method that combines immunoprecipitation-based enrichment and MinION long-read DNA sequencing was developed to solve the challenge of phytoplasma genome studies. This approach produced long reads with high mapping rates and high genomic coverage that can be combined with Illumina reads to produce complete genome assemblies with high accuracy. We applied this method to strain NCHU2014 and determined its complete genome sequence, which consists of one circular chromosome with 635,584 bp and one plasmid with 4,224 bp. Although ‘Ca. P. aurantifolia’ NCHU2014 has a small chromosome with only 471 protein-coding genes, it contains 33 transporter genes and 27 putative effector genes, which may contribute to obtaining nutrients from hosts and manipulating host developments for their survival and multiplication. Two effectors, the homologs of SAP11 and SAP54/PHYL1 identified in ‘Ca. P. aurantifolia’ NCHU2014, have the biochemical activities in destabilizing host transcription factors, which can explain the disease symptoms observed in infected plants. Taken together, this study provides the first complete genome available for the 16SrII phytoplasmas and contributes to the understanding of phytoplasma pathogenicity.

Mungbean (Vigna radiata (L.) R. Wilczek), an important legume crop in Asia, is primarily cultivated in the central-southern region of western Taiwan. In 2020, mungbean exhibiting typical phytoplasma-induced disease symptoms, such as witches’ broom, phyllody, virescence, and proliferation, was observed in Yunlin County, Taiwan. Moreover, the seeds harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbeans demonstrated that the occlusion of sieve tubes resulted from the accumulation of phytoplasma-like bodies in sieve elements along with filament-like structures in sieve pores. The association of phytoplasma in symptomatic mungbean was confirmed by PCR analyses of the 16S rRNA and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma-associated mungbean phyllody disease identified in this study belongs to the 16SrII-V subgroup. BLAST analysis and the phylogenetic analysis indicated that the SAP11-like protein identified in mungbean phyllody disease is identical to PnWB phytoplasma SAP11, which explains the witches’ broom phenotype observed in symptomatic mungbean. The results described in this report confirm that the 16SrII-V phytoplasma, a widely distributed phytoplasma associated with peanut witches’ broom disease in Taiwan, has also infected mungbean. This is not only the first instance of mungbean phyllody disease found in Taiwan, but also the first instance of mungbean phyllody disease causing by 16SrII-V subgroup phytoplasma.

Snake gourd (Trichosanthes cucumerina L.), an annual climbing plant belonging to the family of Cucurbitaceae, is native to Southeast Asia countries, e.g., India, Pakistan, Malaysia, China, and Indonesia. It is commonly consumed as a vegetable and also used as a traditional herbal medicine due to the antidiabetic, anti-inflammatory, antibacterial, hepatoprotective, and cytotoxic activities (Devi 2017). In September 2020, phytoplasma-induced disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom were observed on snake gourd in Yunlin County, Taiwan. The cross-sectional examination of the symptomatic plant by transmission electron microscopy showed typical phytoplasma-like pleomorphic bodies with spherical, oval and tubular shapes in sieve elements. Further examination by nested PCR revealed that a 1.2 kb DNA fragment for 16S rRNA gene was only amplified from symptomatic leaf of snake gourd using the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2. BLAST and iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) analyses on the amplified DNA fragment (accession no. MW309142) revealed that it shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches’ broom (PnWB) phytoplasma, a ‘Candidatus phytoplasma aurantifolia’-related strain (Firrao et al. 2004), and could be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further characterized by western blotting using the polyclonal antibody raised against the Imp of PnWB phytoplasma (Chien et al. 2020a, b). An expected signal of 19 kDa specific for Imp was only detected in the symptomatic snake gourd, but not in healthy snake gourd. Since the disease symptoms caused by phytoplasma infection are highly dependent on the secreted effectors (Namba 2019), phyllogen gene that is responsible for phyllody and virescence symptoms was amplified from symptomatic snake gourd by PCR. BLAST analysis revealed that phyllogen identified in snake gourd is identical with that of PnWB phytoplasma. In Taiwan, species of family Cucurbitaceae such as loofah, bitter gourd, and pumpkin are commonly infected by 16SrVIII phytoplasma (Davis 2017). In this study, we report for the first time that snake gourd, a species of family Cucurbitaceae, was infected by 16SrII-V PnWB phytoplasma in Taiwan.

