The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

OAF is a DAF-like gene that controls ovule development in plants.

We previously found that the RING-type E3 ligase DEFECTIVE IN ANTHER DEHISCENCE1- (DAD1-) Activating Factor (DAF) controls anther dehiscence by activating the jasmonate biosynthetic pathway in Arabidopsis. Here, we show that in Arabidopsis, the DAF ancestor was duplicated into three genes (DAF, Ovule Activating Factor (OAF), DAFL2), which evolved divergent partial functions from their ancestor through subfunctionalization. In this case, DAF-DAD1-JA signaling regulates anther dehiscence, whereas OAF controls ovule development by negatively regulating cinnamyl alcohol dehydrogenase 9 (CAD9) activity and being negatively regulated by miR847 itself in Arabidopsis. Downregulation of OAF or upregulation of CAD9 and miR847 caused similar abortion of ovule formation due to precocious ovule lignification in transgenic Arabidopsis. Interestingly, only one DAF-like gene, PaOAF, exists in the monocot orchids, which has likely evolved through nonfunctionalization and maintains a conserved function as Arabidopsis OAF in regulating ovule development since defective ovules were observed in the virus-induced gene silencing (VIGS) PaOAF Phalaenopsis orchids. The absence of the DAF ortholog and its function in orchids is likely due to the evolution of stamens to a unique pollinium structure that lacks the feature of anther dehiscence. These findings expand the current knowledge underlying the multifunctional evolution and diverse functionalization of duplicate gene pairs within/among plants.

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.

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.

Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of 'Candidatus Phytoplasma luffae' strain NCHU2019 that is associated with witches' broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

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.

Accelerating Complete Phytoplasma Genome Assembly by Immunoprecipitation-Based Enrichment and MinION-Based DNA Sequencing for Comparative Analyses.

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.

First Report of 16SrII-V Peanut Witches' Broom Phytoplasma in Snake Gourd (Trichosanthes cucumerina L.) in Taiwan.

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.

First Report of 16SrII-V Phytoplasma Associated with Green Manure Soybean (Glycine max L.) 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.

Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor.

Amaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

Identification of 16SrII-V Phytoplasma Associated with Mungbean Phyllody Disease in Taiwan.

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 seed harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbean 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 ribosomal RNA (rRNA) and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual restriction fragment length polymorphism 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 peanut witches' broom 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 caused by 16SrII-V subgroup phytoplasma.

Similarities and differences between Mn(II) and Zn(II) coordination polymers supported by porphyrin-based ligands: synthesis, structures and nonlinear optical properties.

Four coordination polymers (CPs) Mn-TMPP (1), Zn-TMPP (2), Mn-THPP (3), and Zn-THPP (4) have been synthesized and characterized (H2TMPP = meso-tetrakis (6-methylpyridin-3-yl) porphyrin; H2THPP = meso-tetrakis (6-(hydroxymethyl) pyridin-3-yl) porphyrin). The one-dimensional (1D) chain compound 1 is formed via a head-to-tail connection of the Mn-TMPP unit, wherein the central Mn2+ features a square pyramidal geometry coordinated by four N atoms from the porphyrin skeleton and one additional N atom from an adjacent Mn-TMPP unit. Compound 2 features an octahedral Zn2+ center associated with four N atoms from the porphyrin skeleton to define the equatorial plane and two additional N donors at the axial positions to give a two-dimensional (2D) CP. The 1D chain of 1 and the 2D layer of 2 possess distinctive molecular structures but nearly identical molecular arrangements in their unit cells viewed along all three crystallographic axes. By contrast, Mn- and Zn-based CPs 3 and 4 supported by the THPP ligand share both identical molecular connectivities and crystal packing. In 3/4, each Mn/Zn center is chelated by four N donors of the porphyrin interior to define the equatorial plane of an octahedron, whose axial sites are occupied by two alcoholic OH groups from a pair of trans-located pyridinemethanol moieties. The third-order nonlinear optical properties of 1-4 investigated using the Z-scan technique at 532 nm revealed reverse saturable absorption and self-focusing effects for all four CPs, with hyperpolarizability values (γ) in the range 1.42 × 10-28 esu to 7.64 × 10-28 esu. These high γ values are comparable to the best porphyrin-based molecular assemblies, demonstrating potential for these materials in optical limiting applications.

Ixeris Chinensis is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan.

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.

Threeflower Tickclover (Desmodium triflorum) is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan.

