First Report of 'Candidatus Phytoplasma australasiaticum' Associated with Phyllody, Virescence, and Witches'-Broom Disease in Chrysanthemum morifolium in Taiwan.

Chrysanthemum morifolium (Asteraceae) is commonly grown as commercial cut flowers or pot mums worldwide. Common diseases of chrysanthemum include bacterial blight, fungal diseases, viruses, and phytoplasmas (Verma et al. 2003; Taloh et al. 2020). In June 2022, C. morifolium plants showing virescence, stunting, witches' broom, and phyllody symptoms were observed in 10 plants representing 10% of the estimated 100 plants in a field in Taichung City, Taiwan (Fig. S1). Three symptomatic samples along with three asymptomatic ones were collected for further study. Nested PCR was performed with two primer sets, P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) to amplify nearly full-length of 16S rDNA from the collected samples. The target 1.2-kb DNA band was only amplified from the symptomatic chrysanthemum plants. The amplicons were sequenced and a representative sequence deposited in GenBank under accession number OR501416. This sequence was used to search GenBank database by the Basic Local Alignment Search Tool (BLAST) program through the web service of National Center for Biotechnology Information (NCBI). In the 16S rDNA analyses, the three randomly picked amplicons from chrysanthemum phyllody phytoplasma (CPP) shared 100% identity with one another, and all shared 99.5% identity with the, 'Candidatus Phytoplasma australasiae' reference phytoplasma strain (Y10097). Further analysis using iPhyClassifier (Wei et al. 2007) revealed that CPP was most similar to the pattern of the peanut witches' broom phytoplasma in the 16SrII-A subgroup (GenBank Acc. No. L33765), with a pattern similarity coefficient of 1.0. For confirmation, the secY gene was amplified by secY-F/R primers (Li et al. 2014), the 1.2-kb band was sequenced and deposit in GenBank (Acc. No. OR508986). BLAST analysis showed that the secY sequence of CPP shared 99.93% of sequence identities to several 'Ca. P. australasiaticum' strains (MN543069, CP097312, CP120449, KC953013, MW085916, MW070030, CP040925). The phylogenetic tree analysis based on the secY gene by MEGA11 employing maximum-likelihood algorithm was performed and the bootstrap value was set as 1000 times for support of the stability for the clades. The result showed that CPP is closely related to other strains in 16SrII group (Fig. S2). Taken together, CPP is a 'Ca. P. australasiaticum' related-strain in 16SrII-A subgroup. This is the first report of chrysanthemum as a host of this phytoplasma in Taiwan, and might have an impact to the horticultural industry and the growers.

First Report of Tomato Leaf Curl Cebu Virus Associated with Stachytarpheta jamaicensis Golden Yellow Mosaic Disease in Taiwan.

