bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.10.21.563392

Recombinase Polymerase Amplification Assay for the Field Detection of Mal Secco Disease by Plenodomus tracheiphilus

Rovetto, E. I.·Garbelotto, M.·Moricca, S.·Amato, M.·La Spada, F.·Cacciola, S. O.
View original source ↗Export metadata ↓

Abstract

In this study, we developed a new diagnostic assay based on the recombinase polymerase amplification (RPA) technology to detect Plenodomus tracheiphilus, the anamorphic fungus responsible for the destructive vascular disease of lemon named mal secco, in infected tissues of host plants. A 142 bp RPA-compatible barcode was sought within the 544 bp Internal Transcriber Spacer (ITS) fragment identified in a previous study and its P. tracheiphilus-specificity was confirmed by BLAST in the NCBI database. This was the premise to design an RPA probe (RPA_Ptrach_Probe). The specificity and inclusivity of the RPA assay were tested on gDNA isolated from tissues of C. limon, isolates of P. tracheiphilus of various origins and axenic cultures of non-target organisms, including fungal and oomycete pathogens typically associated to citrus trees, such as Alternaria spp., Colletotrichum spp., Phyllosticta spp., Penicillium spp., Phytophthora spp. With a detection threshold of 1.0 pg of gDNA the RPA assay proved to be as sensitive as the SYBR(R) Green I Real Time-PCR test included in the diagnostic protocol for P. tracheiphilus of the European and Mediterranean Plant Protection Organization. RPA assay was even more sensitive than Real Time-PCR in tests on DNA samples obtained through a rapid extraction method. In tests, on naturally infected lemon twigs, molecular approaches were comparable to each other and performed better than conventional isolation method. Overall, results of this study demonstrate the potential of RPA for rapid, easy to handle and cost effective in-field diagnosis of mal secco.

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Rovetto, E. I., Garbelotto, M., Moricca, S., Amato, M., La Spada, F., Cacciola, S. O.. 2023-10-21. Recombinase Polymerase Amplification Assay for the Field Detection of Mal Secco Disease by Plenodomus tracheiphilus. https://doi.org/10.1101/2023.10.21.563392

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Trans-branching of polyubiquitin chains orchestrates the DNA replication stress response

Polyubiquitin chain geometry dictates functional consequences of ubiquitylation. Although branched polyubiquitin chains are abundant in cells, little is known about their functions. Here we show that branching on the DNA replication factor PCNA, mediated by the ubiquitin-conjugating enzyme UBE2K and involving lysines 63 and 48 of ubiquitin, orchestrates the sequence of events in response to replication stress. By inducing VCP-dependent extraction of PCNA from chromatin, branching promotes re-priming of stalled forks and necessitates a BRCA1-dependent pathway of daughter-strand gap repair. Our study identifies hyper-accumulation of daughter-strand gaps as the mechanistic basis underlying the toxicity of inhibitors of the PCNA-specific isopeptidase, USP1, in BRCA1-deficient cells. Moreover, an unexpected preference of UBE2K to operate in trans suggests a general timing mechanism to organize hierarchies amongst ubiquitin signals.

molecular biology↗
bioRxiv2026

Impaired proteostasis is an early feature of the diabetic heart in humans and mice

Diabetes and obesity increase cardiac lipid levels leading to cardiomyopathy and heart failure. We hypothesized that intermittent fasting would reduce cardiac lipid levels. Surprisingly, intermittent fasting increased myocardial triglyceride content, but rescued mortality and attenuated cardiomyopathy in mice overexpressing cardiomyocyte acyl-CoA synthetase 1 (MHC-ACSL1). Lipid overload caused cardiomyocyte accumulation of polyubiquitinated protein aggregates containing desmin, a scaffolding intermediate filament protein, which intermittent fasting prevented. Furthermore, intermittent fasting reversed elevated myocardial C16:0 ceramide content, and knockdown of ceramide synthase CerS5 and CerS6 reduced palmitate-induced protein aggregation, highlighting a role for C16:0 ceramides in this pathology. Conversely, impairing aggrephagy with cardiomyocyte-specific p62 ablation induced heart failure in mice fed a high-fat diet, with paradoxically reduced cardiac lipid content. Crucially, non-failing diabetic human hearts also exhibited protein aggregate pathology. Taken together, these results demonstrate that impaired proteostasis characterizes cardiomyopathy from cardiac lipid overload and identify a promising new therapeutic target for this condition.

molecular biology↗
bioRxiv2026

Spatial profiling and neurovascular communication in the developing and adolescent cortex following prenatal alcohol exposure

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used the NanoString Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

molecular biology↗