QING PI DOU, a local variety of soybean (Glycine max (L.) Merrill) with small seed size, is primarily cultivated in the southern region of Taiwan. Due to the advantage of high germination rate, fast growth and high nitrogen fixation capacity, QING PI DOU has widely used as green manure in rotation with rice to increase soil fertility in Taiwan. In the summer of 2020, phytoplasma-induced disease symptoms were observed in QING PI DOU with 23% (18/78) disease incidence in Yunlin County, Taiwan. These plants exhibited severe disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom compared to healthy plants. Leaf samples of the symptomatic plants were subsequently collected and examined through transmission electron microscopy (TEM), PCR, and western blotting analyses. The ultrathin sections of the diseased QING PI DOU were double-stained with uranyl acetate and lead citrate. The typical phytoplasma-like pleomorphic bodies were observed in sieve elements of leaf veins by TEM. To investigate the association of phytoplasma with the diseased QING PI DOU, total DNA extracted by the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) was examined by nested PCR using the phytoplasma universal primer pair P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). The 1.2 kb PCR product specific for 16S ribosomal RNA (16S rRNA) gene was only amplified from symptomatic plants but not from healthy plants. BLAST analysis demonstrated that the sequence (accession no. MW393690) of amplified DNA fragment of 16S rRNA is identical to that of GenBank accession no. NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches’ broom (PnWB) phytoplasma, a ‘Candidatus phytoplasma aurantifolia’-related strain (Firrao et al. 2004). Further analysis on the virtual RFLP pattern of MW393690 generated by iPhyClassifier confirmed that the phytoplasma identified in the diseased QING PI DOU can be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further selected for total cell extracts preparation and characterized by western blotting using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of PnWB phytoplasma (Chien et al. 2020). An expected signal of 19 kDa specific for Imp was only detected in symptomatic plants but not in healthy plants. Moreover, the PCR products encoding SAP11 and phyllogen, the virulence factors responsible for phytoplasma-induced witches' broom and phyllody symptoms (Namba 2019), were also amplified from symptomatic QING PI DOU by PCR using the primer pairs 5’-ATGGCTCCCGAAAAAAATGATAAAGG-3’/5’-TTTTTTAGAATCATCAGGCTTTTTAG-3’ (0.28 kb) and 5’-ATGGATCCAAAACTTCCAGAAACT-3’/5’-GTTTTTTTCATCATTTAAATCAT-3’ (0.27 kb), respectively. Further analysis by BLAST revealed that SAP11 and phyllogen identified in symptomatic QING PI DOU are identical with those of PnWB phytoplasma. To the best of our knowledge, this report is the first to describe phytoplasma-associated soybean (Glycine max L.) witches’ broom disease in green manure soybean in Taiwan.

Lilac tasselflower (Emilia sonchifolia) is an annual herbaceous plant that belongs to the family of Asteraceae. Lilac tasselflower is widely distributed at mid-low altitude regions in Taiwan, and is commonly used as traditional herbal medicine for the treatment of inflammation, rheumatism, dysentery, and analgesic. In March 2020, disease symptoms such as shoot proliferation, phyllody, and witches' broom were observed on lilac tasselflower at the sansheng community park in Mailiao, Yunlin County, Taiwan. Totally, four lilac tasselflower plants were checked and half of them were symptomatic. At the same area, similar symptoms associated with peanut witches’ broom (PnWB) disease were observed (Liu et al. 2015). Samples including one healthy and two symptomatic lilac tasselflower were collected for total DNA and protein extraction used for PCR and western blotting assays, respectively. First, two sets of phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 were used to perform nested PCR for detection of 16S ribosomal RNA (rRNA) gene (Lee et al. 1993). A specific signal of expected size (1.2 kb) for 16S rRNA was only detected in samples of lilac tasselflower exhibiting disease symptoms. The amplified DNA fragment using primer pairs P1/P7 was partially sequenced (accession no. MT420682) with P1 and a nested primer (5’-GGGTCTTTACTGACGCTGAGG-3’). The 1.4 kb nucleotide sequence shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Further analysis by iPhyClassifier, the virtual RFLP pattern of MT420682 confirmed that the phytoplasma detected in symptomatic lilac tasselflower could be classified into the 16SrII-V subgroup. For western blotting, total protein of each sample was examined using the polyclonal antibody raised against Imp protein of purple coneflower witches’ broom phytoplasma (Chien et al. 2020), which shares 100% identity with that (accession no. ADD59806) of PnWB phytoplasma. A specific signal of expected size (19 kDa) for Imp was detected in symptomatic lilac tasselflower, but not in healthy lilac tasselflower. Subsequent PCR, DNA sequencing and western blotting assays further confirmed that the gene encoding a SAP11-like protein detected in samples of lilac tasselflower exhibiting disease symptoms is identical to that (accession no. EMR14684) of PnWB phytoplasma. Our results indicated that lilac tasselflower, which is recognized as a common weed in Taiwan, may facilitate the spreading of phytoplasma disease by acting as an alternative natural host for PnWB phytoplasma.