Plant Disease

Pear decline, associated to ‘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 their potential role in ‘Ca. P. pyri’ spreading under Chilean field conditions.

In July 2025, two loblolly pines (Pinus taeda L.) growing within the 761-acre coastal Carolina Beach State Park (New Hanover County, NC) were observed with conspicuous growth abnormalities. One tree displayed a large witches’ broom arising from the main stem approximately 7 meters above the ground, immediately below the primary canopy, whereas the second showed extensive proliferation of epicormic shoots bearing needles along much of its 12-meter trunk, accompanied by reduced crown development. Needle tissues were collected from both trees, along with samples from four asymptomatic loblolly pines in the same area for the investigation of the possible phytoplasma presence. Total DNA was extracted using a Qiagen DNeasy Plant Pro Kit according to the manufacturer’s instructions. DNA extracts were screened by phytoplasma-specific real-time PCR (Hodgetts et al. 2009), which resulted in detection of phytoplasmas only in samples from the symptomatic trees. The full-length 16S rRNA gene (1,531 bp) was amplified with PCR primers flanking the gene: Tyr-F2 (5’-CAGTCTGTAAAACTGYTCCG-3’) and Ile-R2 (5’-TATCAGGCGTGTGCTCTAAC-3’). Amplicons of the expected size (~1.9 kb) were purified and sequenced using Oxford Nanopore sequencing. Identical consensus sequences were obtained and deposited in GenBank as strains PES-NC3 and PWB-NC4 (accession numbers PZ173280–PZ173281). BLASTn analysis of the 16S rRNA gene sequence showed the highest identity (1,530/1,531 bp; 99.93%) with a ‘Candidatus Phytoplasma pini’-related strain MDPP4 from Maryland (GenBank acc. no. OR147379), and 98.43% identity with the reference strain of ‘Ca. P. pini’ Pin127S from Spain (subgroup 16SrXXI-A; GenBank acc. no. AJ632155). Virtual RFLP analysis of the F2n/R2 region using iPhyClassifier (Zhao et al. 2009) produced a restriction pattern identical to that of the designated reference strain of subgroup 16SrXXI-B (GenBank acc. no. KU242428), supporting assignment to subgroup 16SrXXI-B. To further characterize the phytoplasma strains, a 1960 bp fragment of the secA gene was amplified from each symptomatic tree as previously described by Valiunas et al. (2015). The secA sequences differed by 10 nucleotides and were deposited in GenBank under acc. nos. PZ184669 (PES-NC3) and PZ184670 (PWB-NC4). BLASTn analysis showed 99.64% identity (1,932/1,939 bp) for PES-NC3 and 99.74% identity (1,934/1,939 bp) for PWB-NC4 to the corresponding locus of ‘Ca. P. pini’ strain MDPP (GenBank acc. no. KU242429). Phylogenetic analyses based on both the 16S rRNA and secA gene sequences indicated that the two strains detected in North Carolina cluster with previously reported strains from Maryland, forming a distinct, well-supported clade that is clearly separated from European ‘Ca. P. pini’ strains. This is the first report of ‘Ca. P. pini’-related strains infecting loblolly pine in North Carolina and only the second documented occurrence of this phytoplasma in the United States of America, following its detection in mountain pine (P. pungens Lamb.) in Maryland (Costanzo et al. 2016). The 16S rRNA gene sequences of the Maryland and North Carolina strains differ by a single nucleotide, whereas both U.S. strains differed from the European reference strain of ‘Ca. P. pini’ by 24 nucleotides across the 1,531-bp 16S rRNA gene sequence. These findings expand the known host range and geographic distribution of phytoplasmas associated with pines and suggest that they may be more widespread in the U.S.A. than previously recognized.

