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 'Candidatus Phytoplasma asteris'-related Strain (16SrI group) Associated with Small Leaves, Leaf Yellowing, and Shoot Proliferation of Morning Glory (Ipomoea biflora) in Taiwan.

Ipomoea biflora L., commonly known as morning glory, is an herbaceous vine plant in the Convolvulaceae family and is widespread at low elevations in Taiwan and other East Asian countries. In September 2023, six I. biflora plants exhibiting small leaves, leaf yellowing, and shoot proliferation were observed in a vacant lot in Taiwan Agricultural Research Institute (TARI), Wufeng District, Taichung, Taiwan, representing 100% disease incidence in the area. All the symptomatic morning glory climbed onto Murraya paniculata L. (common jasmine orange) which however showed no similar symptoms. The total DNA (two samples for each plant) from leaf tissues of three symptomatic morning glory plants, two asymptomatic morning glory plants, and one asymptomatic common jasmine orange was isolated by the CTAB method (Fulton et al. 1995) and used for PCR with the universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995), to amplify a fragment containing partial 16S rDNA. Expected 1.8-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no PCR product was detected from that of the asymptomatic I.biflora and M. paniculata plants. Six PCR products were cloned and sequenced in the Biotechnology Center DNA-sequencing facility at National Chung Hsing University, and one representative sequence was selected and deposited in GenBank. BLAST analysis revealed that the obtained 16S rDNA sequence (PP230905) shared 99.92% identity with the following phytoplasma strains: rapeseed phyllody phytoplasma (CP055264), plumbago auriculata leaf yellowing phytoplasma (MN239503), and aster yellows phytoplasma (MK992774), which all belong to the 16SrI subgroup. The query 16S rDNA sequence shares 99.84% identity with that of the 'Candidatus Phytoplasma asteris' reference strain (M30790), suggesting that the phytoplasma is a 'Ca. Phytoplasma asteris'-related strain. A virtual restriction fragment length polymorphism (RFLP) analysis was conducted using iPhyClassifier tool (Zhao et al. 2009), and the pattern derived from the 16S rDNA fragment of the I. biflora phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of 16SrI, subgroup B (onion yellows phytoplasma OY-M; AP006628). Six total DNA samples from symptomatic plants were used as templates to amplify 842 bp secA sequences with SecAfor1 and SecArev3 primers (Hodgetts et al. 2008), and one representative sequence was deposited in GenBank. The partial secA sequence (PP263636) showed 98.22% identity with that of Trema levigatum witches'-broom phytoplasma (MW032212) that also belongs to the 16SrI group (Wan et al. 2021). Phylogenetic analysis of both 16S rDNA and secA confirmed I. biflora phytoplasma as 16SrI, subgroup B. Taken together, we concluded that the morning glory phytoplasma in this study was a 'Ca. Phytoplasma asteris'-related strain belonging to the 16SrI group. To the best of our knowledge, this is the first report of a phytoplasma-infected I. biflora in Taiwan, suggesting morning glory as a new natural host of 16SrI phytoplasmas, alongside other plants like roselle and citrus (Tseng et al. 2014; Feng et al. 2015).

The Efficacy of Orange Terpene and Bacillus mycoides Strain BM103 on the Control of Periwinkle Leaf Yellowing Phytoplasma.

