Biology subjects

Breitbart, M.

Publications and source records attributed to Breitbart, M..

6 recordsLinked to original sources

bioRxiv2025

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid colorimetric detection of pepper mild mottle virus (PMMoV)

Pepper mild mottle virus (Tobamovirus capsica, PMMoV) is a plant virus in the genus Tobamovirus that infects peppers and other members of the family Solanaceae. The virus is transmitted mechanically, poses a significant threat to crops globally, and is one of the most abundant viruses found in human feces and wastewater. Two colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed to detect PMMoV, one targeting the RNA-dependent RNA-polymerase (PMMoV_RdRp) and the other targeting the coat protein (PMMoV_CP). Synthetic gBlock positive controls were used to determine the detection limit of each assay. PMMoV_RdRp detected PMMoV at concentrations greater than or equal to 100 copies/L, the same sensitivity as the RT-qPCR assay for this gene. In contrast, the detection limit of the PMMoV_CP RT-LAMP assay was an order of magnitude greater. Both assays were specific to PMMoV and did not amplify plant host tissue or related tobamoviruses. Since these RT-LAMP assays do not require specialized laboratory equipment and yield positive results within 20-30 minutes, they are advantageous for point-of-use testing. Overall, the RT-LAMP assays described here are sensitive, specific, and more rapid than existing methods for PMMoV detection and quantification and thus have potential widespread applications for agriculture, wastewater treatment assessment, recreational water quality testing, and food safety. HighlightsO_LIRT-LAMP assays were developed for the detection of the PMMoV RdRp and CP genes C_LIO_LIThe RdRp assay matches the detection limit of the established PMMoV RT-qPCR assay C_LIO_LIPositive results are obtained within 20-30 minutes from the reaction start C_LIO_LIThese RT-LAMP assays are specific to PMMoV and do not amplify related viruses C_LIO_LIThese assays are applicable for PMMoV detection across diverse scientific fields C_LI

microbiology↗

bioRxiv2025

Diverse ssRNA viruses associated with Karenia brevis harmful algal blooms in Southwest Florida

Harmful algal blooms (HABs) caused by the dinoflagellate Karenia brevis frequently occur in the eastern Gulf of Mexico, where they negatively impact the environment, human health, and economy. Very little is known about viruses associated with K. brevis blooms, although viral infection of other HAB-forming phytoplankton species can play an important role in bloom dynamics. We used viral metagenomics to identify viruses in 11 pooled seawater samples collected from Southwest Florida, USA in 2021 during a severe, spatiotemporally dynamic K. brevis bloom. Assembled viral genomes were similar to published genomes from the order Picornavirales, family Marnaviridae, and genera Sogarnavirus, Bacillarnavirus, and Marnarnavirus. Viruses from these groups infect bloom-forming diatoms (Chaetoceros sp. and Rhizosolenia setigera) and the raphidophyte Heterosigma akashiwo. We also recovered unclassified Riboviria genomes related to a Symbiodinium positive-sense ssRNA virus sequenced from coral dinoflagellate symbionts. Reverse-transcriptase PCR assays were performed to monitor the occurrence of seven representative virus genomes in these 2021 samples and 43 seawater samples collected during a subsequent, typical bloom between November 2022 and May 2023. Over half of the samples contained multiple viruses, and at least one viral genome was detected in 44 of 54 samples, collected across seasons and years, highlighting the ubiquity of these viruses in this region. Alpha diversity was highest in the summer months and positively correlated with K. brevis cell counts. Multiple regression revealed month and the presence of unclassified Riboviria sequences most similar to dinoflagellate viruses as significant predictors of K. brevis cellular abundance. ImportanceHarmful algal blooms caused by the dinoflagellate Karenia brevis negatively impact the tourism, fisheries, and public health sectors. Anticipated impacts of climate change, nutrient pollution, and ocean acidification may sustain and/or exacerbate K. brevis blooms in the future, underscoring the need for proactive monitoring, communication, and mitigation strategies. This study represents a pioneering effort in monitoring viruses associated with K. brevis blooms. The findings lay the groundwork for studying the effects of environmental drivers on K. brevis blooms and their associated viruses, as well as for exploring the roles of viruses in bloom dynamics and potential applications of viruses as biocontrol agents for K. brevis blooms. Furthermore, the comparison of viral dynamics relative to local and regional bloom dynamics in this study helps inform future monitoring and modeling needs.

