First report of a ‘Candidatus Phytoplasma aurantifolia’-related strain (16SrII-V) associated with phyllody, virescence, and shoot proliferation of sweet William (Dianthus barbatus) in Taiwan

The sweet William (Dianthus barbatus) is an ornamental belonging to the Caryophyllaceae family; the species produces clusters of flowers that comes in various colors and is grown commonly as garden plants (Lim 2014). In February 2021, sweet Williams showing symptoms typical of phytoplasma diseases were found in a garden located in Wufeng District, Taichung, Taiwan (24°04'37.6"N 120°43'20.4"E). Infected plants exhibited virescence and phyllody symptoms and produced an abnormal number of new shoots from the base of the flowers/flower-like structures (Figure S1) as well as the base of the plants. Among the fifteen plants grown in the area, two exhibited such symptoms. The two symptomatic plants, along with five symptomless plants were sampled. Two flower-like structures were collected from each of the symptomatic plants, and two flower samples were collected for each symptomless plant (Figure S2). Total DNA were extracted from each sample using the Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) and subjected to diagnostic PCR using primers P1/P7 (Schneider et al. 1995). All four symptomatic samples produced a 1.8-kb amplicon and the ten symptomless samples did not. The amplification products were diluted fifty-fold and used in a second round of PCR using primers R16F2n/R16R2 (Gundersen and Lee 1996). Again, only the symptomatic samples produced an expected 1.25-kb amplicon. A sample was selected for each plant and the PCR products from the first round of PCR were cloned using the pGEM-T Easy Vector System (Promega Inc.) and sequenced (three clones per sample). Fragments of the 16S rRNA gene (1,248 bp; GenBank accession: MW788688) were analyzed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). Sequences obtained from the two infected plants were identical, and were classified to the 16SrII-V subgroup with similarity coefficients of 1.0; they also shared 98.6% similarity with the sequence of a 'Candidatus Phytoplasma aurantifolia' reference strain (accession: U15442). BLASTn results indicated that the 16S rRNA gene sequences detected were identical to those of 16SrII-V phytoplasmas affecting mungbean (accession: MW319764), lilac tasselflower (accession: MT420682), peanut (accession: JX403944) and green manure soybean (accession: MW393690) found in Taiwan. To corroborate the above results, 16SrII group-specific primers were used to conduct nested and semi-nested PCR targeting the pathogen’s 16S rRNA gene (outer primers: rpF1C/rp(I)R1A; inner primers: rp(II)F1/rp(II)R1; Martini et al. 2007) and immunodominant membrane protein gene (imp; outer primers: IMP-II-F1/IMP-II-R1; inner primers: IMP-II-F2/IMP-II-R1; Al-Subhi et al. 2017). In both assays, the symptomatic samples produced the expected amplicons and the symptomless samples did not. The coding sequence of the imp gene (519 bp; accession: MW755353) was the same among all symptomatic samples, and shared 100% identity with that of the peanut witches'-broom phytoplasma (16SrII; accession: GU214176). To our knowledge, this is the first report of a 16SrII-V phytoplasma infecting sweet Williams in Taiwan. Since 16SrII-V phytoplasmas have also been found infecting mungbeans and peanuts in Taiwan (Liu et al. 2015), the findings here suggest that by serving as a natural host in the field, the sweet William may potentially contribute to the spread of 16SrII-V phytoplasmas to food crops.

Genetic Diversity of Dianthus Plants Revealed by SRAP Markers

Dianthus is a valuable genetic resource for flower breeding. However, study on the genetic similarity of Dianthus plants is rare. In this work, 20 pairs of SRAP markers were used to analyze genetic diversity of 44 Dianthus plants, including 13 lines of wild Dianthus chinensis L., 7 lines of wild Dianthus superbus L. and 24 commercial varieties including Dianthus caryophyllus L., Dianthus plumarius L. and Dianthus barbatus L.. Results showed that precise interspecific and intraspecific genetic diversity was provided in Dianthus plants by using 20 pairs of SRAP molecular markers. The interspecific genetic diversity of Dianthus plants was much abundant and the intraspecific genetic difference of wild Dianthus species was related to their geographical distribution and habitats. In this work, theoretical basis and technical support were provided for crossbreeding and molecular mechanism research of Dianthus plants.

EFFECTS OF WATERING FREQUENCY AND SOIL TYPES ON SEED GERMINATION AND SEEDLING PERFORMANCE OF LESPEDEZA CYRTOBOTRYA AND DIANTHUS BARBATUS