Stachytarpheta jamaicensis (L.) Vahl, also known as snack weed, is an exotic plant in Taiwan. In April 2021, severe golden yellow mosaic leaves (Fig. S1) were observed on S. jamaicensis plants in Taichung City, Taiwan. Samples from eight symptomatic and two asymptomatic plants were collected from the public flowerbed. Total DNA was extracted from each of the collected samples by using a modified CTAB method (Echevarría-Machado et al. 2005). PCR with Begomovirus degenerate primers (PAL1v1978/PAR1c715; Rojas et al. 1993) was conducted. The expected 1.5-kb fragment was amplified only from the 8 symptomatic samples. Two randomly selected amplicons were cloned on pCRII-TOPO TA vector (Invitrogen Co., San Diego, CA, USA) and sequenced with the ABI3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) at National Chung Hsing University (NCHU). After NCBI BLASTn analysis, the sequences were shown to be most closely related to tomato leaf curl Cebu virus (ToLCCeV) isolates (EU487042, EU487025, KU946997), with 92.4-92.5% nucleotide sequence identity by using the CLUSTAL W method of MegAlign program (DNASTAR, Inc., Madison, WI, USA). A ToLCCeV specific primer pair (FJJ2021-165 /166 5'-ACTTACAGGCCCATGTATCG-3' / 5'-GAATGGGTATCCGAGCACG-3') was designed to amplify and sequence the remaining half of viral DNA. The expected 1.6-kb amplicon was amplified only from the symptomatic samples. The full-length of DNA-A consisted of 2.7-kb nucleotides (ToLCCeV isolate stachy, ON525110 and ON525111) and contained six open reading frames (two in viral sense, V1 to V2 and four in the viral complementary sense, C1 to C4) and the conserved nonanucleotide motif (TAATATTAC). The full-length DNA-A of ToLCCeV stachy isolates shared 99.9% nucleotide identity to each other and 91.2-92.4% and 91.3-92.5% nucleotide identities to other ToLCCeV isolates (EU487042, EU487025, KU946997) available in NCBI GenBank. Besides, ToLCCeV is a monopartite begomovirus that harbors no DNA-B. Thus, there were no bands amplified from the degenerate primer pair for DNA-B (DNABLC2 / DNABLV2; Green et al. 2001). Furthermore, the infectious clone was constructed by using phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) for rolling circle amplification (RCA). The RCA product was partially digested with ApaI (NEB) and ligated into the binary vector pCambia0380 (AF234290). The resulting recombinant vector was transformed into Agrobacterium tumefaciens C58. A. tumefaciens C58, containing the infectious ToLCCeV-Stachy DNA-A vector, was grown overnight in LB broth containing kanamycine (50 µg/ml) at 28°C. S. jamaicensis and Nicotiana benthamiana (Nb, four to six leaf stage) plants were agroinoculated to confirm the infectivity of the ToLCCeV clone. The leaf curling and blister symptoms were observed on the Nb systemic leaves 17-day post inoculation (dpi) and the golden yellow mosaic symptom noticed on S. jamaicensis systemic leaves 30-dpi. The presence of the viral DNA in the inoculated plants was confirmed by PCR using the specific primer pair of ToLCCeV. To the best of our knowledge, this is the first report of the monopartite begomovirus, ToLCCeV, associated with golden yellow mosaic disease in S. jamaicensis in Taiwan. The existence of ToLCCeV might severely impact the tomato and pepper industry because they are the natural hosts of ToLCCeV (Tsai et al. 2011) and ToLCCeV may be transmitted by the whitefly, Bemisia tabaci, in Taiwan (Ko et al. 2005).

First Report of Bidens Mottle Virus Causing Mild mottle and Leaf Distortion in Stachytarpheta jamaicensis in Taiwan.

Stachytarpheta jamaicensis, a traditional herbal pharmacological plant in the Family Verbenaceae that produces purplish-blue flowers, is mainly used as a garden plant in tropical and subtropical areas, including Taiwan. A begomovirus, stachytarpheta leaf curl virus (StaLCV) that caused disease on S. jamaicensis, has been reported (Xiong et al. 2005). In 2021, five symptomatic plants with mild mottle and leaf distortion (Fig. S1A, B) and three asymptomatic plants were collected in Taichung City, Taiwan. Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR assays using degenerate primer pairs with expected amplicon sizes of 1.2-1.3 kb (PAL1v1978/PAR1c715; Rojas et al. 1993), 312 bp (dTospo-F2/dTospo-R2; Huang et al. 2018), and 600-750 kb (Hrp5/Pot1; Chen et al. 2006, Colinet and Kummert 1993) for Begomovirus, Orthotospovirus, and Potyvirus, respectively were performed using total DNA and total RNA plant extracts. Results showed the expected fragments were only amplified from the 5 symptomatic plants using Potyvirus degenerate primers. Three out of five randomly picked amplicons, coding the 3'-end of nuclear inclusion b protein (NIb) and 5'-end coat protein (CP) genes, were cloned and sequenced with the ABI3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) in Biotechnology Centre DNA-sequencing facility at National Chung Hsing University (NCHU). After NCBI BLASTN analysis, the sequences were shown to be most closely related to bidens mottle virus (BiMoV). The nucleotide sequence identities analyzed using the CLUSTAL W Methods of MegAlign program (DNASTAR, Inc., Madison, WI, USA), showed the three amplicons shared 95.8-99.8% to each other and 94.3-97.1% with 18 BiMoV isolates available in NCBI GenBank. Further RT-PCR with a specific primer (FJJ2021-278) designed from the CP of previously amplified amplicons, paired with oligo d(T) primer, were used for amplification of the 3'-CP gene and 3'-untranslated region (UTR) from total RNAs purified from symptomatic plants. The full-length CPs (804-nt and 268-aa) of the BiMoV isolates described here (Acc. Nos. OM406329 and OM406330; designated as isolate Stachy3 and Stachy7, respectively) shared 96.5-98.5% nucleotide and 97.8-99.3% amino acid identity to other BiMoV isolates. The isolate used for back-inoculation to S. jamaicensis was selected after the completion of triple single chlorotic local lesion isolation in Chenopodium quinoa. Two mechanically-inoculated S. jamaicensis plants exhibited symptoms 14-16 days post-inoculation similar to those observed in field plants and tested positive in RT-PCR using BiMoV-specific primers. In transmission electron microscopy, crude sap extracted from mechanically-inoculated C. quinoa and stained with uranyl acetate (UA) revealed flexuous filamentous virions of approximately 720 × 12 nm (Fig. S1C). A western blot assay using BiMoV antiserum (Chen and Lee 2012) revealed bands of about 34 KDa only from the mechanically-inoculated C. quinoa and the five symptomatic S. jamaicensis plants collected from the field. Taken together, we believe this is the first report of BiMoV infecting and causing mild chlorotic mottle and leaf distortion on S. jamaicensis. S. jamaicensis may serve as a new alternative host of BiMoV that can spread the disease, and consequently may directly impact the producers of horticultural or economical crops, such as lettuce, calendula, sunflower, lisianthus, and garland chrysanthemum in Taiwan.

