Comparative analysis uncovers the limitations of current molecular detection methods for Fusarium oxysporum f. sp. cubense race 4 strains

Abstract

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) is threatening banana production worldwide. Despite quarantine efforts, the pathogen continues to spread; thus, early diagnosis plays an essential role for the proper execution of contingency plans. Here, we assess the accuracy of four PCR-based molecular methods described in the literature for the identification and detection of race 4 strains, including Subtropical (Foc STR4) and Tropical Race 4 causing Fusarium wilt of banana. We screened a total of 302 isolates using these four markers, and performed phylogenetic analyses, Vegetative Compatibility Group (VCG) testing, sequence comparison, and pathogenicity tests for selected isolates. Our results show that three out of the four markers tested are not reliable for identification of Foc STR4 and TR4, as DNA from isolates from Ecuador, pathogenic and nonpathogenic to banana, obtained from different banana cultivars, displayed cross-reaction with these methods; that is, false positives can occur during the diagnostic process for race 4. Phylogenetic analyses, VCG testing, sequence comparison, and pathogenicity tests suggest the presence of non-target F. oxysporum isolates that share genomic regions with pathogenic strains but lack true pathogenicity to banana. The findings of this work are of foremost importance for international regulatory agencies performing surveillance tests in pathogen-free areas using the current diagnostic methods. We suggest the use of a genetic locus possibly related to virulence, previously identified by T-DNA, and amplified with primers W2987F/ W2987R, for diagnosis of Foc TR4 as the most reliable alternative. We urge the adoption of a more holistic view in the study of F. oxysporum as a plant pathogen that considers the biology and diversity of the species for the development of better diagnostic tools.

Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water

“…Primers developed for VCG 01213/16 were specific to Foc TR4 and did not detect false positives previously reported for other TR4 primers by Magdama et al . (2019) [ 49 ] (results not presented). It also did not amplify other Foc strains causing disease to Cavendish bananas in the tropics, such as the closely related Foc VCG 0121 [ 31 ].…”

Genetic Diversity of Fusarium oxysporum f. sp. cubense, the Fusarium Wilt Pathogen of Banana, in Ecuador

“…EC5-LR-GM1 shared identical TEF sequences with pathogenic isolates from Ecuador in lineage I + IV; but it is closely related to TR4 based on IGS sequences. Previous research showed that EC5-LR-GM1 is not pathogenic to Cavendish ‘Gran Naine’ and ‘Gros Michel’, but along with other endophytes, it shares genomic regions similar to those found in TR4 strains that have been exploited for PCR diagnostics leading to false positives [ 50 ]. Another interesting isolate was EC10-B-GM3, sampled from asymptomatic ‘Gros Michel’, with unresolved phylogenetic placement.…”

First Comprehensive Report of Clinical Fusarium Strains Isolated in the State of Sao Paulo (Brazil) and Identified by MALDI-TOF MS and Molecular Biology

“…Molecular techniques are the most frequently identification methods because of species specificity of this region. The technique is known to provide good resolution at the sub-species level, and thus sequence analysis is a superior choice for phylogeny in Fusarium species complexes [13,36,37].…”

“…clinical isolates. In addition, ITS sequencing is known to provide good resolution and a superior choice for phylogenetic studies in the species complex [13,[37][38][39]. However, the high-throughput sequencing of Fusarium spp.…”