Phytoplasmas are obligate phytopathogenic bacteria belonging to the class Mollicutes. The pathogens, transmitted by insect vectors, associated with hundreds of plant diseases worldwide. Due to the regulation on banning use of antibiotics and limited efficacy of the traditional disease management manners, an eco-friendly alternative is needed. Given that terpene and probiotics have antibiotic activity and the ability to induce systemic resistance, in this study, the effectiveness of orange terpene and a Bacillus mycoides strain, BM103, was evaluated in periwinkle plants infected with periwinkle leaf yellowing (PLY) phytoplasma derived from a shoot-tip tissue culture system. Weekly drenching of 1,000 ppm diluted orange terpene emulsion or pre-activated strain BM103 liquid culture dilution exhibited the ability to inhibit PLY phytoplasma accumulation. The expression of the genes associated with plant defense response and flower development was upregulated after treatment. Moreover, pre-treatment of orange terpene or strain BM103 delayed PLY infection via cleft-grafting inoculation. While orange terpene did not suppress the symptoms, strain BM103 did result in a milder symptom expression that might partially attribute to its plant growth-promoting characteristics. Additionally, the pre-activation of strain BM103 may contribute to its efficacy. Taken together, this research indicates that orange terpene and B. mycoides BM103, with the ability to rapidly induce plant defense responses, could potentially be developed into biological control materials as preventive agents or biofertilizers.

Seed and Pollen Transmission of Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Taiwan Virus, and Tomato Yellow Leaf Curl Thailand Virus in Cucumbers and Tomatoes.

Understanding the seedborne nature of plant viruses is essential for developing disease control strategies and is impactful to the seed market. Here, we investigated seed transmissibility of tomato leaf curl New Delhi virus-cucumber isolate (ToLCNDV-CB) and -oriental melon isolate (ToLCNDV-OM) in cucumber and seed transmissibility of tomato leaf curl Taiwan virus (ToLCTV) and tomato yellow leaf curl Thailand virus (TYLCTHV) in tomato. Parent plants were inoculated using agroinfiltration with virus infectious clones, and virus infection was confirmed by PCR with virus-specific primers. ToLCNDV-CB and ToLCNDV-OM were detected in different parts of the female and male flowers and the fruits of cucumbers. ToLCNDV-CB and ToLCNDV-OM were also detected in cucumber seed coats and seedlings with an infection rate higher than 79%. Similar results were observed with ToLCTV and TYLCTHV as they were detected in different parts of the female and male flowers and fruits of three tomato cultivars. ToLCTV and TYLCTHV were also detected in tomato seed coats and seedlings with an infection rate higher than 36%. In addition, pollen-mediated transmission assays of these four begomoviruses were conducted with pollen derived from virus-infected plants to healthy plants. Results showed that ToLCNDV-CB and ToLCNDV-OM were detected in cross-pollinated cucumber progenies with an infection rate higher than 70%. ToLCTV and TYLCTHV were also detected in cross-pollinated tomato progenies with an infection rate higher than 77%. Our results indicated that ToLCNDV, ToLCTV, and TYLCTHV can be transmitted via seeds or pollens of cucumber and tomato plants. To our knowledge, this is the first report documenting the pollen-mediated transmission of begomoviruses.

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).

TaqMan Quantitative PCR Detection of Xylella taiwanensis in Taiwan.

Xylella taiwanensis (Xt) is a nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, loss of tree vigor, and reduction in fruit yield and quality. No cure for PLSD is available. The only option for growers to control the disease is to use pathogen-free propagation material, which requires early and accurate detection of Xt. Currently, only one simplex PCR method is available for the diagnosis of PLSD. We developed five Xt-specific TaqMan quantitative PCR (TaqMan qPCR) systems (primers-probe sets) for the detection of Xt. The PCR systems target three conserved genomic loci commonly used in bacterial pathogen detection: the 16S rRNA gene (rrs), the 16S-23S rRNA intergenic transcribed sequence (16S-23S rRNA ITS), and the DNA gyrase gene (gyrB). BLAST analysis using the GenBank nr sequence database, including whole genome sequences of 88 Xanthomonas campestris pv. campestris (Xcc) strains, 147 X. fastidiosa (Xf) strains, and 32 Xt strains, showed that all primer and probe sequences were specific only to Xt. Single nucleotide polymorphisms (SNPs) provided the primer/probe specificity to Xt. The PCR systems were evaluated by using DNA samples from pure cultures of two Xt strains, one Xf strain, one Xcc strain, and 140 plant samples collected from 23 pear orchards in four counties in Taiwan. The two-copy rrs and 16S-23S rRNA ITS-based PCR systems (Xt803-F/R, Xt731-F/R, and Xt16S-F/R) showed higher detection sensitivity than the two single-copy gyrB-based systems (XtgB1-F/R and XtgB2-F/R). A metagenomic analysis of a representative PLSD leaf sample detected the presence of non-Xt proteobacteria and fungal pathogens that should be taken into consideration in PLSD, as they might interfere with diagnosis.

