Plant Disease

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.

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).

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.

Citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), is a destructive disease threatening global citrus industry. Although citrus cultivars differ in HLB sensitivity, how infection alters endophytic bacterial communities in cultivars with contrasting susceptibility remains unclear. Here, we compared endophytic microbiome shifts in leaf and root tissues of HLB-susceptible Shatangju mandarin (Citrus reticulata cv. Shatangju) and HLB-tolerant Shatian pomelo (C. maxima cv. Shatian) at 10 months postgrafting with CLas-infected buds. Infected Shatangju mandarin showed severe symptoms and higher CLas levels in leaves than in roots, while infected Shatian pomelo remained asymptomatic with higher CLas in roots than in leaves. High-throughput 16S rRNA gene sequencing revealed that CLas infection restructured the endophytic microbial community in a tissue-specific manner. CLas-infected Shatian pomelo leaves displayed increased microbial diversity and network complexity, while CLas-infected Shatangju mandarin roots harbored more disease-suppressive taxa like Streptomyces. Across both cultivars, network complexity correlated inversely with CLas abundance. Key genera (e.g., Pseudomonas, Streptomyces, and Prevotella) correlated positively with CLas, suggesting roles in mitigating pathogen effects. Predicted functional profiling indicated activated pathways (amino acid metabolism, terpenoid/polyketide biosynthesis, and xenobiotic biodegradation) potentially supporting host defense in infected tissues. The HLB tolerance of Shatian pomelo appeared linked to tissue-specific bacterial restructuring, particularly recruitment of antibiotic-producing bacteria and enhanced metabolic resilience. These findings elucidated cultivar-specific microbial strategies against CLas, offering insights for microbiome-mediated HLB management.

Citrus huanglongbing (HLB), associated with the bacterium ‘Candidatus Liberibacter asiaticus’ and spread by the Asian citrus psyllid (Diaphorina citri; ACP), poses a significant threat to California’s citrus industry. First identified in Los Angeles in 2012, HLB has since spread through residential areas across Southern California. A risk-based survey (RBS) model has been developed to improve HLB surveillance and intervention. Within this framework, model components change as HLB dynamics shift, requiring regular updates to maintain data accuracy and model reliability. Disease spread is influenced by natural factors, such as ACP establishment and confirmed HLB locations, as well as human-mediated factors like global mobility (travel introduction from HLB-infected countries), transportation of citrus materials, nurseries, packinghouses, farmers’ markets, and proximity to private or otherwise inaccessible lands. Human-mediated risk factors account for approximately 26.3% (18.4 to 38.4%) of HLB incidence across different years, and natural causes predominantly explain the remaining 73.7% (61.6 to 81.7%). Notably, global mobility was crucial for early HLB detection in new areas, whereas ACP density strongly correlated with disease spread once established. A retrospective analysis from 2015 to 2022 evaluated the RBS model’s performance, showing a predictive power of 88 to 97%, which confirms its validity for developing targeted interventions and early detection strategies in California.

Areca palm yellow leaf phytoplasma (AYLP) and Areca palm velarivirus 1 (APV1) are two critical pathogens associated with yellow leaf in areca palm, resulting in devastating damage to areca production. However, evidence of their coinfection remains unclear. Areca palms showing yellow leaf symptoms (suspected to be caused by phytoplasma and velarivirus 1) were surveyed and sampled around Hainan Island of China, and the pathogens were identified and analyzed in the study. The results showed that infectious leaf yellowing symptoms of areca palms primarily occurred in eastern and southern cities and counties of Hainan Island. Molecular identification revealed coinfection of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup) and APV1 in individual areca palm samples. Among 520 samples tested by qPCR, AYLP was detected in 23.46% (n = 122), APV1 in 53.46% (n = 278), and a coinfection with both AYLP and APV1 in 10.96% (n = 57). A statistically significant difference in APV1 detection rate was observed between asymptomatic and symptomatic plantations (P < 0.0001), suggesting that APV1 infection likely represents an important risk factor for yellowing development in cultivated areca palm populations. These results confirm the coinfection of AYLP and APV1 on Hainan Island, offering epidemiological evidence for targeted disease management.

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.