Bambusa multiplex, one of the most acclimatized clumping-bamboo species, has been broadly cultivated in China and has significant economical, ecological and ornamental importance (Yuan et al. 2009). In November 2025, more than 30% of B. multiplex plants exhibiting typical witches’ broom, internode shortening, leaf proliferation and clustering of leaves were observed in a horticultural landscaping field in Zhangjiang city, Guangdong Province, China. To confirm the presence of a phytoplasma, total DNA was extracted from fresh leaves of ten symptomatic and four asymptomatic plants using the CTAB method. Conventional nested PCR was performed using primer pairs P1/P7 followed by R16F2n/R16R2 (Gundersen and Lee, 1996), and expected fragment amplified only from the symptomatic plants. The PCR products were sequenced, and deposited in GenBank under the accession number PX998769. BLAST analysis revealed that the sequence shared 99.60% identity with that of the SPLL subgroup reference strain ‘Candidatus Phytoplasma australasiaticum subsp. Ipomoeae’ o7C (GenBank accession no. GCA_024425275.1, 1,244/1,249 bp) (Rodrigues Jardim et al. 2023). Further virtual RFLP pattern analysis of the 16S rRNA sequence by iPhyClassifier (Zhao et al. 2009) verified that the phytoplasma strain, designated as BMW-ZJ, was a member of the 16SrII-A subgroup with a 1.00 similarity coefficient to the reference phytoplasma strain (GenBank accession no. L33765). Moreover, the secY gene was amplified using primer pairs secYwbF1/secYwbR1 followed by secYwbF2/secYwbR2 (Wang et al. 2025), and the sequence(GenBank accession no. PZ135411) shared 100% identity to several ‘Ca. P. australasiaticum’ strains (NTU2011, BAWM-TWN and BAWM-THA-CLP). Phylogenetic analysis based on the 16S rRNA and secY genes conducted by MEGA 12 (Kumar et al. 2024), confirmed that BMW-ZJ was related to ‘Ca. P. australasiaticum’(Bertaccini et al. 2022; Rodrigues Jardim et al. 2023). Earlier, 16SrII phytoplasma associated with bamboo species Dendrocalamus giganteus (Che et al. 2024), and 16SrI phytoplasma associated with Sasa fortunei (Zhang et al. 2009) were reported in Hainan and Shaanxi Province, China, respectively. To our knowledge, this is the first report a ‘Ca. P. australasiaticum’ -related strain associated with Bamboo Witches’ Broom Disease (BWB) in Guangdong, China. The BWB caused by Ca. Phytoplasma (16SrI, 16SrII, 16SrVIII and 16SrXIV group), has been confirmed in several bamboo species in Southeast Asian countries such as India (Yadav et al. 2016; Ravi et al. 2022), Philippines (Dolores et al. 2023), Indonesia (Prasetya et al. 2020) and China (Zhang et al. 2009; Che et al. 2024). This study highlight the need to identify insect vectors and alternative hosts, which will provide theoretical guide to the control of Bamboo Witches’ Broom Disease.

Chokecherry (Prunus virginiana L.) is a native shrub that is widely found across North America and is a host of ‘Candidatus Phytoplasma pruni’ associated with X-disease of stone fruits. This pathogen is of major concern to U.S. cherry and peach growers, with the most recent and ongoing outbreak in the Pacific Northwest causing major economic harm. To investigate the role of chokecherry as a potential reservoir and source of orchard-associated strains, we surveyed wild chokecherry trees across 12 U.S. states, and compared the strains present to those in commercially cultivated Prunus species. Approximately 47% of chokecherry sampled tested positive for a ‘Ca. P. pruni’-related strain and phylogenetic analysis revealed high strain diversity, including six distinct clades and four newly identified lineages. In contrast, ‘Ca. P. pruni’ strains isolated from cultivated cherry and peach trees exhibited lower diversity and were infected by known disease outbreak-associated strains such as NWX. These data suggest limited transmission between wild and cultivated hosts in the western U.S., with patterns indicating spillover from orchards into nearby chokecherry populations rather than the reverse. However, in states east of the Rocky Mountains, chokecherry likely functions as a reservoir for orchard infections.

