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

Abstract

Banana is an important food crop and source of income in Africa. Sustainable production of banana, however, is at risk because of pests and diseases such as Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). Foc can be disseminated from infested to disease-free fields in plant material, water and soil. Early detection of Foc using DNA technologies is thus required to accurately identify the fungus and prevent its further dissemination with plants, soil and water. In this study, quantitative (q)PCR assays were developed for the detection of Foc Lineage VI strains found in central and eastern Africa (Foc races 1 and 2), Foc TR4 (vegetative compatibility groups (VCG) 01213/16) that is present in Mozambique, and Foc STR4 (VCG 0120/15) that occurs in South Africa. A collection of 127 fungal isolates were selected for specificity testing, including endophytic Fusarium isolates from banana pseudostems, non-pathogenic F. oxysporum strains and Foc isolates representing the 24 VCGs in Foc. Primer sets that proved to be specific to Foc Lineage VI, Foc TR4 and Foc STR4 were used to produce standard curves for absolute quantification, and the qPCR assays were evaluated based on the quality of standard curves, repeatability and reproducibility, and limits of quantification (LOQ) and detection (LOD). The qPCR assays for Foc Lineage VI, TR4 and STR4 were repeatable and reproducible, with LOQ values of 10 −3-10 −4 ng/μL and a LOD of 10 −4-10 −5 ng/μL. The quantitative detection of Foc strains in Africa could reduce the time and improve the accuracy for identifying the Fusarium wilt pathogen from plants, water and soil on the continent.

Potential inoculum sources of Fusarium species involved in asparagus decline syndrome and evaluation of soil disinfestation methods by qPCR protocols

“…This result shows irrigation water to be another source of inoculum of pathogenic Fusarium species. In accordance with this result, rivers and irrigation systems have previously been identified as potential sources of inoculum for pathogenic fungi, 20,29 including Fusarium species 30,31 . In this scenario, monitoring for pathogenic Fusarium species in pre‐planting soil, starting plant material and irrigation water prior to the establishment of an asparagus crop is desirable to prevent ADS and replanting problems.…”

Nitrogen fertilizer rate but not form affects the severity of Fusarium wilt in banana

“…Foc abundance was quantified with absolute quantification-based qPCR as previously published (Matthews et al, 2020 ), except for the use of SsoAdvanced SYBR ® SuperMix (BioRad), which has been shown to increase reaction efficiency compared to other qPCR master mixes (Browne et al, 2020 ). Briefly, Foc-specific DNA sequences were targeted using the forward priming sequence (5′-GACATTTGACGACTTTCTGA-3′), the reverse sequence (5′-GACATTTGACGACTTTCTGA-3′) (Matthews et al, 2020 ). Each 10 μl total-volume reaction contained 5 ng of extracted DNA, 0.3 μM of desalt-grade primers (Sigma-Aldrich), 5 μl of SsoAdvanced SYBR ® SuperMix, and 2 μl molecular grade Ultra-Pure H 2 O™ (Invitrogen) water.…”

Genome Sequence Data Reveal at Least Two Distinct Incursions of the Tropical Race 4 Variant of Fusarium Wilt into South America

“…Presence/absence of SIX genes in the Colombian Foc TR4 genomes During the last ten years, several studies have developed molecular markers for detection of Foc TR4 (Matthews et al 2020;Magdama et al 2019;Ndayihanzamaso et al 2020;Dita et al 2010;Aguayo et al 2017;Lin et al 2013;Li et al 2013;Carvalhais et al 2019). This raises an important question: Are the existing molecular markers present in the Colombian Foc TR4 genomes?…”