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.

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.

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.

A 3D Plasmonic Crossed-Wire Nanostructure for Surface-Enhanced Raman Scattering and Plasmon-Enhanced Fluorescence Detection.

In this manuscript, silver nanowire 3D random crossed-wire woodpile (3D-RCW) nanostructures were designed and prepared. The 3D-RCW provides rich "antenna" and "hot spot" effects that are responsive for surface-enhanced Raman scattering (SERS) effects and plasmon-enhanced fluorescence (PEF). The optimal construction mode for the 3D-RCW, based on the ratio of silver nanowire and control compound R6G, was explored and established for use in PEF and SERS analyses. We found that the RCW nanochip capable of emission and Raman-enhanced detections uses micro levels of analysis volumes. Consequently, and SERS and PEF of pesticides (thiram, carbaryl, paraquat, fipronil) were successfully measured and characterized, and their detection limits were within 5 µM~0.05 µM in 20 µL. We found that the designed 3D plasmon-enhanced platform cannot only collect the SERS of pesticides, but also enhance the fluorescence of a weak emitter (pesticides) by more than 1000-fold via excitation of the surface plasmon resonance, which can be used to extend the range of a fluorescence biosensor. More importantly, solid-state measurement using a 3D-RCW nanoplatform shows promising potential based on its dual applications in creating large SERS and PEF enhancements.

Characterization of a New Monopartite Begomovirus with a Betasatellite Associated with Leaf Curl, Yellow Vein, and Vein Enation in Hibiscus rosa-sinensis.

A new begomovirus, tentatively named hibiscus yellow vein leaf curl virus (HYVLCV), was identified in Hibiscus rosa-sinensis plants showing symptoms of leaf curl, yellow vein, and vein enation on the undersides of the leaf in Taiwan. Sequence analysis of the full-length HYVLCV genome from the rolling cycle amplicon revealed a genome of 2,740 nucleotides that contains six open reading frames and a conserved sequence (5'-TAATATTAC-3') commonly found in geminiviral genomes. HYVLCV shares the highest nucleotide identity (88.8%) with cotton leaf curl Multan virus (CLCuMuV) genome, which is lower than the criteria (91%) set for species demarcation in the genus Begomovirus. No begomoviral DNA-B was detected; however, a begomovirus-associated DNA betasatellite (DNA-ß) was detected. The DNA-ß (1,355 nucleotides) shares the highest nucleotide identity (78.6%) with malvastrum yellow vein betasatellite (MaYVB). Because the identity is slightly higher than the criteria (78%) set for the species demarcation threshold for a distinct DNA-ß species, the DNA-ß of HYVLCV reported in this study is considered the same species of MaYVB and tentatively named MaYVB-Hib. An expected 1,498-bp fragment was amplified with two HYVLCV-specific primers from 10 of 11 field-collected samples. Four independent amplicons were sequenced, revealing 100% nucleotide identity with the HYVLCV genome. Agroinoculation of a dimer of the infectious monopartite genome alone to Nicotiana benthamiana resulted in mild symptoms at 28 days postinoculation (dpi); coagroinoculation with the DNA-ß satellite resulted in severe symptoms at 12 dpi. HYVLCV could be transmitted to healthy H. rosa-sinensis by grafting, resulting in yellow vein symptoms at 30 dpi.