In 2024, an infestation of corn leafhoppers, Dalbulus maidis (DeLong) (Hemiptera: Cicadellidae), was detected in several commercial corn fields across Oklahoma (OK) (Faris et al. 2024). Corn hybrids in the infested field showed symptoms resembling maize bushy stunt, including chlorotic striping, reddening of leaves, production of multiple ears, and stunting. Visual disease incidence ranged from 25% to 100% among affected fields. Leaf samples from symptomatic plants were collected from 15 commercial corn fields across seven counties in OK (Payne, Logan, Caddo, Kay, Texas, Pottawatomie, and Grady) during September, October, and November of 2024. Each sample consisted of 3-4 symptomatic corn leaves randomly collected in each field. In addition, 43 corn leafhopper samples were collected from the symptomatic fields using handheld vacuums. Each corn leafhopper sample consisted of a minimum of 10 corn leafhoppers per field. In the laboratory, the corn leafhoppers were identified as D. maidis based on morphological characteristics as described (Faris et al. 2024). DNA from the 15 leaf samples was extracted from the leaf midrib and blade tissues using the DNeasy® Plant Mini Kit. Insect DNA was extracted using a CTAB-based method (Gorayeb et al. 2023), with each sample consisting of five pooled insects per field. DNA samples from plants and insects were initially screened using the R16F2n/R16R2 primer pair (Gundersen and Lee, 1996) for the presence of phytoplasmas. Seventeen insect and five plant samples tested positive for phytoplasma. Subsequent phytoplasma screening was performed on insect and plant samples collected from the same fields on the same dates, comprising a total of four insect and five plant samples. To further characterize the phytoplasma, a semi-nested PCR targeting the 16S rRNA gene was performed on these samples (four insect and five plant samples) using primer pairs P1/16S-SR followed by P1A/16S-SR (Wei et al. 2021), yielding an expected amplicon of approximately 1.5 Kb. An additional gene marker, the ribosomal protein (rp) gene, was also amplified using universal primers rpF1/rpR1 (Lee et al. 1998), producing 1.2 Kb PCR amplicons. PCR products were subjected to Sanger sequencing, and 16S rRNA and rp gene sequences were deposited in GenBank under accession numbers PX927497–PX927505 and PX993703-PX993711, respectively. BLASTn analysis of the 16S rRNA sequences showed 99.14–99.93% nucleotide identity with reference strains of ‘Candidatus Phytoplasma asteris’ associated with maize bushy stunt (accessions MW578284 and CP015149). Similarly, the rp gene sequences shared 99.91–100% nucleotide identity with the same reference strains. Using the iPhyClassifier tool (Zhao et al. 2013), the 16S rRNA gene sequences were classified within 16Sr group I, subgroup B, with a similarity coefficient of 1.0 (AP006628). The pairwise comparison and subsequent phylogenetic analysis showed that the OK isolates clustered within the 16SrI-B clade. This finding represents the first report of a 16SrI-B phytoplasma (‘Candidatus Phytoplasma asteris’) associated with maize bushy stunt in OK. Although currently classified as ‘Candidatus Phytoplasma asteris’, a proposal to establish a novel phytoplasma species, ‘Candidatus Phytoplasma zeae’, has been made based on ecological distinctiveness, vector specificity, whole-genome comparisons, and community consensus (Pellegrinetti et al. 2025).

