Plant Disease

Shorea assamica is an evergreen to semi-evergreen tree in the family Dipterocarpaceae. It is widely distributed in China, India, Myanmar, Malaysia, Indonesia, the Philippines, and other regions. In China, it is found in western Yunnan and southeastern Tibet. The species is valued for its aromatic white resin, which serves as an important chemical raw material (Ang and Maruyama 1997). In May 2025, S. assamica trees exhibiting symptoms of witches'-broom disease and plexus bud disease were found at Mengmao, WanDing, Nongdao, Jiegao, and Jiexiang in RuiLi County (97°31′-98°02′ E, 23°38′-24°14′ N), Dehong Prefecture, Yunnan Province, China. Following disease onset, the overall growth of infected plants was significantly slower compared to healthy individuals. Infected plants exhibited excessive branching, shortened internodes, and a clustered growth symptom. Lateral buds on diseased tree frequently developed into abnormal twigs, with many buds forming dense clusters and only a few producing leaves. The disease was designated as S. assamica witches'-broom (SAWB). Across the seven surveyed areas, over 74% of trees displayed symptoms, while the disease incidence in nursery-cultivated seedlings was 66%. The disease was found to be spreading actively. A total of 56 samples were collected from seven areas, including 42 symptomatic plants and 14 asymptomatic plants. The lateral stem tissues of 30 samples were observed under a scanning electron microscope (Hitachi S-3000N). Spherical bodies were found in the phloem sieve cells of symptomatic plants. Total DNA was extracted from 56 plant samples using the CTAB method (Porebski et al. 1997) and subjected to nested PCR testing. Double-distilled water was used as negative control, and the DNA extracted from Dodonaea viscosa affected by D. viscosa witches’-broom disease was used as a positive control. Nested PCR was employed to amplify the pathogen’s 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993), and 1.2 kb segment was produced (GenBank accessions: PZ025091, PZ025092, and PZ025093). PCR specific to the ribosomal protein (rp) gene yielded a segment of approximately 1.2 kb (Lee et al. 2003) (GenBank accessions: PZ028767, PZ028768, and PZ028769). The fragment size from 31 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of S. assamica witches’-broom phytoplasma showed that it shared 99.68% identity with that of Rice orange leaf phytoplasma (GenBank accession: JQ965690). The rp sequence shared 99.83% identity with that of Salix tetradenia witches'-broom phytoplasma (GenBank accession: KC117314). An analysis with iPhyClassifier (Zhao et al. 2013) showed that the virtual RFLP pattern derived from the 16S rDNA fragment of SAWB phytoplasma was identical (similarity coefficient 0.98) to the reference pattern of 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The SAWB phytoplasma is a variant of 16SrI-B. The phylogenetic tree was reconstructed based on 16S rRNA and rp gene sequences using MEGA (Tamura et al. 2013). The analysis was conducted using the neighbor-joining method with 1,000 replicates of bootstrap analysis. The results indicated that the SAWB phytoplasmas grouped into clades including phytoplasmas belonging to 16SrI-B and rpI-B, respectively. In addition, 1-year-old S. assamica were used for grafting assays in nursery, the twigs from infected S. assamica under natural conditions were used as a scion, and the phytoplasma was detected using nested PCR after grafting for 40 days. To the best of our knowledge, S. assamica is a new host of ‘Ca. P. asteris’-related strain (16SrI-B) in China. The newly emerged disease is a threat to S. assamica.

‘Candidatus Liberibacter solanacearum’ (Lso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major threat to carrot production, particularly in the Mediterranean Basin and the Middle East. We investigated the impact of plant age at the time of inoculation on symptom development and yield under greenhouse and outdoor conditions. In greenhouse trials, 1- and 2-month-old plants were inoculated with psyllids harboring Lso. Both age groups showed reduced taproot weight compared to controls, but younger plants were more severely affected, exhibiting 90% yield reduction, while 2-month-old plants showed a 30% reduction. Witches’ broom symptoms appeared earlier in plants inoculated at 1-month-old (5 weeks post-inoculation); however, within two weeks, plants inoculated at two months reached comparable levels of symptom incidence and severity. Additional greenhouse experiments using Lso-free psyllids confirmed that yield losses and disease symptoms were specifically due to Lso infection, and not psyllid feeding. In outdoor trials, we tested the effects of inoculation at 7, 11, 15, and 19 weeks post-sowing (wps) on disease incidence and yield. Significant yield losses occurred only in plants inoculated at 7 and 11 wps, with up to 45% reduction by harvest time. Symptoms began to appear at 20 wps in plants inoculated at 7 and 11 wps. At 23 wps, symptoms also appeared in plants inoculated at 15 wps, with a similar incidence to that observed in the earlier inoculation groups. Molecular detection of Lso at harvest (23 wps) confirmed that all plants inoculated at 19 wps or earlier were infected. These results demonstrate that carrots are highly susceptible to Lso-induced yield losses at the earlier stages of growth and highlight the importance of managing early-season vector pressure to minimize economic damage.

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.