A Molecular Assay Allows the Simultaneous Detection of 12 Fungi Causing Fruit Rot in Cranberry

Abstract

Cranberry fruit rot (CFR) is arguably one of the most limiting factors of cranberry (Vaccinium macrocarpon) production throughout its growing areas. The disease is caused by a group of closely related fungi that require identification using long and cumbersome steps of isolation and microscopic observations of structural features. The objective of this study was to develop a molecular assay to simultaneously detect and discriminate 12 of the most important fungal species reported to be pathogenic on cranberry fruit to facilitate the diagnosis of CFR. As the first approach, internal transcribed spacers and large subunit regions of all fungi were sequenced and confirmed with sequences available in the NCBI database. These data were used to develop primers able to differentiate seven of the 12 species. The five remaining species, including three in the Phacidiaceae family and two in the Glomerellaceae family, were differentiated on the basis of a more discriminant marker, the translation elongation factor 1-α. Two PCR reactions were optimized to clearly delineate the 12 species. The multiplex test was first validated using pure fungal cultures; it was subsequently validated using fruit collected in cranberry beds in eastern Canada. In the latter case, the test was rigorous enough to clearly discriminate the fungal pathogens from contaminants. Within the tested samples, Physalospora vaccinii and Coleophoma empetri were most commonly found. This molecular test offers scientists, diagnosticians, and growers a powerful tool that can rapidly and precisely identify fungi causing CFR so they can implement appropriate control methods.

Discriminant haplotypes of avirulence genes of Phytophthora sojae lead to a molecular assay to predict phenotypes

“…In recent years, molecular techniques have revolutionized and greatly facilitated the diagnostic of plant pathogens (Kostov et al ., ; Michalecka et al ., ; Reich et al ., ; Silva et al ., ; Burbank and Ortega, ). Among other applications, they have allowed the precise identification of the causal agents of diseases caused by a complex of fungi (Conti et al ., ), the fulfillment of Koch's postulates with closely related species such as Fusarium spp. (Moine et al ., ), and the diagnosis of the presence and identity of a pathogen from levels that were hitherto undetectable (Rollins et al ., ; Haudenshield et al ., ).…”

New real‐time PCR assay for detecting the blueberry and cranberry twig blight pathogen

“…vaccinii has been carried out by the isolation approach followed by morphological screening and sequencing or using an end-point PCR assay (Conti et al, 2019). In the present study, a new TaqMan probebased real-time PCR assay was developed for specific and sensitive detection of D. vaccinii targeting the TEF1 gene region.…”

“…A rapid increase in the movement of plant material worldwide has increased the need for rapid and efficient monitoring of imported plant material for unwanted plant pathogens. To date, the detection of D. vaccinii has been carried out by the isolation approach followed by morphological screening and sequencing or using an end‐point PCR assay (Conti et al, 2019). In the present study, a new TaqMan probe‐based real‐time PCR assay was developed for specific and sensitive detection of D. vaccinii targeting the TEF1 gene region.…”

“…Therefore, PCR‐based testing would be preferred for fast, reliable and efficient detection and identification of D. vaccinii . While a multiplex PCR assay, based on the internal transcribed spacer (ITS) and large subunit (LSU), is available for the detection of D. vaccinii (Conti et al, 2019), real‐time PCR (also referred to as qPCR) assays are more suited for faster sample processing in diagnostic laboratories (Amaral Carneiro et al, 2017).…”

Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant

“…The designated primers successfully amplified a 400 bp fragment from F. buharicum genomic DNA, allowing for the discrimination of F. buharicum from other fungal species. Several species-specific primers, utilizing this gene, have been developed and employed for detecting various plant pathogenic fungi, including Fusarium [10,[31][32][33]. The primer developed in this study demonstrated high specificity for F. buharicum and sensitivity, capable of detecting the pathogen at concentrations as low as 1.0 ng of fungal genomic DNA in conventional PCR assays.…”