Biology subjects

Rotondo, F.

Publications and source records attributed to Rotondo, F..

3 recordsLinked to original sources

bioRxiv2026

Validation of a quantitative PCR assay for the detection and quantification of the nematode Litylenchus crenatae in American beech leaf tissue

Beech leaf disease (BLD), caused by the migratory plant pathogenic nematode, Litylenchus crenatae (LC, Anguinidae) is presently one of the most concerning forest diseases in the United States. BLD has spread rapidly across the landscape and is currently found in 15 U.S. states and Ontario, Canadian, making monitoring a top priority. Here, we report on the development and testing of a probe-based qPCR detection assay that integrates previously reported LC-specific primers with a newly designed probe, enabling sensitive and reproducible quantification of nematodes in plant and non-plant matrices. This assay exhibited strong in silico and in planta specificity and high quantitative performance (R2 = 0.988-0.996), with amplification efficiencies of 90.0-96.8%. The assay also performed consistently on LC-spiked spore trap membranes used to monitor the fungal aerobiome, despite matrix-associated shifts in amplification efficiency. These results support the utility of the assay for monitoring and surveillance. The reliability was further evaluated by two independent diagnostic laboratories, demonstrating high inter-laboratory concordance in detection and strong agreement in relative quantification. The analytical sensitivity and reproducible performance of the assay align with key validation principles outlined by the APS Diagnostic Assay Validation Network (DAVN) and support its application as a molecular tool in experimental, diagnostic, and surveillance contexts, with important implications for national and international LC monitoring and disease management.

molecular biology↗

bioRxiv2026

Metagenomics for bacterial spot pathogen and virulence factor tracking for Ohio fresh market tomato and pepper production

Bacterial spot is a consistent threat to global tomato and pepper productions; however, Ohios fresh market production currently lacks the updated surveillance data necessary to provide accurate management solutions. While traditional diagnostics focus on identification of a single causal agent, shotgun metagenomic sequencing (MGS) offers a comprehensive view of the infection court. An assignment-first MGS workflow was developed and validated in this study, utilizing Kraken2 databases to extract Xanthomonas species associated with bacterial spot and to characterize the microbial communities of bacterial spot in Ohio production systems. Through in silico spiking experiments, thresholds were established for bacterial spot identification. Species and pathovar identification via average nucleotide identity (ANI) remained accurate at abundance as low as 0.1%. A minimum of 2% Xanthomonas reads were required for high genome completeness (BUSCO >90%) and 3% for reliable type III secretion system (T3SS) effector profiling. Analysis of 63 samples from fresh-market production fields identified Xanthomonas hortorum pv. gardneri, Xanthomonas euvesicatoria pv. euvesicatoria, and Xanthomonas arboricola residing in symptomatic samples, alongside other taxa including Pseudomonas and Stenotrophomonas. Phylogenetic comparisons of metagenome-assembled genomes (MAGs) were comparable to whole genome sequences (WGS) from the same samples, supporting the reliability of culture-independent diagnostics. These results provide a robust framework for utilizing metagenomics as a diagnostic tool, expanding our knowledge of bacterial spot population structure in Ohio, and uncovering the bacterial communities associated with bacterial spot.

genomics↗

bioRxiv2024

Intercontinental dissemination and diversification of Xanthomonas perforans in tomato production

Emerging and re-emerging plant diseases continue to present multifarious threats to global food security. Considerable recent efforts are therefore being channeled towards understanding the nature of pathogen emergence, their spread and evolution. Xanthomonas euvesicatoria pv. perforans (Xep), one of the causal agents of bacterial spot of tomato, rapidly emerged and displaced other bacterial spot xanthomonads in tomato production regions around the world. In less than three decades, it has become a dominant xanthomonad pathogen in tomato production systems across the world and presents a model for understanding diversification of recently emerged bacterial plant pathogens. Although Xep has been continuously monitored in Florida since its discovery, the global population structure and evolution at the genome-scale is yet to be fully explored. The objectives of this work were to determine genetic diversity globally to ascertain if different tomato production regions contain genetically distinct Xep populations, to examine genetic relatedness of strains collected in tomato seed production areas in East Asia and other production regions, and to evaluate variation in type III effectors, which are critical pathogenicity and virulence factors, in relationship to population structure. We used genome data from 270 strains from 13 countries for phylogenetic analysis and characterization of Xop effector gene diversity among strains. Our results showed notable genetic diversity in the pathogen. We found genetically similar strains in distant tomato production regions, including seed production regions, and diversification over the past 100 years, which is consistent with intercontinental dissemination of the pathogen in hybrid tomato production chains. Evolution of the Xep pangenome, including the acquisition and loss of type III secreted effectors, is apparent within and among phylogenetic lineages. The apparent long-distance movement of the pathogen, together with variants that may not yet be widely distributed, poses risks of emergence of new variants in tomato production.

microbiology↗