Biological, Pathological, and Molecular Characteristics of a New Potyvirus, Dendrobium Chlorotic Mosaic Virus, Infecting Dendrobium Orchid.

Dendrobium smillieae is one of the popular orchids in Taiwan. This report describes a new potyvirus tentatively named Dendrobium chlorotic mosaic virus (DeCMV) causing chlorotic and mosaic symptoms in D. smillieae. Enzyme-linked immunosorbent assay (ELISA) tests using six antisera against orchid-infecting viruses revealed that only a monoclonal antibody against the potyvirus group reacted positively with crude saps prepared from a symptomatic dendrobium orchid. Potyvirus-like, flexuous, filamentous particles were observed under an electron microscope, measuring approximately 700 to 800 nm in length and 11 to 12 nm in diameter. Sequence analyses revealed that DeCMV coat protein gene shared 59.6 to 66.0% nucleotide sequence identity and 57.6 to 66.0% amino acid sequence identity, whereas the DeCMV complete genome shared 54.1 to 57.3% nucleotide sequence identity and 43.7 to 49.5% amino acid sequence identity with those other known potyviruses. These similarity levels were much lower than the criteria set for species demarcation in potyviruses. Thus, DeCMV can be considered a new potyvirus. The whole DeCMV genome contains 10,041 nucleotides (GenBank accession no. MK241979) and encodes a polyprotein that is predicted to produce 10 proteins by proteolytic cleavage. In a pathogenicity test, results of inoculation assays demonstrated that DeCMV can be transmitted to dendrobium orchids by grafting and mechanical inoculation, as verified by ELISA and western blot analyses using the DeCMV polyclonal antiserum and by reverse transcription polymerase chain reaction using the coat protein gene-specific primers. The inoculated orchids developed similar chlorotic and mosaic symptoms. In conclusion, DeCMV is a novel orchid-infecting potyvirus, and this is the first report of a new potyvirus that infects dendrobium orchids in Taiwan.

Complete nucleotide sequences of M and L RNAs from a new pepper-infecting tospovirus, Pepper chlorotic spot virus.

Pepper chlorotic spot virus (PCSV), newly found in Taiwan, was identified as a new tospovirus based on the molecular characterization of its S RNA. In this study, the complete M and L RNA sequences of PCSV were determined. The M RNA has 4795 nucleotides (nts), encoding the NSm protein of 311 aa (34.5 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1122 aa (127.6 kDa) in the viral complementary (vc) strand. The L RNA has 8859 nts, encoding the RNA-dependent RNA polymerase (RdRp) of 2873 aa (330.8 kDa) in the vc strand. Analyses of the NSm, Gn/Gc and RdRp of PCSV revealed that PCSV is phylogenetically clustered within the watermelon silver mottle virus-related clade. Based on the whole genome sequence, PCSV is closely related to Tomato necrotic ringspot virus and should be classified as a new tospovirus species.

Detection of viruses directly from the fresh leaves of a Phalaenopsis orchid using a microfluidic system.

Early detection of pathogens is crucial for the effective surveillance of diseases. Many efforts have been made to explore methods which can detect these pathogens within a short period of time without requiring a tedious protocol. However, these developed methods have disadvantages such as they are relatively time-consuming or require specialized laboratory facilities. In this work, we have developed an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis from fresh Phalaenopsis orchid leaves. The entire protocol, including ribonucleic acid (RNA) purification, reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection by measuring changes in turbidity was performed on a single chip. This is the first time that an integrated microfluidic system for the detection of viruses infecting the Phalaenopsis orchid has been demonstrated. The sensitivity of the developed system was also explored in this study to validate its performance. FROM THE CLINICAL EDITOR: In this study, the authors report the development of an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis of fresh Phalaenopsis orchid leaves, performing the 3-step protocol using a single chip. Similar methods may find clinical application for fast and accurate detection of viral infections.

Virus-virus interactions alter the mechanical transmissibility and host range of begomoviruses.