First Report of 'Candidatus Phytoplasma asteris' (16SrI group) Associated with Murraya exotica Witches'-Broom Disease in Taiwan.

Murraya exotica L., commonly known as orange jasmine, is an evergreen shrub belonging to the Rutaceae family. It has long been used as traditional Chinese medicine for treating abdominal pain, toothache, scabies, and other disorders (Liu et al. 2018). M. exotica is widely grown as a garden bush in Taiwan. A prokaryotic pathogen, 'Candidatus Liberibacter asiaticus' (Damsteegt et al. 2010), reportedly could infect M. exotica, but there is no reported phytoplasma disease in M. exotica. In June 2020, M. exotica plants exhibiting witches'-broom (WB), leaf yellowing, and small leaves (Fig. s1) were observed in a horticultural landscaping field in Taichung City, Taiwan. It was estimated that more than 70% of M. exotica plants within a single area were affected. DNA was extracted separately from petioles of five symptomatic and one asymptomatic plants using a modified CTAB method (Echevarría-Machado et al. 2005) and used for nested PCR with two universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995) followed by R16F2n/R16R2 (Gundersen and Lee 1996) to amplify a 1.2-kb 16S rRNA fragment. PCR was also conducted by primers, rp(I)F1A/rp(I)R1A to amplify a partial ribosomal protein S3 and L22 (rplV-rpsC) fragment (Lee et al. 2004). Expected 1.2-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no bands were amplified from that of the asymptomatic plant. The amplicons were cloned, sequenced with an ABI 3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) in Biotechnology Centre DNA-sequencing facility at National Chung Hsing University (NCHU) and deposited in GenBank. BLAST analysis revealed that 16S rDNA sequences (MZ373297 and MZ373298) shared 100% identity to each other and both shared 99.4% identity with those of several phytoplasma strains, e.g., rapeseed phyllody phytoplasma (CP055264), Brassica sp. phyllody phytoplasma (MN877914), Plumbago auriculata leaf yellowing phytoplasma (MN239504), and aster yellows phytoplasma (MK992774), which all belonging to the 16SrI group, by using the CLUSTAL W Methods of MegAlign program (DNASTAR, Inc., Madison, WI, USA). Further analysis using iPhyClassifier tool (https://plantpathology.ba.ars.usda.gov) indicated that the virtual restriction fragment length polymorphism (RFLP) patterns derived from the 16S rDNA F2nR2 fragment of the M. exotica WB phytoplasma was most similar to the reference pattern of the 16SrI-B subgroup, with a pattern similarity coefficient of 0.97 and shared 99.3% sequence identity to 'Candidatus Phytoplasma asteris' (M30790). The partial rplV-rpsC gene sequence (OM275408) showed 99.7% of sequence identities to those of rapeseed phyllody phytoplasma (CP055264), plum witches'-broom phytoplasma (MH061366) and oilseed rape phytoplasma (KX551965), by using the CLUSTAL W Methods of MegAlign program. Taken together, we concluded that the phytoplasma strain associated with M. exotica WB disease was a strain belonging to a 16SrI. To the best of our knowledge, this is the first report of M. exotica being infected by a phytoplasma in the aster yellows group, and M. exotica may also serve as an intermediate reservoir host to other plants, e.g., wax apple, periwinkle and roselle, of 16SrI phytoplasma.

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification.

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous plants and seeds intended for import and export.