Lethal wilt (LW), known as “Marchitez Letal” in Colombia, is an endemic disease affecting oil palms (Elaeis guineensis Jacq.) and a leading cause of crop loss. The disease is characterized by the drying of leaflets from the tip to the base, primarily impacting the lower third of the plant and progressively moving upward. This progression leads to physiological disturbances, including necrosis at the tips of immature inflorescence bracts (spines) and the detachment of bunch fruits, ultimately causing wilting. As a phytosanitary measure, infected palms are eradicated to prevent further spread of the disease. The primary goal of this research was to identify the bacteria associated with LW and to validate a molecular detection method. A 16S amplicon-based analysis was employed to identify and compare the microbial diversity in LW-affected palm tissues with those of healthy plants. Among the 16 OTUs corresponding to different bacterial genera found in all LW samples, taxonomic classification and symptomatology suggested that the bacteria closely associated with LW belong to the genus ‘Candidatus Liberibacter’. Further phylogenetic analysis indicated that these bacteria are part of the Rhizobiaceae family, grouping closely with other species of the genus ‘Ca. Liberibacter’. The concentration of the pathogen in different oil palm tissues was determined using droplet digital PCR and quantitative PCR, expressed in copies/μl in the LW samples. This study represents the first report of ‘Ca. Liberibacter’ sp. being associated with lethal wilt in oil palms of the Arecaceae family in Colombia. The findings from this research could contribute significantly to the development of effective management strategies to prevent crop losses. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Insecticide applications are commonly recommended for managing Diaphorina citri, the vector of huanglongbing (HLB), but their effectiveness in reducing transmission of ‘Candidatus Liberibacter asiaticus’ (CLas), especially during continuous psyllid influx and shoot growth, remains unclear. This study evaluated the efficacy of foliar application of thiamethoxam and spinetoram in reducing CLas transmission in sweet orange seedlings. Two experiments were conducted up to 13 days after first spray. In experiment 1, CLas-positive psyllids were confined in seedlings every 2 days. Treatments were: control; S14, one spray at day 0; and S7, sprays at days 0 and 7. In experiment 2, psyllids were released for free choice within a screenhouse every 2 days. Treatments were: control, S14, S7, and S3, sprays at day 0 and every 3 days. Sprays on day 0 were over unfolded leaves (V2 stage). Psyllid mortality was assessed in experiment 1. Psyllid occupancy (percentage of seedlings with at least one psyllid) and abundance (number of psyllids per seedling) were assessed in experiment 2. After 6 months, seedlings were tested for CLas by qPCR. In experiment 1, cumulative psyllid mortality was higher in sprayed treatments than in the control for both insecticides, but HLB incidence was similar. In experiment 2, both psyllid occupancy and abundance decreased with shorter spray intervals, with the lowest HLB incidence in S3. These findings suggest that reducing spray intervals can limit CLas transmission during periods of shoot growth and continuous vector pressure. However, CLas transmission was not entirely prevented, highlighting the need for integrated, area-wide management strategies.

Citrus huanglongbing (HLB) is recognized as the most destructive bacterial disease affecting citrus. Among the causal agents, Candidatus Liberibacter asiaticus (Las) is the predominant species causing HLB epidemics worldwide. Detecting this phloem limited bacterium at early or low titer stages and assessing its cellular activity remain major challenges for HLB research and management. To address these limitations, we developed a one step reverse transcription quantitative PCR (RT qPCR) assay for highly sensitive Las detection using total nucleic acids and the established Li 16S rRNA/rDNA primer–probe set. This method simultaneously amplifies both Las 16S rRNA and rDNA in a single reaction, increasing sensitivity by up to 500 fold compared with qPCR, which targets only 16S rDNA. The improved sensitivity reduced false negatives, expanded sampling capacity, and enabled detection of low titer infections previously undetectable. In addition, the higher abundance of 16S rRNA generated a consistent Ct gap between RT qPCR and qPCR, which we used to estimate relative Las cell activity in vivo in host insects and plants and to identify the most effective inoculum sources. This comparative detection strategy was further applied to evaluate inoculum potential, monitor changes in relative cell activity during in vitro cultures, and assess antimicrobial treatments targeting Las. The persistent detection of long lasting, low titer Las infections underscores the complex biology of the HLB causal agent and highlights ongoing challenges for effective disease management.