Utilization of fast <scp>qPCR</scp> techniques to detect the amphibian chytrid fungus: a cheaper and more efficient alternative method

Kerby, Jacob L.; Schieffer, Alicia; Brown, Jennifer R.; Whitfield, Steven M.

doi:10.1111/j.2041-210x.2012.00263.x

Cited by 26 publications

(17 citation statements)

References 22 publications

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“…To test for presence of Bd and ranavirus, we used qPCR following protocols described previously (Boyle et al 2004, Brunner et al 2004, Forson & Storfer 2006, Hyatt et al 2007, Kerby & Storfer 2009, Whitfield et al 2012a, Kerby et al 2013. For both Bd and ranavirus, we extracted nucleic acids from swabs using Qiagen DNeasy Blood and Tissue spin column kits following the manufacturer's protocols.…”

Section: Pathogen Detectionmentioning

confidence: 99%

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs

Whitfield¹,

Geerdes²,

Chacón³

et al. 2013

Dis. Aquat. Org.

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Section: Pathogen Detectionmentioning

confidence: 99%

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs

Whitfield¹,

Geerdes²,

Chacón³

et al. 2013

Dis. Aquat. Org.

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“…The ITS regions are less conserved than those of the 18S and 28S rRNA genes, allowing for primer design that differentiates among closely related fungal species (15,21). This approach has been successfully used to target and quantify the amphibian chytrid pathogen Batrachochytrium dendrobatidis (22)(23)(24)(25). The isolation procedure and culture maintenance have been described previously (26).…”

Section: Methodsmentioning

confidence: 99%

Enumeration of Parasitic Chytrid Zoospores in the Columbia River via Quantitative PCR

Maier

Peterson

2016

Appl Environ Microbiol

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Through lethal infection, fungal parasites of phytoplankton ("chytrids") repackage organic material from the large, effectively inedible, colonial diatoms they infect into much smaller zoospores, which are easier for zooplankton to consume. However, their small size and lack of distinguishing morphological features render it difficult to distinguish zoospores from other small flagellates in mixed assemblages in the natural environment. In this study, we developed and tested a method to quantify chytrid zoospores in field studies using quantitative PCR (qPCR) targeting the internal transcribed spacer 2 (ITS2) region within the rRNA gene cluster. To achieve accurate quantification, the assay was designed to be highly specific for a parasite (Rhizophydium planktonicum) of the diatom Asterionella formosa; however, the approach is applicable to additional host-parasite systems. Parasitic zoospores were detected and quantified in the freshwater reaches of the lower Columbia River, as well as in the salt-influenced estuary and river plume. The coincidence between zoospore abundances and a prevalence of small zooplankton during blooms of large, colonial diatoms in the spring suggests that Columbia River zooplankton may be poised to benefit nutritionally from chytrid zoospores, thus providing a mechanism to retain organic carbon within the system and reduce losses to downstream export. We estimate that ϳ15% of the carbon biomass tied up in blooms of the dominant diatom species is transformed into zoospores through the parasitic shunt during spring. IMPORTANCEThe small size of the parasitic fungi that infect phytoplankton makes it difficult to identify and quantify them in natural systems. We developed and tested a method to quantify these organisms (chytrid zoospores) using a molecular technique that targets the internal transcribed spacer region within the rRNA gene cluster. Using this method, we quantified the abundance of the motile stage of a specific parasite in the freshwater and saltwater-influenced regions of the Columbia River in the U.S. Pacific Northwest. Parasitic chytrid zoospores were found to be present throughout the year and at higher abundances during the spring, when phytoplankton blooms occur. The presence of these organisms indicates not only that they may be responsible for the death of host phytoplankton cells but that they may also provide a readily available food source to small consumers (zooplankton) in the food web of the Columbia River. Z oosporic fungi (chytrids) are common parasites of phytoplankton in aquatic systems. Unlike most fungi, the infective stage is a free-swimming zoospore that seeks out a host, attaches to it, and from successful attachment grows a mature sporangium within which new zoospores develop (1). Laboratory studies have shown that chytrid parasites of phytoplankton can effectively repackage organic material from large, functionally inedible, colonial diatoms into zoospores, which are easier for zooplankton to consume (2-5). A major limitation for field investig...

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“…As it may be difficult to isolate Phytophthora species using traditional techniques, a real-time quantitative PCR (qPCR) assay based on a genus-specific dual-labelled fluorescent TaqMan probe was developed and optimized. This is a sensitive and highly specific molecular diagnostic technique allowing rapid pathogen quantification, even from asymptomatic tissues (Luchi et al, 2005(Luchi et al, , 2006(Luchi et al, , 2013 or water samples (Aw & Rose, 2012), and is especially useful in the case of pathogens that are difficult to culture (Kerby et al, 2013). The qPCR assay was applied to analyse the presence of Phytophthora species in tissues and soil of potted ornamental plants from retail nurseries.…”

Section: Introductionmentioning

confidence: 99%

The potential of symptomless potted plants for carrying invasive soilborne plant pathogens

et al. 2015

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Aim The most common pathway for the movement of plant pests across borders is the horticultural trade in live plants, especially potted ornamentals. Soilborne pathogens, possibly alien and potentially invasive, have a higher possibility of surviving transportation and becoming established at their destination if they are carried in potted plants. The European Union (EU) has an open‐door phytosanitary system, under which any plant that is not specifically regulated can be imported. Inspections are focussed on a small number of economically important plant pests and even then limited to visual examinations of the aerial parts of the plant. Inspections fail to detect regulated pests or others internal to the tissues, or in the soil, if plants appear asymptomatic, or if incipient symptoms are limited to the roots. Phytophthora, a soilborne pathogen universally infamous for its ruinous outbreaks, but poorly regulated in Europe, was chosen to illustrate the risk inherent in the nursery pathway. The aim of this study was to demonstrate the level of infestation by Phytophthora in ornamental plants largely traded to, from and within Europe. Location European Union. Methods As Phytophthora species are not easily isolated, a real‐time PCR assay was developed, based on a genus‐specific TaqMan MGB probe, to detect the pathogens in plant tissues or soil even when present at low concentrations, and before symptoms occurred. Phytophthora species were identified by isolation and sequencing of the ITS (internal transcribed spacer) region. Results Phytophthora was detected by qPCR in 87% of the tested pots and in 70% of the asymptomatic potted plants. Potted plants in soil carried several Phytophthora species without showing any external symptoms. Main conclusions The results of this study strongly support the case for more rigorous European legislation on the trade of live plants in pots. As eradication of soilborne organisms is difficult, if not impossible, an embargo on plant movements into the EU and between member states is the only advisable measure against the spread of these pathogens.

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Utilization of fast qPCR techniques to detect the amphibian chytrid fungus: a cheaper and more efficient alternative method

Cited by 26 publications

References 22 publications

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs

Enumeration of Parasitic Chytrid Zoospores in the Columbia River via Quantitative PCR

The potential of symptomless potted plants for carrying invasive soilborne plant pathogens

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