Combination of mesenchymal stem cell-conditioned medium and botulinum toxin type A for treating human hypertrophic scars.

BACKGROUND: Both mesenchymal stem cell-conditioned medium (MSC CM) and Botox have demonstrated therapeutic effects for hypertrophic scar (HS). It is unclear whether a synergistic effect occurs when these treatments are used in combination. We aimed to investigate the therapeutic effects of MSC CM and Botox alone when compared with those of a combined regimen on HS. METHODS: Fibroblasts from human HS were isolated and treated with Dulbecco's modified Eagle's medium (DMEM), MSCCM, or Botox alone or a combination of MSCCM and Botox. We also used an in vivo HS-buried null mice model to investigate the efficacy of combination treatment. RESULTS: The results demonstrated that the combination of MSC CM and Botox downregulated both mRNA and protein levels of type I collagen, type III collagen, and alpha-smooth muscle actin (α-SMA) in HS fibroblasts. The combined regimen also suppressed fibroblast proliferative activity, increased apoptosis, and displayed significant inhibitory effects on the contractile ability of HS fibroblasts compared to MSC CM, Botox, or DMEM alone. Using an in vivo HS-buried null mice model, significant scar weight reduction, cell apoptosis, and less α-SMA expression were observed from the combined regimen of MSC CM and Botox compared to those from the other groups. The combined regimen also significantly improved arrangement and deposition of collagen fibers. CONCLUSIONS: This study demonstrates that a combination of MSC CM and Botox exhibited a significant therapeutic effect compared to monotherapy. Clinical translation of this therapy should be further considered.

Bone marrow concentrate-induced mesenchymal stem cell conditioned medium facilitates wound healing and prevents hypertrophic scar formation in a rabbit ear model.

BACKGROUND: Hypertrophic scars (HSs) are formed via an aberrant response to the wound healing process. HSs can be cosmetic or can result in functional problems. Prolonged proliferation and remodeling phases disrupt wound healing, leading to excessive collagen production and HS formation. However, there are currently no satisfactory drugs to prevent HS formation. Mesenchymal stem cell (MSC) conditioned medium (CM) has therapeutic effects on wound healing and preventing HS formation. Bone marrow concentrate (BMC) contains various growth factors and cytokines that are crucial for regeneration and has been applied in the clinical setting. In this study, we evaluated the effects of BMC-induced MSC CM on HS formation in a rabbit ear model. METHODS: We established a rabbit ear wound model by generating full-thickness wounds in the ears of rabbits (n = 12) and treated wounds with MSC CM, BMC CM, or BMC-induced MSC CM. Dermal fibroblasts from human hypertrophic scar were stimulated with transforming growth factor beta 1 (TGF-ß1) for 24 h and cultured in each culture medium for 72 h. We measured the hypertrophic scar (HS) formation during the skin regeneration by measuring the expression of several remodeling molecules and the effect of these conditioned media on active human HS fibroblasts. RESULTS: Our results showed that BMC-induced MSC CM had greater antifibrotic effects than MSC CM and BMC CM significantly attenuated HS formation in rabbits. BMC-induced MSC CM accelerated wound re-epithelization by increasing cell proliferation. Additionally, BMC-induced MSC CM also inhibited fibrosis by decreasing profibrotic gene and protein expression, promoting extracellular matrix turnover, inhibiting fibroblast contraction, and reversing myofibroblast activation. CONCLUSIONS: BMC-induced MSC CM modulated the proliferation and remodeling phases of wound healing, representing a potential wound healing agent and approach for preventing HS formation.

Effect of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts on testosterone release from rat Leydig cells.

Adlay has been used as a traditional Chinese medicine for the treatment of many diseases. However, few studies have reported the effects of adlay seeds on the endocrine system. In the present study, the effects of methanol extracts of adlay hull (AHM) on testosterone synthesis were studied. Rat Leydig cells were incubated with different reagents including human chorionic gonadotropin, 8-bromo-adenosine-3',5'-cyclic monophosphate, forskolin, A23187, progesterone and androstenedione in the presence or absence of AHM. The rat anterior pituitary (AP) gland was treated with gonadotropin-releasing hormone (GnRH) in vitro in the presence or absence of AHM, and the concentrations of luteinizing hormone (LH) in the media were measured. AHM decreased testosterone release via the inhibition of (1) the PKA and PKC signal transduction pathways, (2) 17beta-HSD enzyme activity in rat Leydig cells, and (3) in vitro GnRH-induced LH secretion.