Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL⁻¹ bacterial DNA and 1 × 10² CFU mL⁻¹ (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii.

BACKGROUND: Pantoea stewartii subsp. stewartii and Maize dwarf mosaic virus (MDMV) infections severely affect corn productivity worldwide. Rapid point‐of‐need diagnoses of quarantine pathogens P. stewartii subsp. stewartii and MDMV are required for early detection, timely disease management and ensuring phytosanitary regulations. Recombinase polymerase amplification (RPA) is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating CRISPR/Cas collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays.RESULTS: RPA‐CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1 × 10⁻⁶ ng DNA μL⁻¹ using real‐time fluorescence and blue light observation methods, and 1 × 10⁻⁴ ng DNA μL⁻¹ using the lateral flow dipstick (LFD). Likewise, RPA‐CRISPR/Cas13a assay detected MDMV coat protein (CP) gene with an ultrasensitive detection limit of 3.69 × 10⁻⁷ ng μL⁻¹ using the real‐time fluorescence and blue light observation methods, and 3.69 × 10⁻⁵ ng μL⁻¹ using the LFD. The dual RPA‐CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays.CONCLUSION: The validated assays provide a useful and sensitive molecular tool for detecting two quarantine pathogens of maize within a minimal resource framework suitable for fast‐tracking the containment strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bacterial fruit blotch (BFB) is a highly destructive seed-borne and seed-transmitted disease caused by the Gram-negative bacterium Acidovorax citrulli that has caused substantial economic losses for the cucurbit industry in China. Despite its potential for economic damage, little is known about the bacterium’s molecular mechanisms of pathogenicity. Toxin–antitoxin (TA) systems are critical for the bacterial stress response. These systems are composed of two genes, toxin and antitoxin, that encode a stable toxin protein and a labile antitoxin protein, respectively. In this study, the genes for the putative HipBA TA system were identified in A. citrulli genomes through bioinformatic analysis. A series of molecular biology experiments have demonstrated that the HipBA TA system exists in A. citrulli Aac5. Furthermore, the transcription of hipA and hipB in A. citrulli Aac5 were induced by pH stress, chloramphenicol stress, and during plant infection. Overall, our results have revealed an active type II TA system, HipBA, in A. citrulli Aac5, and provided insights into its biological functions. These findings contribute to a better understanding of TA systems in plant pathogens.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli , is a devastating seed-borne disease of cucurbit crops. The chemical form of nitrogen fertilizer is a key factor modulating host plant defense and pathogen virulence. In this study, we investigated the effects of three nitrogen regimes on watermelon resistance to BFB by assessing disease severity, seedling biomass, stem vascular morphology, expression of defense-related genes, and in planta growth of A. citrulli . Our results showed that sole ammonium nitrogen application led to a significantly higher disease index compared with nitrate nitrogen alone or a 1:1 nitrate-ammonium mixture. Moreover, nitrate nitrogen application enhanced watermelon seedling growth, calcium uptake, development of stem vascular tissue, and expression of disease resistance-related genes. Temporal dynamics revealed that A. citrulli populations in cotyledons were initially lower under sole ammonium but surpassed nitrate and mixed nitrogen treatments at later infection stages. Furthermore, the expression of type III secretion system genes hrpG and hrpE in A. citrulli from watermelon cotyledons initially decreased and then increased over time, with this fluctuating expression pattern accelerating as the ammonium proportion in the fertilizer increased. This finding suggests that A. citrulli establishes infection more rapidly in watermelon seedlings under sole application of ammonium nitrogen. Together, nitrate nitrogen mitigates BFB severity in watermelon, whereas sole ammonium exacerbates disease likely via combined effects of weakened plant vigor, altered tissue nutrition, and accelerated pathogen infection. This study offers a theoretical foundation for nutrient−based management of watermelon BFB.

Codiaeum variegatum is a valuable ornamental plant with distinct bright yellowing and golden spots on dark green leaves, which resemble virus symptoms. To investigate the factors, especially viral agents, associated with the variegated leaf color of C. variegatum, we performed virome profiling of a single C. variegatum ‘Gold Dust’ leaf sample collected from Hainan, China, using ribosomal RNA-depleted total RNA sequencing on an Illumina NovaSeq 6000 platform. Two novel viruses, with two variants each, belonging to the family Closteroviridae, were detected and characterized: Croton golden spot-associated virus C variants 1 and 2 (CGSaVC-v1 and CGSaVC-v2) of the genus Crinivirus and Croton golden spot-associated virus A variants 1 and 2 (CGSaVA-v1 and CGSaVA-v2) of the genus Ampelovirus. Transmission electron microscopy showed long, flexuous, filamentous virus particles approximately 15 nm in diameter and 760 to 770 nm in length. Molecular screening of 97 variegated individual plant leaves showed a high prevalence of CGSaVA-v2 (90.7%), CGSaVA-v1 (75.3%), CGSaVC-v1 (70.1%), and CGSaVC-v2 (47.4%), while asymptomatic leaves near the meristem tip were mostly free of the target viruses. To our knowledge, this is the first study to demonstrate the significant association between closterovirids and the golden spots. The findings provide novel insights into the genetic diversity of the family Closteroviridae and inform future germplasm conservation and new cultivar development of C. variegatum.

Pathogenic bacteria utilize a type III secretion system (T3SS) to inject type III effectors (T3Es) into plant cells, suppressing plant immunity and facilitating colonization. Paracidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of Cucurbitaceae crops, harbors a functional T3SS like many other plant pathogens. The expression of its T3SS and T3Es is regulated by the two-component system response regulators HrpG and HrpX. Here, we demonstrate that the aspartic acid (Asp) residues at positions 52 and 60 in P. citrulli HrpG are essential for its complete function. Plasmid-mediated complementation of the ΔhrpG mutant with hrpG carrying Asp52→alanine (Ala) or Asp60→Ala mutations failed to restore the ability of P. citrulli to induce a hypersensitive response (HR) in tobacco, whereas the Asp46→Ala mutation fully rescued this phenotype. Furthermore, genomic hrpG point mutations generating strains Aac5 (D52A) and Aac5 (D60A) abolish the activation of hrpX transcription, resulting in decreased HrpX accumulation. Collectively, Asp 52 and Asp 60 in P. citrulli HrpG are essential for transcriptional activation activity of hrpX and HR induction, serving as a potential phosphorylation site (Asp 52) for upstream histidine kinases and a Mg²⁺ coordination site (Asp 60). Given that conserved Asp residues often function as phosphorylation sites in two-component system response regulators, this study provides a foundation for identifying upstream histidine kinases that modulate HrpG activity in P. citrulli.