microbiology↗

bioRxiv2023

Degenerate PCR primers for potexvirus detection in seagrasses

2.Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity a?ects host-virus interactions. 3. Impact statementPotexviruses are widespread in terrestrial plants; however, the recent discovery of TVX in the seagrass Thalassia testudinum extends their host range to marine flowering plants. Here we use existing Potex-5 and Potex-2RC degenerate primers to explore potexvirus infections in several seagrass species. TVX sequence variants were detected in T. testudinum collected from the eastern Gulf of Mexico, uncovering previously unknown genetic diversity of this poorly understood virus. 4. Data summaryAll sequence data are available in NCBI GenBank under the accession numbers OR827692-OR827705, OR854648, OR863396, OR879052-OR879056, and PP430548-PP430571. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2023

Transglobal spread of an ecologically significant sea urchin parasite

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s led to a persistent phase shift from coral-to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, where urchins experienced gross signs compatible with scuticociliatosis. However, the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of D. setosum occurred along the Sultanate of Omans coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in D. setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

ecology↗

bioRxiv2023

Design and validation of a PCR protocol to specifically detect the clade of Philaster sp. associated with Diadema antillarum scuticociliatosis

Diadema antillarum scuticociliatosis (DaSc), caused by a scuticociliate closely related to Philaster apodigitiformis, has affected Caribbean long-spined urchins since at least January 2022. Quantitative PCR (qPCR) is currently the standard method for detection of this ciliate in tissue and coelomic fluid samples, yet this method requires specialized equipment and is more expensive than standard PCR methods. The DaSc scuticociliate occurs against a backdrop of endo- and ecto-symbiotic ciliates which complicate detection using universal or pan-phylum PCR primer sets. To overcome these limitations, we designed and validated a sensitive and specific PCR primer (scutico-634F) and nested two-step PCR protocol to detect this taxon, which excludes other ciliates associated with D. antillarum and has poor affinity for other related ciliates. This primer and protocol for the DaSc-associated Philaster clade (DaScPc) allow for widely-accessible investigation of this pathogen in new regions and within environmental reservoirs.

microbiology↗

bioRxiv2022

Prophages regulate Shewanella fidelis 3313 motility and biofilm formation: implications for gut colonization dynamics in Ciona robusta

Lysogens, bacteria with one or more viruses (prophages) integrated into their genomes, are abundant in the gut of animals. Prophages often influence bacterial traits; however, the influence of prophages on the gut microbiota-host immune axis in animals remains poorly understood. Here, we investigate the influence of the prophage SfPat on Shewanella fidelis 3313, a persistent member of the gut microbiome of the model marine tunicate, Ciona robusta. Establishment of a SfPat deletion mutant ({Delta}SfPat) reveals the influence of this prophage on bacterial physiology in vitro and during colonization of the Ciona gut. In vitro, deletion of SfPat reduces S. fidelis 3313 motility and swimming while increasing biofilm formation. To understand the in vivo impact of these prophage-induced changes in bacterial traits, we exposed metamorphic stage 4 Ciona juveniles to wildtype (WT) and {Delta}SfPat strains. During colonization, {Delta}SfPat localizes to overlapping and distinct areas of the gut compared to the WT strain. We examined the differential expression of various regulators of cyclic-di-GMP, a secondary signaling molecule that mediates biofilm formation and motility. The pdeB gene, which encodes a bacterial phosphodiesterase known to influence biofilm formation and motility by degrading cyclic-di-GMP, is upregulated in the WT strain but not in {Delta}SfPat when examined in vivo. Expression of the Ciona gut immune effector, VCBP-C, is enhanced during colonization by {Delta}SfPat compared to the WT strain; however, VCBP-C binding to the WT strain does not promote the excision of SfPat in an SOS-dependent pathway. Instead, VCBP-C binding significantly reduces the expression of a phage major capsid protein. Our findings suggest that SfPat influences host perception of this important colonizing commensal and highlights the significance of investigating tripartite dynamics between prophages, bacteria, and their animal hosts to better understand the gut microbiota-host immune axis.

microbiology↗