Plant Disease

Welsh onion (Allium fistulosum) is an important aromatic vegetable crop, particularly in Asia (Kim et al. 2023). In November 2025, two-month-old Welsh onion plants showing leaf proliferation, little leaf, and yellowing symptoms were found at low frequency (< 1%) in Fangyuan Township, Changhua County, Taiwan. The plot was around 880 m 2 in size and about 7,200 clumps were grown. Five symptomatic and three symptomless plants were sampled (bagged separately). The plants’ basal leaf tissues were subjected to DNA extraction and PCR analysis. DNA was extracted using a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) using filter tips, and the DNA samples’ quality was verified by successful amplification of a plant 26S rDNA fragment (0.7 kb) using primer pair 28KJ/28C (Cullings 1992). Nested polymerase chain reaction (PCR) assays using phytoplasma 16S rDNA-specific primers were conducted. The nested primer pairs used were P1/P7 and fU5/rU3 (Lorenz et al. 1995), respectively. DNA of periwinkle infected with periwinkle leaf yellowing phytoplasma served as a positive control and no-template controls (nuclease-free water) were included in both PCR rounds. In both tests, only DNA from the five symptomatic plants produced the expected amplicons (1.8 kb and 881 bp for the outer and inner assays). The products from the first and second rounds of PCR were sequenced and assembled. Sequencing chromatograms of the PCR products had single, well-resolved peaks across all positions in all the samples, indicating that only one strain was present. Near-full-length (1,466 bp) 16S rDNA sequences of all five samples were identical to that of a reference ‘Candidatus Phytoplasma australasiaticum’-related strain, NCHU2014 (accession no. CP040925, bp 537,765 to 539,230; 16SrII-A; Rodrigues Jardim et al. 2023), and a representative sequence has been deposited in GenBank (accession no. PX696818). A 1,248 bp fragment (accession no. PX696818, bp 84 to 1,331) of the detected 16S rDNA sequence was excised for analysis using the web tool iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and the virtual RFLP patterns obtained were identical to the reference pattern of 16SrII-A (accession no. L33765; similarity coefficient 1.00). Additional PCR tests targeting the protein translocase gene secY and the elongation factor Tu gene (tuf) of 16SrII phytoplasmas were respectively conducted with the infected samples using primer pairs SecYF1(II)/SecYR1(II) and TUF-II-F1/TUF-II-R1 (Al-Subhi et al. 2017; Lee et al. 2010). The secY (accession no. PX737873; 1,263 bp) and tuf (accession no. PX737874; 1,183 bp) sequences were again found identical to those of the reference strain NCHU2014 (16SrII-A phytoplasma; accession no. CP040925, bp 192,846 to 194,108 and bp 139,150 to 140,332). In Taiwan, 16SrII phytoplasma has been reported affecting various plants, such as seseme and purple coneflower (Rodrigues Jardim et al. 2023; Tseng et al. 2014). In A. fistulosum, however, phytoplasma infection has only been associated with group 16SrI (Cho et al. 2019). The present study is the first report of a 16SrII-A phytoplasma affecting Welsh onion and the findings expand the understanding of the width of the subgroup’s host range in the island. Since Welsh onion is an important culinary crop, and the disease significantly affects its morphology and commercial value, ongoing surveillance of its spread in other regions is warranted.

Pear decline, caused by 'Candidatus Phytoplasma pyri', has emerged in Chilean pear orchards in recent years. While several Cacopsylla species are potential vectors of 'Ca. P. pyri', the disease's full epidemiological cycle remains uncertain. Cacopsylla bidens, present in Chilean orchards, has recently been reported as a vector. This study conducted year-long surveys in two commercial pear orchards across different Chilean regions, capturing C. bidens in zones with 'Ca. P. pyri'-infected pear trees. All developmental stages were collected, with peak abundances occurring in March and April. Outside the study zones, C. bidens were found in pine trees but not in adjacent cultivated areas. Two seasonal morphotypes, summer and winter forms, were identified. Molecular analysis detected 'Ca. P. pyri' in a high proportion of insects, with maximum infection rates in March and April. These findings advance our understanding of 'Ca. P. pyri' spatial and temporal dynamics and its potential role in 'Ca. P. pyri' spreading under Chilean field conditions.

