Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

Abstract

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

Tendencias en investigación y desarrollo tecnológico en la cadena productiva de aguacate (Persea americana L.)

“…Los tres autores de mayor relevancia de acuerdo con el número de publicaciones fueron: Randy C. Ploetz, profesor de "Plant Pathology" en el "Tropical Research & Education Center-Institute of Food & Agricultural Sciences (IFAS)", quien investiga temas relacionados con patologías e insectos que afectan a diversos cultivos frutales (Ploetz, et al, 2011;Ploetz, Hughes, et al, 2017;Ploetz, Konkol, et al, 2017). Daniel Carrillo, vinculado con la Universidad de Florida, y el "Tropical Research and Education Center", este investigador trabaja en acarología, entomología y control biológico (Carrillo, Duncan y Pea, 2012, Carrillo, 2014Carrillo, et al, 2020). Y Paul Kendra, investigador del área de entomología del Departamento de Agricultura de los Estados Unidos (USDA) (Kendra, et al, 2012;Kendra et al, 2014;Menocal, et al, 2018) (Tabla 2).…”

Rapid and cost‐effective detection of Fusarium euwallaceae from woody tissues

“…Recent developments for the diagnosis of F . euwallaceae using multiplex PCR provide visual diagnosis using gel electrophoresis without the need for DNA sequencing (Carrillo et al, 2020; Short et al, 2017). With the increased availability of numerous fungal genomes and transcriptomes in public databases, it has become more readily possible to identify novel primer pairs that target genes specific to certain species in silico.…”

Design of a diagnostic system based on molecular markers derived from the ascomycetes pan-genome analysis: The case of Fusarium dieback disease

“…In both cases, a small quantity of DNA is required during the assay. There are several PCR based methods to identify Fusarium species [17][18][19][20][21][22], including methods based on real-time PCR (qPCR) [23] moreover there are some recent approaches based on this technique for the detection of ambrosia fungi species. For example, Carrillo et al [23] developed a multiplex qPCR assay using hydrolysis probes for the detection of the b-tubulin gene.…”