This study was carried out to determine the effect of various watering regimes in different soils on seed germination and seedling establishment for two species (Dianthus Barbatus and Lespedeza cyrtobotrya) The ratio of water used for watering was 16ml water/100g soil. Three watering frequencies (i.e. once daily; once every 2 days; once every 3 days) were applied for two soil types (Leaf mold soil and merchantable soil) for a period of 150 days. The differences in seed germination, seedling survival and seedling growth were monitored in all experimental units. A more regular watering frequency increases soil moisture, and vice versa. The results showed that watering frequency had significant effects on seed germination (p=0.008), number of plant leaves (p=0.05), with visible but statistically insignificant effects on mean plant height (p=0.19) and seedling survival (p=0.3) for two plant species. The total height of plant species also varied greatly within treatments. Positive correlations were recorded between germination rate with leaf number (r=0.79, p=0.0001); plant height with survival rate (r=0.65, p=0.001); plant height with leaf number (r=0.77, p=0.0001); survival rate with leaf number (r=0.66, p=0.0001) etc. Seed germination and aboveground heights of D. barbatus and L. cyrtobotrya decreased at the more regular watering frequency of once daily in both soils. On leaf mold soil, the total height and mean height of L. cyrtobotrya decreased with increased water stress at once every 3 days watering frequency. Also, the total height of D. barbatus decreased with increased water stress at once every 3 days watering frequency on this soil, even though its mean height did not decrease. In contrast on merchantable soil, the total height and mean height of L. cyrtobotrya increased with increased water stress at once every 3 days watering frequency, indicating that this plant is more resistant to stress on this soil. Whereas, the total height and mean height of D. barbatus decreased with increased water stress on this soil. On leaf mold soil, once every 2 days watering was the most suitable watering frequency for both species in terms of achieving best seed germination and seedling establishment. In contrast on merchantable soil, once every 3 days watering frequency was most suitable for L. cyrtobotrya, whereas both once every 2 days and once every 3 days watering frequencies were suitable for D. barbatus on this soil. The results suggest that using the required watering frequency and soil type is imperative to ensure sustainability in plant production and seedling establishment.

Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening

Abstract10 species’ drought resistance cases have been studied, including Paeonia lactiflora, Hemerocallis dumortieri, Physostegia virginiana, Iris lacteal, Hylotelephium erythrostictum, Sedum lineare, Iris germanica, Cosmos bipinnata, Hosta plantaginea and Dianthus barbatus. By researching these drought resistance cases, a suggestion can be given for roof greening. This research sets 3 drought stress scenarios by controlling the soil relative water content (RWC), including moderately drought stress (40%±2% < RWC < 45%±2%), strong drought stress (RWC < 30%±2%) and control group (RWC > 75%±2%). After the seedlings survived the drought stress, the damaging rate of permeability (DRP), total chlorophylls concentrations (Chl), superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (AsAPOD) of seedlings will be measured. Finally, a subordinate function method was applied to assess these species’ drought resistance. Cosmos bipinnata and Physostegia virginiana was dead after having suffered with moderately drought stress and strong drought stress, respectively. Although other species survived, the individual variation was huge especially for physiological and biochemical index. Hemerocallis dumortieri, Iris lactea and Hosta plantaginea’s DRP had little change when they lived in the normal water condition and suffered with drought stress. Most of the species (except Paeonia lactiflora and Sedum lineare) showed a lower SOD activity during moderately drought stress compared with the sufficient soil water condition and strong drought stress condition. The changes of plants’ POD activity and AsAPOD activity are very similar: when drought stress enhanced, the activity of protect enzyme reduced. According to the subordinate function method, the order of plants’ resistance to the drought is as follow: Hosta plantaginea > Sedum lineare > Iris germanica > Hemerocallis dumortieri > Iris lactea >Hylotelephium erythrostictum > Dianthus barbatus > Paeonia lactiflora > Physostegia virginiana > Cosmos bipinnata.

Efecto de tratamientos pregerminativos en semillas de Dianthus barbatus L. cv. ‘Purple’ bajo condiciones controladas

Los claveles son de las flores más apetecidas por sus excelentes características de duración y belleza. Sin embargo, la especie Dianthus barbatus L. cv. ‘Purple’, presenta problemas en la germinación de sus semillas, lo que conlleva incremento de los costos de producción. Por lo anterior el objetivo de la investigación fue evaluar diferentes tratamientos pregerminativos sobre la germinación de semillas de Dianthus barbatus L. cv ‘Purple’. Para esto, se estableció un Diseño Completamente al Azar con diez tratamientos correspondientes a la combinación de tres dosis de KNO3 (150, 200, 250mg L-1) y tres tiempos de imbibición (6, 12, 24 horas) más el tratamiento testigo. Las variables evaluadas fueron: porcentaje de germinación (PG), agua absorbida, velocidad media de germinación (VMG), tiempo medio de germinación (TMG), porcentaje de adaptabilidad (PA). La aplicación de los tratamientos pregerminativos basados en la aplicación de KNO3 combinado con el tiempo de imbibición mostró un efecto positivo sobre el porcentaje de germinación, VMG, TMG, tasa de imbibición y adaptación en semillas de Dianthus barbatus L. cv. ‘Purple’ bajo condiciones controladas. El tratamiento que mejor respuesta generó fue la aplicación de KNO3 en una concentración de 250mg L-1 y seis horas de imbibición el cual presentó diferencias significativas (P≤0,05) frente al tratamiento testigo en la mayoría de las variables evaluadas, esto lo convierte en una alternativa importante en la reducción de costos de producción dentro del proceso de propagación sexual de D. barbatus L. cv ‘Purple’ bajo condiciones controladas.