In 2022 and 2024, field surveys were conducted in the Columbia Basin of Washington State, USA, for pathogens transmitted by the beet leafhopper, Circulifer tenellus, including beet curly top virus (BCTV), ‘Candidatus Phytoplasma trifolii’ (CPt), and Spiroplasma citri (S. citri). For these pathogens, an association of symptoms with a causal agent is often complicated by co-infections and the inability to culture CPt (Hu 2021; Rivedal et al. 2022, 2024; Schoener & Wang 2023). In 2022, 35 hemp (Cannabis sativa) plants were observed at the Washington State University (WSU) Research Station in Othello, WA, with typical symptoms of BCTV (a finding published by Jarugula et al. 2023) and we further tested these samples for C. tenellus-associated bacteria. In 2024, 23 and 15 hemp plants observed in Prosser, WA, from the WSU Irrigated Agriculture Research Center and WSU Roza Farm, respectively, with symptoms of CPt infection, including stem fasciation and purpling. Leaf tissue from each plant was ground in liquid nitrogen, DNA extracted using the DNeasy Plant Mini Kit (Qiagen, Inc.), and a multiplex real-time PCR protocol used to detect the C. tenellus-associated pathogens (Swisher Grimm et al. 2023). CPt was identified in 8.6% and 65.8% of plants in 2022 and 2024, respectively, while S. citri was not detected in 2022 but was found in 15.8% of plants in 2024. Co-infections were common. Nested PCR (primers P1/P7 and R16F2n/R16R2, Crosslin et al. 2006) and conventional PCR (primers Trif-SecY-163F, 5ʹ-AGCAGCTAAAAAAGTTAGAAAAAACCTC-3ʹ/Trif-SecY-1040R, 5ʹ-AAATCTAGCGAAAATGATTTTTTGTTTTCA-3ʹ) targeted the CPt 16S rRNA and secY genes, respectively. S. citri infection was confirmed by PCR targeting the spiralin gene (primers SpiralinF/R, Yokomi et al. 2008). All targets were amplified using PrimeSTAR HS DNA polymerase (Takara Bio Inc.). Thermal cycle conditions for CPt 16S rRNA and S. citri spiralin genes consisted of 30 or 40 cycles, respectively, at 98°C for 10 s, 55°C for 15 s, and 72°C for 10 s, followed by a final elongation at 72°C for 5 min. Conditions for CPt secY consisted of 35 cycles at 98°C for 15 s, 55°C for 30 s, and 72°C for 10 s, followed by a final elongation at 72°C for 5 min. Amplicons of size 1,250 bp, 877 bp, and 675 bp for the 16S rRNA, secY, and spiralin genes, respectively, were visualized on an agarose gel stained with GelRed (Sigma-Aldrich) or Ethidium Bromide. For CPt, sequencing of 16S rRNA from seven infected samples (2 from 2022, 5 from 2024) revealed identical sequences (GenBank Accession PV983691) with 100% identity to CPt in U.S. hemp (OQ597521), and sequencing of secY in one sample from 2022 and one from 2024 revealed identical sequences (PX725980), with 100% identity to CPt in U.S. periwinkle (GU004317). For S. citri, two sequenced samples had identical spiralin sequences (PV955037), matching 100% and 99.85% with S. citri found in Oregon cabbage (PV099668) and hemp (OQ969984), respectively. This is the first confirmed report of CPt and S. citri in C. sativa in Washington state. These findings highlight the need to evaluate effects of mollicutes on hemp and underscore the need to develop integrated pest management strategies to reduce vector transmission. In addition, these findings suggest that hemp could serve as a reservoir of the C. tenellus-transmitted pathogens, leading to higher pathogen prevalence across the region, and negatively impacting economically important vegetable and seed crops grown in Washington state that are susceptible to these pathogens.