Introduction: Begomoviruses are mainly transmitted by whiteflies. However, a few begomoviruses can be transmitted mechanically. Mechanical transmissibility affects begomoviral distribution in the field. Materials and methods: In this study, two mechanically transmissible begomoviruses, tomato leaf curl New Delhi virus-oriental melon isolate (ToLCNDV-OM) and tomato yellow leaf curl Thailand virus (TYLCTHV), and two nonmechanically transmissible begomoviruses, ToLCNDV-cucumber isolate (ToLCNDV-CB) and tomato leaf curl Taiwan virus (ToLCTV), were used to study the effects of virus-virus interactions on mechanical transmissibility. Results: Nicotiana benthamiana and host plants were coinoculated through mechanical transmission with inoculants derived from plants that were mix-infected or inoculants derived from individually infected plants, and the inoculants were mixed immediately before inoculation. Our results showed that ToLCNDV-CB was mechanically transmitted with ToLCNDV-OM to N. benthamiana, cucumber, and oriental melon, whereas ToLCTV was mechanically transmitted with TYLCTHV to N. benthamiana and tomato. For crossing host range inoculation, ToLCNDV-CB was mechanically transmitted with TYLCTHV to N. benthamiana and its nonhost tomato, while ToLCTV with ToLCNDV-OM was transmitted to N. benthamiana and its nonhost oriental melon. For sequential inoculation, ToLCNDV-CB and ToLCTV were mechanically transmitted to N. benthamiana plants that were either preinfected with ToLCNDV-OM or TYLCTHV. The results of fluorescence resonance energy transfer analyses showed that the nuclear shuttle protein of ToLCNDV-CB (CBNSP) and the coat protein of ToLCTV (TWCP) localized alone to the nucleus. When coexpressed with movement proteins of ToLCNDV-OM or TYLCTHV, CBNSP and TWCP relocalized to both the nucleus and the cellular periphery and interacted with movement proteins. Discussion: Our findings indicated that virus-virus interactions in mixed infection circumstances could complement the mechanical transmissibility of nonmechanically transmissible begomoviruses and alter their host range. These findings provide new insight into complex virus-virus interactions and will help us to understand the begomoviral distribution and to reevaluate disease management strategies in the field.

Evaluation of DNA fragments covering the entire genome of a monopartite begomovirus for induction of viral resistance in transgenic plants via gene silencing.

Tomato-infecting begomoviruses, a member of whitefly-transmitted geminivirus, cause the most devastating virus disease complex of cultivated tomato crops in the tropical and subtropical regions. Numerous strategies have been used to engineer crops for their resistance to geminiviruses. However, nearly all have concentrated on engineering the replication-associated gene (Rep), but not on a comprehensive evaluation of the entire virus genome. In this study, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting begomovirus in Taiwan, was subjected to the investigation of the viral gene fragments conferring resistance to geminiviruses in transgenic plants. Ten transgenic constructs covering the entire ToLCTWV genome were fused to a silencer DNA, the middle half of N gene of Tomato spot wilt virus (TSWV), to induce gene silencing and these constructs were transformed into Nicotiana benthamiana plants. Two constructs derived from IRC1 (intergenic region flanked with 5' end Rep) and C2 (partial C2 ORF) were able to render resistance to ToLCTWV in transgenic N. benthamiana plants. Transgenic plants transformed with two other constructs, C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3' end of the C1 ORF), significantly delayed the symptom development. Detection of siRNA confirmed that the mechanism of resistance was via gene silencing. This study demonstrated for the first time the screening of the entire genome of a monopartite begomovirus to discover viral DNA fragments that might be suitable for conferring virus resistance, and which could be potential candidates for developing transgenic plants with durable and broad-spectrum resistance to a DNA virus via a gene silencing approach.

Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct.

Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R(0) plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R(1) progeny of the two resistant R(0) lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R(1) plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

FKBP-type peptidyl-prolyl cis-trans isomerase interacts with the movement protein of tomato leaf curl New Delhi virus and impacts viral replication in Nicotiana benthamiana.

Begomoviruses belonging to the family Geminiviridae are plant-infecting DNA viruses. Begomoviral movement protein (MP) has been reported to be required for virus movement, host range determination, and symptom development. In the present study, the FK506-binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (NbFKPPIase) of Nicotiana benthamiana was identified by a yeast two-hybrid screening system using the MP of tomato leaf curl New Delhi virus (ToLCNDV) oriental melon (OM) isolate (MPOM ) as bait. Transient silencing of the gene encoding NbFKPPIase increased replication of three test begomoviruses, and transient overexpression decreased viral replication, indicating that NbFKPPIase plays a role in defence against begomoviruses. However, infection of N. benthamiana by ToLCNDV-OM or overexpression of the gene encoding MPOM drastically reduced the expression of the gene encoding NbFKPPIase. Fluorescence resonance energy transfer analysis revealed that MPOM interacted with NbFKPPIase in the periphery of cells. Expression of the gene encoding NbFKPPIase was induced by salicylic acid but not by methyl jasmonate or ethylene. Moreover, the expression of the gene encoding NbFKPPIase was down-regulated in response to 6-benzylaminopurine and up-regulated in response to gibberellin or indole-3-acetic acid, suggesting a role of NbFKPPIase in plant development. Transcriptome analysis and comparison of N. benthamiana transient silencing and overexpression of the gene encoding MPOM led to the identification of several differentially expressed genes whose functions are probably associated with cell cycle regulation. Our results indicate that begomoviruses could suppress NbFKPPIase-mediated defence and biological functions by transcriptional inhibition and physical interaction between MP and NbFKPPIase to facilitate infection.

Intelligent image analysis recognizes important orchid viral diseases.

Phalaenopsis orchids are one of the most important exporting commodities for Taiwan. Most orchids are planted and grown in greenhouses. Early detection of orchid diseases is crucially valuable to orchid farmers during orchid cultivation. At present, orchid viral diseases are generally identified with manual observation and the judgment of the grower's experience. The most commonly used assays for virus identification are nucleic acid amplification and serology. However, it is neither time nor cost efficient. Therefore, this study aimed to create a system for automatically identifying the common viral diseases in orchids using the orchid image. Our methods include the following steps: the image preprocessing by color space transformation and gamma correction, detection of leaves by a U-net model, removal of non-leaf fragment areas by connected component labeling, feature acquisition of leaf texture, and disease identification by the two-stage model with the integration of a random forest model and an inception network (deep learning) model. Thereby, the proposed system achieved the excellent accuracy of 0.9707 and 0.9180 for the image segmentation of orchid leaves and disease identification, respectively. Furthermore, this system outperformed the naked-eye identification for the easily misidentified categories [cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV)] with the accuracy of 0.842 using two-stage model and 0.667 by naked-eye identification. This system would benefit the orchid disease recognition for Phalaenopsis cultivation.

Resistance to a DNA and a RNA virus in transgenic plants by using a single chimeric transgene construct.

Tomato leaf curl Taiwan virus (ToLCTWV) and Tomato spotted wilt virus (TSWV) are two major tomato viruses that cause serious economic losses. In this study, a partial C2 gene from ToLCTWV and the middle half of the N gene of TSWV were fused as a chimeric transgene to develop multiple virus resistance in transgenic plants. This construct was introduced into Nicotiana benthamiana and tomato by Agrobacterium-mediated transformation. Several transgenic lines showed no symptom post agro-inoculation with ToLCTWV and displayed high resistance to TSWV. The detection of siRNAs indicated that the resistance was via RNA silencing. This study demonstrated that linkage of gene segments from two viruses with distinct genomic organization, one DNA and the other RNA, can confer multiple virus resistance in transgenic plants via gene silencing.