Sampling and PCR method for detecting pathogenic Fusarium oxysporum strains in onion harvest

Abstract

Significance and Impact of the Study: Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTA TM technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the postharvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion. AbstractFusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onionpathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTA TM filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100 pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%.

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“…Raoudha et al (2012) performed amplifications from 10 ng of DNA of leaf plant Stipa lagascae. In our study, the DNA threshold are much higher than the one obtained by Latvala et al (2019) which is 100 pg of DNA of pathogenic F. oxysporum strains in onion harvest. This difference of detection threshold is linked to the dual PCR protocol, the used primers and the two types of DNA (DNA of onion and DNA of pathogenic F. oxysporum strains in onion) in the reactional medium that these authors used.…”

Pathogenicity of Fusarium oxysporum and Fusarium proliferatum isolates from symptomless onions (Allium cepa) and onions with Fusarium basal rot

“…Of the F. oxysporum isolates from Finland, the isolates causing FBR in onion were found to carry all seven SIX genes and the C5 gene, whereas none of those genes were detected in the nonpathogenic onion-associated F. oxysporum isolates (Haapalainen et al, 2022). In the study by Latvala et al (2020), the presence of the SIX3 gene in F. oxysporum isolates originating from different European countries was shown to correlate with pathogenicity to onion. Thus, SIX3 was chosen as a marker gene to detect latent infections with pathogenic F. oxysporum strains in harvested onions (Latvala et al, 2020).…”

“…FBR reduces onion yield and quality both during the growing season and in storage, frequently causing economic losses (Cramer, 2000). During several months of storage, latent Fusarium infections in healthy-looking harvested onions can develop into disease (Latvala et al, 2020). Latent infections in onion sets cause…”

A fast approach to discard false negative susceptible lettuce genotypes to Fusarium oxysporum f.sp. lactucae race 1

“…The possibility to use a molecular tool to shorten the field or greenhouse evaluation and discarding false negative susceptible lettuce can represent a step forward in obtaining the expected results. qPCR represents an efficient and less time‐consuming way to screen the resistance in lettuce cultivars as well as to evaluate the efficacy of fungicide treatments in early stages (Munkvold & O'Mara, 2002), and the levels of contamination of the seeds and of the soil (Pollard & Okubara, 2019) or latent infections of F. oxysporum strains to manage the post‐harvest losses in onion and for testing batches of onion sets (Latvala et al, 2019). Although an analysis of the costs needs to be done, the method can be modified and validated for other races of the same pathogen, with possible time saving in the screening process, allowing also the detection of the pathogen in plants not yet expressing typical symptoms of Fusarium wilt.…”