Three-flower Tick-clover (Desmodium triflorum) is a perennial herbaceous plant that belongs to the family of Leguminosae. Threeflower tickclover widely grows at mid-low altitude regions in Taiwan and is commonly used as a traditional herbal medicine for the treatment of dysmenorrheal, muscle spasm, cough, pain and poisoning. In March 2020, disease symptoms such as little leaf, phyllody, virescence, and witches' broom were observed on threeflower tickclover at the sansheng community park in Mailiao, Yunlin County, Taiwan. Similar disease symptoms were observed on peanut infected with peanut witches' broom (PnWB) phytoplasma grown in the same area (Liu et al. 2015). Leaf samples collected from the healthy and symptomatic threeflower tickclover were used to extract total DNA and protein for PCR and western blotting assays, respectively. Nested PCR was performed with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 for the amplification of 16S ribosomal RNA (rRNA) gene (Lee et al. 1993). A specific DNA fragment of expected size (1.2 kb) for 16S rRNA was only amplified from leaf samples of symptomatic threeflower tickclover. The nucleotide sequence of the amplified DNA fragment using primer pairs P1/P7 was deposited into the GenBank (accession no. MT452308). Blast analysis revealed that MT452308 shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Based on the virtual RFLP pattern of MT452308 generated by iPhyClassifier, the phytoplasma detected in symptomatic threeflower tickclover could be classified into the 16SrII-V subgroup. For western blotting, 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, was used. An expected signal of 19 kDa specific for Imp was only detected in threeflower tickclover exhibiting disease symptoms. Subsequent assays including PCR, DNA sequencing and western blotting further confirmed that the gene encoding a SAP11-like protein (accession no. EMR14684) identified in PnWB phytoplasma was also found in samples of symptomatic threeflower tickclover, and shares 100% identity with each other. Our results indicate that threeflower tickclover, a common weed in Taiwan, may act as an alternative natural host for PnWB phytoplasma, and contributes to the spreading of phytoplasma disease.

Lilac Tasselflower (Emilia sonchifolia) is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain 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.

Nasopharyngeal Swabs Are More Sensitive Than Oropharyngeal Swabs for COVID-19 Diagnosis and Monitoring the SARS-CoV-2 Load.

Objective: Detection of SARS-CoV-2 by oropharyngeal swabs (OPS) and nasopharyngeal swabs (NPS) is an essential method for coronavirus disease 2019 (COVID-19) management. It is not clear how detection rate, sensitivity, and the risk of exposure for medical providers differ in two sampling methods. Methods: In this prospective study, 120 paired NPS and OPS specimens were collected from 120 inpatients with confirmed COVID-19. SARS-CoV-2 nucleic acid in swabs were detected by real-time RT-PCR. The SARS-CoV-2 detection rate, sensitivity, and viral load were analyzed with regards NPS and OPS. Sampling discomfort reported by patients was evaluated. Results: The SARS-CoV-2 detection rate was significantly higher for NPS [46.7% (56/120)] than OPS [10.0% (12/120)] (P < 0.001). The sensitivity of NPS was also significantly higher than that of OPS (P < 0.001). At the time of sampling, the time of detectable SARS-CoV-2 had a longer median duration (25.0 vs. 20.5 days, respectively) and a longer maximum duration (41 vs. 39 days, respectively) in NPS than OPS. The mean cycle threshold (Ct) value of NPS (37.8, 95% CI: 37.0-38.6) was significantly lower than that of OPS (39.4, 95% CI: 38.9-39.8) by 1.6 (95% CI 1.0-2.2, P < 0.001), indicating that the SARS-CoV-2 load was significantly higher in NPS specimens than OPS. Patient discomfort was low in both sampling methods. During NPS sampling, patients were significantly less likely to have nausea and vomit. Conclusions: NPS had significantly higher SARS-CoV-2 detection rate, sensitivity, and viral load than OPS. NPS could reduce droplets production during swabs. NPS should be recommended for diagnosing COVID-19 and monitoring SARS-CoV-2 load. Chinese Clinical Trial Registry, number: ChiCTR2000029883.

Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein.

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein-peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.

Morphology-dependent third-order optical nonlinearity of a 2D Co-based metal-organic framework with a porphyrinic skeleton.

A two-dimensional (2D) Co-based metal-organic framework (MOF) with porphyrinic skeleton forms crystalline plates, flower-shaped clusters, and ultrathin films under optimized conditions, including the use of polyvinylpyrrolidone (PVP) as a surfactant. Ultrathin films demonstrate the best solution-based third-order nonlinear optical properties, featuring a nonlinear transmittance (T) value of 0.54, absorption coefficient (α2) of 9.5 × 10-10 m W-1 and second hyperpolarizability (γ) value of 1.37 × 10-28 esu, which are slightly better than those of the flower-shaped clusters (T = 0.65, α2 = 7.0 × 10-10 m W-1; γ = 1.27 × 10-28 esu), but marginally better than those of the crystalline thin plates (T = 0.94, α2 = 2.4 × 10-10 m W-1; γ = 0.24 × 10-28 esu).